International Review of Cytology: A Survey of Cell Biology, Volume 48

INTERNATIONAL REVIEW OF CYTOLOGY VOLUME48 ADVISORY EDITORS H. W. BEAMS ROBERT G. E. MURRAY HOWARD A. BERN W. BERN...

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INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME48

ADVISORY EDITORS

H. W. BEAMS

ROBERT G. E. MURRAY

HOWARD A. BERN W. BERNHARD

ANDREAS OKSCHE VLADIMIR R. PANTIC

GARY G. BORISY

DARRYL C. REANNEY

ROBERT W. BRIGGS RENE COUTEAUX

LIONEL I. REBHUN JEAN-PAUL REVEL

MARIE A. DI BERARDINO

WILFRED STEIN

N. B. EVERETT

ELTON STUBBLEFIELD

CHARLES J. FLICKINGER

HEWSON SWIFT DENNIS L. TAYLOR

K. KUROSUMI MARIAN0 LA VIA GIUSEPPE MILLONIG

J. B. THOMAS

ARNOLD MITTELMAN DONALD G. MURPHY

ROY WIDDUS

TADASHI UTAKOJI ALEXANDER L. YUDIN

r

-.

INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

Yerkes Regional Primute Reseurch Center Emory University Atlanta, Georgiu

Worcester Polytechnic lnstitute Worcester, Massachusetts

ASS I STANT EDITOR K. W. JEON Depurtment of’ Zoology University of Tennessee Knoxville, Tennessee

VOLUME48

ACADEMIC PRESS New York

San Francisco London

A Subsidiary vf Hurcourt Bruce Jouanovich, Publishers

1977

COPYRIGHT 0 1977, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART O F THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC O R MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, O R ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC. 111 Fifth Avenue, New York,N e w

York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER:52-5203 ISBN 0- 12-364348-1 PRINTED IN THE UNITED STATES OF AMERICA

Contents LIST O F

CONTRIBUTORS

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ix

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1 2

Mechanisms of Chromatin Activation and Repression NORMANMACLEAN AND

VAUCHAN

A . HILDER

I . Introduction . . . . . . . . . . . . 11. Activation and Repression at the Level of the Whole Chromosome I11. Activation and Repression at the Level of Large Tracts of Chromatin IV . Chromosomes That Are Transcriptionally Active . . . . V . Activation and Repression of Euchromatin . . . . . VI . General Conclusions . . . . . . . . . . VII . Summary . . . . . . . . . . . . References . . . . . . . . . . . .

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LO 13 19 46 47 48

Origin and Ultrastructure of Cells in Vitro L . M . FRANKSAND

PATRICIA

D.

WILSON

. . . . . . . . . . . . I . Introduction . . . . . . . . I1. General Features of Cells itt Vitro . . . . . . . . I11. Special Features of Cells in Vitro . IV . Ultrastructure of Primary Explants and Epithelial Cell Strains from . . . . . . . . . Normal Epithelial Tissues . . . V . Ultrastructure of Mesenchymal Cells from Normal Tissues . . . VI . Ultrastructure of Cells from Brain and Hemopoietic Tissue . . . . . . . VII . Ultrastructure of Tumor Cells in Vitro . . . . . . . VIII . Ultrastructure of Cells in Organ Cultures . . . . . . . . . . . . . IX . Conclusions . References . . . . . . . . . . . . .

55 59 81 91 108 120 121 125 128 131

Electrophysiology of the Neurosecretory Cell KINJI YACI I. 11. 111. IV. V.

VI .

VII .

AND

SHIZUKOIWASAKI

Introduction . . . . . . . . . . . Identification of the NS Cell in Electrophysiological Studies . Electrical Properties of the Membrane . . . . . Characteristic Nature of Electrical Activity . . . . Role of Action Potentials in Endocrine Activity . . . Synaptic Control of the Hypothalamic NS Cell . . . Conclusions . . . . . . . . . . . References . . . . . . . . . . . V

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141 142 145 153 160 166 178 180

vi

CONTENTS

Reparative Processes in Mammalian Wound Healing: The Role of Contractile Phenomena GIULIOGABBIANI AND DENYSMONTANDON

. . . I . Introduction . I1. The Evolution of a Wound .

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. . 111. Epithelialization of a Wound IV. Pathology of Granulation Tissue and Fibromatoses . . . . . . . . . V . Conclusions . References . . . . . . . . .

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187 188 207 209 214 215

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221 226

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Smooth Endoplasmic Reticulum in Rat Hepatocytes during Glycogen Deposition and Depletion ROBERT R . CARDELL.J R. I . Introduction .

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I1. Structure and Function of the SER: A General Concept .

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111. Important Factors in Morphological Studies of Hepatic Glycogen . . . . . . . . . . . . Metabolism . . . . . . . IV . A Controlled Feeding Schedule for Rats . . . . . . V . Hepatic Glycogen Levels in Control-Fed Rats . . . . . . . . . . . VI . The Hepatic Lobule . . . . . VII . Hepatic Glycogen Patterns in Fasted and Fed Rats . VIII . Morphology of Hepatocytes during Glycogen Deposition and De. . . . . . . . . . . . . pletion . IX Fine Structure of Hepatocytes during Glycogen Deposition and Depletion . . . . . . . . . . . . . . X . Morphometric Analysis of Components in Hepatocytes during Glycogen . . . . . . . . . Deposition and Depletion . . . . . . . . . . . XI . Concluding Remarks . Appendix . . . . . . . . . . . . . References . . . . . . . . . . . . .

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234 236 237 238 241 246 247 266 268 271 274

Potential and Limitations of Enzyme Cytochemistry: Studies of the Intracellular Digestive Apparatus of Cells in Tissue Culture M . HUNDGEN

. . . . . . . . . I . Introduction . I1. The Influence of Fixation on the Localization of Enzymes . . . 111. Cytochemical Demonstration of Enzymes . . . . IV . The Intracellular Digestive Apparatus . . . . V . Limitations of Enzyme Cytochemistry . . . . . . . VI . General Conclusions . References

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281 282 290 295 314 317 318

vii

CONTENTS

Uptake of Foreign Genetic Material by Plant Protoplasts E . C . COCKING

I . The Isolated Plant Protoplast System I1 . Uptake of DNA and Viruses . .

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111. Uptake oforganelles and Microorganisms IV . Uptake as a Consequence of Protoplast Fusion References . . . . . . . .

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323 324 330 337 341

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The Bursa of Fabricius and Immunoglobulin Synthesis Hnum GLICK I . Introduction .

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I1 . Origin and Migration of Bursal Lymphocytes

111. IV . V. VI .

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Bursa Kinetics . . . . . . Characterizing Bursal Lymphocytes . . Bursal Regulation of Immunoglobulin (Antibody) Production Concluding Remarks . . . . . . . . . References . . . . . . . . . . .

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SUBJECT INDEX C ONT E N T S OF PREVIOrlS VOLUMES

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345 352 358 361 370 393 394

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403 406

This Page Intentionally Left Blank

List of Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

ROBERTR. CARDELL,JR. (221), Department of Anatomy, University of Virginia, School of Medicine, Charlottesville, Virginia

E. C . COCKING (323), Department of Botany, University of Nottingham, Nottingham, United Kingdom L. M. FRANKS(55), Department of Cellular Pathology, Imperial Cancer Research Fund, London, England GIULIO GABBIANI(187), Department of Pathology, Medical School, University of Geneva, Geneva, Switzerland BRUCE GLICK (345), P o u l t y Science Department, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, Mississippi State, Mississippi VAUGHAN A. HILDER (l), Department of Biology, Southampton University, Southampton, England M. HUNDGEN (281), Department of Zoology, University of Bonn, Bonn, West Germany SHIZUKOIWASAKI(141), Department of Physiology, Tokyo Medical College, Shinjuku-ku, Tokyo, Japan NORMANMACLEAN(l),Department of Biology, Southampton University, Southampton, England DENYSMONTANDON(187),Department of Surgery, HBpital Cantonal, Geneva, Switzerland PATRICIA D. WILSON (55), Department of Cellular Pathology, Zmperial Cancer Research Fund, London, England KINJI YAGI (141), Department of Physiology, Jichi Medical School, Tochigi-ken,Japan

ix


Mechanisms of Chromatin Activation and Repression NORMAN MACLEAN AND

VAUGHAN

A. HILDER

Department of Biology, Southumpton University, Southampton, England

I. Introduction . . . . . . . . . 11. Activation and Repression at the Level of the Whole . . . . . . . . Chromosome . A. Terminology . . . . . . . . B. Alteration in Chromosome Number. . . . C . Sex Chromosomes . . . . . . . D. Autosomes . . . . . . . . . E. T h e Mechanisms Involved . . . . . 111. Activation and Repression at the Level of Large Tracts of Chromatin . . . . . . . . A. Tracts of Constitutive Heterochromatin . . B. Tracts of Facultative Heterochromatin . . C . Mitotic Chromosomes . . . . . D. Position Effects . . . . . . . IV. Chromosomes That Are Transcriptionally Active . A. Giant Polytene Chromosomes . . . B. T h e Lampbrush Chromosome . . . C. T h e Chromomere Concept . . . V. Activation and Repression of Euchromatin . A. Levels of Template Restriction. . . B. The Transcription Mechanism . . . C. The Organization of the Genome . . D. Regulators . . . . . . . VI. General Conclusions . . . . . VII. Summary . . . . . . . . References . . . . . . .

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10 10 11 11 12 13 13 16 17 19 19 28 32 39 46 47 48

I. Introduction The purpose of this article is to consider the range of mechanisms implicated in the activation and repression of chromatin. Whereas the genomic DNA of prokaryotes is neutralized by relatively small molecules such as polyamines, the DNA of eukaryotes is complexed with relatively large amounts of basic protein, normally histone. The molecular complexity of eukaryotic chromatin, and the very large amounts of DNA in the genomes of most eukaryotic organisms, not only suggest that many mechanisms of gene control are unique to higher organisms, but also render the investigation of eukaryotic genetic regulation much more difficult. Although we are primarily 1

2

NORMAN MACLEAN AND VAUGHAN A. HILDER

concerned here with the eukaryotic situation, we refer frequently to observations and experiments on bacteria and viruses from which fundamental features of genetic regulatory mechanisms have emerged. We discuss gene regulation at the different levels at which the activation or repression seems to occur-the whole nucleus, the individual chromosome, the large block of genes, and the individual gene locus. Such an approach helps to reveal the great range of factors and processes involved in eukaryotic gene regulation. The subject is, at the moment, in a period of intense investigation and rapidly changing ideas. Techniques involving radioactive complementary DNA probes render the detection of specific mRNA molecules feasible, some regulatory molecules and control sequences have been characterized, at least in bacteria, and at last light seems to be dawning on the vexing question of the physical organization of DNA-histone complexes. It therefore seems useful and necessary to attempt to review what is known and what is thought in this area at this time.

11. Activation and Repression at the Level of the Whole Chromosome A. TERMINOLOGY An elaborate terminology has arisen in connection with the different functional and structural states of chromatin, and these we briefly discuss. A more extensive review of the topic can be found in Comings (1972). Probably the earliest and most widespread subdivision of chromatin into classes was that involving the terms euchromatin and heterochromatin. In fact, these descriptions were originally applied to whole chromosomes, the sex chromosomes being termed heterochromosomes and the autosomes termed euchromosomes (Wilson, 1925). The sex chromosomes were differentiated in this way because of their tendency to be heteropycnotic (differently stained) during meiosis. Usage of the term heterochromatin to delineate particular areas of chromosomes probably stems from Heitz (1928), who demonstrated that particular parts of individual chromosomes retain the property of an intense staining reaction throughout interphase. Most parts of the chromosomes stained in this way only at or about metaphase. In more recent times heterochromatin has been designated according to three separate criteria. They are: (1)Heterochromatin remains heteropycnotic throughout interphase; (2) heterochromatin is

CHROMATIN ACTIVATION AND REPRESSION

3

genetically inactive; and (3) heterochromatin undergoes DNA replication out of phase with the rest of the cellular DNA, and generally later in the cell cycle. These three criteria are discussed extensively by Comings (1972). By contrast, euchromatin is chromosomal material that does not have these characteristics. As we shall see, some of these characters are easier to measure than others, and the crucial observation about genetic inactivity is particularly difficult to determine. In particular, the question arises whether the genetic inactivity is temporary or permanent, reversible or irreversible. Some investigators have seen fit to use the word heterochromatin to designate material that fulfills only one or at most two of the three propositions listed. Thus most of the chromatin of mature lymphocytes and avian reticulocytes has been designated heterochromatin (Frenster et al., 1963) on the grounds that such chromatin is genetically inactive. If such cells are stimulated to pass through an S phase by the use of phytohemagglutinin or cell-cell hybridization, however, most of the intense staining property of the chromatin is lost, and therefore the first condition for the designation of heterochromatin is not met. It is much better to refer to the chromatin of such cells simply as condensed chromatin. Even when the term heterochromatin is used in its more useful and restricted sense, problems remain, however. Brown (1966) introduced the use of the terms constitutive and facultative heterochromatin, the former occurring on homologous portions of both homologous chromosomes while the latter normally occurs on only one of two homologs, for example, the inactivated human X chromosome in the female. Comings (1972) lists some characteristics of these two subdivisions of heterochromatin. Even this classification does not lead us out of the woods entirely, since the human Y chromosome has no homolog but is heterochromatic. It is not entirely or even mainly composed of simple sequence DNA and does not stain intensely with quinacrine dyes, yet it is constitutive in the sense that it is apparently incapable of reactivation. This problem has been approached by some investigators by using the term semifacultative heterochromatin (Comings, 1972) to describe such intermediate examples as the single X chromosome of certain grasshoppers and the Y chromosome of many male mammals. These chromosomes have chromatin which, although almost entirely genetically inert, is not simple sequence DNA. The present position can be summarized by stating that most constitutive heterochromatin consists of highly reiterated sequences of simple sequence DNA, some of which is located in the region of the centromere, and some of which is distributed throughout the chromosome (intercalary heterochromatin). Other chromatin, found chiefly

4


on certain sex chromosomes including the human Y, is constitutive in its permanence and inactivity but does not meet some of the other characteristics of normal constitutive heterochromatin, such as having an identical homolog and being simple sequence DNA. It may therefore be termed semifacultative. True facultative heterochromatin is present on only one of two homologous chromosomes and is not rich in simple sequence DNA. The human female inactive X is the prime example. All other chromatin is rather loosely referred to as euchromatin although, as we discuss later, many different kinds of organization are found in euchromatin. These include condensed mitotic chromosomes, condensed chromatin in such cells as nucleated erythrocytes, the extended chromatin of polytene chromosome bands and lampbrush chromosome loops, and the entire conglomerate of RNA genes, control genes, spacer sequences, and structural genes that makes up the bulk of most chromosomes and chromatin. Some of the criteria used in this evolved classification of chromatin seem to us to be somewhat irrelevant or confusing, in particular the question whether or not a chromosome has a homolog. We suggest that a more helpful use of the terminology would be to use only three terms-constitutive heterochromatin, facultative heterochromatin, and euchromatin. The first term refers only to a block of chromatin that is never used as a source of genetic information, and the second to a block of chromatin that is temporarily unused as a source of genetic information, but so used in some cells at certain times. Euchromatin is genetically and transcriptionally active chromatin. This usage, although simplified to exclude criteria of staining and time of replication, would be more useful than the present confused terminology. The discussion that follows does, however, utilize some of the existing forms of usage simply to render it presently understandable. We also refer in this discussion to blocks of chromatin. These may of course be entire chromosomes, as further discussed in this section, or quite small regions of chromosomes, even down to the level of the condensed chromomere.

B. ALTERATIONIN CHROMOSOME NUMBER The crudest level of genetic control is the elimination from a cell, or a line of cells, of whole segments of genetic material. Probably the most surprising conclusion to emerge from early studies on differentiation was that the genetic control of differentiation does not in general operate in this way. Many organisms do, however, alter the chromosome complement of cells and tissues, either by chromosome elim-


5

ination or by aneuploidy or polyploidy in certain differentiated cell types. Unequal or partial distribution of the genetic material can be achieved by varying the intervention of meiotic adjustment of chromosome number. Most groups of plants exploit the haploid-diploid alternation of generations in their life cycle to a much greater extent than do higher animals, and some simple plants such as mosses are haploid for the larger part of their life cycle. Even in animals the haploid-diploid difference in genetic complement can be utilized to provide variation in the form of the adult. Thus many hymenopteran insects, including the honeybee Apis, have evolved a system in which the males develop from unfertilized haploid eggs, while females develop from fertilized eggs. In some of these cases the male compensates for the lack of chromosomes by becoming a homozygous diploid, but in others the adult male is haploid in most tissues. Parallel examples of parthenogenesis are found among mites and rotifers. However, numerous organisms have an altered ploidy in only some of their somatic cells, such as the polyploid cells of mammalian liver and of many invertebrate somatic tissues and, in plants, the tetraploid cells in the root nodules of many members of the family Leguminosae and the polyploid cells in the roots of such plants as Allium. Besides cells and tissues having varying overall ploidy within one organism or among organisms, there are also examples of particular tissues or individuals being aneuploid, that is, having an irregular number of one particular chromosome. Thus, in many insects, sex determination is accomplished by the female possessing two X chromosomes and the male only one, no Y chromosome being existent (Lewis and John, 1968). The common mammalian variation in the X and Y chromosome complement is discussed in Section I1,C. We now turn to an examination of a few examples in which actual chromosome elimination is utilized to accomplish a particular genetic balance. In the fungus gnat, Sciuru, the entire paternal set of chromosomes is eliminated in the male, which therefore functions throughout its life with only maternal chromosomes (Crouse, 1943).The phenomenon is presumably related to male haploidy resulting from parthenogenetic development, alluded to above. Other examples of chromosome elimination are found in the nematode worm Paruscaris equuorum, and in the gall midges, Cecidomyiidae. I n the first example, the zygote possesses two very large compound chromosomes. As division proceeds, the germ line alone retains these large chromosomes intact; in the somatic cells they break up into many smaller

6


chromosomes, and large tracts of the terminal parts of the original chromosomes are eliminated from the cells, There is evidence that the eliminated chromatin consists of highly repeated DNA sequences (Moritz and Roth, 1976). A rather similar situation obtains in gall midges, where the germ line retains all the chromosomes but some are eliminated from the somatic cells (see review by Gurdon and Woodland, 1968). Chromosome elimination is also observed in artificially fused cells which are tetraploid following fusion, and the loss of particular chromosomes is often observed to be nonrandom (Migeon and Miller, 1968). All these examples involve actual elimination of chromosomes from the cells or altered chromosome numbers resulting from abnormal meiosis or fertilization. In most cases it is arguable whether the alteration in chromosome number is a device intended to accomplish differentiation, or whether the alteration is itself more a symptom than a cause of differentiation. It is noted that many of the cited examples of unequal chromosome distribution involve reproductive strategies or are used to accomplish sexual dimorphism. In any event it is an unusual mechanism, since most of the controlled gene expression displayed by living cells results from the selective activity of parts of the genome, either one or two copies of each gene and each chromosome being retained by every cell.

C. SEX CHROMOSOMES Although most cells retain all the chromosomes which they receive at mitosis or meiosis, many cells pemianently inactivate entire chromosomes. The best known example of such inactivation is the X chromosome of humans and most other mammals, one copy of which is permanently inactivated in the normal female (for review, see Lyon, 1974). The inactivation is paralleled by intense chromatin condensation. Although this system is found in almost all eutherian mammals and marsupials, in the latter the X chromosome derived from the father is preferentially inactivated (Cooper et al., 1971). XChromosome inactivation has not been observed in monotremes or in nonmammalian vertebrates. Two points are well established with regard to the X-chromosome phenomenon in mammals. The first is that, where inactivation occurs, it generally takes place early in embryonic life, but both X chromosomes are functional during the very earliest stages of female development. This fact is well demonstrated by the abnormality of Turner’s syndrome in humans, which is XO and would present as a typical female if only one X chromosome were active in all stages of normal development. [Actually, in the


7

mouse, the XO female has a normal phenotype, and females of the creeping vole (Microtus oregoni) are normally XO (Lewis and John, 1969),but these examples are exceptional.] The second point is that, with the exception of marsupials, the paternal and maternal X are inactivated randomly in early embryonic life, but clones of cells appear in later life, all with the same X chromosome inactivated. It then follows that animals heterozygous for genes carried on the X chromosome are natural mosaics in the female, and such indeed is conspicuously the case with the tortoiseshell cat, which is invariably female (Lyon, 1970). X-Chromosome inactivation in female mammals has been cited as an example of dosage compensation (Lyon, 1962) on the grounds that, if numerous X chromosomes are present in the karyotype, all but one will be inactivated. In other words, in both normal males and normal females, only one X chromosome is permitted to be active. Curiously enough, in triploids or tetraploids, more than one X may be active, suggesting that the dosage is related in some way to the overall chromosome complement. At the time of writing no really convincing mechanism for establishing the inactivation of X chromosomes has been discovered. There is some evidence suggesting that each X chromosome has one or more inactivation centers which control the activity state of the whole chromosome (Brown and Chandra, 1973; Eicher, 1970; Russell, 1964; Russell and Montgomery, 1965).Such genetic units on the X chromosome are then presumed to be sensitive to cytoplasmic signals determining the X-chromosome dosage. DNA methylation has been suggested as the means by which the inactivation centers are themselves turned off (Riggs, 1975). Once determined, the clones of cells that all have the same X active could result from the late replication of the inactivated X, causing its selective inactivation in each cell following mitosis. Whatever mechanism is involved, the inactivated X chromosome is an example of chromatin permanently condensed and heterochromatic through interphase, but capable of transcription and gene expression in some cellular situations, that is, facultative heterochromatin, as discussed later. As with position effects, discussed in Section II,D the heterochromatization of the inactivated X can repress the activity of genes translocated into its proximity (Eicher, 1970). It is interesting to ask whether, once inactivated, the X chromosome can be reactivated. Certainly, reactivation occurs normally during development of germ line cells in the female. But if cells are derived from females heterozygous for an X-linked deficiency, some of the cells will be phenotypically defective because the wild-type X is inac-

8


tivated. Cells suffering from such a deficiency in the enzyme hypoxanthine-guanine phosphoribosyl transferase cannot be grown in media requiring the wild-type enzyme. Such cells possess the gene in an inactive chromosome and apparently cannot reactivate it (Migeon et al., 1968). Reactivation of restricted portions of the heterochromatic human X has been detected, however, but only as a rare event, in clones of mouse-human cells hybrids (Kahan and DeMars, 1975). The Y chromosome is responsible for male determination in many species and is largely or entirely composed of constitutive heterochromatin, that is, chromatin that is not only condensed and transcriptionally repressed but has no apparent potential for being otherwise. The most completely heterochromatic Y chromosomes are found in such insects as Drosophila, in which species the males are XY, and XO flies are sterile males (see discussion in White, 1973).Spiders, nematodes, and some insects normally have XO males, the Y chromosome being entirely absent. More commonly, however, the Y chromosome is not entirely dispensable and possesses functional significance even if it contains no functional genes. Thus the human Y chromosome is maledetermining, XO individuals presenting as an abnormal female phenotype. Even in Drosophila individuals with extra Y chromosomes have a modified phenotype (Hess, 1970). Since the Y chromosome consists of permanent heterochromatin (as does a small part of the X chromosome), we might expect to find fundamental differences in base sequence or arrangement between Y chromosomes and autosomes. Such indeed is the ease, the Drosophila Y chromosome being particularly rich in AT sequences (Blumenfeld and Forrest, 1971).This finding has been confirmed by the quinacrine fluorescence of this chromosome in other insects (Ellison and Barr, 1972). Only part of the human Y chromosome fluoresces intensely with quinacrine-the distal part of the long arm [and interestingly enough, this identification hallmark indicates that the Y chromosome is often associated with the nucleolus (Bobrow et al., 1971)l.

D. AUTOSOMES Aside from the observations on sex chromosomes, there are two other well-known examples of entire chromosomes being heterochromatic. The first involves the mealybugs, Coccoidea. These insects, which are plant-feeding bugs belonging to the order Hemiptera, have ten chromosomes, all of which are active and euchromatic in the female, but of which five are heterochromatic and inactive in the male (Berlowitz, 1965). Moreover, it is the paternal chromosomes that are selectively inactivated and condensed in the male (Brown and Nur,


9

1964). Although the paternal set of chromosomes behaves in most tissues and most species as if it were entirely inactivated, and synthesizes no RNA, in certain tissues of some species such as the intestinal wall and Malphigian tubule cells, these chromosomes become euchromatic and fully active. Interestingly, the males of these mealybugs are rather small, fragile, and short-lived, which may result from effective haploidy. Attempts have been made to reactivate the heterochromatic mealybug chromosomes by exposure to hypotonic salt solutions. Although decondensation was observed, no increase in RNA synthesis was detected (Pallotta, 1972). The heterochromatization of these chromosomes is obviously facultative, but is none the less extremely stable and relatively resistant to reversal. Other chromosomes that may be entirely inactive are the supernumerary or B chromosomes encountered in maize (Carlson, 1969), rye (Jones and Rees, 1969), mealybugs (Nur; 1969),grasshoppers (Hewitt and John, 1970), and many other plants and animals. A review of B chromosomes has been written by Bataglia (1964). B Chromosomes are extremely variable in size, shape, number, stability, and phenotypic effect. No structural genes have been found to be active on these structures, but they influence such parameters as fertility and chiasma frequ.ency and distribution. Evidence has been presented that at least part of the B chromosomes of grasshoppers is made up of highly repeated sequences and so may be regarded as constitutive heterochromatin (Hewitt, 1972), but contradictory evidence also exists (Dover and Henderson, 1976), and certainly most of their DNA behaves as if it were unique sequence DNA. Since these chromosomes commonly seem to be genetically silent and condensed, we can assume that they are often entirely comprised of heterochromatin, part of which may be constitutive and part facultative. The relationship of B chromosomes to the other chromosomes in the cell is problematical. In plants they do not seem to be homologous to any existing chromosomes, but in some species of insects there is evidence suggesting their homology with sex chromosomes (see discussion in White, 1973). There is also evidence that sizable sections of the B chromosomes of maize may, at least in some tissues, be euchromatic. E. THE MECHANISMSINVOLVED I t is appropriate to conclude this section by pointing out that, at the level of the whole chromosome, distinct categories of chromatin can be recognized. They are: (1)constitutive heterochromatin which consists mainly or entirely of DNA with a highly repeated sequence, for example, Y chromosomes and some B chromosomes; (2) constitutive

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heterochromatin which is never active in any cell at any time, but apparently is not composed of simple sequence DNA, for example, some B chromosomes; (3) facultative heterochromatin which is active in at least some cells and therefore possesses many functional genes, for example, X chromosomes in most species and some autosomes, such as the inactivated autosomes of male mealybugs; (4) normal euchromatic chromosomes which may contain sections of' constitutive or facultative heterochromatin but also possess some transcriptionally active chromatin.

111. Activation and Repression at the Level of Large Tracts of Chromatin

A. TRACTSOF

CONSTITUTIVE

HETEROCHROMATIN

Most constitutive heterochromatin is now known to consist of highly repetitious simple sequence DNA (U'alker, 1971). Such DNA can be isolated from the bulk of the DNA as a satellite in density gradient centrifugation and has characteristics of rapid renaturation and sometimes of peculiar base composition (Rae, 1972).These properties have enabled sensitive radioactive probes to be employed in order to determine the chromosomal location of such chromatin (Jones, 1970; Pardue and Gall, 1970; Jones et al., 1974). Rapidly reannealing DNA (which of course, is not only simple sequence DNA) has been detected in many species of plants and animals, usually accounting for about 10-30% of the total DNA, but more in some organisms such as salmon (60%), onion (70%), and Amphiuma (80%).The chromosomal sites presently determined for constitutive heterochromatin are: (1) in the centromeric region; (2)around the nucleolar organizer region; (3)at or about the site of the 5s RNA cistrons; and (4) at the ends of chromosomes, where it is referred to as telomeric heterochromatin. In general, these localities are termed either centromeric or intercalary, the last category including all the other positions (see review by Yunis and Yasmineh, 1972). It seems unlikely that simple sequence DNA is normally transcribed in the living cell (see reviews by Yunis and Yasmineh, 1972; Rae, 1972). There are, however, no basic reasons why it should not function as a satisfactory template, as indeed it does for its own replication and in in vitro transcribing systems. It therefore follows that its transcriptional inactivity probably follows more directly from its physical packing in the chromatin than from its base composition and sequence.


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Despite many attempts (Walker, 1971; Britten and Davidson, 1971; Rae, 1972), no really convincing account of the function of simple sequence DNA has yet been proposed. A very useful discussion of the problem can be found in Swift (1973). Its very variability, even in closely related species, suggests a relatively unimportant role, and indeed some species of plants and animals are known that possess almost no constitutive heterochromatin, for example, Nigellu damascena (Natarajan and Ahnstrom, 1969) and the rodent Ellobius Zutescens (Schmid, 1967). Although truly constitutive simple sequence DNA is distributed throughout the chromosomes in relatively large blocks, semifacultative DNA is perhaps confined to the Y chromosome. Our ability to detect blocks of inert DNA that is not simple sequence DNA is probably not good, however, and the presence of such DNA, scattered throughout the genome, cannot be ruled out. Although we have already discussed such heterochromatin at the level of the whole chromosome, we should comment here that its genetic inactivity apparently does not stem from its base sequence but presumably from its packing characteristics. This being so, we should emphasize this apparent existence of DNA that is not exclusively simple sequence DNA but is nevertheless never transcribed in any cell at any time. B. TRACTSOF FACULTATIVE HETEROCHROMATIN

If it is accepted that essential features of facultative heterochromatin are a nonheterochroniatic homologous chromosome and late replication, there is no evidence indicating the existence of facultative heterochroniatin below the level of whole chromosomes. Therefore, although the mammalian X chromosome is now thought to possess multiple inactivation centers, there are no grounds for supposing that such inactivation centers are located singly on other chromosomes. The curious example of allelic exclusion in immunoglobulin synthesis might be construed as evidence suggesting limited inactivation of part of one autosome (see discussion in Section IV). C. MITOTIC CHROMOSOMES The point should be made here that there is one time in the cell cycle when all chromatin becomes transcriptionally inert, namely, mitosis. We are not suggesting that the chromatin of mitotic chromosomes be termed heterochromatin-that would simply confuse an overextended terminology further. But it is important to notice that the extreme condensation of chromatin that occurs at mitosis is also correlated with genetic inactivity. All euchromatin is therefore ca-

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pable of condensation into an inert form which, at least for that limited time, takes on some of the important characteristics of heterochromatin. Similar condensation also occurs in more permanent form when nuclei such as those of chicken erythrocytes become relatively inactive in transcription. Total genetic inactivity does not necessarily imply severe condensation of chromatin, and indeed in some examples the genetic shutdown appears to precede the visible condensation (Comings, 1966), but very frequently the two aspects are closely correlated. Moreover, decondensation of condensed chromatin does not necessarily induce reactivation (Pallotta, 1972).

D. POSITIONEFFECTS Some genes undergo changes in their expression if they are translocated to different positions on the same or different chromosomes. One of the earliest recognized examples of this phenomenon was the phenotypic appearance of the Drosophila eye, in which some facets are white rather than red, when one of the eye color genes becomes adjacent to heterochromatin (Lewis, 1950). The eyes of affected flies are actually variegated, some facets being red and some white, and this mode of expression seems to depend on a somewhat variable effect of the heterochromatin on adjacent genes. All the affected genes reside on the Drosophila X chromosome and display variegation as they approach the heterochromatic parts of this chromosome as the result of translocation. The effects do not demand direct proximity, since some of the affected genes studied by Lewis (1965) are actually quite distant from the heterochromatin. A more recent article by Cattanach (1974) explores the mechanism of inactivation involved in autosomal genes translocated onto the X chromosome of mice. We note that in many of these examples the visible phenotypic expression of the position effect is in terms of the activity of recessive genes. If a heterozygous animal has the dominant allele silenced in some cells by association with heterochromatin, then the recessive allele may be expressed in the phenotype of that cell. One of the problems of this system is that the variation in the cellular or clonal phenotype in affected individuals may arise either because of the random inactivation of one or the other of the two X chromosomes, or because of a variable capacity for inactivation on the part of the heterochromatic region. Indeed, both factors might sometimes act together (Cattanach et al., 1972). In Cattanach’s recent article (Cattanach, 1974) he distinguishes between these two aspects of genetic expression and demonstrates that inactivation by the heterochromatic region is indeed variable, both among cells and cell clones, and also,


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with time, within a single cell. In general, this work shows that position-effect variegation is, like X-chromosome inactivation, clonal. This implies that the inactivation of autosomal genes exerted by the heterochromatin is inherited by the daughter cells after mitosis of a similarly affected parent cell. Contrary to earlier ideas in which inactivation was thought to be highly stable, Cattanach (1974)now suggests that gene inactivation by heterochromatin association is inherently unstable, and that during the life of the organism the affected genes tend to recover their potential activity, commencing with those most distant from the heterochromatin.

IV. Chromosomes That Are Transcriptionally Active Perhaps the greatest impediment to studies on chromosome structure and function is that most chromosomes appear when they are relatively inactive-at mitosis-and disappear when they are active-during the rest of the cell cycle. There are no satisfactory ways of visualizing chromatin during interphase-in the light microscope, and electron microscopy of chromatin has not proved particularly useful. Fortunately, there are two special types of chromosomes that can be visualized while involved in transcription, and therefore their contribution to our knowledge of chromosomes has been enormous. These are the giant polytene chromosomes of insects, particularly those of the salivary glands ofDrosophila, and the lampbrush chromosomes of the oocytes of some vertebrates, especially those of the Amphibia.

A. GIANT POLYTENECHROMOSOMES As we have said, these structures are found in certain tissues of dipteran flies and persist through interphase. Their significance and general biology have been discussed by DuPraw (1970) and, more fully, in Developmental Studies on Giant Chromosomes (Beerman, 1972). We need only discuss here their particular contribution to our knowledge of chromatin organization and activity. The polytene chromosome is a multiple structure consisting of between 1000 and 4000 single chromatids lying side by side, with homologous portions in register. Each chromosome can be seen, even after only mild staining procedures, to have alternate dense and less dense regions along its length, these being termed band and interband regions. Bands are also referred to as chromomeres, and interband regions as interchromomeres. The combined chromosome set of

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Drosophila melanogaster displays at least 2000 separate bands and interbands, and the number for other differing species may be as high as 5000 (Beerman, 1972). Although the morphology of banding in these chromosomes has been studied almost exclusively in fixed material, there is no doubt that the same banding exists in living cells. Probably the most important single observation about these chromosomes is that, although the DNA molecules run through the entire length of the chromosome, they are much more condensed in the bands, and indeed 95% of the DNA content is contained in the bands and only about 5% in the interbands, although visually the latter appear to make up the greater part of the chromosome length. A second important observation is that the overall pattern of bands and interbands is the same in all tissues of the same organism and the same species. This implies that the folding of the extremely long strands of DNA cannot be random but must be rigidly determined by some feature of the chromatin, a consideration that strongly influenced the model we propose in Section V,C,3. The appearance of the same band varies, however, in different tissues. The variation involves what is known as puffing, when the condensed chromatin apparently unwinds in one band and the band appears swollen. Puffing is known to be strongly correlated with activity in RNA synthesis (Pelling, 1972), and indeed there is little evidence for RNA synthesis elsewhere in the chromosome (Pelling, 1964). Another important observation on the genetic significance of giant chromosome structure is that no more than one structural gene has so far been located in any one band-interband complex (one band plus one adjacent interband) (Hochman, 1971). The precise location of the structural gene within a band-interband complex is not known, but suggestive evidence exists that the gene is either in the interband region or part of the band immediately adjacent to it (Lefevre, 1971). Since each band-interband unit contains, on the average, about 20,000 nucleotide pairs of DNA (Beerman, 1972), this would provide adequate coding length for at least 20 cistrons, yet only one cistron is so far known to map in any one of these sections. It is therefore possible either that most or all of an interband region, which includes only about 1000 nucleotides on the average, is the structural gene, or that the gene is included within the more tightly packed chromatin of the band. Although some workers favor the idea that the structural gene is present in the interband region (Crick, 1971), most do not and, as we shall see, it is more probably located within the band itself. Now clearly the simplest possible view of the giant chromosome structure is that, taken together, a genome set of these chromosomes


15

includes between 2000 and 5000 structural genes, these genes being fairly evenly spaced along the lengths of the chromosomes and adjacent to almost 10 times as much other DNA, which may largely consist of control genes for the adjacent cistrons. Between these cistrons and control gene groups (the bands) are spacer regions of the DNA, which are interbands and are perhaps not transcribed. Such a concept of the giant chromosome (and, if these chromosomes are taken to be representative, of the chromosomes of all higher organisms) is still supported by only suggestive evidence and is flatly disbelieved by many. But it is probably a fair summary of the presently held view of the majority of geneticists and cell biologists. If it is true, even approximately, it raises several interesting questions related to chromatin activation and repression. The first concerns the function of the interband DNA. Being normally uncondensed, it appears to be available for transcription but is not transcribed. Is it the site of repetitive sequence DNA? Another question raised by this view of the polytene chromosome is why the supposed control gene area, the greater part of the chromosome, needs to be involved in such an escalation of its activities as puffing surely involves. Even if the structural gene is included in the chromomere and not the interband, why should the whole chromomere become decondensed? If the puff, and the RNA synthesis implicated in the phenomenon, involves even most of the chromomere, considerable transcriptional activity on the part of this DNA is implied. A common view of eukaryotic gene control is that the different control genes are not all active at the same time and that many do not function by producing a product but by being sensitive to the presence of a regulatory molecule. However, the heterogeneous nuclear RNA (HnRNA) that is the precursor of at least some of the mRNA, is very large and may well possess repetitious RNA at its 5’ end (Lodish et al., 1974). We return to discuss these interesting questions later. Before leaving the polytene chromosome, it should be pointed out that, if puffs represent active gene loci, the number of puffs may be taken to be roughly indicative of the number of genes active within one tissue. In Chironomus tentans, which possess a total of about 2000 bands, the salivary gland chromosome boasts only about 300 puffs (Pelling, 1964).This implies that only 15%of the genome is active in this tissue. An analysis in a different species, Drosophila hydei, suggested that only about 5% of all puffs are tissue-specific (Berendes, 1966), an observation that clearly places very close limits on the number of possible differentiated cell types-put at 120 by Pelling (1972).

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B. THE LAMPBRUSH CHROMOSOME These structures are essentially diplotene meiotic chromosomes and occur in the oocytes of many and perhaps all vertebrates. They have been intensively studied in the oocytes of newts and other amphibians and are discussed at length by DuPraw (1970) and Lewin (1974). It seems likely that their structure results from the demand for intensive transcription to coincide with meiotic division, as it must do in the rapid growth of the maturing egg. Each chromosome consists of an enormously long structure, up to 800 km long, made up of an extremely thin thread, actually the two DNA duplexes of the two chromatids lying side b y side, and a series of several hundred beadlike chromomers distributed along it. Each bead can in fact be resolved into two bits of chromomere, one for each chromatid, and from each of these chromomeres arises a thin loop of DNA. Loops from sister chromomeres are apparently identical, and so the entire chromosome seems to consist of a string of beads, each bead subtending a pair of identical loops to give the appearance of a Victorian lamp brush. The basic structure was originally discussed by Callan (1963). The similarity between the giant polytene chromosome and the lampbrush chromosome should be noted, in that the number of chromomeres-5000 in the lampbrush haploid set as suggested b y Callan-is roughly similar in the two structures (Callan, 1963). Such a small number of genes may seem surprising to many, but certainly satisfies the important observation of Muller (1967) and Ohno (1971) on the evolutionary consequences, in terms of mutational load, of the size of the genome. An early suggestion of Callan’s was that the DNA in the loops moved, being spun out from one end of the chromomere and presumably spun in by the other. The evidence that suggests such a mechanism is, first, the sequential labeling of some giant loops in these chromosomes (Gall and Callan, 1962)and, second, the observation that the protein and RNA associated with a loop was much more abundant at one end than at the other (Miller, 1965). We are inclined to take the view that movement of DNA in the loop is an unnecessary postulate and that the pattern of incorporation of label is a function of the movement of RNA polymerase molecules and their product. Although it is clear that a segment of condensed chromatin (chromomere) persists at either end of the lampbrush loop, this is not known to be the case for the puffed band of the giant polytene chromosome-the puffs, at least visually, appear commonly to involve all the chromomeric DNA in a decondensed extended form.


17

Another remarkable difference between the two chromosome types is that, while only a few bands are puffed in any giant chromosome set at one time, all lampbrush loops appear to be active in transcription. This has never been adequately explained, but is presumably a peculiarity of the developing oocyte, implying that at this stage in development all or most genes are transcribed. This curious state of generalized transcriptional activity during meiosis has been discussed by Edstrom and Lambert (1975). They propose that this transcription during meiosis involves production of a special class of RNA, complementary to sequences not transcribed at other times and also not translated. The function of this special RNA is visualized by these investigators as being evolutionary, consisting of “copies of genes from homologous chromomeres with which the chromomere has been paired during previous generations.” This theory seems to us implausible simply on the grounds that it would yield hybridization data for, say, the globin gene suggestive of multiple copies. In other words, it is a return to a form of the master-slave model of the genome, with the difference that the slaves are unrectified but silent. It can be simply stated that both polytene and lampbrush chromosomes can be made to fit roughly into the same model (assuming that the structural gene is included in the lampbrush loop and the puffed band), but that in the lampbrush chromosome all genes appear to be active but only in a section of each chromomere, while in the giant polytene chromosome only a few genes are active, but the entire chromomere is involved in such localized activation. Some possible conclusions from this model of the chromosome are: First, the interchromomeric DNA is not condensed but is also not known to be transcribed. Is is constitutive heterochromatin of simple sequence DNA? Second, activation and decondensation seem to be closely linked phenomena in the chromomeres. Third, the distribution of chromosomal protein is not even but is probably chiefly associated with the chromomeres and their extended loops or puffs. This latter conclusion is certainly valid for the lampbrush chromosome (Miller, 1965).

C. THE CHROMOMERE CONCEPT Before leaving the topic of chromosome structure and its implications for gene activity, it is appropriate to ask how easy it is to apply this model of the chromosome to mitotic or interphase chromatin. Midmitotic chromosomes are not noticeably subdivided into chromomeres, and the banding pattern that can be induced or resolved in

18


such chromosomes by treatment with trypsin or acid-saline, followed b y Giemsa staining (Evans, 1973), is different and at a much grosser level than the chromomere distribution already discussed. But chromatin during the leptotene stage of meiosis resembles strings of beads, and indeed the early proposal of Belling (1928) that chromomeres are in fact genes was based on observations on such chromosomes. There is also evidence suggesting that the chromomere may be the chromosomal unit of replication (Pelling, 1966; Huberman and Riggs, 1968) and of recombination (see discussion in Whitehouse, 1973), and some suggestion that even Giemsa banding of extended chromosomes may be related to chromomere distribution (Okada and Comings, 1974; Bigger and Savage, 1975). In brief, there is evidence drawn from many sources that eukaryotic chromatin is often or always organized into chromomeres and that this level of organization is highly significant in terms of the distribution of structural genes and mechanisms of transcription, replication, and recombination. The notion that the chromomere is a universal characteristic of eukaryotic chromatin with a key role in the regulation of transcription is attractive, but not without difficulties. One has recently been highlighted b y Vlad and Macgregor (1975) in their studies on chromomere number in the lampbrush chromosomes of three species of American salamander. These three species are closely related but have very different C values. The number of chromomeres in the chromosomes of these three species has been estimated from light microscope observation and is found to be proportional to genome size. As Vlad and Macgregor (1975) point out, it is scarcely conceivable that the number of structural genes is also proportional to the genome size in these three closely related species. These observations are highly significant, indicating that the chromomere is indeed the pattern in which eukaryotic DNA is normally packaged, even if it is redundant. This does not imply that the chromomere is without a function in transcription, but rather that its function is chiefly structural and that chrornomeres that do not house structural genes may often exist-as may also chromomeres with more than one structural gene. This evidence reinforces our model of chromomere structure and function (see Section V,C,3) as consisting largely of repetitive sequences which exert control over transcription by their structural rearrangement. To quote from Vald and Macgregor (1975), “These sequences can change radically without affecting the expression of neighboring genes that are translated into functional polypeptides.”


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V. Activation and Repression of Euchromatin A. LEVELSOF TEMPLATE RESTRICTION Even within the euchromatic portion of the genome, w e can distinguish template restriction occurring at several different levels and involving a variety of mechanisms, some of the more important of which are outlined-below. 1. Untrunscribed DNA Part of the DNA in chromatin consists of sequences that may never be transcribed in uiuo. Geldennan et al. (1971) found that only 8% of the genome was complementary to the rapidly labeled RNA from whole neonatal and fetal mice. Allowing for possible underestimation of the degree of hybridization, they suggested that transcription occurred from up to 25% of the genome. This is considerably greater than the proportion of the genome required to code for structural genes. The reason for this excess transcription, including the possibility of its involvement in the control of specific gene activity, is discussed later. It is clear, however, that even in this situation, where we would expect the majority of genes to be active in one cell or another, a large part of the DNA apparently remains untranscribed. Into this category of untranscribed DNA fall the sequences of satellite DNA (Flamm et al., 1969), the inactive spacer regions in nucleolar genes (Miller and Beatty, 1969)and between histone genes (Birnstiel et al., 1971), and probably the spacer regions in the interbands of polytene chromosomes (Pelling, 1972). The antimessage strand of the DNA of structural genes is also probably not transcribed. In the experiments of Wilson et al. (1975), transcripts of the antimessage strand of rabbit globin genes could not be detected in erythroid cells by hybridization techniques. It is most unlikely that the antimessage strand could carry a transcriptionally useful base sequence and at the same time be complementary to a gene sequence. The transcription of these strands could be prevented simply by the omission of promoter and initiator sequences from them, which, as a result of the degeneracy of the genetic code, would put little constraint on the coding potential of the message strand. Although these sequences are not transcribed in uiuo, they form satisfactory templates for RNA polymerase in in uitro RNA-synthesizing systems (Jones, 1970; Reeder, 1973).In the case of ribosomal cistrons, the spacer sequences and the antimessage strands are transcribed by Escherichia coli polymerase, but the “correct” strand is still preferen-

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tially utilized. A similar situation is found in the transcription of rabbit globin genes by a eukaryotic polymerase, as well as by the bacterial enzyme (Wilson et al., 1975). DNA has functions other than acting as the template from which the genetic information of the cell is transcribed. It is concerned with the replication of this information for transmission to future generations of cells, and with the maintenance of its integrity within the cell nucleus. We might therefore expect there to be sequences within the DNA that are devoted to these other essential functions and that it would be at least wasteful, and probably undesirable, to transcribe them. The in vivo transcription of these sequences is probably prevented b y some feature of their base sequence, which automatically makes them inaccessible to RNA polymerase in the nuclear milieu, perhaps through their assumption of an unusual secondary structure, through a special type of interaction with nuclear proteins, or simply through the absence of promoter sequences within them.

2. Restriction by Gross Chromutin Condensation The repression of the genetic activity of chromatin that is in a highly condensed state and is associated with mitotic chromosomes and heterochromatin was discussed in a previous section. We have also commented on the fact that specific decondensation seems to be required for the transcription of chromomeres in polytene chromosomes. We believe that the chromomeric organization observed in polytene chromosomes probably applies to all euchromatin, and that chromomeres containing inactive genes are normally condensed (although not to the extent seen in mitotic chromosomes), this condensation itself providing an additional measure of control (see Section V,C,3). The chromatin of nucleated erythroid cells becomes increasingly condensed as they mature, and transcriptional activity declines to an unusually low level. It is probable that this condensation causes a relatively nonspecific repression of all genetic activity in these metabolically inert cells. Decondensation is not enough in itself to cause any major increase in transcription, either in erythrocytes (Hilder and Maclean, 1974) or in facultative heterochromatin (Pallotta, 1972), although it may be an essential preliminary to the reactivation of genes (Harris, 1967; Leake et al., 1972). Histone H1, and the tissue-specific H 5 in erythrocytes, are strongly implicated in chromatin condensation (e.g., Bradbury et al., 1973; Billett and Barry, 1974).There are strong grounds for believing that these histones do not form part of the main histone complexes around which the DNA coils (see Section V,D,l), but are attached to the outside of


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the histone-DNA subunits. Condensation probably results from an alteration in the conformation of these histones, permitting a more intimate H1-H1 interaction and thereby pulling together the regions of DNA to which each is attached. It is easy to envisage that such condensation renders initiation sites inaccessible to polymerase enzymes and/or blocks movement of the polymerases along the template. It has been proposed that condensation into mitotic chromosomes is initiated by the phosphorylation of histone H 1 (Bradbury et al., 1974). Maintenance of the condensed state, however, probably involves other factors stabilizing the H1-H1 interaction, or the partial replacement of H1 with H 5 in erythrocytes. There may be certain genes, particularly those whose products are required in almost all cells, for which this is the only repression mechanism. There are certainly few other situations in which the major ribosomal genes are inactivated. There are, however, some genes that seem to be resistant to repression even by this means, particularly those coding for certain low-molecular-weight nontranslated RNAs (Zylber and Penman, 1971; Maclean et al., 1973).

3 . Tissue-Specific Restriction The expression of particular parts of the genome in some cells, and different, although sometimes partially overlapping, parts in other cells is the chief mechanism in the process of cellular differentiation. It is probable that such differences in expression are largely the result of regulation at the level of transcription, although control of the later stages of gene expression plays a part in some situations. I n many cases, a cell becomes absolutely committed to develop in a particular direction long before overt signs of differentiation are detectable, a process known as determination. A striking feature of this level of gene control is its permanence. Once established, the determined fate is passed on in a stable form through many cell divisions. This is well illustrated by the experiments of Hadorn (1966) on the imaginal discs of Drosophila larvae. The excised discs can be passed through several hundred generations in the hemocoel of adult insects. Differentiation is arrested under these conditions but, on reimplantation of the discs into a larva, they differentiate into the tissue characteristic of the area from which they were originally taken. The process of differentiation can be reversible under certain circumstances. Thus entire carrot plants can b e grown from individual isolated phloem parenchyma cells (Steward, 1958), entire Xenopus can be grown from isolated intestinal epithelial cell nuclei injected into enucleated Xenopus eggs (Gurdon, 1970), and the relatively inac-

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tive nucleus of the chicken erythrocyte can be induced to synthesize large amounts of RNA b y its incorporation into a heterokaryon with an active cell (Harris, 1967). These are responses to highly traumatic and unnatural conditions, and in each of these cases some doubt has been raised as to the extent to which the competent cells are truly differentiated. Certainly, the differentiated state appears to be stable and irreversible under normal conditions. Some aspects of the relationships between, and the stability of, determination and differentiation are illustrated by experiments using the thymidine analog 5-bromodeoxyuridine (BUdR). Incorporation of this nucleoside into DNA appears to inhibit the expression of tissuespecific characteristics at levels that do not affect total DNA, RNA, and protein synthesis (see Wilt and Anderson, 1972). If it is administered during early embryogenesis, presumably prior to a crucial determinative event, complete failure to develop mature differentiated characters will occur. Once cells are determined, however, incorporation of BUdR may inhibit terminal differentiation, but this inhibition is reversed by the replacement of BUdR by thymidine. At some time before the onset of terminal differentiation, cells may become refractile to inhibition by BUdR, even though division, and therefore incorporation of the nucleoside into DNA, may continue. The effects vary considerably, depending on the system used; in some systems, notably those involving glycoproteins (see Levitt and Dorfman, 1974), BUdR may reversibly suppress the expression of an already attained differentiated state. Much of the variability in results is probably due to the fact that the substance has an effect at several different levels, including the cell surface and certain metabolic pathways-particularly those involving glycosyltransferases (Rogers et al., 1975)-as well as in DNA, where it appears to cause increased condensation and alterations in protein binding (Lapeyre and Bekhor, 1974). Once it is clear where the primary site of BUdR action is in a particular system and what the molecular level mechanism is, it should provide a very useful tool for elucidating the regulatory interactions involved in differentiation. During the process of development and differentiation it seems that, as the initially pluripotential cell divides, its daughter cells become progressively more firmly committed to a particular fate, involving a progressive loss of plasticity. Although there are estimated to be about lo4to lo5 structural genes in the eukryotic genome (e.g., Ohta and Kimura, 1971), the end result of differentiation is the production of at most a few hundred different kinds of tissue, even in the most complex organisms. Such considerations led to the proposal that


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the tissue pattern of differentiation is achieved by control of the activity of a relatively small number of genes, for which we propose the term tissue-master genes; the products of such genes would control the activity of large sets of structural genes. Some tissue-master gene products would have a detectable effect only after their interaction with some other factor such as steroid hormones; neither specific gene regulator molecules nor secondary effector molecules would have any genetic effect on their own, and secondary effector molecules would affect only those cells in which the appropriate tissue-master gene product was present. Such a mechanism would provide a basis for the process of determination. An absolutely essential feature of such tissue-master genes is that their regulation must differ from that of most other genes in that it cannot be achieved by a product of the genome. Their activity must be determined by some external effectors which have or acquire asymmetric distribution during the development of the organism. Our tissue-master genes are in many respects similar to the integrator genes proposed by Davidson and Britten (1973)in their model of eukaryotic regulation. These investigators, however, stress the coordinated action of integrator genes in response to external signals for the control of inducible genes. This is an aspect of genetic regulation that has been greatly overemphasized compared to the relatively permanent activation and repression that characterize cell differentiation. Thus it is important to stress, both in nomenclature and description, the overriding role of tissue-master genes, their limited variety, and their extremely stable state once they are turned on or off. Thus there must exist a mechanism for the selective and relatively permanent activation or repression of particular genes. In the case of tissue-master genes this mechanism must involve external effectors, and in the case of most other genes it would involve one or more tissue-master gene products, the effects of which would in some cases be sensitive to modification by external effectors. It is impossible, based on the currently available data, to assess the relative importance of mechanisms of positive and negative control in eukaryotes. This problem is frequently confused by failure to distinguish between control of transcription from DNA, which is a relatively good template, and control of transcription from chromatin, which has a much lower intrinsic template activity. As the natural template in eukaryotes is in the form of chromatin, it has been popular to suppose that the major control mechanism must be by activators acting selectively on specific tracts of repressed DNA. This, however, presupposes that all regions of the DNA in chromatin are inhibited to the same extent, and by the

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same nonspecific means, unless specifically activated. It may be significant that most prokaryotic regulator molecules, and the only one so far purified from a eukaryote (Crippa, 1970), are specific gene repressors. The problem of the nature and mode of action of regulator molecules and effectors is discussed further in Section V,D.

4. Temporal Restriction within a Cell A further level of restriction is that at which particular genes may be active in a given cell at certain times but not at others, usually in response to a specific signal. This is presumed to be a phenomenon distinct from the nonspecific repression of gene activity during times of major chromatin condensation such as during mitosis and the latter part of the final interphase of erythrocytes. Induction of the lac operon in E . coli by lactose and its analogs is an example of this type of regulation in prokaryotes, but examples in eukaryotes are rather more difficult to find. Perhaps the best examples are the responses of certain target tissues to hormones. In several such systems, alterations in the overall rate and specificity of transcription have been shown to be a basic feature of the response to hormone treatment (see Tata, 1966; Tomkins and Martin, 1970). The response frequently involves the activation of specific genes, and the specificity of the response is dependent on the prior determinative differentiation of the cell. A question that applies to any of the levels of control we have discussed, but which becomes particularly acute here, is whether control is achieved by simple on-off switching or by modulation of the rate of transcription. There are, unfortunately, very few cases in which the primary product of a particular gene can be identified (by virtue of some unique feature of its structure or our ability to purify it and therefore prepare a complementary DNA probe), and therefore in which the activity of a particular gene can be followed against the background of the overall genetic activity of the cell. One such case is rRNA and tRNA genes. The rate at which rRNA and tRNA are produced varies in certain situations (Scharff and Robbins, 1965; Miller, 1967), and the stimulation of rRNA synthesis during hormone induction is associated with an increase in the activity of nucleolar RNA polymerase (Lukacs and Sekeris, 1967; Yu and Fiegelson, 1970). The presence of multiple copies of these genes, however, means that we cannot distinguish between alterations in the overall rate of synthesis due to alterations in the number of unmasked copies of the genes or the rate at which they are transcribed. In recent studies of the induction of ovalbumin synthesis in the oviduct by estradiol, ovalbumin mRNA was undetectable in the unin-


25

duced cells (Cox et al., 1974; O’Malkey et al., 1975).It is of course impossible to eliminate entirely the possibility that ovalbumin mRNA was produced at a very low basal rate in the uninduced cells. On the whole, however, the data tend to suggest that genes are effectively either “on” or “off” in a particular cell. Different genes are clearly transcribed at different rates, as is shown by autoradiographs of Drosophila polytene chromosomes labeled with tritiated uridine. Such autoradiographs demonstrate that a chromosome has many more transcriptionally active sites than visible puffs, and that the activity of these sites is extremely variable, some being very heavily labeled and others only slightly (Zhimalev and Belyaeva, 1975). Most genes that are active appear to be transcribed throughout interphase, but the activity of certain genes may be linked to a particular phase of the cell cycle. This would clearly be advantageous where the gene product is essentially involved in only a small part of the cell cycle, such as DNA synthesis or mitosis. The best example of this type of temporal control is provided by histone genes, whose expression is confined to the limited S phase of the cell cycle (Bloch et al., 1967; Robbins and Borun, 1967). Histone mRNA is detectable in the cytoplasmic RNA of HeLa cells only during S phase (Stein et al., 1975). Even in this case, and more so in studies on the variation in enzyme activity during the cell cycle (see Halvorson et al., 1971),the possibility of a posttranscriptional control mechanism has not been entirely excluded. Halvorson and his colleagues (1971) have proposed a model to explain the periodic control of transcription based on the linear reading of genes in the order in which they occur in linkage groups. RNA polymerase is assumed to move along the chromosome, and each gene is transcribed, or transcribable, only at that point in the cell cycle when the polymerase reaches its locus. Some support for this model has been found in prokaryotes and budding yeasts, but its applicability to higher eukaryotes, where gene loci are widely spaced and RNA polymerase relatively abundant, is questionable. An alternative model is that of oscillatory repression, reviewed by Mitchison (1971). This model is based on end product inhibition of transcription, whereby a gene not specifically repressed by some other means remains active only as long as its product is removed by involvement in other metabolic processes. Again, this model was developed to account for certain features of gene expression in prokaryotes and lower eukaryotes, and its applicability to higher eukaryotes is difficult to test. End product inhibition provides an excel-

26


lent method of coordinating the synthesis of individual components of a multimeric complex, and the relationship between the synthesis of the various components of ribosomes may be explained in these terms. The induction of enzymes not linked to the cell cycle is probably best explained by a mechanism similar to that in the lac and ara operons of E . coli (see Section, V,B,2).

5 . Amplification and Magnification of Genes As pointed out in Section II,B, alteration in the DNA content of a cell is a relatively uncommon phenomenon. The only examples in which it is known to occur at a level finer than those already discussed concern ribosomal cistrons. There is a dramatic increase in the number of copies of the major rRNA genes during maturation of the oocyte in a wide range of animal species (see Birnstiel et al., 1971). These additional rRNA cistrons are located in numerous nucleoli, many of which are apparently quite free from the chromosomes, and disappear very early during embryogenesis. The other example in which the number of copies of these genes is altered involves the bobbed mutants of Drosophila (Ritossa, 1973). These mutants have a partial deletion of the rRNA gene block, but the progeny of flies that are homozygous for the deletion show a high proportion of apparently spontaneous reversion to the normal wild-type rRNA gene number (hence the term magnification). A related rectification of the number back to normal occurs in flies with increased rRNA due to genetic duplication of the nucleolar region. The mechanism by which specific regions of the DNA can be amplified or deleted is unknown. It has been suggested that an RNAdependent DNA polymerase (reverse transcriptase) may be involved in gene amplification (Crippa and Tochinni-Valentini, 1971), but the original experiments have not been satisfactorily substantiated. Amplification has been observed in no other genes, not even in 5s rRNA cistrons. It is one of several respects in which ribosomal genes are atypical, although some of the other features, such as multiple copies and a specific RNA polymerase, are found in other genes with nontranslated products or which are subject to transient high demands. These unusual features are probably determined by two main factors: the almost universal requirement for these gene products, and the fact that opportunities for posttranscriptional control are much more limited with nontranslated transcripts. In connection with the first point, it may be that control via an independent RNA polymerase is more appropriate for these genes than mechanisms involving tem-


27

plate repression. It is worth noting that bobbed Drosophila with only half the normal number of rRNA genes produce normal amounts of rRNA, suggesting that normally the nucleolar RNA polymerase level may be limiting, and that stimulation of the activity of this enzyme is frequently an early event in hormone induction. In connection with the second point, it is noted that a transcript from a ribosomal cistron yields only one molecule of each of its final products, whereas, for example, a molecule of ovalbumin mRNA is translated about 5 X 10" times on the average (O'Malkey et al., 1975). Posttranscriptional control mechanisms may play some part in the regulation of nontranslated RNAs; the maturation of the 45s rRNA precursor can follow at least two distinct pathways which are of different importance in different situations (Purtell and Anthony, 1975), but this is unlikely to be a major factor in determining the quantity of these molecules in the cell.

6. Allelic Exclusion A final level of control which we consider is that in which only one of a pair of alleles at a particular locus is expressed. This is termed allelic exclusion and is known to occur only in genes on the X chromosome during X inactivation (see Section II,A), in immunoglobin genes (see reviews by Hertzenberg et al., 1968; Stevenson, 1974), and perhaps in ribosomal genes of interspecific Xenopus laevis-Xenopus mulleri hybrids, (Honjo and Reeder, 1973). Although few examples are known, and some genes definitely do not demonstrate allelic exclusion (e.g., both p and S alleles of human /3 globin are expressed in all erythrocytes of individuals heterozygous for the sickle cell gene), there are relatively few loci at which it has been sought, and so it may be a rather more widespread phenomenon than is generally believed. I n true allelic exclusion, as seen in the X chromosome and immunoglobulin system, the choice of which allele is expressed in a particular cell appears to be random. Once established, however, it is stably passed on in cell division, There is as yet no satisfactory explanation of this phenomenon. In their study on the expression of the ribosomal genes in hybrid Xenopus from reciprocal X . laevis x X . mulleri crosses, and crosses with X . laevis carrying a rDNA deletion, Honjo and Reeder (1973) demonstrated that the presence of X . laevis rDNA permanently represses the expression of X . mulleri rDNA. This differs from the classic examples of allelic exclusion in that it is always the same allele that is expressed, and thus is explicable in terms of a greater sensitivity ofX. mulleri rDNA to some inhibitory factor, possibly rRNA itself.

28


B. THE TRANSCRIPTION MECHANISM

1. TheEnzyme In order to develop ideas on how transcriptional control might be achieved, it is necessary to understand something of the enzymic machinery involved. The details of the reactions involving the RNA polymerase of E . coZi are much better known than in any eukaryotic system (see Kornberg, 1974), but there is no reason, based on the currently available evidence, to suppose that the situation is fundamentally different in eukaryotes. Perhaps the most important steps in the reaction at which control can be exerted are the binding of the enzyme to DNA and the initiation of transcription, since this is where selection occurs of those parts of the genome that which are to be transcribed and, in a steady state, the rate of completion of transcripts depends solely on the rate of initiation. RNA polymerase binds with a low affinity to any DNA (Richardson, 1966; Jones and Berg, 1966) but, under appropriate circumstances, binds a few sites, the promoters, with a very high affinity 1970; Hinkle and Chamberlin, 1970). Interaction of the (Zillig et d., polymerase with a promoter is a prerequisite for the initiation of RNA synthesis on native DNA templates, although these two functions are not always closely linked spatially (Blattner et d.,1972). Thus the inactivation or masking of promoters and the alteration of the promoter specificities of the polymerase are among the simplest methods of modulating transcription. The discovery of the sigma factor, which stabilizes the promoter-polymerase complex in E . coli, led to the proposal that transcription can be controlled by a series of such factors with different promoter specificities. The search for heterogeneity of sigma factors has, however, yielded negative results (Bautz and Bautz, 1970), which is perhaps not surprising since the sigma factor appears to function by decreasing the affinity of the core polymerase for bulk DNA rather than increasing it for promoter DNA (Mueller, 1971).There are, however, many instances in which initiation specificity in prokaryotes is modulated by the interaction of auxiliary factors with the core polymerase or by the modification or replacement of certain core polymerase subunits. In general, control at this level appears to be rather crude, such as the distinction between rRNA and mRNA synthesis (Travers and Buckland, 1973) and during the major and irreversible changes in gene expression accompanying sporulation and bacteriophage infection (see Travers, 1970). Most eukaryotic cells have multiple nuclear RNA polymerases


29

which can be distinguished from one another on the basis of several criteria, such as their binding to ion-exchange columns, ionic optima, thermolability, and sensitivity to the fungal toxin a-amanitin, but probably the essential differences between them reside in their different nuclear localization and template specificities (see Biswas et al., 1975). RNA polymerase type A is localized in the nucleolus, whereas types B and C are bound to the nonnucleolar chromatin and free in the nucleoplasm (e.g., Tata and Baker, 1974). Type-A polymerase is responsible for the synthesis of rRNA, type B for DNA-like RNA, and type C for low-molecular-weight (4and 5 s ) RNA (Price and Penman, 1972). There appears to be inore than one type of RNA polymerase C (Hossenlopp et al., 1975), so it is possible that there is a separate enzyme for each class of low-molecular-weight RNA. There are several instances in which changes in the relative activity of the different polymerases (which in some cases have been shown to be due to the interaction of an auxiliary factor with the enzyme) are paralleled b y changes in the relative rate of synthesis of the major species of RNA (e.g., Lukacs and Sekeris, 1967; Versteegh et al., 1975; Guilfoyle et al., 1975). Thus we have the same type of crude control mechanism as that seen in prokaryotes. As the enzymes are differentially sensitive to ions, modulation of the nuclear ionic milieu could also exert some control over the relative rate of synthesis of rRNA and mRNA. There is no evidence that the initiation specificity of these polynierases, in particular of polymerase B, can be altered by the binding of a factor to the enzyme, and it seems that we must look to the template for finer regulatory mechanisms.

2. The Template It is likely that most gene activation and repression occurs through factors interacting primarily with the template, rather than with the polymerase. It is at this level that the classic example of gene regulation, the operon model proposed by Jacob and Monod (1961) for the control of the lac cistrons in E . coli, is applicable. By and large, eukaryotic genomes do not appear to be organized into operons, with functionally related genes structurally linked into a polycistronic transcription unit, although there may b e some such structures in lower eukaryotes (Arst and MacDonald, 1975). One of the features of the operon model is the presence of a sequence near the promoter, preceding the structural genes, to which a protein can bind, thereby facilitating or inhibiting one of the early steps of the polymerase reaction. In the lac operon, this protein is a repressor, as it is in most of the operons known to date. There are some

30


operons that are subject to positive control, the best characterized being the U M operon of E . coli (see Englesberg, 1971). Another feature of this system, particularly important from the point of view of the organization of the eukaryotic genome, is that a gene that is not closely linked to the main operon is also under the control of the operator binding protein (presumably through having a similar operator), thereby allowing coordinated control of unlinked genes. There may be more than one control site within the operon, which permits the coordinated control of operons in different sets in response to different conditions. The lac operon, and several others, display catabolite sensitivity, as the result of another regulatory protein binding site near the promoter (DeCrombrugghe et d . ,1971),and there may be yet another distinct control locus in this region (Crepin et al., 1975). Although operatorlike regions and corresponding regulator proteins have yet to be identified in eukaryotes, their existence is widely assumed.

3. The Recognition Problem An important point to be considered is the manner in which regulatory molecules and RNA polymerases recognize specific regions of the DNA. In normal double-helical conformations, the structural information about the base sequence available from the grooves is severely limited (von Hippel, 1969). Unique control sequences in eukaryotes are expected to be at least 10 nucleotides long. The existence of a class of molecules that could coil around the helix in one of the grooves and make such a number of very fine discriminations is therefore rather unlikely. The required specific information could be derived from a DNA structure in which the helix is locally distorted as the result of a particular base sequence. Evidence suggesting that DNA can assume a number of different sequence-determined helical structures in which the helical pitch and/or the base stacking angle is unusual has been presented (Bram, 1973), but it is not widely accepted that such structures occur to any significant extent in viuo. Alternatively, specific regions of the DNA may assume nonhelical configurations involving hairpin loops and cloverleaf formations (Gierer, 1966). Such structures are commonly found in RNA (e.g., Holley et al., 1965; Min-Jou et al., 1972; Wellauer and Dawid, 1974), and so the DNA from which these are transcribed must also be able to assume these configurations as an alternative to the normal double helix. These structures would be less stable than the double-helical

CHROMATIN ACTIVATION A N D REPRESSION

31

form in normal solution (since they involve fewer bases in pairing), but they would be readily recognized, and perhaps, stabilized by proteins. Regions that could form such structures would automatically have twofold symmetry in their base sequences. Such twofold symmetry is observed in several regions of DNA known to be involved in specific interactions with particular molecules, such as the lac repressor (Gilbert and Maxam, 1973; Maizels, 1973)and various restriction endonucleases (Kelly and Smith, 1970; Hedgpeth et al., 1972; Sugisaki and Takanami, 1973). Moreover, as more E . coli initiation sequences are determined, a lack of similarity in their primary structure makes recognition on the basis of a higher-order structure more likely (Sekiya and Khorana, 1974). Sobel (1973) has proposed that sequence symmetry must be involved in the sequence-specific binding of multimeric molecules which themselves showed symmetry. The lac and lambda repressors and RNA polymerase have been shown to bind to DNA as multimers, although there is as yet no information available concerning the relationship between the subunits. The multimeric histone complex (Thomas and Kornberg, 1975), which is likely to be symmetric, has been shown to bind randomly to DNA (Zimmerman and Levin, 1975; Lacy and Axel, 1975), but in this case interaction is probably with the sugar phosphate backbone of the DNA. The importance of symmetry in so many biological processes (see Engstrom and Strandberg, 1967) makes its involvement in genetic regulation an attractive proposition. The other conformation DNA may assume is as localized single strands. In this state, the full informational content of its base sequence would b e exposed, although under normal circumstances such structures would be very much less stable than base-paired helices. Tritium-exchange studies show that a constant opening and closing of DNA helices occurs (Printz and von Hippel, 1965), and the helices formed by certain base sequences are intrinsically less stable than others. It may be expected that localized melting of particularly unstable sequences occurs more frequently, and for longer periods of time, than in bulk DNA. During these “open” periods, regulatory molecules may slip in and hold the strands apart. Some recent studies on the lac operon by Dickson et al. (1975) suggest that such a site may indeed be present in the promoter region, permitting the RNA polymerase to enter and begin to open u p further sections of the helix. Comparisons of thermal hyperchromicity (Frenster, 1965a) and antiDNA antibody binding (Kunkel and Tan, 1964) between isolated ac-

32

NORMAN MACLEAN AND VAUGHAN

A. HILDER

tive and repressed chromatin indicate a significant degree of strand separation in the former. Models for the control of genetic activity emphasizing localized single-stranded recognition sites have been proposed by Frenster (1965a,b) and Crick (1971).

c.

THE ORGANIZATION OF THE GENOME

1. Sequence Organization Our knowledge of the arrangement of the functionally different kinds of sequences in the eukaryotic genome is severely limited, largely because of the unsuitability of this type of cell for the sort of fine genetic analysis that led to the elucidation of the organization of the bacterial genome. Some progress has, however, been made in the analysis of sequences distinguished according to other criteria, some of which can be linked to functional differences. The DNA of the eukaryotic genome differs from that of prokaryotes not only in size, but also in that a large part of it consists of base sequences which occur many times, in addition to those present as single copies. The frequency with which particular sequences occur, their length, and the degree of sequence homology have been deduced from measurements of the kinetics, and fidelity, of the reassociation of various-sized fragments of denatured DNA under carefully controlled conditions (see Britten and Kohne, 1968). Three broad categories of sequences may be distinguished based on these criteria: (1) highly repetitive sequences, which occur as lo5 to lo6 copies per genome (Kit, 1961; Sueoka, 1961; Arrighi et al., 1970); (2) intermediate repetitive sequences which occur as 102 to 104 copies per genome (Britten and Kohne, 1968; Walker, 1971); and (3) nonrepetitive sequences which occur as one, or perhaps a few, copies per genome. These different kinds of sequences are distributed throughout the genome in a detectable pattern. The highly repetitive sequences occur as clusters of very closely related short sequences, mainly in centromeric regions, and comprise the untranscribed sequences of satellite DNA. In all the species so far studied, most of the intermediate repetitive sequences do not occur as clusters but are interspersed with nonrepetitive sequences. In a wide range of species, these intermediate repetitive sequences have an average length of approximately 300 nucleotides, and they alternate with nonrepetitive sequences of between a few hundred and more than 10,000 nucleotides. A major part of these genomes is in the form of a 300-base-pair intermediate repetitive region adjacent to a 700- to 3000-base-pair nonrepetitive region (see


33

Davidson et d,,1975).The exception to this arrangement is found in Drosophila, where both the intermediate repetitive and nonrepetitive components are much longer (about 5000 and 13,000 nucleotides, respectively) (Manning et al., 1975). The significance of this exception is far from clear (although one fact that does stand out is that the Drosophila genome is by far the smallest investigated), but it serves to emphasize that caution is required in interpreting these findings. It is unlikely that such an orderly arrangement is fortuitous, but we cannot as yet specify its functional significance. There is a considerable body of evidence that the majority of structural genes occur only once per haploid genome and that these are therefore part of the nonrepetitive sequences (Bishop et al., 1972; Greenberg and Perry, 1972; Harrison et aZ., 1972; Suzuki et al., 1972; Goldberg e t al., 1973). Consideration of the size distribution of polypeptides, and the known sizes of mRNAs, leads to the conclusion that any nonrepetitive sequence of between a few hundred and many thousands of nucleotides may be a coding sequence (Davidson and Britten, 1973), although the overall size of the genome implies that many of them cannot be. Notable exceptions to the single-copy generalization are provided by the genes coding for rRNAs (Wallace and Birnstiel, 1966; Brown and Weber, 1968), tRNAs (Ritossa et al., 1966), histones (Kedes and Birnstiel, 1971), Balbiani ring proteins (see Edstrom and Lambert, 1975), feather keratin (Kemp, 1975),and certain low-molecular-weight nuclear RNAs (Busch et al., 1971). These genes are present as multiple copies and therefore form part of the intermediate repetitive fraction, albeit a minor one. In addition, genes coding for proteins with highly repetitious structures, such as fibroin (Suzuki et al., 1972) and keratin (Kemp, 1975), have hybridization characteristics suggestive of repeated sequence DNA. An insight into the function of the bulk of the intermediate repetitive sequences is crucial in our assessment of models proposed for the organization and control of the eukaryotic genome.

2. The Transcription Unit With the exception of ribosomal cistrons, whose unique features make them easier to investigate (e.g., Weinberg and Penman, 1970; Miller and Beatty, 1969), the structure of the eukaryotic transcription unit is rather ill-defined. The evidence is fairly conclusive that polysoma1 mRNA is monocistronic (see Davidson and Britten, 1973) and contains relatively few transcribed nucleotides in addition to the coding sequence. The primary transcript, of which mRNA forms a part, is, however, in the form of HnRNA, which ranges in size from

34


about lo6to 1.5 x lo' daltons, with a considerable excess of large molecules over what would be required for exclusively monocistronic transcription. HnRNA contains both intermediate repetitive sequences and nonrepetitive sequences (Firtel et al., 1972),and a large part of it is rapidly degraded in the nucleus (Soeiro et al., 1968). Messenger sequences have been shown to be present at the 3' end (Imaizumi et al., 1973), and no more than one messenger sequence has been shown to be present in any particular HnRNA. The function of the excess sequences in HnRNA remains a matter of speculation. It has been suggested that they constitute transcribed control sequences preceding one or more messenger sequences (Georgiev, 1972). In this case one would expect there to be less of an excess in the less complex lower eukaryotes, as is observed (Prescott et al., 1971a,b; Firtel, 1972; Firtel et nl., 1972). The amount by which they exceed the coding sequences in higher eukaryotes, however, suggests that many more individual regulatory events than would be required for a tissue pattern of control must take place if control is by operatorlike elements, as envisaged by Georgiev. Davidson and Britten (1973)have expressed doubts as to the generality of very large transcripts and propose that the rapidly degraded nuclear RNA is largely composed of sequences which are, or code for, regulatory molecules. Perhaps the greatest attraction of HnRNA as the primary transcript is that such a model is strongly suggested by the chromomeric organization stressed earlier in this article. In terms of the model we present below, HnRNA is interpreted as being the result of transcription of structural, regulatory, and coding sequences which make up the chromomere.

3. General Models of Eukaryotic Regulation In light of these facts we now consider some of the features required by models of the organization and regulation of the eukaryotic transcription unit. All models require that the coding sequences (structural genes) be preceded by regulatory sequences which may interact with specific factors to determine whether the unit is transcribed or not. In addition to promoter and operatorlike sites, we also expect to find sequences concerned with the posttranscriptioiial processing of the transcript, such as endonuclease sites and ribosome binding sites, in the region preceding the coding sequence. Many of these sequences might be expected to be repetitive.


35

In some models, genetic regulation is regarded as being essentially identical to that known to be adequate for bacteria; the large apparent excess of DNA in eukaryotes is explained by the supposed presence of additional copies of structural genes, as in the master-slave hypothesis of Callan (1967) and in Edstrom’s model (see Edstrom and Lambert, 1975), or is described as ‘3unk” DNA (Comings, 1972). This latter term is a little misleading, since it suggests that the DNA is quite functionless (and therefore a very expensive burden in terms of energy expenditure), while in fact several rather nonspecific functions are ascribed to it. It is not appropriate to discuss here all the evidence that relates to the master-slave hypothesis (see discussion in Comings, 1972), but it is reasonable to state that most of it now argues against such a model. In particular, hybridization studies on gene frequency (Harrison et al., 1972; Bishop and Rosbach, 1973) and mutation data suggest that there are only one or two copies of most structural genes in the genome. These data are b y no means so damaging to Edstrom’s model, since in this case only one copy of a gene is normally active, the additional “memory” copies being primarily of evolutionary significance, for which purpose they would tend to be as sequencedivergent as the degeneracy of the genetic code would permit for isocoding genes. On this basis it is proposed that the apparently excess DNA has a role in increasing the efficiency of eukaryotic evolution. This interesting model largely ignores the vastly increased organizational complexity of higher eukaryotes over prokaryotes, and this requires regulatory mechanisms on a much larger scale, or of a different kind (or both), in the former. Georgiev (1969, 1972) has proposed that the regulatory region is very long, comprising many operator sequences, and that its transcription is necessary for transcription of the contiguous coding sequence, thereby accounting for the excess length of HnRNA over mRNA. Davidson and Britten (1973) have objected to the distance over which control would have to be effective if a large number of regulatory sequences is involved and have disputed the generality of large primary transcripts. The RNA polymerase-template complex is very stable once initiation has taken place, so control over a large distance presents no problem, and the evidence in favor of large primary transcripts cannot be discounted. The Georgiev model implies, however, that far more regulatory events are involved than would be expected from the tissue pattern of differentiation. We have proposed a model that stresses the chromomeric organization of the genome, with the promoter located at one end of the chromomere and the coding sequence at the other. The intervening DNA

36


A

B

f

C

FIG. 1. Condensation and decondensation of the chromomere. Regions of the deoxyribonucleohistone containing packing DNA are shaded, and those parts of it responsible for the base sequence-specific interactions on which condensation depends are clear. (A) Condensation results directly from an alteration in the secondary structure of these sequences. An attractive way in which this could occur is by the formation of Gierer loops (Gierer, 1966), although there are many alternatives, such as localized strand separation (Frenster, 1965b). (B) Condensation results from interactions between these sequences, stabilizing folds in the intervening regions. (C) A similar mechanism but with the interaction mediated by proteins (€4).


37

contains sequences involved in the essential function of packing the DNA into the characteristic condensed chromomeric structure and a limited number of operatorlike sequences. We envisage the packing DNA as being of two types; one, which would be engaged in the interactions that maintain the condensed state of the chromomere, may perhaps consist of multiple copies of related sequences, forming part of the intermediate repetitive DNA fraction; but the bulk of the packing DNA would be required to provide adequate separation of these regions, and its sequence would not be important for this purpose (see Fig. l). This might explain much of the interspersion of repetitive and nonrepetitive DNA sequences in the eukaryotic genome and, as there would presumably be an optimal length for such elements to produce a stable structure, might account for the frequency with which repeat units of a particular size are found. Decondensation, due to an alteration in the interaction between packing DNA sequences, would be a prerequisite for transcription of the chromomere, but this would be only a crude level of control above which control by the operatorlike elements would apply. Partial transcription of chroinomeres that were decondensed, but repressed at an operator, might give rise to the class of HnRNA that does not receive a poly-A tail (see Fig. 2). To permit different unlinked structural gene sequences to be controlled together, most models have assumed that such genes have sequences in common, capable of interaction with a particular regulatory molecule. These sequences are therefore likely to be repetitive. The activation or repression of genes in different combinations under different conditions can he achieved by the introduction before the coding sequence of a control sequence for each set of which the gene is a member. Davidson and Britten (1973)introduce the essential feature that the regulatory molecules are the products of genes, the activity of which is determined by external factors. These, called integrator genes by Davidson and Britten, have most of the features of tissue-master genes referred to earlier in this article. The product of a specific integrator gene binds to a particular repetitive sequence and contributes to the control of any gene preceded by that sequence. The coordinated control of genes in different partially overlapping sets can therefore be achieved either by having polycistronic integrator genes that code for regulator molecules specific for different repetitive sequences, or by having multiple and differing control sequences preceding the coding sequence of the structural gene. In other words, redundancy of sequences involved in control must occur

38

NORMAN MACLEAN AND VAUGHAN A. HILDER D

0,

0,

e

sg a t

D

01

0,

e

sg a t

A

t B

C

D FIG.2. Sequence organization and transcription of the chromomere. Regions of the deoxyribonucleohistone containing packing DNA are shaded, and the other sequences included are the promoter (p), operators (ol, 02), a restriction endonuclease site (e), a structural gene (sg), a poly-A attachment site (a), and a transcription termination site (t). These elements are represented within the packing DNA, fulfilling not only their usual roles but also that of separating the condensation directing sequences. Some or all of them, however, could lie outside the packing DNA region. (A) The chromomere is fully decondensed, and the operators derepressed (or bound by an activator if they are subject to positive control). Thus the entire chromomere between the promoter and terminator is transcribed, yielding avery long RNA molecule (dashed line). Poly A is attached to the attachment site at the 3' end, and the activity of a restriction endonuclease at (e)


39

either at the level ofthe integrator genes themselves, or at the level of the control sequences associated with any one structural gene. The model of Davidson and Britten (1973)favors the former level of redundancy, and that of Georgiev (1972) the latter. The chromomeric model we have proposed can accommodate redundancy at either level. It should be noted, however, that in our model the chromomere must be decondensed before these operatorlike control mechanisms become significant. The sequences that determine chromomere condensation, which we term packing DNA, would themselves be multiple within the chromomere and probably common to several chromomeres. It may be that certain tissue-master gene products influence the interaction between some of these sequences, but it is more likely that they are directly sensitive to external effectors such as ions. The presence of this separate level of control reduces the extent to which redundancy of the fine-control elements is necessary. D. REGULATORS

1. Histones Since such prokaryotic gene regulators as have been identified are proteins (Gilbert and Muller-Hill, 1966; Ptashne, 1967; Emmer et al., 1970), it is on the chromatin-associated proteins that the search for eukaryotic regulators has centered. Quantitatively, the major class of these are the histones-relatively small, very basic proteins, divisible into five major groups on the basis of their electrophoretic mobility (see Phillips, 1971). These proteins have had a checkered career as contenders for the role of specific gene regulators since the possibility was suggested by Stedman and Stedman (1951).They clearly inhibit transcription (e.g., yields mRNA from the 3’ end, the 5’ end being degraded. (B) The chromomere is fully condensed, but the presence o f a repressor (R,) on the operator (ol) prevents transcription beyond 0,.Thus the transcript is shorter than in (A) (although it may still be considerably longer than the average mRNA) and lacks a messenger sequence and poly-A attachment site. The blocking operator site in this figure is distant from the promoter and therefore permits a short transcript. If the blocking operator were next to the promoter, however, then complete decondensation of the chromomere might occur without any ensuing transcription, a situation probably analogous to many experimental observations on chromatin decondensation (Pallotta, 1972; Hilder and Maclean, 1974). (C) The chromomere is partially condensed. Transcription can occur only between the promoter and the condensation site, yielding a product similar to that in (B). (D) In the fully condensed chromomere, the promoter is inaccessible to RNA polymerase, and the movement of any polymerase already bound to the template is blocked.

40


Huang and Bonner, 1962; Paul and Gilmour, 1966), but their striking uniformity in quantity and composition in active and inactive chromatin (e.g., Allfrey and Mirsky, 1962; Comings, 1967), different tissues (e.g., MacGillivray, 1968; Boulanger et al., 1969), and even different eukaryotic (taxonomic) kingdoms (DeLang et al., 1969) suggests that they fulfil some essential function common to all eukaryotic cells as opposed to a specific regulatory function. There are certain exceptions to the normal pattern of histone distribution; in particular, unusual basic proteins may be found in lower eukaryotes, in germ line cells (see Bloch, 1969), and in nucleated erythrocytes (Neelin, 1964). The proportions of the major histone classes change during maturation of the nucleated erythrocyte (Ruiz-Carrillo et al., 1974), and cells of the erythropoietic series contain a tissue-specific histone (Neelin, 1964). The high degree of repression in these cells cannot, however, be directly linked to these changes (Bolund and Johns, 1973) which are probably involved in maintaining the unusually high degree of interphase chromatin condensation. The observation that a few of the amino acid residues in histones may be differentially modified by enzymic methylation, acetylation, and phosphorylation after their synthesis (e.g., Allfrey et al., 1964; Kleinsmith et al., 1966; Ord and Stocken, 1966) revived their candidacy as specific regulators. Alterations in the extent of histone acetylation and phosphorylation have been shown to parallel changes in genetic activity in such systems as the phytohemagglutinin-induced transformation of lymphocytes (Pogo et al., 1966; Kleinsmith et al., 1966), liver regeneration (Pogo et al., 1968), erythrocyte maturation (Allfrey, 1970; Tobin and Seligy, 1975), and in several cases of target tissue response to hormones (e.g., Allfrey, 1966; Libby, 1968; Langan, 1969; Takaku et al., 1969). A casual relationship cannot, however, be established in any of these cases. The change in genetic activity induced in lymphocytes b y phytohemagglutinin is blocked by the administration of cortisol, but the increase in histone acetylation still occurs under these circumstances (On0 et a1., 1969). It has recently become clear that the histone-DNA complex is a core of histones H2a, H2b, H3, and H4 (two molecules each) around which the DNA coils and interacts with the basic regions of the histone molecules, producing beadlike structures which have been termed nucleosomes (Komberg and Thomas, 1974; Baldwin et id., 1975).The conservatism of histone sequences suggests that almost all the residues are essential for the correct interaction between histones and between histones and DNA (since any slight change in the conformation of the complex, repeated throughout chromatin containing a


41

mutant histone, would result in massive disruption of chromatin packing). Localized modification of the histones may therefore alter this conformation sufficiently to permit, though probably not cause, specific gene activation. The model we have proposed (Section V,C,3) suggests an interesting reinterpretation of the role of histones in chromatin. It is possible that binding of histones to DNA may not in itself inhibit transcription, but that only when the DNA is in the form ofdeoxyribonucleohistone can the packing DNA sequences interact to condense the chromomeres. It might be this condensation, automatic in the presence of histone, that is largely responsible for the reduced template activity of DNA in the presence of histones. This suggestion applies principally to those histones that provide the core around which the DNA coils. Histone H1, which is attached to the outside of the nucleosomes, may be more directly involved in tight condensation, as in the mitotic chromosome (e.g., Bradbury et d.,1974).

2. Nonhistone Proteins The remaining nuclear proteins, collectively known as nuclear nonhistone proteins, are a much more likely source of specific regulatory moIecules. This is a very heterogeneous fraction, functionally as well as structurally, and includes structural proteins-actin, myosin, tropomyosin, and tubulin have been identified as major components of the salt-soluble nonhistone proteins of rat liver chromatin (Douvas et al., 1975)-and many proteins with enzymic activities, such as RNA and DNA polymerases and various kinase, methylase, acetylase, and other enzymes. Some of the features of this fraction which suggest that part of it is concerned with specific gene regulation are reviewed below. Polyacrylamide gel electrophoresis of nuclear nonhistone proteins yields complex patterns which may have over 100 bands (e.g., Garrard et al., 1974). It may be argued that the degree of heterogeneity usually found is insufficient to account for the number of regulatory events that must occur in higher eukaryotes, even with a tissue pattern of control. It must, however, be borne in mind that the number of bands observed does not always equal the number of proteins, since some may be outside the size range of the gels, some may have such similar molecular weights or isoelectric points that they run as a single band, and some may be present in quantities below the limit of detection on the gels. It has also been suggested that, by analogy with the lac repressor ofE. coli, which is present as about 10 molecules per cell on the average (Gilbert and Muller-Hill, 1966), the quantity of any specific regulatory molecule per cell may be so low as to make its de-

42


tection on such gels unlikely. Other prokaryotic repressors, however, have dissociation constants much higher than that of the lac repressor. In view of the greater volume of the eukaryotic nucleus [let alone the cytoplasm, in which these proteins are synthesized (Stein and Baserga, 1971)], the greater amount of DNA and basic protein with which nonspecific interactions can occur [such nonspecific interaction has been shown to be important in the kinetics of repressor and sigma-factor action (von Hippel et al., 1974)],the possibility that a particular regulator may be involved in the control of many loci (see Section V,C,3), and the possibility that fhctionally related peptides may be structurally so similar as to run as a single electrophoretic band, a regulatory function cannot be automatically ruled out for any of the bands seen on polyacrylamide gels. The banding pattern of nuclear nonhistone proteins shows considerable differences between species and, within a species, between different tissues (e.g., Platz et al., 1970; Chytil and Spelsberg, 1971; Barrett and Gould, 1973)and at different periods of the cell cycle (e.g., Stein and Baserga, 1971; Stein and Borun, 1972). Changes in the amounts of particular components parallel changes in genetic activity during the response of target tissues to hormonal stimulation (Shelton and Allfrey, 1970; Cohen and Hamilton, 1975).In general, they are present in higher amounts in active cells (e.g., Arnold et al., 1973; Ruiz-Carrillo et al., 1974) and in active regions of chromatin (Frenster, 1965a; Berkowitz and Doty, 1975). These observations tend to suggest that, if most of the nonhistone proteins are involved in genetic regulation, they must function, on the whole, as activators. The presence of a particular band in the nonhistone proteins of mature erythrocytes of Xenopus, which is not detectable in immature erythroid cells (Hilder et al., 1975), and of a particular subfraction bound specifically to repressed DNA (Pederson and Bhorjee, 1975)means, however, that the possibility of an important negative control function for part of this fraction cannot be eliminated. Part of the nonhistone protein fraction binds to DNA and shows some specificity for homologous DNA (Teng et al., 1971; Chiu et al., 1975). The best evidence of a regulatory role for these proteins comes from experiments on the in vitro transcription of partially reconstituted chromatin. Reassociation of nonhistone proteins with DNA yields a template with a higher transcriptional activity than naked DNA. This argues strongly in favor of a role in the positive control of transcription from DNA. Evidence for specificity comes from experiments in which chromatin is reconstituted from DNA, nonhistone pro-

CHROMATIN ACTIVATION A N D REPRESSION

43

teins, and histones derived from cells with different transcriptional specificities. The transcripts from such templates are characteristic of the cells from which the nonhistone proteins were derived, and are independent of the source of DNA and histones (Barrett et al., 1974; Gilmour et d . , 1975; Stein et d . , 1975). Many components of the nonhistone protein fraction undergo extensive enzymic phosphorylation (chiefly of serine residues) after synthesis. The extent of this phosphorylation is correlated with changes in genetic activity in several systems such as the phytohemagglutinin stimulation of lymphocytes (Kleinsmith et al., 1966), testosterone stimulation of prostate cells (Ahmed and Ishida, 1971), and during the cell cycle of tissue culture cells (Platz et al., 1973; Karn et al., 1974). Phosphorylated nonhistone proteins are among the most favored for the role of gene activator (see Kleinsmith, 1975). Thus there is strong evidence that some nuclear nonhistone proteins, and their differential phosphorylation, are involved in genetic regulation. It is likely that tissue-specific differences in template restriction are a function of the nonhistone protein complement of a tissue, and that changes in transcription within a tissue may arise from secondary modifications of its nonhistone proteins. The models so far proposed for nonhistone protein action at the molecular level are based on the suggestion that their negatively charged groups, in particular their phosphate groups, interact with histones to relieve the histone inhibition of transcription from DNA (Kleinsmith et al., 1966; Kleinsmith and Allfrey, 1969; Kaplowitz et aZ., 1971). Such models provide for the positive control of transcription from chromatin, but do not account for the apparent reactivation of transcription from DNA or for specific repression. The development of satisfactory models for nonhistone protein action probably must await the preparation of homogeneous fractions with more closely defined effects.

3. Base-Modification Enzymes Holliday and Pugh (1975) discussed the proposal that differentiation and a cellular time base (i.e., a mechanism by which cells can “count” the number of mitoses they have undergone) may be due to enzymic modification of specific bases in the DNA. It is supposed that the susceptible bases are located at protein binding sites such as operators and promoters, and that modification alters the affinity of such sites for their binding proteins. Specificity is introduced into the reaction by having a recognition sequence for the modification enzymes in the vicinity of the relevant bases. Modifications would be

44


heritable to differing extents, depending on the reaction involved and on whether it affects bases in both or only one strand of the DNA duplex. Unusual bases, such as 5-methylcytosine and 6-methyladenine are present in eukaryotic DNA (Doskocil and Sorm, 1962; Gorovsky et al., 1973), and preferential methylation of bases in the promoter proximal region of the transcription unit has been found in HeLa cells (Volpe and Eremenko, 1974). Direct evidence for the involvement of such a mechanism in gene control is, however, lacking. One attraction of this model is that it readily accounts for the stability of the determined state of cells. It is not clear, however, how the activity of the modification enzymes is itself controlled. 4. RNA Some models for eukaryotic regulation have been proposed in which a particular species of RNA functions as the regulator (Fenster, 1965a; Britten and Davidson, 1969). RNA is a constituent of chromatin, and a species of low-molecular-weight RNA is associated with the salt-dissociable chromosomal proteins (Huang and Bonner, 1965). This has been called chromosomal RNA (cRNA) and is reported to be of general occurrence (Benjamin et al., 1966), of heterogeneous sequence (Bonner and Widholm, 1967) and, to some extent, tissuespecific (Mayfield and Bonner, 1971). Template specificity in reconstituted chromatin is achieved only when reconstitution is carried out under conditions that permit DNA-RNA hybridization to occur (Beckhor et al., 1969) and is diminished if zinc nitrate (which degrades RNA) is included (Huang and Huang, 1969).These results are, however, open to several interpretations, and it has been suggested that cRNA is in fact degraded tRNA (Heyden and Zachau, 1971). The significance of this species of RNA must therefore remain a matter of speculation.

5. External Effectors All the potential regulatory factors discussed so far are themselves products of the genome they are proposed to regulate. For anything other than unidirectional changes in genetic activity, transcription must be amenable to modification by some external factors. These may act either directly on the template, or indirectly by modifying the interaction of some template-bound regulator. It has been suggested that hormones act as such external effectors (e.g., Davidson and Britten, 1973). The activation of specific genes plays an important part in the response of many target tissues to hor-


45

mones (although some responses definitely do not involve transcription) (see Tata, 1966; Toinkins and Martin, 1970). Some hormones, particularly polypeptides and glycoproteins, act at the cell surface, where they activate the membrane-bound enzyme adenyl cyclase. This leads to an increase in the intracellular level of cyclic 3’-5’-AMP, and it is this that is responsible for the intracellular events associated with these hormones. Certain nuclear protein kinases are stimulated by cyclic AMP, and so such hormone responses may be mediated by the phosphorylation of chromatin proteins. Steroid hormones enter the cells and nuclei of their target tissues in association with a specific receptor protein (see Jensen et ul., 1971). The specificity of response appears to be dependent on the presence of these receptor proteins in the cell. Thus the response to hormones is determined by the prior genetic activity of the cell, and it therefore cannot be the sort of external effector for which we are looking. Simple inorganic ions more nearly fulfil the criteria for the required external effectors. The distribution of ions across biological membranes is (usually) selectively anisotropic, and this anistropy is actively maintained. Shifts in the concentration of ions on either side of a membrane and alterations in membrane permeability or the energy available for active ion transport can lead to different steady-state conditions without any prior genetic activity. Evidence that changes in the nuclear ionic concentration are related to decondensation of specific chromomeres and changes in genetic activity have been reviewed by Lezzi (1970). The maintenance of the highly charged DNA structure, and histone-DNA interaction, are dependent on ionic bonds and are therefore sensitive to alterations in the nucleoplasmic ionic concentration. If certain promoter regions, for example, are particularly sensitive, changes in ionic concentrations might cause conformational changes in them during active and inactive states, thereby determining the activity of a related structural gene. Such a transition, in response to temperature and potassium ion concentration, has been shown to take place in the rRNA promoter of E . coli (Travers et al., 1973). It is clear that such a system would have a very low specificity in response to individual ions. This is, however, just the sort of control we would expect to determine the condensation and decondensation of chromomeres where the cumulative, and possibly cooperative, interactions between many sites means that a certain ambiguity in individual interactions would not affect the net result. The ion-induced puffing at specific loci (see Lezzi, 1970)is very suggestive of a mechanism such as we have proposed.

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VI. General Conclusions DNA is affected in its genetic activity by many different factors, and it is convenient to group these in the way in which they influence the DNA. The first factor is the primary base sequence of the DNA itself. Much of the DNA of eukaryotes apparently consists of highly repeated short sequences, often grouped in blocks and sometimes making up most or all of certain chromosomes. The base sequence of such DNA seems to ensure that it is genetically inactive in uivo. Second, there is the possible activation or repression of transcription, as a result of the DNA itself adopting an unusual tertiary structure, forming loops, hairpins, or more complicated configurations. Perhaps some accessory molecules may be implicated in the formation or persistence of such tertiary structure, and often such structures may themselves permit or prevent recognition of particular sites on the DNA by regulatory molecules. Third, there is the possibility of permanent or temporary modification of DNA base sequence by enzymes, thus permitting differentiation to become fixed by the obliteration ofcertain gene sequences previously present. All these mechanisms just cited depend on structural features of the DNA itself. Other levels of control are clearly exerted by the attachment of specific molecules to the DNA, especially of histones and nonhistone molecules of a regulatory type. The very nature of eukaryotic chromatin itself suggests genetic regulation at this level. Such large molecules may often act simply by impeding the passage of the RNA polymerase enzyme along either the structural gene or the relevant regulatory sequences preceding it. It seems likely that the chromomere is a key unit of eukaryotic genetic function, and we have suggested that much of its length is probably explained by the existence of packing DNA, the association of which with histone and other proteins determines the state of condensation or decondensation of the chromomere. This property of the chromomere is particularly sensitive to ionic concentration in our view, thus implicating these small particles in some central aspects of gene regulation. Lastly, the activity of DNA can be affected by availability or modification of suitable polymerase enzymes, although the importance of this factor in general eukaryotic gene regulation remains uncertain. It is necessary to emphasize that all these mechanisms cited as important in regulating gene activity may affect the structural gene itself, preceding regulatory gene sequences, or both. Satisfactory transcription of a structural gene seems to require the prior transcription of preceding sequences, hence the large size of the HnRNA that constitutes the normal primary gene product in eukaryotes.


47

VII. Summary 1. Most gene regulation involves selective activity and repression of different parts of the genome, and only very rarely elimination or destruction of unwanted genetic material. 2. At the level of the whole chromosome, sex chromosomes are often genetically inactive. The male Y chromosome owes its genetic inactivity largely to its DNA base sequence, while the inactivated X chromosomes found in many female mammals are rendered inactive by permanent chromatin condensation. 3. Large tracts of chromatin within chromosomes may be inactive. Thus centromeric and intercalary heterochromatin is interspersed with euchromatin on the same chromosome. Such heterochromatin may influence the expression of neighboring genes by a position effect. Large tracts of facultative heterochromatin are not known to exist below the whole-chromosome level. 4. The dipteran polytene chromosome is a remarkable example of chromatin which can be visualized during the transcriptional activity of interphase. Observations on these chromosomes suggests that the chromomere is a basic unit of genetic structure and function, a conclusion that also fits with observations on the active lampbrush chromosomes of amphibian oocytes. 5. We endorse the view that the chromomere is a length of DNA which includes only one structural gene, but is itself many times longer than the structural gene. The large size of eukaryotic HnRNA suggests that entire chromomeres are usually transcribed, the HnRNA being trimmed down within the nucleus to yield functional mRNA. 6. We suggest that the large size of the chromomere and the HnRNA is accounted for partly by control sequences which precede the structural gene sequence, but more extensively by the existence of packing DNA which determines the condensed or extended form of the chromomere by its variable association with chromatin proteins. Such variable condensation would be frequently modulated by ionic concentrations and would provide, along with the other control sequences in the chromomere, a regulatory influence over the transcription of the chromomere and its structural gene. 7. We also propose the existence of tissue-master genes. Such genes would be sensitive to effector molecules from outside the cell, would control the determined or differentiated state of the cell, and would explain the striking, although less than absolute, stability of these states. These tissue-master genes are in many ways analogous to the integrator genes proposed by Davidson and Britten (1973),but in our terminology and model their key role is given greater weight.

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8. The probable importance of the DNA adopting unusual tertiary structures, often with the assistance of other accessory molecules, has been discussed and emphasized as a regulatory factor. ACKNOWLEDGMENTS

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Origin and Ultrastructure of Cells in Vitro L. M. FRANKS AND

PATRICIA

D.

WILSON

Department of Cellular Puthvlogy, Imperial Cancer Research Fund, London, England

I. Introduction . . . . . . . . . . The Origin ofTissue Culture Cells . . . . . 11. General Features of Cells in Vitro . . . . . A. Shape and Surface Morphology of Cells in Culture . B. Surface Coat and Plasma Membrane of Cells in Vitro . C. Cell Contacts, Junctional Complexes, and Cell Sub. . . . . . . . strate Adhesion D. The Filament-Microtubule System. . . . . E. Mitochondria . . . . . . . . . F. Cytoplasmic Inclusions . . . . . . . G . Viruses and Viruslike Particles . . . . . H. The Nucleus . . . . . . . . . I. Enzyme Changes in Cells in Vitro. . . . . J. Degenerative Changes in Cells in Vitro . . . . . . . . 111. Special Features of Cells inVitro. A. Morphology of Differentiated Cells . . . . R. Aging in Vitro . . . . . . . . . C. Ultrastructure of Hybrid Cells . . . . . D. Ultrastructural Features of Neoplastic Transformation . IV. Ultrastructure of Primary Explants and Epithelial Cell . . . . Strains from Normal Epithelial Tissues A. Explants of Fetal Salivary Gland , . . . . B. Explants of Adult Salivary Gland , . . . . C. Primary and Transferable Cultures from Other Organs . D. Conclusions , . , . . , . . , V. Ultrastructure of Mesenchymal Cells from Normal Tissues . A. Differentiated Mesenchymal Cell Strains . . . B. Undifferentiated Mesenchymal Cell Strains and Lines C. The Origin of Mesenchymal Tissue Culture Cells . VI . Ultrastructure of Cells from Brain and Hemopoietic . . . . . . . . . , Tissue . VII. Ultrastructure of Tumor Cells in Vitro. . . . . VIII. Ultrastructure of Cells in Organ Cultures . . . . IX. Conclusions . . . . . . . . . . References . . . . . . . . . .

55 56 59 60 62 64 69 74 76 76 79 79 81 81 81 84 84 85 91 92 97 103 107 108 108 109 117 120 121 125 128 131

Did I say so? . . . to be sure if I said so, it was so (Goldsmith, 1760).

I. Introduction In 1945, Porter, Claude, and Fullam used an electron microscope to look at whole cells from chicken tissue cultures. Since sectioning 55

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L. M. FRANKS ANDPATRICIA D. WILSON

methods were not available, their observations were restricted to those areas of the cell that were thin enough to allow the passage of electrons. Even using this relatively crude technique it was obvious that a new era in cytology was beginning. Within the next few years progress was rapid. By 1953 (review by Selby) sectioning of plastic-embedded material had allowed the identification of most cell organelles and some viruses in tissues and in cultured cells. Since 1953, improvements and simplifications in methodology have been rapid, and it seems likely that the technical limits of resolution of the microscope have almost been reached. Recording the ultrastructure of the cell in vitro is now commonplace. Even to do it well is no longer a major scientific achievement. The value of the technique depends on the interpretation, and it is now obvious that for many purposes simpler methods are not only easier but better. The light microscope, using phase- or interferencecontrast, or old-fashioned histochemical stains answer many questions of identity and organization more effectively than the electron microscope but, when used to answer the right type of question, it can provide an answer that cannot easily be obtained in any other way. Morphology is a useful guide to identity, but in many cases the pattern is as important as the detailed structure of the individual components. Thus morphology allows us to identify most tissues with certainty, but the identification of an individual cell from any given tissue may not be possible unless it has some clearly recognizable marker. A major contribution the electron microscope can make to in oitro experiments is the recognition of subcellular markers and the alteration in their structure, distribution, and functional activity. In this article we concentrate on those features that are particularly suitable for electron microscope investigation, and in particular the changes in ultrastructure due to in vitro culture. Areas that have been described extensively elsewhere, for example, cell division, chromosome structure, virus replication, antibody distribution, and so on, and which show no features specifically related to cell culture, are not considered. We make no attempt to provide an exhaustive review of ultrastructural studies on mammalian cells in vitro but give a sufficient number of examples to illustrate the general patterns that may be found.

THE ORIGIN OF TISSUECULTURE CELLS Before describing the ultrastructure of the cells it is necessary to recognize the nature and origin of cells that can be maintained in vitro. It is also helpful to define two terms-epithelial and fibro-

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blastic-often used in tissue culture circles. True epithelium is defined as the cell types that cover or line surfaces or glands developing from them, and may be derived from any of the three primitive germ layers (Ham, 1969).The two exceptions to this general rule are the cells lining blood vessels (endothelium)and coelomic cavities such as peritoneum and pleura (mesothelium). These two cell types are derived from mesenchyme (connective tissues), even though they may adopt an epithelial form. They retain their mesenchymal potential, which is often expressed in vivo, particularly under pathological conditions. For example, they may produce large quantities of collagen in some disease processes. Most mesenchymal cells, including smooth muscle and vascular precursor cells, may retain the capacity to produce collagen, elastic tissue, and connective tissue mucopolysaccharides such as chondroitin sulfate and hyaluronic acid. In cell culture, regular hexagonal cells with well-defined margins, growing in closely packed sheets, are usually described as epithelial. The other common cell type, usually spindle-shaped and growing in parallel bundles and forming a meshwork, is described as fibroblastic, because it resembles a tissue fibroblast, although a wide range of variation in appearance occurs depending on medium, substrate, cell number, and so on. Early cell culturists were aware that the terms were inappropriate but convenient (see, for example, Willmer, 1958, for review) for descriptive purposes. It has been known for many years that specialized differentiated epithelial cells from normal tissues die out after a relatively short period in culture and are replaced by undifferentiated cells, usually regarded as fibroblasts, which have no tissue-specific markers, although some cell lines derived from tumors may retain both functional and morphological differentiated characters (e.g., Davidson, 1964; Wigley, 1975; and others). I n earlier articles we have shown (Franks and Wilson, 1970; Franks, 1972; Franks and Cooper, 1972) that cells that can be established in cultures from normal tissues are derived from the vasoformative mesenchymal cells of small blood vessels, a fact noted incidentally by Porter and his colleagues (1945). The evidence for this was mainly morphological, since there is no other method for positive identification, and we could not exclude the possibility that the cells may have been derived from specific parenchymal cells but that all had adopted a similar form because of the tissue culture conditions. If there is a selection of preexisting cells rather than a structural change affecting differentiated cells, the cells that are eventually selected should be present in the starting tissues and in the initial cell suspensions. I n a detailed study of cell suspensions from mouse tissues (Franks and Wilson,

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1970) and of human embryo lung tissues (Franks and Cooper, 1972), cells with morphological and functional characters of the tissue culture cells were identified as endothelial cells and pericytes in the starting tissue. Although groups of parenchymal cells survive in primary or secondary cultures for at least 6 months (Wigley and Franks, 1976), they usually have an organized form and are easily recognized as epithelial. In many cultures foci of cells with an epithelial morphology are found and are usually regarded as relatively undifferentiated epithelial cells. We have shown that many of these cells are derived from nervous tissue-Schwann cells and perineural fibroblasts (E. Hamilton, L. M. Franks, and V. J. Hemmings, unpublished). Cultures established from tumors show a similar pattern in that in primary cultures both the tumor cells and the mesenchyme proliferate. In many cases the tumor cells die out, as in cultures from normal tissues, leaving a culture of similar mesenchymal cells. I n some instances there seems to be a more rapid outgrowth of these cells from tumor explants (unpublished observations). In a small number of cases-about 1 in 18-in a series of cultures from bladder cancers (Rigby and Franks, 1970) the tumor cells predominate and eventually seem to populate the entire culture, although it is probable that a small number of mesenchymal cells persist and are transferred at each subculture, since cells of mesenchymal form are often seen in late uncloned cultures of tumor cells. Cells of neural origin have not been seen so far in cultures derived from tumors. The fate of the mesenchymal cells in cultures from normal and tumor tissues depends on the species of origin (Macpherson, 1970). From human and avian tissue the cells all die out eventually, after a variable number of transfers, that is, they have a typical Hayflick-type limited life-span. Those from mice usually transform spontaneously (Sanford, 1965; Franks and Henzell, 1970). This type of spontaneous transformation probably accounts for the so-called sarcomatous transformation that sometimes occurs when established tissue culture cell lines derived from epithelial tumors are reimplanted in syngeneic hosts (Sanford et al., 1952, 1961; Franks and Hemmings, 1976). The discussion so far has concerned cells and tumors derived from epithelial organs, but it seems likely that cells from mesenchymal organs and tumors may behave similarly, although it may not be possible to distinguish between specific mesenchymal cells and cells derived from multipotential mesenchymal cells which may have the capacity to differentiate and produce other mesenchymal products or structures.


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The evidence on which these statements are based and descriptions of the ultrastructure of primary cultures and established cell lines from normal tissues and tumors are given in subsequent sections of this article. 11. General Features of Cells in Vitro

The adaptation of cells to growth in vitro requires a modification of cell function to allow survival under conditions that differ greatly from those in vivo, and in general this involves a loss of organized structure to a greater or lesser degree, usually accompanied by an increase in cell mobility. The nutrient and gas exchange systems are also less closely regulated than in vivo. Cells that survive in vitro are therefore required to adopt certain common metabolic and functional patterns which differ from those found in viuo. These are mirrored by structural changes and, since the tissue culture conditions are usually standarized, the cells, whatever their origin, adopt a standard undifferentiated pattern. Thus most tissue culture cells are similar in surface structure and mitochondria1 pattern and show an increase in pinocytosis and phagocytosis and an alteration in the distribution of intracellular filaments associated with attachment and cell movement. The majority of cells seen in sections from cultures have this undifferentiated pattern and, whatever their origin, cannot easily be distinguished from each other. Because of the physical requirement that the cells grow in flat sheets, even differentiated characters that may be retained by some cells, for example, specialized junctional complexes or secretory products, may show considerable modification. Since many of the specialized structures are limited to small areas of the cell surface, the chances of finding such specialized features in sections are relatively small. Only a small number of cells showing these features is likely to be found unless there is deliberate selection. Thus in mixed cultures it is not possible to identify the majority of cells with any certainty. Finally, cells in culture are usually selected by their ability to proliferate in vitro. Consequently, not only are mitotic cells common, but cells in an active growth cycle are less likely to demonstrate differentiated characters. All the structural components found in cells in vivo are present in vitro, and only those features that are altered in any significant way are discussed. The effects of fixation and of different fixatives on various cell components are also similar, but the distribution of cell organelles, and particularly the intracellular filaments, junctional

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complexes, and surface pattern, are influenced by the state of attachment of the cells at the time of fixation (see, for example, Lucky et al., 1975).

A. SHAPE AND SURFACEMORPHOLOGY OF

CELLSIN CULTURE

The shape of cells in culture is best appreciated with the light microscope, since in ultrathin sections the apparent shape is due largely to the orientation of the cells and the plane of section. The use of replica techniques and, more recently, scanning electron microscopy, has provided useful information on cell shape and surface morphology. The degree of attachment of cells to the substrate also has considerable influence on cell shape. The preparation of cell suspensions, particularly by trypsin or other proteases, causes striking changes in surface morphology. The surface features described are microvilli, marginal ruffled membranes (as seen in the light microscope), blebs, and filopodia-long thin extensions of cell cytoplasm. The effects on intracellular ultrastructure of the release of cells from their substrates is discussed elsewhere (see Section II,D), but Dalen and Todd (1971) have also reported on changes in surface morphology after trypsinization of Chang human embryo liver cells. During the rounding-up process of flattened cells long cytoplasmic retraction processes with terminal swellings were left attached to the substrate. These resembled mitotic filopodia (see elsewhere in this section) but developed much more rapidly (1-2 minutes). Microvilli present on the untreated cells disappeared within the first 5 minutes of trypsinization, but others (Cooper and Fisher, 1968; Follet and Goldman, 1970)have shown that these return very rapidly. Cooper and Fisher (1968) found that the distribution of microvilli in several different mammalian cells varied from less than 2 to more than 10 per 100 pm. Follet and Goldman (1970), using BHWC13 cells, found that the number of microvilli present on the cells was related to the phase of the cell cycle, the number increasing when the cell rounded up, and decreasing as the cell spread out on the substrate. In a study using scanning electron microscopy and transmission electron microscopy on replicas, Pugh-Humphreys and Sinclair (1970) found that microvilli measuring 0.1-0.2 pm in diameter and up to 5 pm in length were present on Landschutz ascites tumor, HeLa, and Madin dog kidney cells, but absent from chick mesenchyme cells. There was

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a fairly wide range of variation in number between cells in the cultures, perhaps related to the physiological state of the cells. This was also shown b y Hodges and Muir (1972), who compared the density, distribution, and morphology of the surface cytoplasmic projections (microvilli) of HeLa, BHK, and baby mouse kidney cells (Franks and Henzell, 1970) maintained in different culture media. There was considerable variation in the density and morphology of the microprocesses among the three lines cultured under similar conditions. Numerous processes were found on HeLa cells and on cultured baby mouse (CBM 17) polygonal cells, but few on CBM 17 “fibroblastic” cells and were rarely seen on BHK cells. At mitosis, the BHK, HeLa, and CBM cell lines lost their microvilli, and the membrane became deeply folded in the rounded cells. Porter et nl. (1973) described the changes that occurred throughout the cell cycle in CHO cells. In G, the cells were spherical and covered with many microvilli. By mid-GI the cells had flattened and the microvilli were almost entirely replaced by groups of blebs. These disappeared at the transition into S, and ruffled membranes became more common. During S microvilli were almost absent, particularly in very flattened cells. During G, the number of microvilli and amount of marginal ruffling increased. In late G, the long filopodia, which are the predominant processes present in the mitotic cell, began to appear. Changes have also been described in human skin epithelium during differentiation in nitro. Hashimoto and Kanzaki ( 1974), using replicas, found that the basal cells had many slender microvilli on the surface and at the advancing borders. As the cells matured to become Malpighian-like, the number of villi decreased and the villi became shorter. Completely keratinized cells were polygonal and scalelike, usually lying on top of less differentiated cells. The scales had few or no villi, but the surface was covered with ridges and furrows. Fibroblasts in the same culture had some long threadlike villi at the edges but few on the surface. A common feature of many cells in culture are surface protrusions filled with small vesicles which usually give an intensely positive reaction for cell surface enzymes such as alkaline phosphatase. The nature of these bodies is unknown. It is possible that these structures may be associated with mycoplasma infection, but this is not certain (Haguenau, 1973; Schneider et al., 1973).Many cell lines are thought to be infected with niycoplasmal species, and their exact effects are not known.

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B.

SURFACECOAT AND PLASMAMEMBRANE OF CELLS in Vitro

The boundary zone between the cell and its environment is usually thought of in terms of its individual components, but it is best considered a unified functional complex made u p of glycocalyx, plasma membrane, and submembraneous cytoplasm (see Benedetti et aZ., 1973; Emmelot, 1973; Bretcher and Raff, 1975; and many others for reviews and references). There are many reports on the structure and function of individual components, but the available methods are such that we still have only a superficial understanding of the relationship between structure and function. Since many cell surface properties are altered during the cell cycle, the growth stimulation induced by the culture conditions must also affect the results obtained using mass populations. Of the many functions ascribed to the cell surface (beyond the limits of this article) some are known to be localized to specific sites, while others are thought to be diffusely distributed over the cell surface. This is mirrored by a structural heterogeneity of all components. In addition to the local specialization of plasma membranes associated with cell contacts there are membrane heterogeneities on a fine level, as shown by histochemistry and freeze-fracture (see Benedetti et d., 1973, for review). Regions of plasma membrane where specialized functions are carried out also have altered structural characters (e.g., De Camilli et al., 1974). The glycocalyx too varies over different areas of the cell. In vivo all cells have a thin glycoprotein coat which is uniform in leukocytes, fibrocytes, and most other mesenchymal cells, including endothelium and muscle. The coat is also present in neurons. In simple epithelia the coat is thicker at the apical (luminal) surface than at the lateral and basal edges. At the basal surface the coat separates the plasma membrane from the basal lamina. It is absent at tight junctions (Rambourg et al., 1966; Rambourg and Leblond, 1967). A more detailed survey of luminal surfaces shows that in many organs, particularly in the alimentary tract, fine strands of material are also present. These appear to be inserted into the outer lamella of the plasma membrane (see Fig. 46). The cell coat material has been shown to be a carbohydrate-rich glycoprotein and can be stained by any of the usual electron stains for this material, including ruthenium red (RR), colloidal thorium and iron, and Alcian blue (see Martinez-Palomo, 1970, for review and references).


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Kelley and Lauer (1975)showed that the cell coat material of human embryo fibroblasts in culture reacts with colloidal thorium and RR and with a lectin probe (concanavalin A, Con A). All reagents demonstrated an even layer of material (30-35 nm) over the whole cell surface. It consisted of an electron-dense zone closely applied to the plasma membrane, covered by a flocculent, less dense inaterial. Pretreatment of the cells with hyaluronidase prevented RR staining, suggesting that the material is a mesenchymal acidic glycosaminoglycan. Focal areas could still be stained with thorium and Con A after this treatment. Treatment with neuraminidase gave a granular reaction. Pronase removed most of the stainable elements but left small localized areas of surface material. Trypsin decreased the thickness of the surface layer, leaving only the narrow membrane-associated zone. EDTA had little effect on the surface material. The results obtained with proteolytic enzymes suggested that proteins also play a structural role in maintaining the cell coat. The patchy distribution of staining also suggested that there may have been differences in the glycoproteins similar to those seen in uivo. The direct interpretation of these and similar results may be influenced by two factors usually not taken into account. Rowlatt et al. (1972) showed that glycoprotein derived from tissue culture medium is deposited on the substrate, and it seems highly probable that similar material is also bound to the cell surface. We have also shown (Franks and Wilson, 1970; Franks and Cooper, 1972) that many mesenchymal cells including human embryo “fibroblasts” in culture produce a fibrillar extracellular material which is closely applied to the cells. This material is trypsin-soluble. It is possible that the flocculent component described by Kelley and Lauer (1975) is made of this material, and that the dense zone represents the true glycocalyx. The distribution of some surface enzymes too varies in different areas of the same cell. There are many examples in v i m , in differentiated cells in liver, kidney, mammary gland, and so on. I n general, the activity at the luminal borders differs from that in the lateral and basal areas (see De-ThA, 1968, for references). The effects of cell culture on the heterogeneity of the various components of the cell surface complex has not been examined extensively, particularly at the ultrastructural level. Functional changes in the cell surface have been discussed extensively in the recent reviews previously cited. Ultrastructural studies have been mainly concerned with the changes that accompany or follow neoplastic transformation. These are considered later.

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C. CELL CONTACTS,JUNCTIONAL COMPLEXES, AND CELL SUBSTRATE ADHESION Recent work, particularly using freeze-fracture methods in conjunction with thin-section electron microscopy and the use of tracers, has clarified our knowledge of the structure and function of cell contacts and junctional complexes. In vitro studies have been concerned with the development of the structures and the alterations they may undergo during or after neoplastic transformation, but they can also be used as markers to identify specific cell types. Unfortunately, it is not always possible to make a positive identification on a thin section alone, since tracers and specific stains and fixations are required to distinguish among different types.

1. Normal Cell Contacts Confusion has been further compounded by the wide variety of names suggested for the different types of junctions. A brief description of their structure, function, and distribution (mainly based on Staehelin, 1974), and of preferred names, is given, but the reviews cited should be consulted for fuller information (see Friend and Gilula, 1972; Staehelin, 1974; Campbell and Campbell, 1971, for reviews and references). Four main types have been described; tight junctions (zonula occludens), spot desmosomes (macula adherens), continuous and discontinuous intermediate junctions (zonula and fascia adherens, belt desmosomes), and gap junctions. These specialized junctions are of course found only between like cells, for example, between two epithelial cells or two endothelial cells. Tight junctions act as watertight seals between different compartments; spot desmosomes are concerned with structural stability of epithelial cell complexes; intermediate junctions are probably involved in some way in cell movement, and gap junctions are concerned with small-molecule transfer. Tight junctions are found between a wide variety of cells, including most epithelia, endothelium, liver cells, mesothelium, and heart cells of chordates. Nonvertebrates are said to lack tight junctions (Satir and Gilula, 1973). Spot desmosomes are confined to epithelial cells and cardiac muscle, although desmosomelike contacts have been described in normal synovial membranes in the rat (but not other species) and in some diseased human synovia (Ghadially, 1975). Continuous and discontinuous intermediate junctions are found in epithelia, muscle, mesothelium (Cotran and Karnovsky, 1968; Fedorko and Hirsch, 1971), and endothelium. Gap junctions are found between most cell types. Junctional complexes made u p of tight junction, inter-


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mediate junction, desmosome, and gap junction, in that order, from the apex (lumen) to the base of the cells, are found only in epithelia. Tight junctions are formed by the apparent fusion of the outer lipid lamellae of two apposing membranes. The central fused lamella may be continuous or made up of rows of small particles. The entire width of the two fused junctional membranes is usually less than the total width of the two contributing unit membranes. Similar junctions are present between endothelial cells in some venules, probably as discontinuous bands, but freeze-cleaved preparations suggest that the lamellae may not be actually fused (Staehelin, 1975). Continuous and discontinuous intermediate junctions are similar to each other in ultrastructure, but the former are found as complete bands surrounding epithelial cells below tight junctions. Discontinuous junctions are usually found as plaques between cardiac and smooth muscle cells. In thin sections they appear as regions in which the plasma membranes of contiguous cells are parallel and separated by a 15-to 25-nm space (compared with 22-35 nm for spot desmosomes). The space is usually filled with very fine filamentous material. No dense stratum has been described in the interspace (except in the pigeon heart; McNutt and Weinstein, 1970). Filaments 7 nm in diameter run into a filamentous mat closely applied to the cytoplasmic side of the apposed plasma membranes. These filaments have been shown to be F-actin filaments. Spot desmosomes are disc-shaped and about 0.2-0.5 p m in diameter. Plasma membranes are separated b y a space usually about 22- to 35-nm wide, filled with filamentous material which is trypsin-soluble and can be stained with RR. Midway between the plasma membranes there is a dense stratum; in some desmosomes similar but thinner dense strata are also present and lie near each plasma membrane. Cross-bridges are often present between the strata. Dense plaques are attached to the cytoplasmic lamella of the plasma membrane which is often more densely stained than the outer lamella in this area. Bundles of 10-nm filaments appear to loop through these plaques. The central intercellular dense stratum, which is the most easily recognizable feature of the desmosome, does not extend over the whole area of the disc, so that in some planes it may not be visible and the distinction between a desmosome and an intermediate junction must be based on the width of the junction and the thickness of the associated filaments. If a tilting stage is available, a central stratum lying out of the horizontal plane can sometimes be demonstrated. Hemidesmosomes are found between the basal areas of epithelial cells and the

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basal lamina. In vitro such structures are sometimes found between the cells and the extracellular material on the substrate. Gap junction fine structure depends on the fixation and staining methods used. Three-layered gap junctions are seen in lead-stained, osmium-fixed tissues. Five-layered junctions are seen in tissues fixed in potassium permanganate, osmium tetroxide, or glutaraldehyde-osmium tetroxide and stained after sectioning with lead citrate and uranyl acetate. The five-layered appearance is due to the apparent fusion of the outer lamellae of two adjacent trilaminar unit membranes. The appearance is similar to that of a tight junction, but the central line is usually thicker-about two lainellae of the unit membrane. In tissues fixed in osmium tetroxide, with or without previous fixation in glutaraldehyde, and stained en bloc with uranyl acetate before dehydration the 2- to 3-nm gap between the plasma membranes can be demonstrated (Revel and Karnovsky, 1967). The frequency with which the different types of junctions occur in vitro depends on the tissue of origin of the cells, the degree of differentiation, especially in tumor cells, and the methods used in establishing or transferring the cultures. As might be expected, they are found most frequently in cultures of differentiated normal or tumor cells in a slow growth phase and are most easily demonstrated b y fixation and embedding of cells in situ. In rapidly growing cultures, particularly if cells are fixed after removal from the substrate by trypsinization or similar procedures, specialized contacts are not often seen.

2. Temporary Junctions or Attachment Plaques These junctions have been described in several tissues during embryonic development. Pannese (1968)described two types of junctions in developing chick spinal ganglia, which were present in the early and intermediate stages but disappeared in the late stages. One type resembled intermediate junctions and the second (and less frequent) tight junctions, although it is possible that they may have been gap junctions. The development of similar tight and intermediate-type junctions in cells in vitro has been described by Flaxman et al. (1969) and Abercrombie and collaborators (1971; Heaysman and Pegrum, 1973a,b), using cine and electron microscopy of cells in movement. Within 20 seconds of normal chick cells coming into contact, specialized areas appeared in the cortical cytoplasm at points of close apposition of the unit membranes. Within 60 seconds filaments appeared in these areas, lined up parallel to the long axis of the cell, and plaquelike thickenings resembling intermediate junctions appeared. Flaxman


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et al. (1969) described one area in which a tight junction developed within 10 minutes of contact. It seems likely that these junctions are temporary and disappear as cell movement continues. 3. Cell Substrate Adhesions Attachment plaques may also be seen between cells and their substrates. The whole of the undersurface of the cell is not closely attached to the substrate, but small protrusions of the lower surface form points of adhesion (Brunk et al., 1971). The ultrastructure of these plaques has not been described in detail for different cell types (see Revel and Wolken, 1973; Revel et al., 1974). Flaxman et al. (1968) compared the attachment of skin epithelial cells and fibroblasts to nitrocellulose-coated cover slips. Beneath the epithelium was a continuous layer of extracellular material with two components. Immediately beneath the cells was a moderately electron-opaque layer 45 nm thick, and beneath this a denser component about 5 nm thick. Localized thickenings in the plasma membrane, with associated densities in the extracellular material, were also present. These were indistinguishable from hemidesmosomes. Beneath the fibroblasts the 45-nm component was always present, but the dense 5-nm layer was sometimes absent and hemidesmosomes were not seen. Cornell (1969a), using mouse embryo cell strains, found that there were limited areas of contact between the cells and substrate, and that in these areas of apposition there was always a gap of about 10 nm. The difference in the size of the gaps described by Flaxman et al. (1968) and Cornell (1969a) may be due to a difference in the thickness of the cell coat material in the cells examined. In a more detailed study Stamatoglou (1976)showed that the substrate and the cells are coated by a material partly derived from the serum component of the medium (Rowlatt et al., 1972). RR staining showed that the cell coat material under some plaques approached very closely but did not fuse with the RR stained substrate coat at the attachment sites, leaving a gap of about 10 nm, but in some no gap was present. In some plaques, areas of membrane density associated with cytoplasmic filaments were also observed. Between the plaques he found that, in some cell strains (young cells from human embryo lung mesenchyme) fibers about 9 nm in diameter ran from the cell surface to the substrate. These fibers were not present in older cultures. (Some of these appearances are illustrated in Fig. 1-5.) 4. Modified Cell Contacts in Vitro

All types of junctions can b e found in cells in uitro, but for technical reasons already discussed it is not always possible to identify the

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types of individual contacts in thin sections. A further complication is that specialized contacts may be disrupted either in the preparation of tissues for culture or by the growth and movement of cells under conditions that do not allow normal cell development. Consequently, incomplete junctions are often found. The most easily recognized are altered desmosomes. After mechanical separation of cells the whole of a junctional complex may be found attached to the plasma membrane of a single cell (Franks et al., 1970b).Desmosomes are particularly persistent after damage (see Campbell and Campbell, 1971, for references). Overton (1968), in a study of the regeneration of desmosomes in chick tissues after trypsinization, showed that cells separate as a result of digestion of the extracellular material in the desmosome, leaving the cytoplasmic and membrane portions on the cell surface. These are later incorporated into intracytoplasmic vacuoles. Such intracytoplasmic desmosomes, which are often seen in some tumors (e.g., Caputo and Prandi, 1972), are sometimes seen in tumor cells in vitro. Overton (1973) and Lentz and Trinkaus (1971) have described the development of desmosomes. Cytoplasmic densities appear opposite each other on adjacent plasma membranes, and electron-dense material develops between the membranes. Ten-nanometer filaments enter the areas of cytoplasmic density as the plaques enlarge, and finally the central dense stratum forms. All stages in the development can be seen in cells in vitro, and in the early stages it may not be possible to separate a developing desmosome from intermediate junctions or from the temporary junctions already described. D. THE FILAMENT-MICROTUBULE SYSTEM

The filament-microtubule system is complex and not completely understood (for recent reviews, see Inoue and Stephens, 1975; Soifer, 1975). All eukaryotic cells seem to contain microtubules of variable FIGS. 1-5. Sections of human embryo lung cells (HE 104) fixed and sectioned in situ at right angles to the substrate. Figs. 1-4, RR-stained sections; Fig. 5, lead citrateand uranyl acetate-stained. (Courtesy S. Stamatoglou.) FIG. 1. Undersurfice of cell showing fine strands connecting RR-stained cell coat to RR-stained material on substrate. x 42,000. FIG.2. Several strands attached to a central globule. x 118,000. FIG.3. Attachment plaque showing close apposition of RR material on cell coat and substrate in some areas, but gaps remain in others. x 118,000. FIG. 4. Upper s u r f k e of cell showing irregular surface layer of RR material. x 118,000. FIG. 5. Attachment plaque (lead citrate- and uranyl acetate-stained section) showing cytoplasmic filaments and increased density of extracellular material. x 118,000.

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length, filaments of 10-nm diameter (tonofilaments), and filaments of about 7-nm diameter, thought to be fibrous actin (F-actin filaments). In addition, finer filaments of 4-nm (Franks et al., 1969) or 5-nm (Spooner et al., 1971) diameter have been described. The filaments and microtubules are found in most tissue culture cells examined by Buckley and Porter (1967). Franks et a2. (1969), Goldman (1971), Spooner et al. (1971), De Brabander et al. (1975),and many others. I n cells in suspension, particularly after trypsin treatment, all the elements are scattered throughout the cell, but in cells fixed in situ there is an orderly pattern. The 7-nm filaments are usually arranged around the periphery of the cell in a thin sheath which in places is thickened into bundles. Periodic densites ranging from 600 to 1200 nm apart are sometimes found scattered along the sheath (Spooner et al., 1971; and others). These are similar to the densities seen in smooth muscle and thought to be the equivalent of Z bands in striated muscle. Individual filaments in the sheath are usually parallel to the long axis of the cell, although short, connecting cross-filaments are sometimes seen (Spooner et al., 1971). The thicker bundles correspond to the stress fibers described by many workers (Buckley and Porter, 1967) and can be traced into the cell processes. In some cells the sheath is made up of two components with the filament groups arranged in two parallel planes (Fig. 6), with the constituent filaments at right angles to each other (Franks et al., 1969). In favorable sections these filaments can be seen to have a substructure made up of elongated units about 4 nm in diameter, possibly arranged in a helix (Franks et al., 1969), resembling that described in whole (Hanson and Lowy, 1963) or sectioned (Panner and Honig, 1967) actin filaments (Fig. 6, inset). Spooner et aZ. (1971)also described a second class of microfilaments about 5 nm in diameter immediately beneath the plasma membrane, particularly in the actively motile regions of the cell such as the undulating membranes. These are arranged as a network of short interconnected segments and sometimes appear to be inserted into the inner surface of the plasma membrane. These filaments are usually seen in tangential or cross section and are difficult to resolve. Accurate measurement of their diameter is not possible in routine sections, but it seems likely that these filaments may represent a single strand of actin subunits. There seems to be no doubt that both these classes of filaments are F-actin from morphological (including heavy meromyosinbinding), biochemical (Pollard and Weihing, 1974), and immunofluorescent evidence using antiactin antibodies (Lazarides and Weber, 1974). The filaments have also been demonstrated in the mi-

FIG.6. T h e edge of a cultured human colon himor cell showing actinlike filaments arranged in two planes parallel to the cell surface. T h e filaments nearer the surface are cut transversely, while the deeper layer is cut along the length of the fibers. x 45,000. The inset shows the twin strands making u p a single fiber (arrows). ~800,000.(From Franks et al., 1969.)

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totic apparatus (Hinkley and Telser, 1974), and in association until one class of specialized cells contacts the belt desmosome or intermediate junction (see Staehelin, 1974, for review). The filaments seem to be associated with cell movement and, from a considerable body of work on muscle, it seems likely that myosinlike molecules must be associated in some way with actin to generate such activity. Myosin has been demonstrated in cells (see Pollard and Weihing, 1974; Ostlund et al., 1974) biochemically. Using specific antimyosin antibodies, Weber and Groschel-Stewart (1974) showed that the myosin was closely related to the actin microfilaments. In BHK 21 cells Rash et al. (1972) described 15- to 18-nm-thick myosinlike filaments with periodic lateral projections interdigitating with 6-nm thin filaments. Ordered thick and thin Z-like assays resembled first stages of myofibril assembly in embryonic skeletal and cardiac muscle, and Gwynn et al. (1974) also demonstrated the presence of myosin associated with the plasma membrane in trypsin-dissociated embryonic chick smooth muscle cells. The 10-nm tonofilaments are present at four main sites-in close association with microtubules, in broad bands or scattered fibers around the nucleus, in small bundles scattered through the cytoplasm, particularly in epithelial cells, and in association with a type of specialized contact, the spot desmosome. These filaments are similar in morphology and do not bind heavy meromyosin; that is, they are not actin; but their chemical nature is not known, and it is not known whether all types are identical. Microtubules are present in all cells, and their morphology, and the chemical structure of a major component (tubulin), have been described by many workers (e.g., Tilney, 1971; Olmstead and Borisy, 1973).The tubules are about 25 nm in diameter, are of variable length, and are present throughout the cell. They are made up of 13 protofilaments each about 4 nm in diameter. The distribution of the filaments described above is that found in cells fixed in situ, the orientation of the 7-nm filaments in particular being directly associated with cell attachment. Separation of the cells from each other and from their substrate by proteolytic enzymes leads to a general disorganization of the pattern, perhaps associated with the retraction of cell processes, but there may also be a direct effect of the enzymes. The actinlike filaments and the microtubules can be disrupted selectively b y cytochalasin B and antitubulins (colchicine, vinblastine, and vincristine), respectively. The ultrastructural changes are described by Spooner et al. (1971), Goldman (1971), and De Brabander et al. (1975).Cytochalasin caused an almost immediate


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cessation of cell movement. After 5-15 minutes, local retractions appeared at the cell margin, and after 6-12 hours there was extensive retraction, and the central areas of the cells rounded up, although peripheral processes remained. Ultrastructurally, the drug appeared to affect the fine network of 5-nm filaments which were converted to dense masses of short-filament segments and granular material. Ribosomes and other cell organelles approached the plasma membrane more closely in these cells. The filaments in the sheaths and bundles, including those in the cell processes, were unaffected, as were the 10-nm filaments and the microtubules. The effects were reversible within 1 hour of withdrawal of the drug. The antitubulins produced strikingly different effects. Within 10-80 minutes (depending on the dose), the cells flattened and adopted a more “epithelial” form. Movement of the whole cell was greatly reduced, but the membrane activity (ruffling) at the leading edge continued. Ultrastructurally, the microtubules disappeared, and there was a loss of intracellular orientation and compartmentalization (De Brabander et al., 1975).This was seen most clearly in the Golgi zone, which normally was perinuclear and organized around the centrioles. In treated cells it “exploded,” and individual Golgi organelles were distributed over the entire cytoplasm. The centrioles were often peripheral. The rough endoplasmic reticulum increased, and annulate lamellae appeared. Within 5 hours the 10-nm filaments increased in number. By 24 hours large bundles and whorls were present. The actinlike filaments and other organelles were unaffected. With vinblastine and vincristine at dose levels of about 1puglml large crystalline aggregates with a tubular substructure also appeared. These are known to be composed of tubulin (Dales et al., 1973). The antitubulin-induced changes are also reversible, and within 30 minutes of removal of the drug microtubules had begun to reappear. By 24 hours the Golgi zone and centrioles had returned to their normal position, but 10-nm filament whorls were still present after 48 hours. The general conclusions drawn from work on the filamentmicrotubule system in cells in vitro are that the 5-nm filaments are associated with cell movements, particularly of the ruffled membrane. The 7-nm fibers are concerned with cell adhesion and the structural integrity of cell processes and may be involved in movements of the whole cell. The microtubules and 10-nm filaments are closely associated and seem to be responsible for maintenance of cell shape, and intracellular organization and transport. I n some functions, particularly whole-cell movement and shape, it is possible that there is some overlapping.

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The filament-microtubule system has been examined in complete detail in only a small number of cell types, mostly mesenchymal, but there seems to be little doubt that the conclusions drawn will be found to apply to most cells in vivo and in vitro. E. MITOCHONDRIA The mitochondria in cells in vitro show considerable variation in structure, presumably a response to changes in respiratory pathways. The organelles are usually larger than in vivo (Armiger et al., 1975), and their overall shape is extremely variable, ranging from short rods to long filaments with occasional branching (Soslau and Nass, 1971). The most bizarre shapes have been found in normal human diploid fibroblasts (WI-38) and in SV40-transformed lines (Lipetz and Cristofalo, 1972; Lipetz, 1973). In HeLa cell cultures this heterogeneity in mitochondrial shape was more pronounced among different cells than within a single cell (Posakomy et al., 1975). The arrangement of cristae also shows different degrees of alteration in the mitochondria of cultured cells. There may be slight disorganization (King and King, 1971) or considerable irregularity of cristal pattern, as in dog myocardium (Armiger et al., 1975) and human liver (Chang) cells in which the cristae are of irregular length, are not parallel, and may be longitudinally oriented (Jagendorf and Eliasson, 1969). Cristae that extend across the complete transverse width of the mitochondrial matrix have been seen frequently in mouse fibroblasts (Soslau and Nass, 1971) and in normal and SV4O-transformed human WI-38 cells (Lipetz and Cristofalo, 1972; Lipetz, 1973). Specialized tubular and vesicular arrangements have also been described in WI-38 cells and in rat adrenal cortex cultures (Kahri, 1971). Matrix granules are rarely a prominent feature in mitochondria of cells in vitro, although in some instances abnormal granules have been reported to accumulate, probably as a result of cell degeneration (Armiger et al., 1975). Homogeneous inclusion bodies, sometimes almost entirely replacing the mitochondria, are often seen in damaged cells, particularly in the mouse. Although they are often seen in carcinogen-treated cells (Knowles et al., 1972), they are found in nonneoplastic cells (Tarin, 1971; Horvath et al., 1973). Knowles et al. (1972) showed that the inclusions contain large amounts of calcium. The appearance of two different types of mitochondrial inclusions has been reported in Chinese hamster fibroblasts after treatment with ethidium bromide. The first appears within 4 hours, is similar in appearance to condensed chromatin, and has a helical structure. This is


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thought to be mitochondrial DNA (McGill et al., 1973).The other inclusions are smaller and appear later when mitochondrial swelling has appeared. These are thought to be divalent cation granules. The electron density of the matrix also varies. In some cases a fairly normal moderate density is found (Kahri, 1971; King et al., 1972), but more frequently the matrix is light, with focal clear patches of variable size (Jagendorf and Eliasson, 1969; King and King, 1971; Soslau and Nass, 1971; Armiger et al., 1975). Changes in mitochondrial ultrastructure of a similar or even more extreme nature have been found in tumor cells in vitro (Rigby and Franks, 1970; Singh et al., 1974), and in cells that have been transformed in vitro, either spontaneously (Franks and Wilson, 1970)or by a virus (Bosmann and Myers, 1974). A variety of possible causes and effects of these changes has been suggested, including accumulation of lactic acid, peroxidation of unsaturated fatty acids to form malonaldehyde effecting a type of in situ fixation, and changes in the rate of mitochondrial DNA, RNA, and protein synthesis. In most cases the configuration of mitochondria in cells in vitro conforms to the orthodox” state indicative of unstimulated endogenous respiration. Condensed configurations have also been found in intact cells, including rat adrenal cortex cultures (Kahri, 1971), long-term mouse cell cultures probably of vascular origin (Franks and Wilson, 1970),and tumor cells in vitro (Laiho and Trump, 1975).Hackenbrock et al. (1971) showed that the transition from orthodox to condensed configuration of isolated mitochondria accompanies a change in energy state of the mitochondria, resulting from the transition from state-4 respiration (in the presence of substrate and inorganic phosphate) to state3 respiration (in the presence of additional ADP). Recent studies have also shown that a similar situation exists in intact cells. The appearance of the condensed configuration indicates the initiation of oxidative phosphorylation and is related to the induced synthesis of ATP in the mitochondria (Andrews and Hackenbrock, 1975). Laiho and Trump (1975) showed that inhibition of ATP synthesis or increased cell membrane permeability induced by a nonpenetrating membrane-damaging agent leads to the rapid appearance of condensed mitochondria. It should be mentioned that, in most studies of the ultrastructure of mitochondria in cultured cells, the fixation procedures used have not been ideal for preserving mitochondria. In most instances 2.5% glutaraldehyde alone has been used as a primary fixative, and osmium for “

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postfixation, whereas more rapid penetration is effected b y mixed fixatives such as those containing paraformaldehyde and glutaraldehyde, which give better preservation of mitochondrial structure. Pilstrom and Nordland (1975) described the effects of variations in osmotic pressure and temperature and of concentration of fixative on mitochondrial ultrastructure in perfused liver. Hypotonic solutions caused mitochondrial swelling, while hypertonic solutions gave constant mitochondrial volume and surface. Increasing concentrations of glutaraldehyde gave larger amounts of cristal membranes. While it is true that mitochondrial ultrastructure is greatly influenced by the type of fixative used, osmolarity, and pH, and that many of the abnormal features of mitochondria found in tissue culture cells may be fixation artifacts, it is also true that there is considerable variation in mitochondrial structure within a culture and even within single cells. This can be correlated with evidence of oscillating respiratory function in cultures (Werrlein and Glinos, 1974). It may be that tissue culture cell mitochondria are more sensitive to fixation than those in uiuo, possibly as a result of membrane changes not yet determined.

F. CYTOPLASMIC INCLUSIONS Most cells cultured in uitro contain inclusions. Many are similar to those found in uiuo and include typical membrane-bounded lysosomes, multivesicular bodies, specific endothelial granules (WeibelPalade bodies, Haudenschild et al., 1975), and specific secretory products. Lipid droplets, usually not membrane-bounded, are common in some cultures (see Section I1,J). Cells in uitro are actively phagocytic, and many bizarre inclusions can be regarded as secondary lysosomes developing around phagocytosed debris from dead cells and from the medium. Crystalline inclusions of various types are not uncommon. Some may be material phagocytosed from the medium. G. VIRUSESAND VIRUSLIKEPARTICLES

Virus particles and mycoplasma have been found in many cell cultures. An extensive list is given by Seman and Dmochowski (1975) in a review on the ultrastructure of human tumors in uitro. The structure of the virus particles in the many groups examined is as described in standard texts, for example, Dalton and Haguenau (1973). In cells deliberately infected with a known virus, ultrastructural identification presents few problems, even though all particles may not be identified with certainty because of the sampling problem implicit in sectioned material. Problems may arise in cultures not


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known to be infected. C-type particles are often found in cultured cells, particularly from mouse tissues, although they also are found in human and feline cell cultures. The structure of the particles varies from species to species, mainly in thickness and in the interrelationships of the intermediate layer lying between the nucleoid and the envelope provided by the host plasma membrane. The structure is described in detail by Dalton (1972a,b), who also discusses the detailed structure of type-A and -B particles. Several intra- and extracellular structures in cell cultures have been mistaken for virus particles. The serum used in tissue culture media may contain elementary bodies of mycoplasma, bacteria, or bacteriophages. These are described in detail by Haguenau (1973)and Dalton (1975). Other particles are also present in fetal and newborn calf serum (de Tkaczevski, 1968; Dalton, 1975). These are round or elongated, 30-60 nm in diameter, and have a trilaminar envelope and a moderately dense core. Dalton (1975) suggests that these particles may be derived from vesicles of multivesicular bodies, microvesicles associated with secretory epithelial cells, or the breakdown of normal cell components. Some are thought to be derived from degenerating mitochondria1 cristae, the unit membranes of which have the same thickness as that of the particles-approximately three-quarters that of the plasma membrane in the cells described by Dalton (1975). Vesicles filled with these particles are sometimes found in cells, and many mimic virus-forming units (Fig. 7). Some are probably formed by phagocytosis. Haguenau (1973)has listed other possible sources of error including nuclear pores, certain types of small regular secretion granules, mycoplasma elementary bodies, and small coated or smooth pinocytotic vesicles. Such viruslike particles have been described in many cell cultures, including human lymphoblastoid lines (Moses et al., 1968) and a human transitional cell cancer line (Elliot et al., 1974). Rather smaller particles 10-12 nni in diameter have been described by Rounds et al. (1975) and Narayan and Rounds (1973) in culture media of some human tumor cell lines and human skin. These ringshaped particles contain RNA and DNA. Their exact nature and origin are not known. The routine identification of virus particles in cultured material by electron microscopy alone is not always possible unless there is an absolute correspondence in structure to a known virus. Naked virus particles can sometimes be identified because of the characteristic structure of the capsid layer, that is, the number and structure of the capsomeres, but this may not be possible for members of the C-type RNA group (Dalton, 1975).It must also be remembered that the morphol-

FIG. 7 . Part of a human kidney cell with particle-filled vesicles mimicking virusforming units. ~30,000. The insets show details of some particles. ~225,000.


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ogy of virus particles may be affected by the method of preparation for electron microscopy (Sarkar et al., 1975). H. THE NUCLEUS The nuclear and nucleolar structure of interphase and mitotic cells in vitro show no fundamental variations from that found in vivo (see, e.g., Wischnitzer, 1973; Busch, 1974, for reviews), but the nuclear outline is often more convoluted and nuclear bodies (Bouteille et al., 1967; Dupuy-Coin et al., 1972) are seen more frequently, particularly if antibiotics have been used in the culture medium.

I. ENZYMECHANGESIN CELLS in Vitro Little is known about the alterations in enzyme patterns that occur when cells are adapted to in uitro life. Studies on the effects of culture on some human tumor cell lines such as HeLa (Bottomley et al., 1969) and H. Ep. No. 2 (Miedema, 1969),and on mouse tumors such as thioacetamide-induced hepatoma (Bhide, 1970) and Crocker mouse sarcoma (Biesele, 1951), have revealed some enzyme changes, particularly in alkaline phosphatase, glucose-6-phosphate dehydrogenase, (GGPDH), lactate dehydrogenase (LDH), glucose-6-phosphatase, fructose 1,6-diphosphatase, ornithine transcarbamylase, arginase, and xanthine oxidase. Similarly, after human and chick embryo tissues were cultured, acid phosphatase (Cristofalo et al., 1968), alkaline phosphatase (Rossi et al., 1959), and LDH isoenzymes (Philip and Vesell, 1962) were altered. In some cases enzyme changes have been associated with neoplastic transformation (Sanford et al., 1970). Similarity between embryonic and malignant tissues has been suggested by studies of antigens (Stanislawski-Birencwajg et al., 1967) and enzymes, including glucose-6-phosphatase, phosphohexose isomerase (Weber and Cantero, 1957), tRNA methylase (Riddick and Gallo, 1970), lactic dehydrogenase (Goldman et al., 1964; Stanislawski-Birencwajg and Loisillier, 1965), and aldolase (Schapira, 1966) in rat and human tissues. Wilson (1973) compared the biochemical pattern and cytochemical distribution of a variety of enzymes in mouse and human embryo tissues and cell strains derived from them. Similar experiments were carried out on four tumors, cell lines derived from them, and tumors established from the same cell lines reimplanted to syngeneic hosts. The tumors were male and female mammary carcinomas, a muscle sarcoma, and a uterine sarcoma. The effect of culture on tumor and embryo tissues appeared to be complex and diverse. The removal of a tumor or embryo tissues from

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their normal environment to an artificial one induced an increase or decrease in DNA, protein, and enzyme levels. Although there were wide variations in enzyme pattern among the tumors and among the embryo tissues, the pattern in tissue culture cells, whatever their origin, tended to be similar. This suggested that the environmental conditions induced the cells to adopt similar enzyme patterns. Sat0 et al. (1960) found a similarity in antigenic specificity, amino acid requirements, enzyme levels, carbohydrate metabolism, and sensitivity to chemotherapeutic agents among cultures derived from different tissues. Some enzyme systems and some tumors appear to be less susceptible to changes induced by culture than others. Cell lines derived from less susceptible tissues and tumors should be more suitable for testing carcinogens or screening chemotherapeutic agents. In the tumors studied by Wilson (1973) the mitochondria1 respiratory enzymes succinic dehydrogenase (SDH) and cytochrome oxidase and the surface enzymes alkaline phosphatase and 5'nucleotidase seemed most susceptible to change in culture. However, on reimplantation of tumor cells in syngeneic hosts many enzyme levels tended to return to approximately their original levels. In some cases, however, an irreversible change apparently resulted from the culture conditions. The most striking example of this was the loss of alkaline phosphatase activity from muscle sarcoma and female mammary carcinoma implants. This enzyme was also absent in tumors derived from normal young and old mouse cells of various tissues after spontaneous neoplastic transformation in vitro. It is known that alkaline phosphatase levels in vitro can be altered by many factors such as cysteine concentration, glucocorticoid hormones (Cox and MacLeod, 1964), serum factors (Herz et al., 1969), osmolarity (Nitkowsky et al., 1963), and the method of removal of cells from their containers (Westfall, 1967). The basically similar levels of most enzymes in the original and in the tissue culture-derived male and female mammary carcinomas and muscle sarcoma suggested that the culture process caused little irreversible biochemical change in these tissues. In the uterine sarcoma, however, more permanent changes in enzyme pattern developed. In nonneoplastic cells the mitochondria1 respiratory enzymes SDH and cytochrome oxidase were particularly susceptible to change, as shown by their large alterations in cultures of both human and mouse embryo tissues (Wilson, 1973). SDH decreased in mouse embryo cultures but increased in human embryo cultures, so that both types of tissue culture cells had similar levels of SDH activity. A similar leveling off of activity was found to a smaller extent in many other en-


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zymes in these tissue culture cells. The enzymes of the cell surface were also susceptible to change. Alkaline phosphatase showed large alterations, and Snucleotidase was increased in both cultures. Acid phosphatase was localized on the cell surface of cultured mouse embryo cells, an unusual site for this enzyme (Fig. 8).This suggested a change in the membrane active transport mechanisms in these cultured cells.

J. DEGENERATIVE CHANGESIN CELLSin Vitro Degenerative changes in cells in vitro are similar to those found in vivo. The most detailed descriptions are those of Trump and his colleagues (e.g., Trump et d.,1965a,b,c; Ginn et d.,1968; Laiho and Trump, 1975), who described the effects of anoxia and chemical toxins on organized tissues. The changes that follow lethal injury are illustrated by Ginn et aZ. (1968). The earliest change is a dilatation of the endoplasmic reticulum, followed by contraction of the inner compartment of the mitochondria and a general increase in cell fluid as shown by an increase in cell size and separation of cell organelles. The plasma membrane outline becomes simplified, and surface blebs appear. Mitochondria1 changes become more marked, with increasing density and distortion. Ribosomes are lost from the endoplasmic reticulum, and polyribosomes disappear. Large or small cytoplasmic vacuoles may develop from the endoplasmic reticulum or from swollen mitochondria. In the final stages all mitochondria are swollen and contain flocculent or microcrystalline deposits. The nucleus shows karyolysis, lysosomes disappear, and the plasma membrane shows points of interruption. Sublethal damage may be general, as shown by intercellular edema or by the appearance of lipid droplets, usually not membrane-bounded. Focal cytoplasmic degeneration (Hruban et al., 1963)may also occur. This has been described after the addition of antisera (e.g., Goldberg and Green, 1959) and in untreated cultures. I n the early stages areas of cytoplasmic matrix are cleared of all particulate components. Cytoplasmic vacuoles may be present in some of the larger cleared areas. Later autophagic vacuoles appear, sometimes containing damaged mitochondria and other cell components. 111. Special Features of Cells in Vitro

A. MORPHOLOGYOF DIFFERENTIATEDCELLS Identification of specialized cells by morphology alone is not always possible, and there are many recorded examples of specialized

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FIG. 8. Non-tumor-producingcell line (eighteenth transfer) from old niouse liver (COM 5/liver/18, Franks and Henzell, 1970) showing positive acid phosphatase reaction at cell surface (Gomori’s method). x 10,000.


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function in cells in vitro although morphological differentiation has been lost. The structure of cells derived from differentiated tissues is considered in more detail later, but there are special features common to many of these cells whether derived from normal or tumor tissue. In general, epithelial cells can be recognized by their organized structure. Even cells derived from epithelial tumors tend to grow in an acinar or tubular pattern, but epithelial cells in pure culture may not produce basal laminae. In longitudinal sections, junctional complexes may be found, and the apposing cell walls beneath the complexes show a characteristic series of interdigitations, although in vitro the spaces between the interdigitations are much larger than in the tissues in vivo. These processes do not have the central core of microfilaments found in epithelial cell microvilli. Spot desmosomes are characteristic of epithelial cells. In the tubules the cells retain their polarity, and in secretory cells secretion droplets may be found near the luminal border. This border of the cell often has a normal pattern of microvilli with central filament bundles. The presence of glycoprotein strands attached to the outer lamella of the plasma membrane of the microvillus is characteristic of some epithelial cells, for example, colon (Mukherjee and Staehelin, 1971; and see Fig. 46), salivary gland (Knowles, 1976), human uterine cervix (Auersperg, 1969), and some bladder cells (Hicks et al., 1974; L. M. Franks, unpublished observations). The formation of blisterlike “domes” is a characteristic feature of epithelial cells in culture. Pickett et al. (1975) have described the ultrastructure of these domes in cultures from normal prelactating mouse mammary gland and from mouse mammary tumors. They found that the roof of the dome was identical in structure and continuous with the cells in the surrounding sheet, with microvilli and junctional complexes toward the free surface. There were no differences in structure between normal and tumor cells. The formation of a specific epithelial product such as keratin or melanin is satisfactory evidence for the epithelial origin of cells but, in early cultures the possibility of phagocytosis of epithelial products by mesenchymal cells must be considered. The increased production of an epithelial product in response to a specific stimulus, for example, adrenal secretion in response to ACTH, is also a useful positive finding. Differentiated mesenchymal cells can be identified only if they have a specific structure or produce a specific mesenchymal product or marker, for example, chondrocytes, embryonic cardiac muscle cells, and endothelial cells with specific Weibel-Palade bodies. Since the

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majority of mesenchymal cells in vitro are multipotential, positive identification of the tissue of origin is not always possible. These cells can generally be recognized b y the absence of a tubular pattern and junctional complexes, a close relationship to collagen fibers, if present, and the formation of extracellular material, some of which may resemble basal laminae. The production of collagen or apparently specific glycosaminoglycans has been demonstrated in many cell lines and can no longer be regarded as evidence for the origin of the cells from “fibroblasts,” or synovial or cartilage cells (see Wigley, 1975, for review and references).

B. AGING in Vitro The ultrastructure ofin vitro aging in cells with a finite life-span has been described b y Robbins et al. (1970) and Lipetz and Cristofalo (1972) in human embryo fibroblasts, Brunk et al. (1973) and Brunk (1973) in human embryo glial cells, and Brock and Hay (1971) in chick embryo cells. With increasing age the cells enlarge, and nuclear abnormalities appear. The most consistent change is the appearance of large residual bodies and secondary lysosomes. Brunk (1973) and Brunk et al. (1973) showed that the accumulation of these structures is a consequence of the failure or delay in cell division that accompanies in vitro aging. Robbins et al. (1970) also found that there was a decrease in the number of polyribosomes and an increase in glycogen, but these changes occurred shortly after the initiation of the cultures and preceded the decline in growth rate. Brock and Hay (1971)described mitochondrial changes. Although mitochondria1 changes in aging cells have been found in vivo (see Wilson and Franks, 1975, for references), the changes described by Brock and Hay (1971) are probably a consequence of the culture conditions.

c.

ULTRASTRUCTURE OF

HYBRIDCELLS

Schneeberger and Harris (1966) described the process of fusion of HeLa and Ehrlich ascites cells, mouse lymphocytes, hen red cells, and cells of HeLa-HeLa hybrids. Cytoplasmic bridges were seen, but the actual mechanism of virus-plasma membrane interaction could not be visualized. Daniels and Hamprecht (1974) described the ultrastructure of mouse neuroblastoma-rat glial cell hybrids, and Kilarski (1975)the cell surface changes in normal and SV40-transformed human fibroblasts and mouse macrophages. Azarnia et al. (1974) reported on the loss of gap junctions from hybrids of human


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Lesch-Nyhan cells which normally have such junctions, and mouse cI-ID cells which do not. As the hybrids lost human chromosomes, clones of cells without gap junctions also appeared. D. ULTRASTRUCTURAL FEATURES OF NEOPLASTICTRANSFORMATION

The usual pathological criteria of neoplasia-cell dedifferentiation, mitochondrial, nuclear, and chromosomal abnormalities, and invasion-cannot be applied to cells in uitro, since all may appear very rapidly in non-tumor-producing cultures established from normal tissues. Several reports have appeared describing ultrastructural differences in general, and cell surface morphology, glycocalyx, plasma membrane, and enzyme patterns. Some examples are discussed below, but it must be emphasized that most are reflections of an alteration or loss of differentiated characters rather than specific indicators of neoplastic change. All can be induced by noncarcinogenic agents. Many reports are based on changes induced by viral agents in mesenchymal cells, and these may differ from those in tumors induced by other agents. There are few changes in general morphology that are characteristic of neoplastic change in uitro. In a comparison of spontaneously transformed (tumor-producing) and nontransformed mesenchymal cells (Franks and Wilson, 1970) the only feature common to some of the transformed cells in this system was the presence of large cytoplasmic glycogen deposits. The cytoplasmic accumulation of glycogen in the tumor lines suggests a disturbance in glucose metabolism. Similar glycogen accumulation has been found in some hepatic adenomas (Garancis et al., 1969), and Sanford and her colleagues (Woods et al., 1959; Sanford et ul., 1969) found biochemical evidence of altered glycolytic activity in some transformed cells. Structural changes suggesting other metabolic disturbances were found in some transformed cell lines, including nuclear bodies (Bouteille et al., 1967), nuclear fibrils (Lane, 1969), dense mitochondria (Jagendorf and Eliasson, 1969; Goyer and Krall, 1969), and large mitochondria of normal density. Other mitochondrial changes in tumor cells were described earlier. None of these features were present in all transformed cells, and none are absolutely indicative of transformation. Fusenig et al. (1973)described the inhibition of ultrastructural differentiation in primary cultures of mouse skin epithelium after treatment with a chemical carcinogen (DMBA). One culture line, after a single treatment, has been maintained for at least 2 years and has produced squamous carcinomas when reimplanted in syngeneic hosts.

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This loss of differentiated characters in vitru, as in vivo, may be associated with neoplasia but again is not an absolute feature. There have been few detailed comparative studies on the ultrastructural changes induced by oncogenic viruses. McNutt et al. (1971) described nuclear and cytoplasmic changes after SV40 transformation, but most of these have been seen in nontransformed cells, except for the distribution of cytoplasmic filaments discussed below. Lipetz (1973) found few significant differences between young and old WI-38 cells transformed by SV40 virus. Schidlovsky et al. (1972) described changes following treatment with R-35 virus (a C-type virus), and Petursson et al. (1969) the ultrastructure of cells transformed by adenovirus 12. None of the changes are specific. Changes in the quantity and distribution of microfilaments have also been associated with transformation. Pollack et al. (1975), using inimunofluorescent antibodies to actin and myosin, confirmed earlier electron microscope findings (McNutt et al., 1971, 1973) that dense sheets of actin filaments were redistributed in transformed cells, and that this change was related to loss of anchorage-dependent growth control. These workers used rat embryo and mouse 3T3 cells transformed with SV40 virus. In these cells there did not seem to be any alteration in the total amount of actin or myosin, but others (e.g., Gabbiani et al., 1975) found that there was an increase in contractile proteins in human skin and mammary carcinomas. These results are based on visual estimates of fluorescence intensity and cannot be regarded as absolute. Porter and his colleagues (1974; Porter and Fonte, 1973; Williams et al., 1973)have described some differences found when comparing the cell surface morphology of tumor cells in vitro with normal cells (Porter et al., 1974; Porter and Fonte, 1973; Williams et al., 1973). HeLa S, cells had microvilli, but these occurred on free surfaces only and were distributed in an uneven pattern. Their length varied considerably from 0.2 to 6 pm, and they were straight or bent and of constant diameter or formed local swellings. These irregularities in form contrasted greatly with the structure of the surface of columnar cells from normal human cervix in which the microvilli were much more closely packed, uniform in diameter, and did not exceed 2.0 pm in length. Wilbanks (1975) compared the surface morphology of normal and premalignant cells (carcinoma in situ) from the human cervix in primary culture. Actively growing normal epithelium had regular short microvilli which were absent from the differentiated surface cells.


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The surfaces of the premalignant cells were much more complex, with many long, folded microvilli, resembling those found in carcinoma cells. Hodges et al. (1973) have also described changes in wiwo and in uitro in bladder cells treated with carcinogens. Vesely and Boyde (1973) review previous work and described the scanning electron microscope appearance of normal, Rous virus-transformed, and cheniically transformed rat embryo fibroblasts and of macrophages. In two lymphoblastic cell lines Nilsson and Sundstrom (1974) found that, although the surf'ace structure of each was characteristic, one having many microvilli and the other having a smooth surface with relatively few villi, the processes themselves were short and irregular. Blebs were sometimes found in one line. Thus in the majority of tumor cells examined a constant feature is the presence of irregular microvilli, although the nuinber present on different tumor cells may vary widely. Although chemical analysis of surface material from virustransformed cells showed that the amounts of some sugars in the glycoprotein and glycolipids were reduced, light and electron microscope observations have produced conflicting results (see Dermer et ul ., 1974, for references). Morphological changes in RR-stained cell surface material after viral transformation were reported by MartinezPalomo et al. (1969). They compared cultures of normal rat and Chinese and Syrian hamster embryo cells with Chinese and Syrian hamster cells transformed by adenovirus 12, Syrian hamster cells transformed b y SV40 virus, and spontaneously transformed BHK 21 and rat fibroblast cells. In all the transformed cells the RR layer was considerably increased. In some recent work Deriner et ul. (1974) found that, in subconfluent cultures, when both normal and mouse sarcoma virus-transformed rat kidney cells were dividing at an equal rate, there were no differences in the surface coats. With RR there was a continuous, thin, electron-dense layer, but with phosphotungstic acid the staining was spotty. In confluent cultures cell growth was greatly reduced in normal cells but continued in transformed cells. Under these conditions coat thickness was greatly increased in the noi-mal cells. RR-binding material is also present on the surfaces of many epithelial tumor cells in uitro (Staniatoglou, 1976), but it is not possible to make direct comparisons of thickness, since normal cells for comparison cannot be maintained in uitro. When similar methods were applied to uncultured nornial and malignant human breast epithelium, Dermer (1973) found that the cell coat was thicker in the nornial cells, than in the tumor cells. Kim et al.

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(1975), using carcinogen-induced rat mammary tumors, found that nonmetastasizing tumors had a thick glycocalyx, but that spontaneously metastasizing tumors had little or no demonstrable glycocalyx. There was also a direct relationship between coat thickness and immunogenicity, which could be quantitated by measurement of a plasma membrane enzyme 5’-nucleotidase. The absence of a glycocalyx from the metastasizing cells seemed to be due to its dissociation from the plasma membrane, since it was present in the blood of the metastasizing tumor-bearing animals. The conclusions to be drawn from in uiuo and in uitro experiments are these. The thickness of the glycocalyx in vitro is related to the growth rate of the cells, although it may be thicker in normal cells. This may in part be due to the increased production of extracellular fibrillar material by mesenchymal cells (see Section V,B). In both normal and transformed cells the thickness of the cell coat may be increased by material deposited from the medium (Rowlatt et al., 1972). In some tumor cells coat material may be less firmly attached and consequently released more easily into the medium, resulting in a thinner coat, although the production rate may not be altered. Changes in cell coat morphology in vitro is thus not an absolute indicator of malignancy. Many normal cells in culture have been shown to possess a large external transformation-sensitive protein (LETSP) on their plasma membrane, which can be identified after lactoperoxidase-catalyzed lZ5Iiodination (Hynes, 1973;Wickus et al., 1974) or a glactose oxidase, tritiated borohydride method (Critchley, 1974). LETSP has a nominal mass of 250,000 daltons and is a glycoprotein, but is not collagen or mucopolysaccharide (Pearlstein and Waterfield, 1974; Hynes and Humphryes, 1974). Although no specific function has yet been ascribed to LETSP, it shows many interesting characteristics. Its levels increased at confluency in many cells (Pearlstein and Waterfield, 1974; Hynes and Bye, 1974; Yamada and Weston, 1974) and decreased when confluent cells were changed into fresh media. Of particular interest was its disappearance from the external plasma membrane during mitosis (Pearlstein and Waterfield, 1974; Hynes and Bye, 1974). In a survey of the plasma membrane composition of a variety of mammalian cells in culture from different lengths of time (Pearlstein et al., 1976), electron microscope autoradiography confirmed that lactoperoxidase-catalyzed iodination labeled surface material only. The protein (LETSP) was present in primary explants of human prostate, calf bladder, and rat mammary gland, and in mouse whole embryos as well as in a variety


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of non-tumor-producing rodent and human mesenchymal cultured cell lines. It was not lost in long-term culture. Neoplastic transformation in uitro, whether spontaneous or induced by a chemical carcinogen or virus led to a loss of LETSP in most but not all cases. The protein was also absent from all the carcinoma cell lines tested but was present in some of the sarcoma cell lines. It could be removed from some cells b y proteases, but electron microscope autoradiography showed that, although LETSP was removed, other iodinated surface proteins remained (L. M. Franks, unpublished observations). Another indicator of cell surface change was the increased agglutinability of virus-transformed cells by lectins (Burger and Goldberg, 1967).It was suggested that this was due to an increase in the number of lectin-binding sites in transformed cells. Nicholson (1971), using iron-labeled Con A, and Smith and Revel (1972), using hemocyaninlabeled Con A, studied this ultrastructurally. The binding sites in the cells studied, 3T3 and SV 3T3 (Nicholson, 1971), and BHK 21, SV 3T3, and rat and human red and white blood cells (Smith and Revel, 1972), were found to be unevenly dispersed over the surface. Nicholson (1971) found that the number of sites was the same in 3T3, and its transformed variant, but that the total surface area of the transformed cells was about half that of the normal cells. H e suggested that the increased agglutinability was due to closer clustering of the binding sites. Similar studies on epithelial cells have not been described. Aggarwal et al. (1975) used platinum-pyrimidine complexes to demonstrate cell surface changes. These compounds bind specifically to nucleic acids but, when they were applied to several tumor cell cultures, electron-dense patches were found at the cell surface. No staining was found in cultures from several normal tissues. I n two cell lines examined staining was prevented by pretreatment with DNase I or neuraminidase. They conclude that tumor cells have patches of DNA at the surface. Surprisingly, the patches were not found on HeLa cells but were present on a nontransformed rat embryo skin cell line. The possibility that DNA may have been adsorbed onto the cells from the serum in the medium or from dead cells was not excluded. Plasma membrane changes have been described in specialized contacts and in the membrane itself. Both Martinez-Palomo et al. (1969) and Demier et nl. (1974) found that intermediate and tight junctions were present in nontransformed cells, but after transformation normal tight junctions were rarely found. In epithelial cells from a Novikoff hepatoma, Johnson and Sheridan (1974) found that gap junctions and intermediate junctions were present, but that true tight junctions and

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desmosomes were rare. This is a common but not invariable finding. Stamatoglou (1976), using five different human bladder tumor cell lines, could not demonstrate tight junctions, but in a tumor-producing transformed epithelial line derived from mouse salivary gland (Knowles and Franks, 1976) tight jimctions and desmosomes were present. Scott et (11. (1973; Furcht and Scott, 1974) described differences in the internal structure of the membranes of normal and transformed cells. Using freeze-fracture, they found that at high cell densities the intramembrane particles were aggregated in normal cells but dispersed in transformed cells. Gilula et al. (1975) found that, if the cells were examined without or with mild glutaraldehyde fixation, there were no striking differences in particle distribution. Aggregation was induced in normal chick embryo fibroblast and mouse 3T3 cells, but not in their Rous virus- or SV40-transformed variants after treatment with glycerol. The effects on epithelial cells have not been reported. Changes in enzyme levels and distribution have been reported in many tumors in vivo and in vitro (see Wilson, 1974, for references). Wilson (1974) compared enzyme patterns by quantitative biochemical assay and optical and electron microscope histochemistry of seven spontaneously transformed and five nontransformed cell lines from various organs of C57BL and C3H mice (Franks and Henzell, 1970). The activity of the surface enzymes was strikingly changed in the tumor-producing lines. Alkaline phosphatase was absent or at very low levels; GGPDH, LDH, and 5’ -nucleotidase levels were low, and P-glucuronidase levels were high in the transformed cells. Histochemistry did not give information about quantities but showed significant alterations in the distribution of the enzymes, particularly with SDH, GGPDH, and LDH. Some cells in the same culture showed an intense reaction for these enzymes, while adjacent morphologically identical cells showed no reaction at all. Four of the transformed cell lines and four of the nontransformed lines also showed a localization of acid phosphatase at the cell surface, as well as at the more usually lysosomal sites. The most consistent changes to be detected on the ultrastructural level were in alkaline phosphatase activity. This enzyme has been reported to decrease after long-term culture of both adult and embryo mouse cells (Westfall, 1967; Sanford et al., 1970)and, even after shortterm culture of mouse and human embryo cells, both alkaline phosphatase and 5’-nucleotidase were significantly altered (Wilson, 1973). In some human embryonic epithelial-like cell lines, low levels of alkaline phosphatase were correlated with a decrease in chromosome number (De Carli et al., 1964).


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Although in one report of in vitro tramformation of mouse cells in the presence of horse serum alkaline phosphatase was reported to increase (Farnes et d.,1968), in most instances the levels of this enzyme, for example, in SV4O-transformed WI-38 cells (Cristofalo et d., 1968), transformed Chang liver cells (Nitowsky and Herz, 1961), spontaneously transformed mouse mesenchymal cells (Wilson, 1974), and chemically transfomied hamster cells in vitro (Sela and Sachs, 1974), it seems to fall after in vitro transformation. Wilson (1974) found that alkaline phosphatase was reduced to extremely low levels or was completely absent in seven tuinorproducing cell lines derived from a variety of nomial mouse tissues (probably vascular in origin) which had undergone spontaneous transformation in vitro. Five ultrastructurally similar but non-tumorproducing long-tenn cultures had significant levels of alkaline phosphatase. In epithelial tumors the presence or absence of alkaline phosphatase depends in part on the presence of the enzyme in the tissue of origin and partly on the degree of differentiation of the tumor. In five human bladder tumor cell lines (Benham et ul., 1976), the total amount, the ultrastructural distribution of the enzyme, and the isoenzyme profile varied considerably among the cell lines. By using specific inhibitors such as phenylalanine and Levamisole, which seem to inhibit particular isoenzymes, it has been possible to localize the possible sites of these isoenzymes under the electron microscope. By using similar methods a placental type of alkaline phosphatase has been demonstrated in a human gastric choriocarcinoma cell line (Kameya et al., 1975).

IV. Ultrastructure of Primary Explants and Epithelial Cell Strains from Normal Epithelial Tissues Rose (1970) illustrated the morphological changes and ultrastructure of many tissues in primary culture, and there are several reports on the ultrastructure of differentiated epithelial cells after primary isolation with or without short-term culture, but there are few detailed descriptions of the cellular changes that take place. In this section, the pattern of morphological change is illustrated in cultures of salivary gland, and some additional points of special interest are considered in descriptions of cultures from several other organs. Cells damaged during the preparation of explants die rapidly but, as already noted, the surviving differentiated epithelial cells in explants usually live for a varying period of time, sometimes for many months-but are usually overgrown by mesenchymal cells or are lost

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after transfer. In some tissues, particularly in explants from embryonic organs, many of the parenchymal cells survive in the explant (Fig. 9). In others, particularly in explants from adult tissues, most of the parenchymal cells die, and the cultures are repopulated from a surviving stem cell population [see, for example, Defries and Franks, 1976 (adult colon); Wigley and Franks, 1976 (adult salivary gland; Rowden et ul., 1975 (skin)]. As well as epithelial cells, scattered mesenchymal cells and blood vessels are also present. A striking feature in many cultures in the early stages is the presence of large amounts of collagen. The amount is often much larger than would be expected to lie present in the original explant and may be due to polymerization of preexisting precursor molecules. Within the first 24-36 hours most of the explants are covered by a layer of flattened cells. Many of the cells can be identified as epithelial by electron microscopy, but in some organs, particularly those in which mesothelium is present, it is not always possible to distinguish between undifferentiated epithelium and mesothelium. When the explants attach to the substrate-a process that may take from less than 24 hours to over 14 days-similar cells grow out onto the substrate in small groups usualIy surrounded by mesenchymal cells.

A. EXPLANTSOF FETALSALIVARYGLAND Some of these changes are illustrated in primary cultures of human embryo salivary gland (Knowles, 1976). Figure 9 shows the whole of an attached primary explant of human embryo salivary gland after 14 days in culture. The remains of a large degenerating acinus can be seen in the center, but the surviving glands are filled with irregular proliferating cells. Figure 10 shows an acinus (A of Fig. 9) filled with a solid mass of cells. The pattern of cell packing and interdigitation, the surrounding basal lamina, and the presence of myoepithelial cells with hemidesmosomes, e.g., Fig. 10 (bottom left) and Fig. 11, establish these cells as epithelial. The pale areas in the cells are glycogen. Figure 12 shows another acinus (B of Fig. 9) more easily recognized as epithelial because of the characteristic microvillar pattern of the lumenal epithelium (Fig. 13). There is a mitotic cell on the left. Mesenchymal cells and collagen fill the spaces between the acini. Many of the cells surrounding the explant (e.g., bottom right) can be identified as epithelial, because of the arrangement of the cells and their microvillar border (Fig. 14). At higher magnifications junctional complexes can be recognized. In this area the subepithelial cells can be identified as mesenchymal, particularly by their relationship to the FIG.9 Section of 14-day-old culture of whole primary explant of human embryo salX 155. (Courtesy Mrs. M. Knowles.)

ivary gland; see text for details.


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FIG. 10. The acinus (A) from Fig. 9, with a myoepithelial cell below; see text for details. x 1500. FIG. 11. The myoepithelial cell from Fig. 10 showing basal lamina and hemidesmosomes. x 66,000. 94

FIG.12. Acinus ( B ) from Fig. 9, with central lumen and mitotic cell; s e e text for details. x 1500. FIG. 13. Luminal border of acinus showing microvilli with attached glycoproteiri strands and jiinctional complexes. x 30,000. 95

FIG. 14. Epithelial cells at edge of explarit from area marked with arrow in Fig. 9, with mesenchymal cells in collagen. x 1500. FIG.15. Cells from acinus (C) in Fig. 9. Some can be recognized as epithelial by tubular pattern (arrow) and microvillar borders. x 1500.


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collagen bundles. At the top of the explant another acinus (C of Fig. 9) can be seen. Cells from this acinus are growing into the outgrowth as a sheet in which many of the cells cannot be identified easily (Fig. 15). Some cells can be recognized as epithelial where a tubular pattern is retained, but individual cells cannot be identified with certainty, particularly when they are closely associated with mesenchymal cells. Some of the cells are illustrated in Figs. 16 and 17 and classified as epithelial if there is evidence of a residual tubular pattern, a basal lamina, secretory droplets, interdigitating cell processes, and specialized contacts. Although such distinctions are apparently based on faith, they in fact represent previous experience of pathologists in observing the appearance of similar cells under abnormal conditions. There seems to be little doubt that this pattern of degeneration and irregular proliferation-probably a repair reaction-occurs in explants from most tissues.

B. EXPLANTS OF

ADULT

SALIVARYGLAND

A similar series of changes has been described in explants of adult salivary gland (Wigley, 1974; Wigley and Franks, 1976). The growth pattern of cells in the outgrowth from these cultures varies from culture to culture. In most, the epithelial and mesenchymal cells become inextricably mixed and cannot easily be distinguished from each other. In others, there may be a complex reorganization of the epithelial cells to form localized areas in which differentiated cells survive for a considerable time. In many cases, the first cells to migrate were fibroblastic or epithelioid mesenchymal cells. Subsequently, in many explants, this was followed by the growth of a contiguous sheet of epithelial cells. In multilayered areas adjacent to the explant, whole ductlike structures migrated out and then flattened onto the substrate. This pattern was frequently seen in explants after about 3 weeks in vitro. Once in the monolayer, epithelial cells were often grouped into orientated units, with a central “lumen.” Occasionally, these structures were large enough to be seen clearly at the light microscope level. After 1-2 months, this pattern was usually observed only in later cultures, where migration had almost ceased and proliferation within the outgrowth had slowed to a very low turnover level (as shown by whole-cell autoradiography). Mesenchymal cells within the outgrowth frequently migrated a considerable distance from the explant and often also overlaid epithelial cells in multilayered areas. Their proliferation appeared to be limited in the early stages in primary cultures that eventually consisted largely of epithelial cells. The proliferation of epithelium appeared to

FIG.16. Epithelial cells from same area, lying inside a basal lamina (arrow).Cell interdigitations and contacts (arrow) can be seen. x 7000. 98

FIG.17. Epithelial cells with lateral interdigitations above basal lamina and mesenchymal cells. x 7000. 99

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L. M. F R A N K S ANDPATHCIA D. WILSON

be correlated inversely with the size of the explant. Explants less than 0.5 mm in diameter often showed an earlier attachment to the substrate and a more prolific yield of epithelium. Primary explant cultures usually remained healthy for at least 6 months. Toward the end of this time period mesenchymal cells showed some signs of degeneration, but epithelium appeared unchanged (Figs. 18 and 19).

1. Ultrastructural Characteristics of Cells in the Explant During the first few days of culture, explants were composed largely of degenerating cells, with a basal lamina outlining the former position of acini and ducts. Basically, ultrastructural evidence only confirmed findings at the light microscope level, acinar cells showing early degeneration and eventually being entirely lost from the explant. Myoepithelium survived slightly longer. Many duct cells also showed signs of necrosis, with intercellular edema, loss of secretory granules, and reversion to a more cuboidal form. Groups of apparently viable cells were often found among degenerating ones, all enclosed by a basal lamina showing the outline of a granular tubule. Duct cells with these features, but retaining a few secretory granules, were often seen on the second and third days. Large lipid droplets appeared in many surviving cells during the first week of culture, sometimes distorting the nucleus. Frequently, these cells showed little sign of further damage and were apparently capable of proliferation. Mitotic figures were occasionally seen at this stage. Over the period extending from about day 3 to day 10, explants showed increasing cellularity, and the intercellular spaces became filled with mature banded collagen fibrils. The duct cells had by this time acquired a superficial resemblance to undifferentiated embryonic gland cells but were organized around more-or-less well-defined lumina. Cells were low columnar or, more usually, cuboidal in shape. The addition of insulin and hydrocortisone to the medium increased the height of most duct cells. Nuclei were generally round or ovoid and had a thin peripheral layer of chromatin with a few dense blocks of chromatin. No particular cytoplasmic organelles were especially well-developed, although mitochondria were relatively abundant. Endoplasmic reticulum was present as undilated membranes studded with ribosomes, and Golgi zones were occasionally seen around the nucleus. Autophagic lysosomes were often present in the cytoplasm. Where cells were polarized around a lumen, typical junctional complexes, luminal microvilli, and occasional intercellular canaliculi were found. The lateral plasma membranes were thrown into inter-


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FIG. 18. Araldite-embedded whole culture of adult mouse salivary gland after 6 months i n citro. Almost all the cells have formed well-differentiated tubules. x 17. FIG. 19. The area marked in Fig. 18 showing epithelial tubule. ~ 3 7 7 5 .

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locking folds, usually filling an enlarged intercellular space. Myoepithelial cells did not appear to surround tubules at this stage, but the basal lamina was still intact. Occasionally, mesenchymal cells were found in the collagen matrix.

2. Ultrastructural Characteristics of Cells in the Outgrowth Where cells were arranged in a ductlike pattern around a lumen within a monolayered or multilayered outgrowth, even if the “duct” were an incomplete structure, they were easily recognizable as epithelial. It became more difficult to distinguish epithelial cells from epithelial-like mesenchymal forms in sheets of closely packed monolayered cells. Two features in particular were found to be associated only with epithelial cells: well-defined desmosomes and bundles of tonofilaments. These were both present in all cells identified as epithelial on other grounds. Monolayer cells considered epithelial in origin often showed increasingly pronounced desmosomes and tonofilaments at later times in culture, giving them the appearance of squamous cells. In fact, in most sheets of monolayered cells, some evidence of the formation of small lumenlike structures was evident. Other ultrastructural features of these cells were similar to those of the duct cells in the explant. In addition, a few small, dense, round or elongated granules were scattered throughout the cytoplasm of some cells. Occasionally, cells with tightly packed mitochondria were found. Cells in an epithelial sheet were frequently arranged into welldefined ductlike structures, with radial polarization around a lumen, in the plane of the monolayer. This ability to organize into organotypic structures was unaffected by any hormone supplement. The resemblance to cells in the explant is marked, and there can be little question of their epithelial derivation. Numerous desmosomes and tonofilaments were seen, especially near the substrate below the level of the nucleus. A second supranuclear zone of filaments and associated desmosomes was often seen in vertical sections. Lateral cell membranes were often interlocking, as is found in submandibular duct cells in vivo. Occasionally, atypical desmosomes were seen between epithelial cells in a sheet. No dense central line was seen between the cells, but parallel transverse densities were present instead.

3. Ultrastructure of Transferred Cells When well-established primary cultures with a high proportion of epithelium were trypsinized and transferred to a secondary culture,


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many sheets of epithelium adhered to the substrate. The simultaneous transfer of a proportion of mesenchymal cells seemed unavoidable and, in secondary cultures of dispersed cells, they started to divide and eventually took over the culture. No such burst of proliferation was seen in the patches of epithelium and, although they survived transference, they did not contribute to the establishment of subsequently passaged lines. In a further series of experiments using a similar system, Knowles (1976) established epithelial cell strains from adult salivary gland cultures, using scraping to remove the mesenchymal cells. After 6 months or more in culture transferable epithelial cell cultures were established. The cells were identified as epithelial by their ultrastructure. Although they grew very slowly at first, they produced epithelial tumors on reimplantation in syngeneic hosts. Proliferating cells in the outgrowth in salivary gland cultures were shown by histochemical methods to have been derived from granular tubular cells but had lost their specific secretory capacity at a very early stage in culture, although they could still be recognized as epithelial.

c.

PRIMARY AND TRANSFERABLE CULTURES FROM OTHER ORGANS

This loss of function and of ordered structure is commonly found in cultures from many normal epithelial organs and may lead to difficulties in identification. Wigley (1975), in an extensive critical review on differentiated cells in uitro, concluded that in the great majority of reported long-term lines derived from normal epithelial tissues the criteria put forward for their identification are inadequate. In most, specialized function is lost shortly after initiation of the cultures. There are several possible explanations. Many cultures may be overgrown by meseiichyinal cells. In others, the cells may be parenchymal cells that have failed to differentiate and function because of the absence of necessary growth factors.

1. Prostate and Breast In the human prepuberal prostate, Webber (1975)found that epithelial cells could b e maintained for up to 21 days, but the cells resembled those found in castrates. In cultures from adult prostate, separated epithelium, although ultrastructurally well preserved, could not be maintained in cultures (Franks et al., 1970b), although mixed cell populations could be maintained for over 16 months. Cultures of cells from postweaning human breast fluid (Russo et al., 1975) and prelac-

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tating normal mouse mammary glands (Pickett et al., 1975) have also been described. These cells were maintained for up to 3 weeks or more and resembled the normal luminal epithelial cells of the secreting breast. The cells were cuboidal and had the usual junctional complexes, interdigitations, and luminal microvilli. Finely granular material in the Golgi vesicles was very similar to milk casein in size and structure. The cultures eventually died out. E. V. Gaffney (personal communication) has established a continuously transferable human breast cell line (HBL 100) from a reduction mammoplasty specimen. These cells are epithelial, since they have desmosomes and show a tendency to differentiate if treated with prolactin or estradiol. Since the cells are not derived from normal tissue, can be maintained continuously in uitro, and form colonies in agar, Gaffney does not feel that the cells can be regarded as normal human breast cells.

2. Thyroid,Adrenal, Pituitary, and Testis The involution found in prostatic and mammary cells may be due to the absence of necessary hormones. This has been demonstrated in cultures from other endocrine-dependent organs. In the thyroid, Lissitzky and his colleagues (Lissitzky et al., 1971; Fayet et al., 1971) showed that trypsin-dissociated pig thyroid cells, cultured in medium without added thyrotrophin, grew as a two-dimensional monolayer, although some junctional complexes were present. When thyrotrophin was added to cultures with a high cell concentration, the cells aggregated and formed three-dimensional follicles, with an ultrastructure resembling that seen in the normal thyroid. The ultrastructural changes were mirrored b y functional changes. In monolayer cultures the cells lost the capacity to concentrate iodide between the first and second days in uitro, whereas the cells in the follicles maintained their capacity to concentrate iodide, iodinate thyroglobulin, and synthesize thyroid hormones. Although there are some reports on the maintenance of functioning normal thyroid cells for several months (see Wigley, 1975, for references), there are no reports of permanently transferable cell lines. In the adrenal the picture is more confusing. Gyevai et al. (1972) described the ultrastructural changes in cultures of embryonic human, rat, and cat adrenals, and Kahri et al. (1972) described the effects on adrenal cell mitochondria. The cultures were established from glands that contained differentiated hormone-producing cells resembling some zona fasciculata cells. Within the first 4-6 days the cells lost this differentiated character in all the species examined. The addition of adrenocorticotrophin (ACTH) to the medium led to a restoration of


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differentiated cell ultrastructure and a great increase in corticosteroid production, but in some cultures the corticosterone response occurred without a change in fine structure, that is, biochemical redifferentiation occurred without complete structural differentiation. O’Hare and Munro Neville (1973)maintained confluent monolayers of zona fasciculata and reticularis cells from adult rat adrenal for up to 4 months, but proliferating mesenchymal cells eventually overgrew the adrenal cells. Cells grown without ACTH spread rapidly to form a monolayer but had the ultrastructural features of adrenocortical cells found in hypophysectomized rats. ACTH or cyclic A M P inhibited cell spreading, but the ultrastructure of the treated cells was similar to that of normal adrenocortical cells. The cells secreted adrenocortical steroids. Isolated zona glomerulosa cells (Hornsby et al., 1974) were also maintained under similar culture conditions. These cells produced aldosterone in response to different levels of potassium, and serotonin, ACTH, and cyclic AMP depressed aldosterone secretion at first but later stimulated the production of corticosterone. At this time the ultrastructure of the cells changed from the glomerulosa type to the fasciculata-reticularis type. In particular, the mitochondria1 cristae changed from a tubular to a vesicular form. There are several reports on the ultrastructure of pituitary cells in culture. Petrovic (1963), using guinea pig and rat pituitary cultures, reported that each of the cell types present in the normal gland retained its specific ultrastructural features, although secretory droplets disappeared rapidly. Pasteels ( 1963), however, described the progressive atrophy and disappearance of all cell types other than prolactinproducing cells in cultures of human and rat pituitary. The structural changes were accompanied b y a rise in prolactin levels in the medium. In other experiments, Hartemann et al. (1973) claimed to have maintained soniatotropin-secreting pituitary cells on a collagen substrate for many months. In these cultures the function was said to be unrelated to morphology or growth rate. This pattern is similar to that found in a hormone-producing cell line (GH 3) (Tashyian et d., 1970) derived from an estrogen-induced rat pituitary tumor. The cells produce both prolactin and somatotropin, but the ultrastructure of the cells is described as embryonic (Gourdji, 1972) and without specific secretory granules. In two important and extensive reviews, TixierVidal and her colleagues (Tixier-Vidal, 1975a; Tixier-Vidal et al., 1975) summarize the available data from their own and other work and conclude that there is a good correlation between ultrastructural appearance and hormone secretory activity of pituitary cells in cell and organ cultures. Prolactin-secreting cells predominate in the cul-

106


tures, but ultrastructural studies have shown that several other cell types may survive in small numbers for at least 2 months. Normal cultures are eventually overgrown by fibroblasts, but functioning tumorderived cell lines can be maintained indefinitely. Chen et al. (1975) cultured cells from human testes removed from patients with prostatic cancer. Cells with the ultrastructural characters of Sertoli cells were maintained for 4 months, but again the cultures were overgrown by fibroblasts.

3. Liver and Other Organs Cell lines established from liver have been intensively studied and illustrate many of the problems involved in the identification of cells in vitro. Some of these are discussed in detail in Gene Expression and Carcinogenesis in Cultured Liver (Gerschenson and Thompson, 1975) and in Wigley (1975).Although freshly isolated adult liver cells retain an ultrastructure almost identical to that of normal liver cells (see, for example, Phillips et al., 1974; and others), after a short time in culture there are no absolute structural criteria not present in other cells (see, for example, Gerschenson et al., 1972; Weinstein et al., 1975; Iype et al., 1975). Identification depends on the retention of specific liver functions and the production of liver-type tumors after neoplastic transformation. A discussion of this problem is outside the scope of this article, but the reviews by, for example, Le Guilly et al. (1973a,b)and Wigley (1975) consider the validity of some of the criteria used. Perhaps the most important feature of these experiments is that most of the established lines from normal liver were established from “epithelial” cells selected for growth by cloning (e.g., Coon, 1968) or by enzyme separation, for example, Williams et al. (1971) and Iype (1971).Williams (1975)applied enzyme methods to adult liver and obtained a high yield of differentiated liver cells, but only a small proportion of these cells were capable of sustained replication in culture. Two strains were maintained for 4 and 13 months, respectively, in vitro and had a near diploid karyotype range of 41 to 46, modes 43 and 44, but with about 25% structural abnormalities. The cells have not been tested as yet for tumorigenicity. The cell lines described by Iype (1971) and the two lines described by Williams et al. (1971) were not tumorigenic in the test systems used. Diamond and her colleagues (1973)have also described the establishment and ultrastructure of cell lines (WIRL 3) from the liver of a weanling rat, using Coon’s method. These cells retained ultrastructural and some biochemical features of


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liver cells, but all transfornied spontaneously after about the twentieth transfer and produced “epithelial” tumors after reimplantation. The cells could also be transformed by SV40 virus. Using an enzyme selection technique, with collagenase and selective trypsinization of cultures to remove fibroblasts, Owens et al. (1974) established mouse cell strains from the liver (three), mammary gland (one), ovary (four), and skin (one). The ultrastructure of the cultured cells suggested an epithelial origin, but none has remained completely normal in culture. All but one were tuimorigenic when reimplanted in newborn isogeneic mice, and all contained a minority population of cells with chromosomal abnormalities. One of the most interesting lines, from liver strain N MuLi, produced benign cystic lesions in newborn mice when lo6 cells were inoculated, but areas of adenocarcinoma developed when 8 x lo6 cells were used (Anderson and Smith, 1975). Eight clones were derived from these cells and all behaved similarly, that is, small numbers produced benign lesions and large numbers produced tumors. This suggests that tumors did not arise from a small population of neoplastic cells in the original cultures, but that all the cells were altered and potentially neoplastic. At least three other liver cell lines derived from normal mouse (Evans et d.,1958), mouse embryo (Waymouth et al., 1971; Rhim et al., 1974), and adult rat (Weinstein et al., 1975) have undergone spontaneous neoplastic transformation. Neoplastic transformation has also been reported in an epithelial cell line established from normal rat uterus (Sonnenschein et al., 1974) by collagenase treatment. The details are not clear in the article, but it seems likely that the cells were derived from only one culture flask out of an unspecified number.

D. CONCLUSIONS The conclusions to be drawn are that many epithelial cells can be maintained in primary culture for a variable period of time. In most cases the differentiated cells present in the original explants die, and the cultures are repopulated by cells derived from a stem cell population. These cells may retain some but not all their differentiated characters. The ultrastructure of the cells may resemble that of the organ of origin, although they may not respond to normal stimuli. Others may lose their normal ultrastructure but still retain a capacity to respond to stimuli, for example, specific trophic hormones. Transferable cell strains or lines from normal epithelium have almost all been established by selection for growth from clones or selec-

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tive enzymic dispersal. The majority are tumorigenic. Others have chromosome abnormalities. It is doubtful that many of these cells can be regarded as normal. The failure of differentiated epithelial cells to survive in uitro may be due to cell damage during preparation of the cultures, to the inability of normal cells to adapt metabolically to in uitro conditions, to deficiencies in the medium, or to a requirement for a stromal product. Tumor cells that can grow in vitro may be more resistant to damage, more capable of metabolic adaptation, or more able to synthesize essential nutrients. The problem of cell damage is well known (see, e.g., Franks et al., 1970b) and need not be discussed further. Another explanation for failure to survive and proliferate may be loss of stroma. The importance of the stroma in embryonic growth and development has been recognized for many years (Grobstein, 1964), and work such as that of Wessells (1963) suggests that embryonic epidermis cannot incorporate thymidine when separated from the dermis. Little is known about the mutual interdependence of the stroma and epithelium in the adult, but there is some evidence to suggest that both epithelium and stroma are necessary for normal growth and function in the cornea (Herrmann, 1960), breast (Lasfargues, 1957), and mouse prostate (Franks and Barton, 1960; Franks, 1963). Autoradiographic evidence suggests that RNA synthesis in the prostate can proceed in the absence of stroma, but that DNA synthesis cannot (Franks et al., 1970b). A final point is that many adult epithelial cells may have a finite lifespan and may be able to go through only a small number of division cycles. The number of stem cells capable of continuous division in each culture may be very small.

V. Ultrastructure of Mesenchymal Cells from Normal Tissues Differentiated mesenchymal cells have also been maintained in culture, but the specific differentiated characters are usually lost after a relatively short time. The difficulties involved in identification have already been referred to (see Wigley, 1975, for discussion), the main problem being that the mesenchymal cells that usually take over the cultures are multipotential and capable of producing a wide range of mesenchymal products.

A. DIFFERENTIATED MESENCHYMALCELL STRAINS There have been relatively few reports of differentiated mesenchyme in culture. The reports of Gimbrone and Cotran (1975) and


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Haudenschild et al. (1975) describe the ultrastructure of human vascular endothelium and smooth muscle in transferable cell strains. The endothelial cells had a specific ultrastructural marker-the Weibel-Palade body (Weibel and Palade, 1964). Heart cell cultures have also been identified ultrastructurally, for example, by Polinger (1973),but no transferable strains have been established. Several workers have described the appearances of smooth muscle cells in culture. Campbell et al. (1971)described the ultrastructure of embryonic chick gizzard muscle cells after trypsinization and shortterm culture. Ross (1971)gives a detailed and well-illustrated account of smooth muscle cells derived from the inner media and intima of guinea pig aorta. These cells retained the morphology of smooth muscle cells at all phases of their growth in culture but also produced extracellular material, some resembling the basal lamina and some the microfibrillar component of elastic tissue both in structure and amino acid composition. Finally, Schubert et al. (1974) described a transferable cell line derived from an intracerebral tumor induced by nitrosoethylurea. The tissue culture cells resembled smooth muscle closely in their ultrastructure. They were contractile, had electrically excitable membranes capable of generating action potentials, responded to acetylcholine and norepinephrine in the same way as smooth muscle, but secreted collagenlike proteins into the medium. B. UNDIFFERENTIATED MESENCHYMAL CELL STRAINS AND LINES We have examined over 30 mouse cell lines (Franks and Wilson, 1970, and nnpublished) and over 50 human embryo cell strains Franks and Cooper, 1972, and unpublished) established in our laboratories from many different organs, and cell lines established in other laboratories, including BALB/c 3T3 (Aaronson and Todaro, 1968), hamster NIL 2E cells and NIL 8 cells (Diamond, 1967; McAllister and Macpherson, 1968), Kaighn’s rat liver cell lines (provided by Dr. B. Weinstein), and several secondary mouse and hamster embryo cell strains. Many of the mouse cell lines had undergone spontaneous neoplastic transformation (Franks and Henzell, 1970). The basic ultrastructure of all the cells was similar, although there were variations in the proportion of the different cell types present. In all the cultures examined two main types of cells were identified. Type-1 cells (Figs. 20 and 21) usually had a large rounded or beanshaped nucleus with a very thin layer of condensed chromatin against the nuclear membrane. Small clumps of chromatin were associated with a prominent, usually single, nucleolus. The cytoplasm contained

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some rough endoplasmic reticulum, many free ribosomes, often in rosettes, a recognizable Golgi zone, and relatively few lysosomes, autophagic vacuoles, and mitochondria. Cell processes were short and few in number. Microvilli were very scarce. Type-2 cells (Figs. 22 and 23) had a more convoluted nucleus in which the peripheral chromatin layer was thicker and small clumps of chromatin were scattered throughout the nuclear matrix and associated with prominent nucleoli. The difference in nuclear pattern was the most distinctive feature of the two cell types. The cytoplasm usually contained a large Golgi zone, many free ribosomes and, particularly in the peripheral zone, many lysosomes, autophagic vacuoles, and mitochondria. The rough endoplasmic reticulum was less abundant than in type-1 cells, but the cisternae were often distended with finely granular material. The cell surface was irregular, with many thin convoluted processes. Sheetlike cytoplasmic fringes often extended for a considerable distance from the cells. In suspensions prepared by scraping they were usually oriented along a layer of extracellular fibrillar material (Fig. 22) and often interdigitated with similar processes from other cells. Microvilli were present in some cells but absent from others. In suspensions prepared b y trypsinization many of the cells were rounded, but others still had many long processes (Fig. 23).A single intracellular cilium, projecting into a small vacuole, was found in type-2 cells in some cultures. Centrioles were seen only occasionally. Although most cells could be easily classified as type 1 or 2, there were some atypical cells in most cultures. Most of these had the cytoplasmic characters oftype-1 cells, but the nuclei were much more convoluted, although the chromatin pattern was similar to that normally found in type-1 cells. Other cells also had more cytoplasmic organelles than a typical type-1 cell, suggesting that there may have been a transition from type-1 to type-2 cells. Occasional giant cells, sometimes multinucleate but usually with a single nucleus, were also found. Only their size distinguished them from other type-1 and -2 cells. All cell types had several features in common. Nuclear bodies simi-

FIGS.20 and 21. Type-1 cells from a spontaneously transformed mouse kidney cell line (CBM 17/64) showing round or bean-shaped nucleus with thin peripheral layer of condensed chromatin. The cytoplasm contains some rough endoplasmic reticulum, inany free ribosomes, and relatively few organelles except for mitochondria. One mitochondrion which has a very dense matrix lies next to a swollen mitochondrion. Fig. 20: ~ 2 8 9 0Fig. . 21: x 14,450. Figs. 20-33 from Franks and Wilson, 1970.


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lar to those described by Bouteille et ul. (1967)(types 1 , 2 , 3 , and 4 in their classification) were seen in many cells. Occasional nuclei contained lamellar structures resembling myelin figures. Bundles of intranuclear fibrils about 4-5 nm in diameter were found in one cell line. Intranuclear cytoplasmic inclusions were common. Pinocytosis was frequent, and the cytoplasm contained many large and small smooth membrane-bounded vesicles. Phagocytosis of cell debris or sometimes of whole cells was also found frequently but was more common in type-2 cells. Glycogen deposits were found in most transfer generations of some tumor-producing mouse cell lines as aggregates of single, roughly isodiametric beta particles 15-30 nm in diameter. Long, dense mitochondria were often present beside the glycogen deposits. The appearance of the mitochondria varied considerably. Normal mitochondria with a comparatively light matrix were found in the cells of most cultures. In some cases these mitochondria were greatly swollen or contained very short cristae. Dense mitochondria were also abundant in all cultures. These were characterized by a very dense matrix and distorted cristae, sometimes in a tubular pattern. Both normal and dense mitochondria were found within the same cell. Intracytoplasmic filaments were present in most cells, usually randomly distributed but sometimes arranged in broad bundles beneath the plasma membrane or next to and parallel with the nuclear mernbrane. Most individual filaments were about 7.5 nm in diameter, and in favorable sections could be seen to have a substructure resembling that of the actinlike filaments described in many other cells. Other filaments about 3.5-4 nm were also present in some cells. Fusiform dense bodies resembling those seen in smooth muscle cells were sometimes seen. These were more frequent in cells sectioned in situ. The filaments were most abundant in type-2 cells. Bundles of 10-nm tonofilaments were present in some cells but were not a prominent feature. A complex system of microtubules was also present. There was no regular arrangement of the microtubules in cell suspensions, but in FIG.22. Type-2 cell tioin a non-hiinor-producingline (COM 4/5/bladder). The cell suspension was prepared by scraping. The nucleus is convoluted and has a thick peripheral layer of' chromatin. There is a thick layer of extracellular fibrillar material. x 2890. FIG.23. Type-2 cell from a spontaneously transformed mouse cell line (COM 26/8). The cell suspension was prepared by trypsinization. The upper cell is rounded, and the i i ~ i c l e convoluted; ~i~ the cytoplasm contains dilated cisteniae of rough endoplasmic reticulum and many cytoplasmic inclusions. The convoluted process of another cell can be seen below, but there is little extracellular filamentous material. x 2890.

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FIG. 24. An intermediate junction between two type-1 cells from a tumor line (CBM 17/64 kidney). X 14,450. FIG.25. The junction shown in Fig. 24. x52,OOO.


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cells sectioned i n situ they ran along the long axis of cytoplasmic projections. Specialized cell contacts (Figs. 24-27) between like cell types (Figs. 24-26) were found in most cultures, but contacts between type-1 and -2 cells were also seen. The majority resembled intermediate junctions, but a few atypical junctions (Fig. 27) were also found. Condensations of amorphous material (Fig. 28) on the inner aspect of the plasma membrane, resembling, but smaller than, the attachment bodies of smooth niuscle, were not infrequent and were often associated with extracellular niaterial resembling the basal lamina (Fig. 28). This material was associated with many type-2 cells and some type-1 cells in all cultures, but in no case was a cell completely surrounded. The dense component, varying in width but usually about 30-40 nm, had a finely filamentous substructure and was separated from the cell by an electron-lucent zone about 15-20 nm wide. Other extracellular material was found in all cultures. The amount present was not related to the age of the culture but appeared to be increased in the presence of type-2 cells. The material was made up of two components, an amorphous material and fine fibrils about 10 nm in diameter. On cross section these had a tubular appearance with an electronlucent core and a denser outer rim (Fig. 29). Small amounts of banded collagen with a repeat period of ca. 53 nm (Fig. 30) were found in some cultures. This periodicity is normal for collagen in our embedded material. Viruslike particles were present in many mouse cell lines. Most were C particles and were extracellular, in cytoplasmic vacuoles, or developing from a plasma or vacuole membrane (Fig. 31). Occasional intracytoplasmic A particles were seen. In one series of cultures from an old mouse (COM 5 peritoneum) intranuclear, cytoplasmic, and extracellular particles of a polyoma type were found (Figs. 32 and 33). Except for the presence of glycogen in some tumor lines, there were no morphological differences or differences in the proportion of the two cell types between tumor and nontumor lines from young and old mice, or among different transfer generations, or among lines derived from different mouse strains. There have been few other detailed reports on the ultrastructure of FIG.26. An intermediate junction between two type-2 cells from a nontumor line (CBM 15/6/kidney). x 19,250. FIG. 27. An atypical tight junction between two type-2 cells from a tumor line (CBM 17/43/kidney). There is n o intercellular space in this region. A group of cytoplasniic fibrils can be seen in the cell o n the left. X44,275.

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undifferentiated cell strains or lines. Cornell (1969b)described the ultrastructure of cells involved in spontaneous neoplastic transformation in mouse cell cultures, and Soto and Castejohn (1969) gave what appears to be the first report on the ultrastructure and normal morphology of BHK cells. Comings and Okada (1970) and Lucky et al. (1975) described human skin fibroblasts, Robbins et al. (1970) and Lipetz and Cristofalo (1972) human embryonic fibroblasts (including WI-38 cells), and Brunk and his colleagues (Brunk, 1973; Brunk et al., 1973) glial cells. Brock and Hay (1971) described the ultrastructure of cultures of chick “fibroblasts.” The appearances of these cells are all consistent with those we have described. Most of the investigators describe two cell types in the cultures, although some, for example, Comings and Okada (1970), suggest that this variation in structure is related to the growth phase of the cells.

c. THE ORIGIN OF MESENCHYMALTISSUECULTURE CELLS The presence of specialized cell contacts, the formation of material resembling the basal lamina, and the fact that the majority of the extracellular material formed in the cells we have examined is not collagen (although this is sometimes formed in small quantities) suggest that the cells are not fibroblasts. The nature of cells that can be maintained in vitro has been a topic for discussion since tissue cultures were first initiated (see Willmer, 1958, 1965, for full discussion). Although at least six different cell types can be recognized in primary explant cultures, one curious feature of pure cell strains is that at any one time there often, if not always, appear to be two morphological classes of cells in them, the spherical or somewhat spindle-shaped type on the one hand and the extended, flattened type on the other” (Willmer, 1965).The finding in our experiments of two main cell types in cultures derived from a wide range of organs-kidney, lung, heart, spleen, bladder, prostate, tongue, spinal cord, and peritoneum-suggest that the cells may originate from a tissue common to all. Alternatively, the appearance of the two cell types may be a direct consequence of the tissue culture environment and may represent a “

FIG. 28. A cell from a tumor line (COM 4/5/bladder) showing small areas of increased density on the plasma membrane (arrow) and a layer of extracellular material resembling a basal lamina. ~69,300. FIG. 29. Fibrillar extracellular material from the same grid as in Fig. 13 showing longitudinal and transverse sections of fibrils. The electron-lucent core and denser outer rim can be seen in transverse sections (arrow). x69,300. FIG. 30. Banded collagen fibrils from a tumor line (CBM 17/Zl/kidney). ~69,300.

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structural modulation rather than a specific selection of two cell types from the original mixed-cell starting inoculum. However, modulation seems unlikely, since some cells similar in morphology to the tissue culture cells can be seen in noncultured cell suspensions or tissues (Franks and Wilson, 1970; Franks and Cooper, 1972). The possibility that one cell type niay be derived from the other cannot be excluded, particularly a s type-1 cells seem to be less coininon in the original suspensions. The ultrastructure of the cells in our cultures differs from that of typical fil>roblasts(Movat and Fernando, 1962; ROSS,1968) and, although cell contacts resembling intermediate and tight junctions have been reported between embryonic fibroblasts (Ross and Greenlee, 1966) and between adult guinea pig “fibroblasts” in culture (Devis and James, 1964), they are rarely, if ever, found between adult connective tissue cells in vivo (Ross and Greenlee, 1966). The relatively frequent occurrence of these contacts and of basal laminae support the suggestion that the tissue culture cells are not fibroblasts. The greater part of the extracellular material is not collagen and has some of the morphological appearances of elastic tissue. The extracellular fibrils are similar to the 10-nin microfibrillar component of elastic fibrils (Greenlee et ul., 1966; Ross and Bornstein, 1969; Fernando et ul., 1964) reported to be present during the early development of elastic tissue. The ultrastructural characters of the cells we have described correspond well with two cells derived from the blood vascular system, the endothelium and the endothelial pericytes, both of which have specialized cell contacts and produce basal laminae. The normal appearances of these cells are described in detail by, for example, Rhodin (1968), and Majno (1965) and Wiener et al. (1969) have described the ultrastructure of proliferating endothelial cells in viuo. Ashton and his group (Ashton and d e Oliveira, 1966; Shakib and de Oliveira, 1966; Ashton, 1966, 1968) studied the einbryological development, structure, and function of these cells in some detail. They conclude that both cell types are derived from a common primitive vascular mesenchyme. Many other workers (e.g., Ehrlich, 1956; Wissler, 1967) believe that the pericyte should be regarded as a multipotent primitive FIG.31. Mutrtro and devcloping C particles in intracytoplasmic vacuoles in a type-1 cell from a notitumor line (CBM 23/15/lrtng). x52,000. FIG. 32. htraiiuclear virus particles of polyoma type in a type-1 cell f ~ O t 1 1a nontitinor line (COM q5/3/peritone~~tn). There is also an extracellular group of similar particles. ~ 5 2 0 0 . FIG.33. Higher magnification ofthe extracellular particles in Fig. 32 showing crystalline array. ~ 3 4 , 6 5 0 .

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mesenchyme cell, capable of differentiating into smooth muscle and of producing collagen. Both endothelial cells and pericytes develop from a common precursor cell (Ashton, 1954a,b), so that these functions may be present in both tissue culture cell types. Possible transition forms between the cell types are not unexpected. The cell strains described by Ross (1971) and Ross and Klebanoff, 1971) were established from tissues in which pericytes are not found, but the cells of origin in the aorta have a similar embryological derivation, so that a similar function is again not unexpected. Since the pericytes are located at a site in the microcirculatory bed capable of rapid growth during embryogenesis (Clark, 1936),and in wound repair (Cliff, 1963; Schoefl, 1963), it would not be surprising if these cells and their associated endothelial cells had a great potential for growth in vitro. The tissue culture cells have a strong morphological resemblance to pericytes and endothelial cells, and the scanning electron microscope appearances of some lines, for example, 3T3 cells (Porter et al., 1973), and some mesenchymal mouse cell lines (Hodges and Muir, 1972) are similar to those of endothelial cells. Morphology alone cannot prove this derivation. We have seen no definite rod-shaped Weibel-Palade bodies (Weibel and Palade, 1964)or cross-striated fibrils (Rohlich and Olah, 1967) similar to those found in human or rat endothelial cells. The morphology and ultrastructure of the mesenchymal tumors derived from these cells transformed spontaneously or by viruses or chemical carcinogens are similar and resemble those reported as hemangiopericytomas with both light and electron microscopy (see Franks et al., 1970a; Wilson and Franks, 1972, for descriptions and references).

VI. Ultrastructure of Cells from Brain and Hemopoietic Tissue There are several reports on the ultrastructure of nerve cells in

uitro, but these are mostly concerned with cultures of organized tissue (see Section VIII) or with cells derived from neuroblastomas (see Section VII). Bendaet al. (1975)have recently established monolayer cultures from the hypothalamus of 14- to 20-day-old mouse embryos and described the ultrastructure of aggregation and differentiation which took place in the cultures over 9-60 days. With increasing in vitro age cells with the ultrastructural characters of astrocytes and ependymal cells appeared. Later clumps of primitive neuroepithelial cells, mature neurons, and neurosecretory cells appeared, and axons and neuronal processes developed. Most reports on glial cells in culture are on tumor-derived cells, for example, that by Ryter and Benda


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(1972). Ponten and his colleagues (Brunk et al., 1971, 1973) have described cells derived from normal human embryo brain, but the cells have no absolute ultrastructural features of glial cells. Lines of cells have also been established from the human hemopoietic system, but there are no microscopic or ultramicroscopic characteristics that permit separation of cultured human lymphoblastoid cells derived from normal individuals and from patients with leukemia, Burkitt’s lymphoma, and infectious mononucleosis (Moore et al., 1968; see also Seman and Dmochowski, 1975, for review and references). These lines carry E B virus and cannot be regarded as normal. VII. Ultrastructure of Tumor Cells in Vitro There are numerous reports on the ultrastructure of tumor cells in vitro, many of which have been already cited, and an extensive review on the fine structure of human tumor cells has recently been published (Seman and Dmochowski, 1975). Only a few points of general interest are considered here. Most tumor cells retain some of their differentiated characters in vitro. In the majority of tumor cultures, as with cultures from normal tissues, the differentiated tumor cells, whether epithelial or mesenchymal, survive in primary culture, but the proportion that gives rise to established cell lines is very small, although many differentiated tumor cell lines have been reported (see Wigley, 1975). Most primary cultures are eventually overgrown by mesenchymal cells. The ultrastructure, in many such cultures w e have examined, reflects this picture of a mixed cell population in primary cultures. The mesenchymal cells do not differ in ultrastructure from those already described in cultures from normal tissues. In a few cultures tumor cells take over, for no obvious reason, sometimes in primary cultures (e.g., Rigby and Franks, 1970), and sometimes after several subcultures (e.g., Franks et al., 1976). These cultures remain mixed, that is, there is no evidence that the mesenchymal cells are ever completely lost. In cultures from human tumors the mesenchymal cells have the characteristic Hayflick limit in doubling potential and eventually die out, but in cultures from rodent tumors spontaneous neoplastic transformation of stromal cells may occur at any transfer stage. The sarcomatous transformation of epithelial tumor cells in vitro is probably due to a change of this type. We have a welldocumented example of a mouse male mammary tumor line which gave rise to two sublines, one of which produced typical mesenchymal tumors and the other epithelial tumors. The ultrastructure of

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FIG. 34. A group of cells grown in serum-free medium CMT 64X, third transfer, showing alveolar pattern. Junctional complexes are just visible as dark areas between


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these sublines was as expected, one being typically inesenchymal and the other resembling the structure of mammary tumor cells (but see Rockwell et a1., 1972, for discussion and alternative explanations). The ultrastructure of mouse mammary tumor cell lines and of the production of mammary tumor viruses are described by Owens and Hackett (1972), Yagi (1973), and others. The isolation of epithelial and mesenchymal cell lines from a human breast carcinoma is described by Plata et al. (1973). A characteristic feature of mammary tumor cells and of all other tumor cell lines described is that ultrastructural identification in most cases is dependent on the pattern of cell growth unless a specific cellular marker for example, a melanosome in a melanoma cell, is present. It is impossible to distinguish with absolute certainty individual tumor cells, for reasons considered earlier (see Section 11),but the pattern may be distinctive. This is illustrated in a mouse lung tumor line CMT 64 (Franks et al., 1976). The mass of this tumor in vitro is made up of undifferentiated cells, particularly when the cultures are not confluent, but within the mass differentiation takes place, and acini with typical ultrastructural features of respiratory epithelium may be found (Figs. 34-38). Other examples from mouse and human rectal tumor cell lines are shown in Figs. 39-43. In both, most of the cells show no distinguishing features, but in some areas a few cells show differentiated gut epithelial characters. The human tumor line also shows intracytoplasmic lumen formation (Fig. 44), a feature seen in other tumors, particularly those of the breast (Battifora,

1975). The degree of differentiation that may occur in tumor cell lines is illustrated in cultures derived from a mouse neuroblastoma cell line (Augusti-Tocco and Sato, 1969). Clones of these cells have been hybridized with L cells, and some of the hybrid lines have been shown to express some neuronal characters to a greater extent than the neuroblastoma parent lines (Minnaet al., 1971).The ultrastructure of similar hybrids derived from neuroblastoma clones and rat glioma cells is described by Daniels and Hamprecht (1974). the cells near the lumen, seen at higher magnification in Fig. 35. X2310. Figs. 34-38 from Franks et al., 1976. FIG. 35 A typical desmosome from a junctional complex as seen i n Fig. 34. x 62,370. FIG.36. Bifurcate lunienal microvilli, CMT 64X cells. X21,OOO. FIG.37. Short swollen cilia with fibrils inserted into a web of actinlike filaments. Part of a junctional complex appears on the right. CMT 64X cells. x21,OOO. FIG.38. Characteristic osmiophilic lamellar inclusions, CMT 64X cells. x 15,400.

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VIII. Ultrastructure of Cells in Organ Cultures A general survey of the ultrastructure of organ cultures is beyond the scope of this article, but the system allows the effects of tissue dissociation and isolated cell growth to be compared with the changes that occur in organized tissues under similar in vitro conditions. An extensive review, with descriptions and references to ultrastructural changes in organ cultures of germ cells, pituitary, lung, pancreas, skin, nervous tissues, and colon, is given in Organ Culture in Biomedical Research (Balls and Monnickendam, 1976). The effects of radiation and of virus and mycoplasma infection are also described. Rose (1970) maintained a wide range of organized tissues (15 different organs), some for several months, in a rather complex circumfusion system designed to circulate a constant flow of medium through a small culture chamber. The electron micrographs show that under these conditions many of the differentiated cells were well preserved, but almost all the cultures were established from fetal tissues. With simpler systems there are reports on the good ultrastructural preservation of some embryonic tissues including nervous tissue (e.g., Lumsden, 1968; Aparicio et al., 1976), but in others degenerative changes soon appear. Masters (1974),for example, found with cultures from fetal mouse lung, that, although norinal development occurred in cultures from 11- and 15-day-old embryos, cultures from 18-day-old mice showed ultrastructural evidence of epithelial cell degeneration, as did cultures from adult mouse lung. H e also showed that differentiation did not proceed in the absence of stroma. In organ cultures from the pituitary (see Tixier-Vidal, 1975a, for review) there was atrophy of most of the glandular cells, but ultrastructural normality could be restored by the addition of hormones. In adult organs Franks and Barton (1960), using cultures of adult mouse prostate, showed that both the hormonal environment and the normal relationship of epithelium to stroma was required for the maintenance of normal ultrastructure and function. In the prostate organ cultures-and most others-the main mass of the culture retains its normal ultrastructure, but around the edges there is an outgrowth of cells (both epithelial and stronial) growing in a thin sheet as in a cell culture system. CulFIG.39. Mouse colon tumor cells (CMT93, fourth in oitro transfer), with acinus formation (top right). x 3000. FIG. 40. The same as in Fig. 39, showing junctional complexes and glycoprotein strands just visible around villi. x 30,000. FIG. 41. Cross section of villi showing central fibril bundles. Glycoprotein strands are just visible in the background. x 108,000.

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FIG. 42. A group of undifferentiated human colon tumor cells (HT 29, onehundred-twenty-ninth in vitro transfer). x 4430. FIG.43. A group of HT 29 cells showing acinus formation. ~ 4 4 3 0 .


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tures maintained in control medium showed degenerative changes which could be prevented by the addition of testosterone. Cells in the outgrowth were grossly abnormal but showed no degenerative changes and failed to respond to testosterone. It was suggested that the failure of the cells in the outgrowth to respond may have been due to the absence of a stromal component necessary for normal function. In adult mouse bladder organ cultures G. M. Hodges (unpublished, 1976) showed that epithelium separated from its stroma survives but does not differentiate normally and produce characteristic, asymmetric, surface plasma membrane. Rowden et al. (1975) maintained human skin in organized culture for up to 18 weeks. After an initial period of degeneration new foci of epidermal cells appeared at the dermoepidermal junction and formed a complete epidermal layer beneath the original epithelium, but normal keratinization did not occur. The individual keratinocytes were ultrastructurally similar to fetal skin cells. In adult mouse colon cultures, Defries and Franks (1976) found that cells in the outgrowth retained their distinctive glycoprotein surface strands but lost most other differentiated characters, although these were retained in the organized areas of the culture (Figs. 45 and 46). These results suggest that a normal endocrine environment and probably a stromal component are necessary for normal function, but there is no evidence to show that their absence leads to cell death.

IX. Conclusions Ultrastructural evidence, confirmed by functional studies and animal inoculation of cultured cells has shown that differentiated epithelial and mesenchymal cells can be maintained in primary culture or in organ culture for many months. As a rule, there is a gradual loss of differentiated function, particularly if its maintenance is dependent on the endocrine environment, but differentiated structure may sometimes be retained. Most, if not all, epithelial cell strains or lines established from normal tissues, usually by enzyme selection techniques, produce tumors on reinoculation into animals. Most mesenchymal cell lines or strains established from normal tissue are similar to each other in ultrastructure. Two cell types predominate in these cultures, although transition forms may occur. The proportion of the different cell types may vary between cell lines. Depending on species of origin these cells may undergo neoplastic transformation or in vitro senescence. The cells are probably derived from endothelial cells and pericytes. Mesenchymal and epithelial cells with differentiated char-

FIG.45. Organ culture of colon; 6-day-old culture from 8-month-old C57BL mouse showing well-preserved epithelium, stroma, and blood vessel. x 2000.

L . M. FRANKS ANDPATFUCIA D. WILSON

FIG.46. Luminal surface of organ culture in Fig. 45 showing glycoprotein strands attached to microvilli, and part of a junctional complex. x 60,000.

acters can be maintained as cell lines from a small proportion of tumors, so that there is some as yet unexplained relationship between neoplastic change and the ability to survive in uitro. The identification of individual cells by electron microscopy is not always possible. Adaptation to growth in uitro usually involves a loss of organized form, and in sections most cells in a culture have a similar ultrastructure, the usual subcellular components being present. The distribution of these components, particularly of the microfilamentmicrotubule system, are dependent on the degree of attachment of the

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cells to the substrate. In some areas, depending on the degree of differentiation of the cells and/or the plane of section only a few cells with recognizable structural markers may be found. Only these cells can be identified with certainty. The ultrastructure of the subcellular Components, particularly the cell surface-plasma membrane complex, the mitochondria, and specialized contacts, are altered by the adaptation to in vitro life and by neoplastic transformation, the progression being toward a less well-organized structure. There are no absolute ultrastructural indicators of neoplastic transformation. Although many markers have been described, their significance cannot be assessed, since lines of normal cells maintained under identical conditions are not available for direct comparison. Ultrastructural studies on cells in vitro were started by Porter and his colleagues (1945), using whole cells. The introduction of highvoltage electron microscopy (e.g., Parsons et al., 1974) and the use of critical-point drying of whole cells (e.g., Buckley and Porter, 1975) may now allow us to use the electron microscope to analyze the spatial distribution of subcellular components. ACKNOWLEDGMENTS

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Electrophysiology of the Neurosecretory Cell YAGI

KINJI

Depzrtiiient of Physiolog?l,Jichi Medical School, Tochigi-ken,Jupccn AND

SHIZUKOIWASAKI Depnrtnient of Physiology, Tokyo Medical College, Shinjuku-ku, Tokyo, Jupun

I. Introduction

.

.

.

.

.

.

.

.

.

141

11. Identification of thc NS Cell i n Electrophysiological

Studies . . . . . . . . . . . . . 111. Electrical Properties of the Membrane . A. Generation and Conduction of Action Potentials . B. Electrical Parameters of the Membrane . . . C. Ionic Mechanisms for Resting and Action Potentials IV. Characteristic Nature of Electrical Activity . . . A. Duration of Action Potentials . . . . . B. Endogenous Pacemaking Activity and Bursting . . . . . . . . Discharges . C. Two Components of the Action Potential . . V. Role of Action Potentials in Endocrine Activity . . A. Synthesis ofNeurohormone . . . . . B. Axonal Transport of Neurohormone . . . . C. Release of Neurohormone. . . . . . VI. Synaptic Control of the Hypothalamic NS Cell . . A. Effects of a Putative Neurotransmitter on NS Neuron . . . . . . . . . Activity B. Recurrent Inhibition of the NS Neuron Activity . C. Recurrent Facilitation of the NS Neuron Activity . VII. Conclusions . . . . . . . . . References . . . . . . . . .

142 145 146 147 147 153 153 154 159 160 160 161 162 166 167 169 173 178 180

I. Introduction

A large number of studies has provided morphological and biochemical evidence for neurosecretion. It is now well established that in the central nervous system of vertebrates and invertebrates the cells that have morphological characteristics of neurons secrete humoral factor(s) into the blood or hemolymph. This article deals with the neurosecretory (NS) cell which has been proved to possess the unique characteristic that at least one of its axon collaterals terminates in the neurohemal structure. Since NS cells have morphological features of neurons, the question naturally arises whether or not 141

142

KINJI YAGI AND SHIZUKO IWASAKI

they can generate and conduct action potentials. Cross and Green (1959) made the first attempt to record action potentials in rabbit supraoptic and paraventricular nuclei which include NS cells. The cells they studied, however, have not been identified as NS cells. Morita et al. (1961)demonstrated for the first time that the caudal NS cell of the eel, identified under a microscope, is electrically excitable and can be activated transsynaptically. Since then many studies have demonstrated the ability of NS cells to generate and conduct action potentials in vertebrates (the hypothalamic NS system and the caudal NS system of fish) and in invertebrates (the NS systems of annelids, molluscs, crustaceans, and insects). This article aims to discuss rather comprehensively the electrophysiological evidence so far reported on (1)electrical properties of the NS cell membrane, (2) the role of action potentials in the endocrine activity of NS cells, and ( 3 ) synaptic control of NS cells.

11. Identification of the NS Cell in Electrophysiological Studies When electrophysiological techniques are employed, it is very important to identify the cell under study as a NS neuron. When a cluster of neuronal cell bodies or axon terminals in a specialized anatomical region has been proved morphologically with the aid of either a light or an electron microscope to belong to NS neurons exclusively, these morphologically identified NS neurons have been useful for electrophysiological studies in leeches, Aplysia, snails, insects, crayfish, crabs, and fishes (Table I). In addition to their location, their external appearance, especially their color, also has been used as one of the criteria for the identification of NS cells. The method used for identifying NS neurons mainly in the mammalian hypothalamus is to demonstrate in the cell under study an antidromically conducted action potential after stimulation of the neurohemal area. By this technique Yagi et al. (1966) first identified NS neurons in rat supraoptic nucleus. Since then many electrophysiological studies have been conducted on antidromically identified NS neurons in the supraoptic and paraventricular nuclei of mammalian species (Table I). This electrophysiological technique has also been employed to identify tuberoinfundibular NS neurons in rat hypothalamus (Yagi and Sawaki, 1970; Makara et al., 1972). Widely used criteria for the antidromic identification of NS neurons have been carefully examined by Sawaki and Yagi (1973).According to them, the cell being observed is identified as a NS neuron only when all of the following criteria are satisfied (Fig. 1). The observed action potential

ELECTROPHYSIOLOGY OF NEUROSECRETORY CELL

A

C

143

E

FIG. 1. Criteria of antidromic identification of a hypothalamic NS neuron. In this and subsequent figures (Figs. 6-14) uuit spikes were recorded in the arcuate nucleus of the hypothalamus in female rats. (A) Constancy in latency of the unit spike produced by antidromic stimuli given to the median eminence. T e n responses are superimposed. (B) Responsiveness of the unit to 100-Hz repetitive stimuli in one-to-one fashion. (C) and (D) Cancelation of the induced unit spike by collision with a spontaneously occurring spike. Oscilloscope sweeps were triggered by a spontaneously occurring spike seen at the left en d of the oscilloscope trace. When time intervals between spontaneous spikes and antidromic stimuli were shorter than a critical length, antidromically evoked spikes were canceled (D).Antidromic stimuli given at a interval longer than the critical length of time always produced a spike response (C). T e n sweeps were superimposed in (C) and in (D). (E) Relative refractoriness following a spike response. When the unit was tested by a stimulus given 5 msec after a conditioning stimulus of the threshold intensity for producing an antidromic spike, the threshold was higher and the latency was longer than those observed following the conditioning stimulus. (F) Absence of temporal facilitation after a conditioning stimulus of subthreshold intensity for producing an antidromic spike. Threshold for the testing stimulus given 3 or 5 msec after the conditioning pulse and latency of the spike response were same as those observed after a single pulse stimulation. Upward deflections are positive in this and subsequent figtires.

must exhibit (1) constancy in latency of the unit spike produced by antidromic stimulation of the neurohemal area, (2) responsiveness of the unit to high-frequency repetitive stimuli of 100 Hz in a one-to-one fashion, ( 3 ) cancelation of the induced unit spike by collision with a spontaneously occurring spike, (4)an increase in threshold as tested b y a stimulus given at an interval of 5 msec following a suprathreshold stimulus for producing the unit spike, and (5) absence of facilitation when the cell was tested by an antidromic stimulus given 5 msec after a subthreshold conditioning stimulus. (Fig. 1). Each neuron pool shown to contain NS neurons in the vertebrate central nervous system appears to include many non-NS interneurons as well. Therefore it is important to identify the cell under study as a NS neuron in an electrophysiological study dealing with NS neurons, which are neuroendocrine transducers.

144

KINJI YAGI AND SHIZUKO IWASAKI TABLE I ANIMALS,NS ORGANS OR NS NEURONGROUPS I N WHICH ELECTROPHYSIOLOCICAL STUDIES HAVE BEEN MADE

Animal

NS organ or NS neuron group

Theromyzon (leech) Aplysia

Supraesophageal ganglion Abdominal ganglion

Otulu (snail)

Right parietal ganglion

Helix (snail)

Right parietal ganglion

Sarcophagu (fly) Periplunetu (cockroach) Schistocerca (locust) C d i p h o r u (blowfly)

Corpus cardiacum system Pars intercerebralis medialis Metathoracic ganglion

Caruusius (stick insect) Procumbarus (crayfish) Libiniu (crab)

Corpus cardiacum system, brain NS cell Transverse and link nerves X organ Pericardial organ

Cordisomu (crab) Anguilla (eel)

Sinus gland Caudal NS cell

Ruju (skate) Purulichtys (fluke) Tilajh

Caudal NS cell Caudal NS cell Caudal NS cell

Goldfish

Preoptic NS neuron

Goosefish

Hypothalamic neuron

Bullfrog

Preoptic NS neuron

References Yagi e t ul. (1963) Strumwasser (1965, 1967, 1968) Frazier et ul. (1967) Jahan-Parwar et (11. (1969) Carpenter and Gunn (1970) Kupferman and Kandel(l970) Mathier and Roberge (1971) Eaton (1972) Boisson and Chalazonitis (1973) Junge and Stephens (1973) Parnas et al. (1974) Barker and Gainer (1975a,b,c) Smith et al. (1975) Gainer (1972a,b,c) Barker and Gainer (1973, 1974a,b, 1975a,b,c) Smith et (11. (1975) Kerkut and Meech (1966, 1967) Kerkut and Gardner (1967) Wilkens and Mote (1970) Gosbee et ul. (1968) Cook and Milligan (1972) Hoyle (1974) Normann (1973) Finlayson and Osborne (1970) Iwasaki and Satow (1971) Cooke (1964) Berlind and Cooke (1968, 1971) Cooke (1967, 1971) Morita et al. (1961) Ishibashi (1962) Bennett and Fox (1962) Bennett and Fox (1962) Yagi and Bern (1965) Fridberg et al. (1966) Kandel (1964) Hayward (1974) Bennett et al. (1968) Potter and Loewenstein (1955) Koizumi et 01. (1973)


145

TABLE I (Continued) I I

Animal Rat

,

NS organ or NS neuron group Supraoptic and paraventricular neurons

I

Tuberoinfundibular neuron

Rabbit

Supraoptic and paraventricular neurons

Cat

Supraoptic and paraventricular neurons

Dog

Supraoptic and paraventricular neurons Supraoptic nucleus and perinuclear zone neurons

Monkey

References Yagi et 01. (1966) Dyball and Koizumi (1970) Ishida (1970) Kelly and Dreifuss (1970) Dyball (1971, 1974) Dreifuss et (11. (1971, 1974) Moss et al. (1971) Wakerley and Lincoln (1971, 1973a,b) Dreifuss and Kelly (1972a,b) Negoro and Holland (1972) Nordmann and Dreifuss (1972) Dyball and Pountney (1973) Lincoln and Wakerley (1974, 1975) Walter and Hatton (1974) Yagi and Sawaki (1970, 1975a,b) Makara et al. (1972) Sawaki and Yagi (1973) Harris and Sanghera (1974) Mandelbrod et al. (1974) Geller (1975) Moss et al. (1975) Cross et al. (1969) Sundsten et a1. (1970) Moss et al. (1972a,b) Novin and Durham (1973) Yamashita et al. (1970) Barker et a1. (1971a,b) Nicoll and Barker (1971) Koizumi and Yamashita (1972) Sakai et a1. (1974) Koizumi and Yamashita (1972) Vincent et al. (1972a,b) Hayward and Jennings (1973a-d) Arnauld et nl. (1974)

111. Electrical Properties of the Membrane As morphological evidence has indicated that the NS cell is a specialized neuron possessing glandular properties, it is natural to consider the ability of NS neurons to generate and conduct action poten-

146

KINJI YAGJ AND SHIZUKO IWASAKI

tials. In fact, since the end of the 1950s successful recordings of action potentials from NS neurons have been reported for various NS systems of a variety of animal species. In this article we describe and discuss the electrical characteristics of the plasma membrane of NS neurons as reported in these studies.

A. GENERATION AND

CONDUCTION OF

ACTION POTENTIALS

The types of NS neurons for which action potentials have been recorded are listed in Table I. The conduction velocities of NS axons have been estimated in some species and appear in Table 11. As clearly shown in Table 11, it is concluded that NS neurons in general generate and conduct action potentials. The conduction velocity of NS axons in vertebrates is very slow and corresponds approximately to that of unmyelinated C fibers. Conduction velocity has been estimated in most cases by dividing the distance between the stimulating electrode and the recording electrode by the latency of the induced action potential. Therefore the estimated value represents the mean conduction velocity of the NS axon between the sites of stimulation and of recording. But it probably differs from site to site along the NS axon, since Bennett and Fox (1962) found in the caudal NS neuron of the fluke that conduction velocity was as slow as 0.05 m per second in NS axons within the neurohemal region, while it was about 1 m per second in NS axons within the spinal cord. Consequently, it is apparent that the best way to estimate conduction velocity is to obtain the relationship of the latency to the distance between the recording and stimulating electrodes from measurements made at several sites along the NS axon. The conduction velocity is represented by the gradient obtained graphically at a certain point on the NS axon. As the stimulus intensity was increased, the latency of an antidromically conducted spike following stimulation of the neurohypophysis was demonstrated to shorten stepwise in NS neurons of cat supraoptic and paraventricular nuclei (Barker et al., 1971a), of goldfish preoptic nucleus (Hayward, 1974), and of the fish caudal NS system (Bennett and Fox, 1962). This phenomenon has been explained by the presence of bifurcations of NS axons near the soma (Hayward, 1974). However, it is also possible that, as the intensity of the stimulus applied to the neurophemal area is increased, the stimulating current may spread to the more proximal parts of the NS axons which have a faster conduction velocity than the NS axons within the terminal region, as discussed above, and as a consequence the latency of the antidromic spike shortens discontinuously.


B. ELECTRICAL PARAMETERSOF

THE

147

MEMBRANE

In various invertebrate species the electrical properties of the plasma membrane of NS neurons have been extensively investigated by means of intracellular recording techniques. However, in vertebrate NS neurons the electrophysiological data so far reported are insufficient for generalizing about membrane characteristics at present. Therefore this and subsequent sections of this article are concerned with electrical parameters of the NS membrane reported mainly in invertebrate NS neurons. The electrical parameters, such as amplitude of resting and action potentials, critical membrane potential for the initiation of action potentials, input resistance, and duration of action potentials in NS neurons are listed in Table 11. It appears that NS neurons cannot be characterized by unique values for these parameters as compared to those reported for invertebrate (Bullock and Horridge, 1965) and vertebrate (Eccles, 1957) non-NS neurons. The duration of the action potentials of NS neurons is discussed in detail in Section IV,A.

c.

IONIC MECHANISMSFOR RESTING AND ACTION POTENTIALS

Since Hodgkin and his colleagues (Hodgkin and Katz, 1949; Hodgkin and Hiixley, 1952; Hotlgkin and Keynes, 1955) made a quantitative analysis of electrical properties of the squid giant axon membrane in resting and active states, ionic mechanisms for the generation of membrane potentials in either state have been made clear in a variety of excitable membranes, and in several types of synaptic membranes (for review, see Eccles, 1957). It seems very likely that these ionic mechanisms also function in NS neurons. In the following discussion electrophysiological studies on the ionic mechanisms in NS neurons are considered.

1. Resting Potentiul The magnitude of the resting potential in NS neurons of various animal species was reported to range from -40 to -80 mV with respect to the outside of the cell, as indicated in Table 11. It was shown b y Kerkut and Meech (1967) that the resting potential of Helix NS neurons depolarizes as predicted by the Nernst equation when the extracellular potassium concentration is higher than 25 mM, while the potassium concentration has less influence on the magnitude of the resting potential in range below 25 mM. In Otala NS neurons the membrane potential was demonstrated to depolarize as much as 33 mV when the potassium concentration was made 10 times higher than

TABLE I1 ELECTRICAL PROPERTIES OF NEUROSECRETORY NEURONS

Animal Leech Aplysia

Neuron" Supraesophageal ganglion NSN White cells Rid

Helix

Crayfish

X organ

Land crab Cockroach

Sinus gland Brain medial NSN

Locust

Metathoracic ganglion NSN Brain medial NSN Corpus cardiacum Corpus cardiacum axon

Blow fly

Resting potential (mV)

-

-

Critical Action depolarConduction potential ization Resistance Duration velocity (mV) (mV) (Ma) (msec) (mlsec) -

6

-

38

74b 806

-

8ob

3ob

-

26b 30-150

-

-

-

40-50

80

47 (active) 51 (dormant) 51

81 83 706

45 50-70

85 60-90

40 55

55 60

30

-

2-3 90

20-51 33

20

-

64

5.4'

-

1-1.5

10 5

-

3-7 3-7 2.5-7

-

References Yagi et al. (1963) Frazier et al. (1967) Boisson and Chalazonitis (1973) Carpenter and Gunn (1970) Strumwasser (1967) Kupfermann and Kandel (1970) Gainer (1972b) Kerkut and Meech ( 1967) Standen (1975a,b) Iwasaki and Satow (1971) Cooke (1971) Cook and Milligan (1972) Gosbee et al. (1968) Hoyle (1974) Normann (1973) Normann (1973) Normann (1973)

Fly

Brain NS

20-40

10-40

5

-

40-60

0.5-1.0

10-20

-

Goldfish

Terminal in corpus cardiacum PO NSN

51 47 65

74 66 -

3.5 3.9 10

0.46 0.52 -

Kandel (1964) Hayward (1974) Bennett et u / . (1968)

-

50-60

77* 85” 60-70

4-10 5 8-10

1.o 0.6

SO NSN SO and PV NSN

-

-

-

0.5 0.4-1.3

SO and PV NSN SO and PV NSN

-

-

-

0.69, 1.64 1.0

SO and PV NSN

-

-

-

03-0.7

SO and PV NSN

-

-

-

1.0

PV NSN SO and PV NSN

40-80

40-80

5

0.7 0.4-0.9 0.5 -

36

45 -

6 -

0.8

Bennett and Fox (1962) Bennett and Fox (1962) Morita et u1. (1961) Ishibashi (1962) Yagi ef (11. (1966) Dyball and Koizumi (1969) Ishida (1970) Dreifuss and Kelly (1972a) Negoro and Holland (1972) Wakerley and Lincoln (1973a) Sundsten et ul. (1970) Yamashita et (11. (1970) Barker et al. (1971a) Koizumi and Yamashita (1972) Sakai et u1. (1974) Hayward and Jennings (1973b)

Goosefish Skate Fluke Eel Rat

Rabbit Cat

Hypothalamic NSN Caudal NSN Caudal NSN Caudal NSN

Cat and Dog SO and PV NSN

Dog Monkey

I‘ ‘I

SO NSN SO NSN

-

-

NSN, Neurosecretory neuron; PO, preoptic; SO, supraoptic; PV, paraventricular Measured from the article. Half duration. Calculated from the article.

-

Wilkens and Mote (1970) Wilkens and Mote (1970)

Y

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KINJI YACI AND SHIZUKO IWASAKI

the original value in a bathing medium either devoid of sodium and chlorine ions or containing dopamine (Gainer, 1972b). In rat neurohypophysis in vitro an increase in the potassium concentration of the medium was shown to abolish compound action potentials evoked by stalk stimulation, probably because of significant depolarization due to the excess potassium (Yagi et al., 1966; Ishida, 1970; Nordmann and Dreifuss, 1972). Therefore in both invertebrates and vertebrates it is reasonable to conclude that potassium ions play a predominant role in generation of the resting potential of NS neurons. In snail (Otala)NS neurons seasonal variations of the resting potential were found by Kerkut and Meech (1967)and Gainer (1972b). The magnitude of the resting potential recorded in the snail in an activated state (47 mV) was shown to be significantly smaller than that recorded in the animal in a dormant state (51rnV). Membrane resistance was reported to be about three times higher in the former state than in the latter. These investigators also demonstrated that in the snail, in an activated state, cyclic decreases in potassium permeability induce cyclic depolarizations which initiate cyclic bursting discharges and result in explosive releases of neurohormone. The fact that a decrease in potassium pernieability produces membrane depolarization seems to indicate involvement in generation and rnodulation of the resting potential by other ions such as sodium ions. 2. Action Potential In the NS neurons studied so far it seems likely that ionic mechanisms for the generation of action potentials are different in different parts of a NS neuron. Therefore ionic mechanisms for the generation of action potentials in the soma, the axon, and the axon terminal of NS neurons are considered separately in the following discussion. a. The Somu. In invertebrate NS neurons it has been shown that action potentials recorded in the soma depend not only on the sodium but also on the calcium concentration of the medium. Neurosecretory neuron somata of the crayfish X organ have been demonstrated to induce action potentials in either a sodium-deficient medium or in a medium containing tetrodotoxin (TTX)which is known to block the sodium-activating system in an excitable membrane. Iwasaki and Satow (1970, 1971) showed that the amplitude of action potentials recorded for NS neurons of the crayfish X organ depends on the sodium concentration in a medium devoid of calcium (Fig. 2, right). However, even in a sodium-deficient medium the membrane of the NS neuron soma still generates action potentials, the amplitude of which depends on the calcium concentration (Fig. 2, left). The peak level of the

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FIG. 2. Dependence of action potential on extracellular sodium and calcium ions in NS neurons of the crayfish X organ. Left: Calcium ion concentration was changed in a sodium-deficient (3 mM) medium. The peak potential level (active membrane potential) was plotted against the calcium concentration of the medium. T h e slope for a 10-fold change in the calcium concentration was 29 mV in this particular NS neuron. In the inset, action potentials elicited by stimulating currents with a l-second duration are shown for the different calcium concentrations. Right: In another NS neuron of the X organ, active membrane potentials were plotted against the sodium concentration in a calcium-deficient (4 mM) medium. At a sodium concentration above 70 mM, the peak of the action potential increased linearly. Deviation from a straight line at the lower sodium concentration may be due to the contribution of the calcium component (Iwasaki and Satow, 1971).

action potential induced by a stimulus of transmembrane current pulse was plotted against either sodium or calcium concentration of the medium (Fig. 2). The peak changed with either ion concentration in good agreement with the slope predicted by the Nemst equation. Similarly, in identified NS neurons of Helix (Gainer, 1972c; Barker and Gainer, 1974b, 1975a,b; Standen, 1975a,b) and of Aplysia (Carpenter and Gunn, 1970), the cell body membrane has been demonstrated to generate both types of action potential which depend on sodium and calcium concentrations in the medium. It is therefore concluded that the soma membrane of invertebrate NS neurons can generate action potentials as the result of an increase in permeability to either sodium ions, calcium ions, or both. The soma membrane of unidentified mollusc neurons, at least some of which are probably NS cells, has been also shown to have the ability to generate calcium dependent spikes as well as sodium-dependent spikes: Aplysia (Geduldig and Junge, 1968; Geduldig and Gruener, 1970; Kado, 1973), Onchidiuin (Oomura et al., 1961), and snails (Kerkut and Gardner,

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KINJI YAGI AND SHIZWKO IWASAKI

1967; Meves, 1968; Krishtal and Magura, 1970; Wald, 1972, 1973; Kostyuk et al., 1974a,b; Sattelle, 1974). Therefore it is very likely that an increase in calcium permeability occurs during an action potential in the NS neuron and that a considerable number of calcium ions flows into the cytoplasm along its electrochemical potential gradient across the membrane of the NS neuron soma. b. The Axon. It appears that the membrane of the NS axon generates sodium-dependent action potentials. Compound action potentials recorded for rat neurohypophysis in vitro have been demonstrated to disappear reversibly in a sodium-deficient medium (Yagi et al., 1966; Ishida, 1970; Nordmann and Dreifuss, 1972). In crustacean NS neurons, Iwasaki and Satow (1971) and Cooke (1971) demonstrated that the NS axon requires the presence of extracellular sodium ions for the generation of action potentials, as shown in Fig. 3. In unidentified mollusc neurons, some of which are probably NS cells, the axon membrane develops an action potential that depends solely on extracellular sodium ions, whereas the soma membrane generates an action potential that depends on both sodium and calcium ions in the extracellular fluid (Wald, 1972; Kado, 1973). It is therefore concluded that in the NS axon action potentials are generated by the action of the sodium-activating system of the membrane, as in the case of the nonNS axon. c. The Axon Terminal. Only an article by Cooke (1971) reported the effect of alterations of the extracellular ionic composition on excitation of the NS axon terminal. He recorded membrane potentials from the NS axon terminal in crab sinus gland and found that the membrane of the axon terminal can generate action potentials in either sodium- or calcium-free medium, while the axon membrane cannot develop an action potential in sodium-free medium. These results appear to indicate that the NS iaxon terminal generates the action potential that depends on both sodium and calcium ions in the extracellular fluid. Calcium influx, which is brought about by the action potential of the NS axon terminal, has been shown to serve as a trigger for exocytotic neurohonnone release in excitation-secretion coupling, as discussed in Section V,C. With regard to non-NS axon terminals, Katz and Miledi (1969a) demonstrated calcium-dependent spikes in the presynaptic terminals of a squid giant synapse bathed in a medium containing TTX and tetraethylammonium ions. They also found in the frog neuromuscular junction that neurotransmitter release from motor nerve endings depends on the extracellular calcium concentration and increases abruptly when the presynaptic axon terminal is depolarized (Katz and Miledi, 1969b). It is therefore concluded that depo-


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FIG.3. Different ion dependency in the soma and the axon action potential. (A) In normal medium, soma action potentials recorded intracellularly (upper) and axon action potentials recorded extracellularly (lower, dotted spikes) were observed simultaneously on direct stimulation applied to the soma. (B)In a TTX medium, in which sodium-dependent action potentials had been blocked, a stimulating current evoked a soma action potential while it failed to generate an axon action potential. The small, slow change in the lower record may be due to electrotonic spread of the soma action potential. Calibration, 50 mV and 20 msec.

larization of the membrane of NS axon terminals, as in the case of the NS neuron soma, causes increases in permeability to sodium and calcium ions and influxes of both ions along their electrochemical potential gradients, resulting in an action potential.

IV. Characteristic Nature of Electrical Activity NS neurons have been found to exhibit electrical activities which are seemingly unique and characteristic. Among other electrical properties of the NS neuron membrane, characteristic activities are long duration of action potential, periodic bursting discharges, and dissociation of an antidromically conducted action potential into A and B spike components when recorded in the soma region. In this section we review the observations reported on these electrical activities of NS neurons. A.

DURATION OF ACTION POTENTIALS

The duration of action potentials recorded from the NS neuron soma and NS axon terminals have been shown to be longer than that observed in non-NS neurons in the leech (Yagi et al., 1963; see Table 11). In Aplysia, white cells (&to RI5)and bag cells, both ofwhich are NS in nature, have been found to exhibit action potentials two to five times longer than those of non-NS neurons (Frazier et al., 1967). A longer

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duration of action potential (3-7 msec) has been reported in NS axons of the corpus cardiacum of a blowfly than for non-NS neurons (0.6-2.5 msec) (Normann, 1973). In caudal NS neurons of the skate the duration of the action potential recorded from the soma has been found to be 4-10 msec-longer than that of spinal motoneurons of the animal (2 msec) (Bennett and Fox, 1962). From these results it seems likely that action potentials of prolonged duration are characteristic of the electrical activity of NS neurons, as already pointed out by Bern and Yagi (1965). With regard to the mechanism for the prolonged duration of the action potential, Iwasaki et al. (1973) and S. Iwasaki and T. Kuroda (unpublished data) observed in the NS neuron soma of the crayfish X organ that the duration of the action potential is shortened by the introduction of manganese ions into the medium, which are known to block the calcium activating system of the excitable membrane, or by the application of a hyperpolarizing current or of hypertonicity (Iwasaki and Kuroda, 1974), which reduces considerably the inward calcium current during an action potential in the NS cell as a consequence of potassium activation, as shown in Fig. 4. They also observed that a medium with low calcium concentration the duration of the action potential is shorter than in the control medium and is not affected by the above treatments. It is therefore concluded that the prolonged duration of the action potential observed in NS neurons is attributed to the slower inward calcium current. The physiological significance of calcium entry during the action potential in the NS neuron soma is discussed in Section V,A in relation to the control of neurohormone synthesis. However, the question of the function of calcium ions entering during action potentials remains open. PACEMAKING ACTIVITYAND B. ENDOGENOUS BURSTINGDISCHARGES

1. Endogenous Pacemaking Activity Periodic bursting discharges have been reported in the NS neuron soma of the crayfish X organ (Iwasaki and Satow, 1969, 1973), in Helix NS neurons (Gainer, 1972c), and in granule-containing neurons of abdominal ganglion of Aplysia (Arvanitaki and Chalazonitis, 1964; Strumwasser, 1965, 1967; Frazier et al., 1967; Mathier and Roberge, 1971; Junge and Stephens, 1973). Periodic bursting discharges have been attributed to an intrinsic pacemaking function of the NS membrane, since (1) the periodicity depends on the level of the membrane potential of the NS cell being observed, (2) an abrupt change in the

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FIG.4. Duration of action potential in the X organ NS neuron. Inset (left): The duration of the action potential decreased when the membrane potential was shifted from 69 mV (solid line) to 77 mV dotted line). At the hyperpolarizing membrane potential level, the calcium action potential is blocked because of increased potassium activation (Iwasaki et al., 1973).Inset (right):The duration of the action potential decreased markedly with the addition of manganese ion (10 mM). In the manganese medium, the calcium component in the action potential is blocked. In the lower graph, the relationship between the conditioned membrane potential and the half-duration of the action potential is shown in the normal medium (solid circles and broken line) and in the medium containing 10 mM manganese (open circles and solid line). The half-duration in the TTX medium in which the sodium component had been blocked is shown by the triangles. It can be inferred that the longer duration of the action potential in the X organ NS neuron of the crayfish is due to the contribution of the calcium component to the membrane permeability change (S. Iwasaki and T. Kuroda, unpublished data).

membrane resets the periodic discharges, (3)postsynaptic potentials responsible for periodic discharges are not observed (Fig. 5 ) , (4) periodic fluctuations in the membrane potential are observed in the NS neuron when sodium-dependent action potentials are blocked by TTX, and (5)periodic discharges also take place in a NS neuron soma completely isolated from an axon with approximately the same frequency as that observed in an intact ganglion (Chenet al., 1971; Gainer, 1972~). Two possible explanations have been proposed for the ionic mechanism of the endogenous pacemaking activity of invertebrate NS neurons. One of them is based on the observation of spontaneously occurring cyclic decreases in potassium permeability of the NS mem-

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KINJI YAGI A N D SHIZUKO IWASAKI

FIG.5. Endogenous bursting discharge in the X organ NS neuron. Interburst interval, which is defined as the period between the first spikes of every burst, changed with the membrane potential level shifted by the current injected through the neuron membrane. It decreased when the membrane was depolarized and increased when the membrane was hyperpolarized. Initiation of the bursting was reset by changing the membrane potential as seen in the upper inset. Six-tenths of a second after the beginning of each record, the membrane potential was shifted to the new potential level noted at the left in each case. The interburst intervals decreased when the membrane potential was depolarized, leaving no noticeable change in the spike pattern of a burst. In the lower inset, two bursting discharges have been enlarged to show that no synaptic potentials trigger the initiation of bursting discharges. These results indicate that the bursting discharge appearing in the NS neuron are due to the endogenous pacemaking activity of the NS neuron membrane. Calibration for the lower inset is 1 second. The arrow on the membrane potential axis designates the resting potential level.

brane. A decrease in potassium permeability would produce membrane depolarization and result in bursting discharges, provided sodium permeability is sufficiently great in the membrane during an interburst period, as in the case of the pacemaker cell of the vertebrate heart (Gainer, 1972c; Junge and Stephens, 1973; Barker and Gainer, 1975a; Smith et al., 1975). However, Strumwasser (1965, 1968) pro-


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posed that cyclic changes in the rate of an electrogenic sodium pump produce periodic membrane depolarizations and result in periodic bursting discharges, based on his observations of the effect of metabolic inhibitors and extracellular anion concentrations on the periodic fluctuations of membrane potentials in Aplysia NS neurons. However, the latter hypothesis does not explain the periodic change in membrane conductance observed by other investigators. In mammalian hypothalamoneurohypophysial NS neurons periodic bursting discharges have been found to occur spontaneously in rat supraoptic and paraventricular nuclei (Dyball, 1971; Dyball and Dyer, 1971; Wakerley and Lincoln, 1971, 1973b; Dreifuss and Kelly, 1972b; Negoro and Holland, 1972; Dyball and Pountney, 1973; Walter and Hatton, 1974), in rabbit paraventricular nucleus (Sundsten et al., 1970), and in monkey supraoptic nucleus (Hayward and Jennings, 1973a,b; Arnauld et al., 1974). It is uncertain at present whether or not periodic bursting discharges in mammalian NS neurons depend on intrinsic membrane properties similar to those of invertebrate NS neurons discussed above. In mammalian non-NS neurons periodic bursting discharges have been recorded for respiratory neurons in the medullary reticular formation and have been attributed to intrinsic neuronal properties (Hukuhara, 1973, 1974). As another possible explanation, Dyball(l971) and Dreifuss and Kelly (1972b) pointed out the possible significance of the recurrent inhibition found in mammalian hypothalamic NS neurons, as discussed in Section VI,B. As another alternative, it may be that a neural mechanism of recurrent facilitation which has been found in mammalian hypothalamic NS neurons (see Section VI,C), functions as a reverberating neural circuit and as a result produces such periodic bursting discharges. The excitatory effect of iontophoretically applied oxytocin on the activity of hypothalamic NS neurons (see Section VI,A) provides another possible explanation, namely, that the positive feedback action of neurohormone may lead to periodic bursting discharges.

2 . Control of the Pacemaking Activity The endogenous pacemaking activity of invertebrate NS neurons has been found to be influenced by various physiological conditions. According to Gainer (1972b), the majority of Helix NS neurons are either “silent” or fire irregularly and not in bursts during the dormant period, and they exhibit characteristic periodic discharges in an activated state. He also reported that in these NS neurons electrical resistance of the membrane is higher and the magnitude of the resting membrane potential lower in summer than in winter. His conclusion

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was that the membrane of the NS neurons in an activated state reduces its potassium permeability and as a result is depolarized. A kind of diurnal rhythm of the pacemaking activity of NS neurons has been observed in Helix (Gainer, 1972c) and in Aplysia (Strumwasser, 1965, 1967, 1968). In Helix NS neurons the magnitude of the “pacemaking potential” has been found to depend on extracellular calcium concentration, with an optimal calcium concentration for the development of the potential (Barker and Gainer, 1973, 1974b71975b).Seasonal variations in calcium concentration in Helix heniolymph were also mentioned by Barker and Gainer (1973). These observations lead us to consider the possibility that the extracellular calcium concentration may play a key role at least in part in the development of either seasonal or diurnal variations in the pacemaking activity of bursting NS neurons, which possess an ability to burst their discharge, although it seems premature at present to suggest this possibility. Recently, endogenous peptides or proteins of low molecular weight have been studied in relation to control of the endogenous pacemaking activity in NS neurons. Gainer (1972b) reported that a specific H a peptide of increase in the rate of incorporation of l e ~ c i n e - ~into about 5000 daltons occurs in accordance with the appearance of bursting discharges in Helix NS neurons. RNA-dependent protein synthesis was found by Strumwasser (1973) to occur specifically in the bursting NS neurons ofAplysia. He also showed that the content of the specific protein in the bursting NS neurons decreases as bursting activity is inhibited by treatment of the chlorine-deficient medium. Strumwasser (1965) demonstrated that actinomycin D injected into a particular neuron soma in Aplysia inhibits endogenous bursting activity for a period of time and then resets the diurnal rhythm of the endogenous pacemaking activity. Wilson (1971) reported that a protein of 12,000 daltons synthesized in an Aplysia N S neuron (R15)is specific to this NS neuron. These results appear to support the hypothesis that the endogenous pacemaking activity of invertebrate NS neurons is controlled by certain peptide(s) or protein(s) of low molecular weight. Barker and Gainer (1974a, 1975b) reported that periodic bursting discharges can be initiated in identified mollusc NS neurons by either an iontophoretic technique or addition to the bathing medium of certain exogenous peptides. They demonstrated that vasopressin and its analogs induce characteristic bursting discharges in the NS neuron which is in an inactive state either because of dormancy or because of addition of cobalt ions to the medium, and that this effect of the peptides is restricted to the NS neurons C,,in Helix and R1, in Aplysia.


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Their analysis of a current-voltage relationship disclosed that the exogenous peptides produce a decrease in the anomalous rectification of the NS cell membrane, which indicates a decrease in potassium permeability. A possible mechanism was discussed in the preceding section, in which cyclic decreases in potassium permeability lead to depolarization and, as a result, cyclic bursting discharges in the invertebrate NS neuron. Barker and Gainer (1974a) commented in their unpublished results that a peptide fraction isolated from snail brain is similar to vasopressin in its effects on the NS cell C,, and is found in the same fraction as vasopressin after column chromatography. The physiological significance of the periodic bursting discharges produced b y the endogenous pacemaking activity of NS neurons is still unknown.

c.

TWO COMPONENTS OF THE ACTION POTENTIAL

The dissociation of an antidroniically conducted action potential into A and B spike components has been found to occur in the soma of neurohypophysial NS neurons of cat supraoptic and paraventricular nuclei (Yamashita et al., 1970; Barker et al., 1971a,b; Koizumi and Yamashita, 1972), rabbit paraventricular nucleus (Novin et al., 1970; Sundsten et al., 1970; Novin and Durham, 1973), rat supraoptic and paraventricular nuclei (Dreifuss and Kelly, 1972a; Negoro and Holland, 1972), and goldfish preoptic nucleus (Kandel, 1964; Hayward, 1974). It was reported that the antidromic spike is occasionally separated completely into two components, while the action potential induced by orthodromic stimuli dissociates only slightly into components and has a small notch in its rising phase. Antidromic spikes induced in tuberoinfundibular NS neurons have been shown to dissociate, although incompletely, into A and B components (Fig. 7; Sawaki and Yagi, 1973). In non-NS neurons only a small notch, if any, has been found in the rising phase of antidromically conducted action potentials (Coombs et al., 1957; Fuortes et al., 1957; Phillips, 1959; Kandel et al., 1961; Bishop et al., 1962; Patton et al., 1962). Therefore the complete separation of an antidromic spike seems to be characteristic of the neurohypophysial NS neuron. With regard to the mechanism underlying the dissociation of antidromic action potentials, recurrent inhibition of the NS neuron provides a possible explanation. Sawaki and Yagi (1976) recently found that an invasion of the soma and dendrites of a tuberoinfundibular NS neuron by an antidromically conducted spike can be blocked b y recurrent inhibition (see Section VI,B and Fig. 7). Therefore complete separation of an antidromic spike is probably due to the powerful

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blocking action of recurrent inhibition in the case of the neurohypophysial NS neuron. An alternative explanation may be derived from axon bifurcations which have been reported by Hayward (1974).The possible explanation is that, when an action potential is conducted antidromically along one of the axon collaterals, the safety factor is too low at the bifurcation of the axon for the antidromic spike to be conducted into the other two axon parts because of an increment in the surface area of the axon member in the resting state, and this decrease in safety factor brings about a delay in the invasion of the soma by the antidromic spike, leaving an electrotonic spread of the antidromic spike which reaches the bifurcation.

V. Role of Action Potentials in Endocrine Activity Biosynthesis, storage, and release of neurohormone have been extensively studied mostly in the mammalian hypothalamoneurohypophysial system. Morphological and biochemical evidence has accumulated supporting the following hypotheses (for reviews, see Sloper, 1958,1966; Bargman, 1966; Sachs, 1967,1969; Sachs et aZ., 1969). It is now widely believed that (1) neurohypophysial hormones and neurohormone-binding proteins (neurophysins) are synthesized in the perikarya of hypothalamic NS neurons by RNA-dependent biosynthetic mechanisms, (2) NS materials associated with peptide hormones and neurophysins are packed into NS granules in the Golgi apparatus, (3) the granules move down to the neurohypophysis in the NS axons of the hypothalamoneurohypophysial tract by an axonal transport mechanism, and (4) neurohormones and neurophysins are released from NS granules stored in the NS axon terminals within the neurohypophyis into the extracellular space by exocytosis induced by an excitation-secretion coupling mechanism. In the following discussion we deal with the question whether the biosynthesis, axonal transport, and release of neurohormone are controlled by electrical activity of NS neurons in mammalian species.

A.

SYNTHESISOF NEUROHORMONE

There is no available evidence at present either for or against the hypothesis that the rate of neurohonnone synthesis in NS neurons depends on the electrical activity. However, an increase in the rate of synthesis of antidiuretic hormone (ADH) has been found following osmotic stimuli such as dehydration or salt loading which is known to stimulate ADH secretion by the neurohypophysis. Cytological and ultrastructural alterations suggesting an increase in metabolic activity


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have been demonstrated in the supraoptic nucleus neuron of rats subjected to dehydration (Zambrano and de Robertis, 1966; Watt, 1970). Dehydration or salt loading has been shown to enhance the rate of synthesis of ADH (Takabatake and Sachs, 1964) and neurophysins (Norstrom et al., 1971a; Norstrom and Sjostrand, 1972a) in hypothalamoneurohypophysial tissue. Incorporation studies with isotopelabeled amino acids have further disclosed that the biosynthesis of ADH and of neurophysins are two closely related events (Sachs et al., 1971) and can be blocked by puromycin or an inhibition of RNA synthesis (Sachs and Takabatake, 1964; Pearson et al., 1975).Puromycin has been shown to prevent the ultrastructural changes indicative of enhanced metabolic activity from being induced by dehydration in supraoptic NS neurons (Zambrano and de Robertis, 1967). Both intracarotid injection of hypertonic saline solution and salt loading have been demonstrated to increase the electrical activity of some identified NS neurons of the rat hypothalamoneurohypophysial system (Dyball and Koizumi, 1969; Dyball, 1971; Dyball and Pountney, 1973). On the basis of their observation that the incorporation of labeled amino acids into vasopressin was not enhanced either shortly after hemorrhage or following electrical stimulation of hypothalamus-median eminence tissue in vitro, Sachs et al. (1969) concluded that the enhanced rate of ADH synthesis after chronic stimuli with dehydration or salt loading could not be the direct consequence of an increased rate of firing but may be attributed to some kind of adaptive change in biosynthetic processes. It has been demonstrated that a particular metabolic pathway of the mammalian peripheral nerves is stimulated by intracellular calcium ions which move in across the membrane as a result of action potentials (Landowne and Ritchie, 1971). The soma membrane of invertebrate NS neurons has been demonstrated to generate action potentials which depend partly on the calcium influx induced by the calcium activation system described in Section II1,C. Therefore the question whether or not intracellular calcium ions moving into the cytoplasm of perikarya following an action potential control the rate of neurohormone synthesis is of interest and remains to be answered. B. AXONAL TRANSPORTOF NEUROHORMONE Sloper et al. (1960)demonstrated for the first time axonal transport in the NS neuron by the method of radioisotope uptake. Later, axonal transport in NS neurons was shown by observation of stainable NS material with the light microscope in goldfish NS neurons (Jasinski et al., 1966)and by means of electron microscope radioautography in rat

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supraoptic neurons (Nishioka et al., 1970). Substances that incorporated either tyro~ine-~H or ~ y s t e i n e - ~were ~ S isolated from the mammalian neurohypophysis and identified as vasopressin, oxytocin, and neurophysins which are synthesized in the NS neuron soma (Fawcett et al., 1968; Pickering and Jones, 1971). Immunoreactive neurophysins have been demonstrated to be transported along the hypothalamoneurohypophysial tract (Fawcett et al., 1968; Alvarez-Buylla et al., 1973). Incorporation studies have further disclosed that the velocity of axonal transport of neurohormone and binding proteins ranges between 2 and 3 mm per hour in mammalian hypothalamoneurohypophysial NS neurons (Norstrom and Sjostrand, 1971a,b; Pickering and Jones, 1971; Jones and Pickering, 1972). Colchicine was found to block the axonal transport of vasopressin, proteins, and neurosecretory granules into which ~ y s t e i n e - ~had ~ Sbeen incorporated, but to produce no significant change in microtubules (Norstrom et al., 1971b; FlamentDurand and Dustin, 1972; Pearson et al., 1975). As far as we know, there has been no reported study on the relationship between action potentials and the axonal transport of neurohormone or binding proteins. However, it has been demonstrated in rat hypothalamoneurohypophysial NS neurons that physiological stimuli for neurohormone release such as dehydration, salt loading, or suckling, all of which are also known to enhance electrical activity, do not significantly change the velocity of neurophysins but increase the rate of axonal transport (Norstrom et al., 1971a; Norstrom and Sjostrand, 1972b). In cat spinal motoneurons, Lux et al. (1970) reported that repetitive antidromic stimulation increases the rate of incorporation of intracellularly injected labeled glycin into proteins in the soma and the amount of protein transported per unit of time along the axon but does not alter the velocity (40 mm per day) of axonal transport. A possible explanation for these observations concerning the rate of axonal transport may be that an impulse being conducted along the axon increases the affinity between the NS granules to be transported and the transport machinery as a consequence of changes in the intracellular ionic environment following an action potential.

c.

RELEASE OF NEUROHORMONE

1. Unit Response in Relation to Neurohomone Release Electrophysiological studies on the relationship between electrical activity and neurohormone release have been concerned in most cases with unit responses expressed as changes in the firing rate re-


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corded for mammalian hypothalamic NS neurons. The first attempt to investigate unit responses to the stimuli known to cause neurohormone release from the neurohypophysis was made by Cross and Green (1959) on the supraoptic and paraventricular nuclei of rabbit hypothalamus. Since then many studies have been made and reviewed (Cross and Silver, 1966; Beyer and Sawyer, 1969; Cross, 1973). As discussed by Cross (1973), the activity of the hypothalamic neurons responsive to these stimuli may not necessarily reflect the activity of an NS neuron but may be derived from an interneuron as well. However, this difficulty has been overcome by the method of antidromic identification of NS neurons. Since Yagi et al. first demonstrated in 1966 antidromically conducted unit spikes in rat supraoptic nucleus following single stimuli given to the neurohypophysis, extensive studies have been conducted on the effect of such stimuli as intracarotid injection of hypertonic saline solution, water deprivation, salt loading, suckling, and vaginal distension on firing rates of antidromically identified supraoptic and paraventricular NS neurons of the mammalian hypothalamus (Dyball and Koizumi, 1969; Wakerley and Lincoln, 1971; Vincent et al., 1972a,b; Dyball and Pountney, 1973; Hayward and Jennings, 1973a-d; Negoro et al., 1973b; Amauld et al., 1974). A good correlation has been found between the unit response to an intracarotid injection of hypertonic solution or suckling stimuli in lactating animals and the simultaneously measured release of vasopressin and/or oxytocin (Dyball, 1971; Wakerley and Lincoln, 1973a; Lincoln and Wakerley, 1974, 1975). These electrophysiological studies produced the following interesting hypotheses or observations with regard to the activity of hypothalamic NS neurons. First, it has been claimed that the osmoreceptor cells that specifically control antidiuretic hormone release are distinct from NS neurons in the mammalian hypothalamus (Hayward and Vincent, 1970). This hypothesis is based on the fact that NS cells that release neurohormone from axon terminals in the neurohypophysis are antidromically identified in the supraoptic nucleus and respond specifically, with excitation followed by inhibition, to intracarotidly injected hypertonic sodium chloride but not to arousing stimuli such as sound, light, or touch. The osmoreceptor cells presumably located in the perinuclear zone responded specifically to osmotic stimuli with monophasic excitation or inhibition and were not identified antidromically (Hayward and Vincent, 1970; Vincent et al., 1972a,b; Hayward and Jennings, 1973a,d). Second, considerable percentages of identified NS neurons were shown to repeat periodically a characteristic bursting discharge of a duration of several tens of seconds (Wa-

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kerley and Lincoln, 1971; Dreifuss and Kelly, 197213; Hayward and Jennings, 1973a,b; Arnauld et al., 1974). Third, electrophysiological evidence was provided to support the view that the supraoptic as well as the paraventricular nucleus includes NS cells that synthesize and release oxytocin (Dyball, 1971; Lincoln and Wakerley, 1974).This hypothesis is supported by observations indicating the synthesis of oxytocin in supraoptic nucleus neurons (Sokol, 1970; Burford et al., 1974; Dyball and Henry, 1975). A number of studies have been conducted on the activity of hypothalamic and preoptic neurons of mammalian species in relation to adenohypophysial functions (for review, see Beyer and Sawyer, 1969; Cross, 1973; Sawyer, 1975).Tuberoinfundibular NS neurons that control adenohypophysial functions have been identified antidromically (Makara et al., 1972; Sawaki and Yagi, 1973). However, electrical activity of identified tuberoinfundibular neurons has not been related definitively to the release of any particular one of at least six releasing and inhibiting factors. Studies on unit responses in relation to neurohormone release have produced a few interesting hypotheses. However, it should be noted that an increase in unit activity following physiological stimuli that induce neurohormone release supports but does not prove the hypothesis that action potentials in the NS axon terminal directly control neurohormone release.

2. Excitation-Secretion Coupling The release of NS materials from axon terminals in the fish urophysis has been demonstrated morphologically in response to electrical stimuli that induce action potentials in urophysial NS neurons (Fridberg et al., 1966). Harris and Ruf (1970) reported the electrical stimulation of hypophysiotrophic areas of rat hypothalamus results in an increased level of luteinizing hormone releasing factor in hypophysial portal blood. In the mammalian hypothalamoneurohypophysial system electrical stimulation of the supraoptic and paraventricular nuclei, the NS axons in the pituitary stalk, or the neurohypophysis in uiuo has been shown to cause the release of neurohypophysial hormones (Harris et al., 1969; Sundsten et al., 1970; Bisset et al., 1971). Isolated neurohypophyses of mammalian species have been demonstrated to release neurohormone in response to electrical stimuli (Haller et al., 1965; Mikiten and Douglas, 1965; Sachs et al., 1967; Sachs and Haller, 1968; Ishida, 1970; Dreifuss et al., 1971; Nordmann and Dreifuss, 1972). Local anesthetics have been found to block the evoked hormone output (Haller et al., 1965; Mikiten and Douglas,

ELECTROPHYSIOLOCY OF NEUROSECRETORY CELL

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1965). Evidence cited above indicates that action potentials conducted along the NS axon trigger the release of neurohormone from the NS axon terminal, as in the case of presynaptic axon terminals. Excitation-secretion coupling between membrane depolarization of NS axon terminals and neurohormone secretion has been extensively studied, mainly in the mammalian neurohypophysis (for review, see Sachs, 1969; Poisner, 1970, 1973; Douglas et al., 1971; Livingston, 1971).Incubation of an isolated neurohypophysis in a medium with a high potassium concentration has been demonstrated to evoke neurohormone secretion (Douglas and Poisner, 1964a; Dicker, 1966;Thorn, 1966; Daniel and Lederis, 1967; Ishida, 1967,1968;Sachs et al., 1967; Fawcett et al., 1968; Sachs and Haller, 1968; Warberg and Thorn, 1969; Dreifuss et al., 1971; Norstrom, 1972). Calcium in the medium has been found to be required for hormone release from NS axon terminals evoked by either an increase in extracellular potassium (Douglas and Poisner, 1964a; Dicker, 1966; Ishida, 1968; Warberg and Thorn, 1969) or electrical stimuli (Mikiten and Douglas, 1965; Nordmann and Dreifuss, 1972). Both high potassium concentration and electrical stimuli have been shown to increase calcium uptake b y the neurohypophysis in uitro (Douglas and Poisner, 1964b; Ishida and Yoneda, 1974). Calcium activation in the NS axon terminal is considered to depend on depolarization, since TTX, which is known to block specifically sodium activation in the generation of action potentials was shown to block both compound action potentials and hormone release following electrical stimulation of the isolated neurohypophysis but not to depress hormone output after treatment with a medium containing excess potassium (Dreifuss et al., 1971), although Ishida (1967) reported the opposite data on the effect of TTX on hormone release following potassium treatment. Russel et al. (1974) found that calcium ionophores, as well as calcium fluxes, increase vasopressin release from the neurohypophysis in uitro. Calcium inactivation associated with a decrease in neurohormone release has been demonstrated in rat neurohypophysis incubated in a medium with excess potassium (Nordmann, 1975). Simultaneous release of neurophysin with neurohormone from the neurohypophysis, both in uitro after treatment with excess potassium (Fawcett et al., 1968; Uttenthal et al., 1971; Norstrom, 1972) and in uiuo following hemorrhage or during parturition (McNeilly et al., 1972a,b), has been demonstrated to occur. These results suggest an exocytotic mechanism for neurohormone release. In regard to excitation-secretion coupling in NS cells the experimental results cited

166


above suggest that depolarization of NS axon terminals due to action potentials conducted along the axon induces an increase in calcium permeability, resulting in a calcium influx along the electrochemical potential gradient, and that intracellular calcium ions in turn evoke exocytotic release of contents of NS granules from the terminals. The mechanism by which intracellular calcium ions evoke exocytosis still remains to be studied in detail. In in uitro studies calcium ions have been shown to inhibit the binding of neurohormones to isolated neurophysins (Smith and Thorn, 1965; Ginsburg et al., 1966). This observation supports the view that an increase in calcium ion level converts bound neurohormone to the free form in the cytoplasm of NS axon terminals and facilitates its release. Poisner (1970, 1973) put forward the hypothesis that calcium ions moving into the cytoplasm cause adhesion of NS granules to the membrane of the axon terminal and activate a contractile protein presumably contained in the granular membrane, and as a result exocytosis occurs. The observation of an inhibitory effect of calcium on neurohormone binding is not incompatible with the hypothesis of exocytotic release.

VI. Synaptic Control of the Hypothalamic NS Cell It is of great interest and importance to know how a NS neuron is synaptically controlled, since a NS neuron functions as the transducer that converts neural signals conveying neuroendocrine information processed in the central nervous system into endocrine signals. Various putative neurotransmitters and antagonistic substances have been demonstrated to alter neurohormone release when either injected into cat supraoptic nucleus (Milton and Paterson, 1974) or added to the organ culture medium of hypothalamic tissues including NS neurons (Daniel and Lederis, 1967; Eggena and Thorn, 1970; Nordmann et aZ., 1971; Grimm and Reichlin, 1973; Bradbury et al., 1974; Simonovic et al., 1974; Hillhouse et al., 1975). These observations strongly suggest that the particular substance acts directly on the postsynaptic membranes of NS neurons, but they do not exclude the possibility that neurohormone release may be caused indirectly as a result of activation of intemeurons by the substance, since two-thirds of the 596 synaptic boutons, on the average, impinging on each supraoptic neuron have been shown to be of intranuclear origin (Lbrimth et al., 1975) and synaptic transmission in NS neurons of organ culture has also been proved (Sakai et aZ., 1974; Geller, 1975). Bloom et al. (1963) first demonstrated that iontophoretic applications of putative neurotransmitter substances provide satisfactory evidence

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for the sensitivity of a hypothalamic neuron under study to the substances although they did not attempt to identify the cell. Recently, a method comprising both techniques of antidromic identifiction of NS neurons and iontophoretic application of drugs has produced electrophysiological evidence for the synaptic control of NS neurons.

A.

EFFECTSOF A PUTATIVE NEUROTRANSMITTER ON NS NEURON ACTIVITY

1. Acetylcholine Iontophoretically applied acetylcholine (ACh) has been demonstrated to facilitate unit activity of antidromically identified NS neurons in supraoptic and paraventricular nuclei (Barker et al., 1971b; Moss et al., 1971, 1972b; Dreifuss and Kelly, 197213). It was also reported that iontophoretically applied nicotine excited some of the identified NS neurons that were facilitated in response to ACh, and that dihydro-P-erythroidine antagonized the effects of ACh and nicotine. It was also demonstrated that iontophoretically applied ACh inhibits unit activity of some of the identified NS neurons. Barker et al. (1971b) found that iontophoretically applied carbachol and acetyl-P-methylcholine also inhibit NS neurons that are depressed by ACh, and that the inhibitory response to the cholinergic drugs is blocked by atropine. They also showed that in both types of AChsensitive NS neurons physostigmine produces the responses to be expected according to its inhibitory action on acetylcholinesterase. The above results on iontophoretic applications of drugs suggest the existence of two types of cholinergic mechanisms. One produces facilitation of neurohypophysial NS neurons by nicotinic action of ACh, and the other evokes inhibition b y muscarinic action. However, Nordmann et al. (1971) suggested that ACh causes neurohypophysial hormone release in vitro through muscarinic action, on the basis of their observations of the blocking action of atropine and the ineffectiveness of nicotine and D-tubocurarine on the release. The discrepancy between the observations in vitro and in vivo remains to be explained. 2. Catecholamines Iontophoretically applied norepinephrine and dopamine have been found to inhibit unit activity of all (Barker et al., 1971b) or a majority (Moss et al., 1971, 1972b) of the antidromically identified supraoptic and paraventricular NS neurons that responded to putative neurotransmitter substances.

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Recently, Moss et al. (1975) reported that iontophoretically applied norepinephrine and dopamine either facilitated or inhibited unit activity of antidromically identified tuberoinfundibular NS neurons, that the catecholamines produced differential responses in each one of the responsive neurons, and that none of the identified neurons showed the same type of response, either excitation or inhibition, to each one of these two compounds. In comparison with these observations and the conclusion drawn by Grim and Reichlin (1973), based on the results of their in vitro experiments, it is worthwhile noting that the stimulatory effect of dopamine on the release of thyrotrophin releasing hormone is mediated via norepinephrine converted from dopamine added to the incubation medium.

3. Other Putative Neurotransmitter Substances It has been demonstrated by the method of iontophoretic drug application that glutamate facilitates, glycine, y-aminobutyric acid (GABA), and 5-hydroxytryptamine (5-HT) inhibit, and histamine does not alter unit activity of identified supraoptic NS neurons (Barker et al., 1971b; Moss et al., 1971; Nicoll and Barker, 1971).The inhibitory action of glycine and GABA have been shown to be antagonized b y strychnine and b y picrotoxin and bicuculline, respectively (Nicoll and Barker, 1971). Moss et al. (1971, 1972b) demonstrated that glutamate excites, GABA inhibits, and 5-HT either excites or inhibits paraventricular NS neurons. Although iontophoretically applied vasopressin was reported to inhibit supraoptic NS neurons (Nicoll and Barker, 1971), Moss et al. (1972a) showed that vasopressin does not excite NS neurons sampled from supraoptic and paraventricular nuclei, while oxytocin excites paraventricular NS neurons. Positive results obtained by iontophoretic applications of a putative neurotransmitter substance and of its antagonist certainly indicate the presence of a receptor for the substance in the NS neuron and therefore strongly suggest the existence of a synapse involving the putative neurotransmitter substance. However, other criteria for a substance to be identified as an actual neurotransmitter should also be satisfied. For example, the candidate substance must be synthesized and metabolized locally, and contained in and released from presynaptic axon terminals impinging on the NS neuron by activation of presynaptic pathways. It must also be shown that the induced synaptic transmission is blocked by a substance known to antagonize the putative neurotransmitter. Apparently, we need much more experimental evidence before the chemical nature of the synaptic transmission controlling NS neuron activity can be determined.


B. RECURRENT INHIBITION

OF THE

169

NS NEURON ACTIVITY

1. Evidence for Its Occurrence Kandel (1964) found that antidromic stimulation of the goldfish neurohypophysis produces an inhibition of spontaneous unit activity of preoptic magnocellular NS neurons. Since then recurrent inhibition has been demonstrated in hypothalamoneurohypophysial NS neurons of rats (Kelly and Dreifuss, 1970; Dreifuss and Kelly, 1972a; Negoro and Holland, 1972; Negoro et aZ., 1973a; Dreifuss et aZ., 1974; Dyball, 1974), of cats (Barker et al., 1971a; Nicoll and Barker, 1971), and of cats and dogs (Koizumi and Yamashita, 1972). Recurrent inhibition has also been demonstrated in antidromically indentified tuberoinfundibular NS neurons in response to antidromic stimulation of rat median eminence (Yagi and Sawaki, 1975a,b). In these studies repetitive stimuli of 100 Hz given to the median eminence were shown to inhibit antidromically identified tuberoinfundibular NS unit (Fig. 6A). Single-pulse stimuli also produced an inhibi-

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FIG.6. Inhibition of spontaneous unit firing following antidromic stimulation of the median eminence in antidromically identified tuberoinfundibular neurons. (A) A unit that showed inhibition of spontaneous firing after repetitive stimulation with 100-Hz pulses for 1second. Arrow indicates stimulation. (B) Superimposition of 1000 responses to single-pulse stimuli. Note remarkable inhibition following the stimuli (arrow). Stimulus intensity was equal to the threshold for producing an antidromic spike in this unit. (C) A poststimulus histogram obtained simultaneously with oscilloscope recordings from the same unit as in (B) by compilation of spontaneous firings during 1000 sweeps into 200 bins of a digital computer. The length of each time bin was 5 msec. The time course of inhibition during the poststimulus period is clearly seen. Stimuli were given at time 0. (D) Effectiveness of stimuli of subthreshold intensity for an antidromic spike in producing an inhibitory response. The unit is the same as that in (C). Stimulus intensity was 0.8 of the threshold for an antidromic spike.

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FIG.7. Recurrent inhibition demonstrated by paired pulse stimulation. The antidromic spike induced by a testing stimulus given a few milliseconds after a conditioning stimulus of subthreshold intensity for an antidromic spike failed to invade the soma and dendrite of an antidromically identified tuberoinfundibular neuron. The results indicate that the subthreshold stimuli given to the median eminence inhibit the somatic-dendritic region ofthis tuberoinfundibular neuron. Time noted on each record is the interval between the two stimulating pulses. The results indicate that the difference in latency between an antidromatically conducted spike and antidromic inhibition is not longer than 2.1 msec. (From Sawaki and Yagi, 1976.)

tory response lasting for several hundreds of milliseconds during the poststimulus period in the majority of the identified tuberoinfundibular units examined (Fig. 6B, C, and D). Subthreshold itensity for evoking an antidromic spike was sufficient to produce the inhibitory response (Fig. 6D). An antidromically conducted spike evoked by a testing stimulus given several milliseconds after a subliminal conditioning stimulus for an antidromic spike was occasionally observed to fail to invade the soma-dendritic region (Fig. 7; Sawaki and Yagi, 1976).These observations clearly demonstrate the existence of recurrent neural pathways comprised of tuberoinfundibular axons, which inhibit tuberoinfundibular NS neurons. Morphological evidence has shown the existence of axon collaterals of neurohypophysial NS neurons (Hayward, 1974) and of tuberoinfundibular neurons containing materials immunoreactive to luteinzing hormone releasing factor (Barry et al., 1974; Barry and Dubois, 1974). Axon collaterals of identified tuberoinfundibular neurons have been also demonstrated electrophysiologically to terminate in structures


171

other than the external layer of the median eminence (Harris and Sanghera, 1974). 2. Interneurons An inhibitory postsynaptic potential (IPSP, Kandel, 1964) and a hyperpolarizing respose which was probably an IPSP (Koizumi and Yamashita, 1972) were demonstrated in neurohypophysial NS neurons to be evoked by antidromic stimulation. Monosynaptic mediation of the recurrent inhibition was claimed by Kandel(l964) on the basis of short latency of the IPSP. Nicoll and Barker (1971) reported an inhibitory effect of iontophoretically applied lysine vasopressin on the spontaneous activity of identified cat NS neurons. However, subsequent studies provided evidence against the hypothesis that the recurrent inhibition of NS neurons is monosynaptically mediated by neurohypophysial neurohormones. Moss et al. (1972a) found that iontophoretically applied oxytocin does not inhibit but facilitates unit activity of identified paraventricular NS neurons. In supraoptic and paraventricular nuclei, Koizumi and Yamashita found the presumed interneurons which were not identified antidromically and responded to single-pulse stimulation of the neurohypophysis with high-frequency repetitive discharges of the Renshaw cell type. Recently, the recurrent inhibition of identified supraoptic NS neurons has been demonstrated in rats which hereditary diabetes insipidus, which cannot synthesize and release antidiuretic hormone (Dreifuss et al., 1974; Dyball, 1974).This is a very interesting observation, since it excludes the possibility that antidiuretic hormone serves as a neurotransmitter at the terminal of an NS axon collateral which mediates recurrent inhibition. In the tuberoinfundibular NS system the latency of the recurrent inhibition induced by antidromic stimulation of the median eminence was not much longer than the latency of the antidromic spike produced by a single stimulus in the unit under study, as shown in Fig. 7. The difference between the two latencies was shorter than 2 msec. The tuberoinfundibular tract was shown to consist of unmyelinated axons of approximately the same size (Monroe, 1967). These observations allow only a very few interneurons to be intercalated in the recurrent inhibitory pathway. Intravenously injected picrotoxin blocked the recurrent inhibition of all the tuberoinfundibular neurons tested, while strychnine did not influence the inhibition appreciably (Fig. 8). Picrotoxin and strychnine are known to antagonize the inhibitory action of GABA and glycine, respectively, on neurons in the mammalian central nervous system. Therefore it is reasonably con-

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FIG. 8. Effect of intravenously injected strychnine (STR) and picrotoxin (PTX) on recurrent inhibition of tuberoinfundibular NS neurons. (A) No significant alteration is noted in the inhibitory response before and after a strychnine injection (0.2 mg/kg). Stimulus intensity used and the threshold for an antidromic spike were 0.5 and 0.3 mA, respectively, in this unit. (B) Poststimulus histograms obtained from another tuberoinfundibular unit before and after a picrotoxin injection (4 mg/kg). Stimulus strength used and the threshold for an antidromic spike in this unit were 0.5 and 0.33 mA, respectively. (From Sawaki and Yagi, 1976.)

cluded that impulses conducted along the tuberoinfundibular axon collaterals activate GABA interneurons which in turn inhibit tuberoinfundibular neurons (Fig. 9).

3. Physiological Significance Dyball(l971) and Dreifuss and Kelly (1972b) pointed out the possibility that negative feedback actions of recurrent inhibition may produce the periodicity of spontaneous unit activity observed in many neurohypophysial NS units. This hypothesis is compatible with the conclusion drawn by Dreifuss and Kelly (1972a) that the recurrent inhibitory pathways converging on each supraoptic NS neuron are restricted to the axon of the cell under study and to only a few axons lying close to it. Intrinsic pacemaking ability, which produces periodic bursting discharges in invertebrate NS neurons (see Section IV,B), provides an alternative explanation for the periodic unit activity reported in mammalian neurohypophysial NS neurons. The tuberoinfundibular system includes at least six kinds of functionally distinct NS neurons. Recurrent inhibition was observed in almost all the tuberoinfundibular neurons studied. Therefore it is concluded that its physiological significance does not involve a specific relation to any one adenohypophysial hormone. A possible explana-

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A MEDIAN EMINENCE

FIG. 9. A hypothetical neural circuit which mediates recurrent inhibition and recurrent facilitation of tuberoinfundibular NS neurons. This illustration is highly schematized (see text). CA, Catecholaminergic neuron; GABA, GABA-releasing neuron; TI, tuberoinfundibular neuron.

tion for the inhibition produced in tuberoinfundibular neurons by antidromic stimulation of the median eminence is recurrent reciprocal inhibition between two or more distinct pools of NS neurons with different functions. The possibility that presynaptic pathways inhibit tuberoinfundibular neurons belonging to the same NS neuron pool as the recurrent axon collaterals stimulated is not excluded. Further investigations appear to be required for an exact assessment of the physiological role.

c.

RECURRENT FACILITATION OF

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1. Evidence for I t s Presence Koizumi et al. (1973) provided electrophysiological evidence for the existence of recurrent facilitatory pathways in bullfrog neurohypophysial NS neurons. Yagi and Sawaki (1975a,b) also found recurrent facilitation of tuberoinfundibular NS neurons in anesthetized rats. In their study repetitive stimuli of 100 Hz given to the median eminence for 1 second were found to be occasionally followed by a facilitation of spontaneous firing of certain antidromically identified tuberoinfundibular neurons (Fig. 10A). Single stimuli also produced the facilitatory

174


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FIG.10. Facilitation of tuberoinfundibular units following antidromic stimulation of the median eminence. (A) A unit that showed a response to repetitive stimulation with 100-Hz pulses for 1 second. Note that the firing rate increased and the amptitude of the spike decreased following the repetitive stimuli. (B) Another example of units that showed transitory facilitation during recurrent inhibition of the poststimulus period following single stimuli. The oscilloscope record shows 1000 superimposed responses.

response (Fig. 10B). Subthreshold stimuli for producing antidromic spike in the unit are effective in inducing the facilitation, and stimulus strength remarkably influences the duration and intensity of the facilitatory response (Fig. 11). Recurrent inhibition is not always accompanied by the facilitatory response, and the latter can be observed without the former (Fig. 12). Therefore the facilitatory response does not reflect a rebound excitation which might have occurred after the tuberoinfundibular neuron was relieved of recurrent inhibition. These results clearly demonstrate the presence of neural pathways mediating recurrent facilitation of tuberoinfundibular NS neurons. In the non-NS neural system recurrent facilitatory pathways also have been found in gracile nucleus neurons (Gordon and Jukes, 1964) and between neurons of the nucleus interpositus of the cerebellum and the nucleus reticularis tegmenti pontis (Tsukahara et al., 1973). 2. lnterneurons At present we are far from being able to depict completely the neural circuit that mediates the recurrent facilitation of NS neurons. However, Yagi and Sawaki (1975a,b) and Sawaki and Yagi (1976) recently examined several kinds of drugs, each of which is known to block specifically synaptic transmission mediated by a particular transmitter substance. After an intravenous injection of picrotoxin recurrent inhibition disappeared and recurrent facilitation appeared in some tuberoinfundibular neurons of rats anesthetized with urethan, paralyzed with gallamine triethiodide, and respirated artificially (Fig. 12A). Some of the identified tuberoinfundibular units sampled after an intravenous in-


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jection of strychnine also displayed the facilitatory response to antidromic stimulation of the median eminence (Fig. 12B), and the percentage of the units that showed the facilitatory response was not significantly different from that observed in control animals. It is supposed that synaptic transmission by the nicotinic action of ACh and/or glycine is not involved in mediation of recurrent facilitation, since gal-

Time after stimulus (msec) FIG. 12. Effects of intravenously injected picrotoxin (PTX) and strychnine (STR). (A) A unit that showed recurrent facilitation only after a picrotoxin injection. Note that the facilitatory period does not coincide with the inhibitory period observed before the injection. (B) A unit sampled after an intravenous injection of strychnine exhibited a response to antidromic stimulation in rats and that previously injected with picrotoxin.

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lamine is known to block the nicotinic action of ACh. Recurrent inhibition reduced or almost canceled the facilitatory response (Fig. 11). However, these investigators found that the time of appearance of facilitatory response observed after a picrotoxin injection did not always coincide with that of the recurrent inhibition (Fig. 12A). It is therefore very likely that recurrent facilitatory pathways are depressed by GABA neurons at some other site of action as well as at the site of action involved in recurrent inhibition (Fig. 9). Intravenously injected a-methyl-p-tyrosine (a-MPT), which is known to inhibit catecholamine biosynthesis, has been found to reduce markedly or block recurrent facilitation in the units that had shown it before the injection (Fig. 13A; Yagi and Sawaki, 1975b). In rats pretreated with intraperitoneally injected a-MPT none of the 9 tuberoinfundibular units showed recurrent facilitation after picrotoxin (Fig. 13B). However, 8 of the 11 units tested after picrotoxin were facilitated following antidromic stimulation of the median eminence in rats pretreated with intraperitoneal L-tyrosine (Fig. 13C and D).


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FIG.14. A characteristic field potential consisting o f a small positive wave followed by a negative wave, which was induced by antidromic stimulation of the median eminence after a picrotoxin injection. (A) A unit that showed the field potential in response to antidromic stimulation with a rather constant latency. A dotted line indicates that the latency of the positive wave of the field potential is constant after single-pulse stimuli. Stimulus intensity was just above threshold for an antidromic spike, except for the lowermost record obtained with subthreshold stimulus for an antidromic spike. The expanded sweep in each record displays the antidromic spike. Upward deflections are positive. Note that bursting discharges occasionally occur in coincidence with the negative wave component. (B) A poststimulus histogram obtained from the same unit as in (A). The latency of the facilitatory response to antidromic stimulation is approximately equal to the latency of the field potential induced by antidromic stimulation (Sawaki and Yagi, 1976).

Antidromic stimulation of the median eminence was found to induce a characteristic field potential consisting of a small positive wave followed by a negative wave after an intravenous picrotoxin injection (Fig. 14). Convulsive discharges were very frequently found to occur in coincidence with the negative wave of the evoked field potential. Subthreshold stimuli for an antidromic spike are also effective in producing a field potential, as shown in the lowermost record of Fig. 14A. These results clearly indicate that the negative wave reflects excita-

178


tory postsynaptic activation of the tuberoinfundibular neuron, and that the preceding positive wave is derived from presynaptic volleys converging on the cell. The latency of the negative wave was found to be much longer than that of an antidromic spike and approximately the same as that of the facilitatory response to antidromic stimulation observed in poststimulus histograms obtained simultaneously in each unit (Fig. 14). It is therefore suggested that the recurrent facilitation of tuberoinfundibular neurons is not a kind of disinhibition but is mediated by the excitatory presynaptic volleys converging on a particular NS cell.

3. Physiological Significance Yagi and Sawaki (1975b)and Sawaki and Yagi (1976) described the recurrent neural pathways converging on a particular tuberoinfundibular neuron that mediate a facilitation, involve catecholaminergic neurons in the mediation, and are normally in a depressed state as a result of the action of GABA-releasing neurons. If GABA-releasing neurons were inhibited by physiological causes, this neural mechanism would be disinhibited and would probably function as a reverberating neural circuit. (Tsukahara et al., 1973). On the basis of the facts that, first, an elevated plasma estrogen level is a prerequisite for a surge of lutenizing hormone (LH) to occur on the day of proestrus in cyclic female rats, second, plasma estrogen produces a stimulatory feed-back action on the so-called ovulation center (see Yagi and Sawaki, 1973) and, third, catecholaniinergic neurons are involved in the neural mechanism that induces an ovulatory surge of LH (Yagi and Sawaki, 1975b) speculated on the possible physiological significance of recurrent facilitation suggesting that the recurrent facilitatory pathways of tuberoinfundibular NS neurons may be the neural mechanism essential for producing the surge of luteinizing hormone releasing factor that eventually induces ovulation. Many more studies are required for the physiological significance of recurrent facilitation to be elucidated.

VII. Conclusions In electrophysiological studies of NS neurons it is extremely important to identify definitively a cell under study as a NS cell either morphologically and/or electrophysiologically. All the identified NS neurons so far examined in invertebrate and vertebrate species have been proved to generate and conduct action potentials. Ionic mechanisms for resting and action membrane potentials have


179

been studied mostly in invertebrate NS neurons. Resting potentials depend predominantly on potassium ions in the extracellular fluid. Action potentials in the NS cell soma and axon terminal are generated by the action of both activating systems for sodium and calcium ions, while only a sodium-activating system works for the NS axon in developing conducting action potentials. The duration of action potentials recorded from the soma and the axon terminal of NS neurons has been found to be longer than that of non-NS neurons. It was postulated that their long duration is attributable to the calcium-activating system of the membrane. Some NS neurons periodically generate characteristic bursting discharges. The bursting discharges in invertebrate NS neurons appear to depend on endogenous pacemaking activity. The resting potential of NS cells showing bursting discharges oscillates as a result of the characteristic membrane properties of relatively large permeability to sodium ions and cyclic fluctuations in potassium permeability. These NS neurons exhibit circadian and seasonal variations in endogenous pacemaking activity. Neurosecretory neurons in the mammalian hypothalamus also display periodic bursting discharges. The underlying mechanism, however, remains uncertain. In vertebrate NS neurons the antidromic unit spike dissociates characteristically into A and B spike components, and the large B component is occasionally abolished. The rate of neurohormone synthesis in the NS neuron soma has been shown to vary in response to various physiological stimuli causing neurohormone release. Although direct evidence has not been reported for the hypothesis that action potentials control the rate of neurohormone synthesis, the possibility has been pointed out that intracellular calcium brought about by a calcium influx during an action potential of the soma may control this rate. Studies on responses of mammalian neurohypophysial NS neurons to the physiological stimuli that cause neurohypophysial hormone release produced the following three interesting observations. (1) Osmoreceptor cells are distinct from supraoptic NS neurons; (2) many of the identified NS neurons generate periodic bursting discharges; and (3)oxytocin is produced by NS neurons of supraoptic as well as paraventricular neuclei. The relationship between action potentials and axonal transport of neurohormone and of binding proteins is not clear as yet. However, in consideration of evidence reported on non-NS axons it seems probable that action potentials conducted along NS axons do not accelerate the velocity but increase the amount of NS material transported per unit of time. A number of studies have depicted the mechanism of excitation-se-

180

KINJI YACI AND SHIZUKO IWASAKI

cretion coupling as follows. Depolarization of NS axon terminals, which is produced physiologically by action potentials conducted along the NS axons, induces an increase in calcium permeability of the terminal membrane, and as a result causes a calcium influx along the electrochemical potential gradient. Intracellular calcium ions in turn evoke the exocytotic release of neurohormones and binding proteins. Studies employing both techniques of antidromic identification and iontophoretic drug application have provided suggestive evidence for a particular substance to be a neurotransmitter which directly controls the activity of NS neurons in the mammalian hypothalamus. However, none of these putative neurotransmitter substances has been definitively identified at present as the neurotransmitter that mediates synaptic transmission of neural inputs from presynaptic pathways of identified origin. Recurrent inhibition of NS neurons has been demonstrated in neurohypophysial and tuberoinfundibular NS neurons. It seems likely that at least one interneuron is intercalated in the recurrent inhibitory pathways. In the tuberoinfundibular system GABA-releasing neurons have been suggested to be involved in recurrent inhibition. In this system recurrent reciprocal inhibition between distinct NS neuron pools with different functions was postulated to be the physiological significance. I n neurohypophysial NS neurons it has been claimed that negative feedback action of recurrent inhibition produces a periodicity in electrical activity. Recurrent facilitation has been found in frog neurohypophysial NS neurons and rat tuberoinfundibular NS neurons. In the latter it was suggested that the neural pathways mediating recurrent facilitation are normally depressed by the action of GABA-releasing neurons and that catecholaminergic neurons are involved in the mediation. The physiological significance of recurrent facilitation of tuberoinfundibular NS neurons has been discussed in relation to the surge of luteinizing hormone at ovulation. ACKNOWLEDGMENTS The authors are very grateful to Miss Yukiko Sawaki for her assistance in preparing the manuscript and to Dr. Seiji Ozawa for critical reading of and valuable comments on the manuscript. This work was supported by the grants from the Ministry of Education of Japan.

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Reparative Processes in Mammalian Wound Healing: The Role of Contractile Phenomena GIULIO GABBIANI Depurtitaent of Puthology, Medical Sclaool, Universitcl of Geneva, Geneva, Switzerland AND

DENYSMONTANDON Departnaent of Surgery, HGpital Caritonul, Geneva, Switzerland

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11. Th e Evolution of a Wound

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I. Introduction

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A. Heniostasis and Inflammation B. Granulation Tissue Formation . . . . . . . . 111. Epithelialization of a Wound . IV. Pathology of Granulation Tissue and Fibromatoses V. Conclusions . . . . . . . . References. . . . . . . . .

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I. Introduction Wound formation and repair are pathological phenomena which can b e traced far back in human history (Majno, 1975). They have been one of the major concerns of medicine in the past and still have an important place in the praxis of general surgery and in the management of traumatic and vascular diseases. The purpose of this article is not to review the entire field of wound healing but rather to summarize the major advances in the understanding of several aspects of reparative processes made in the last few years. Humans, as well as other vertebrates, do not possess a great power of cell or organ regeneration when compared with lower species, and repair of a loss of tissue is essentially made by the synthesis of connective tissue which abrogates the volume discontinuities and keeps together the margins of the remaining parenchymas. Only some tissues, such as epidermis, are capable of regenerating over the scar and reconstituting the continuity lost with the wound. In general (Van den Brenk, 1956; Ross, 1968a), the healing of a wound consists of two interconnected phenomena: (1) synthesis and/or regeneration of tissues which replace the damaged ones, and (2)remodeling of these tissues to restore the form of the body. The rel187

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GIULIO GABBIANI AND DENYS MONTANDON

ative importance of these phenomena may vary. In cutaneous linear wounds or in wounds of areas firmly adherent to deeper planes, the reduction in spatial discontinuity is d u e to the local growth of granulation tissue and epithelium, whereas in large wounds of mobile areas of the skin, or in ischemic lesions of the myocardium, the reduction in spatial discontinuity is mostly due to wound contraction. The reparative events are generally preceded by hemostatic and inflammatory phenomena which may in turn influence the final result of wound healing. 11. The Evolution of a Wound

The body’s reaction to the formation of a wound is now well known. The damaged area is invaded by plasma components (e.g., fibrin) and circulating blood cells (e.g., neutrophils, monocytes) which constitute a passive and active barrier against the invasion of foreign material. When an efficient defensive reaction has been organized, the reparative phenomena start with the synthesis of new connective tissue (Ross, 1968a) (Fig. 1). A. HEMOSTASISAN11 INFLAMMATION Among the early effects of a traumatic agent is hemorrhage with accumulation of fibrin; this is generally the first structure connecting the margins of a wound. Hemostasis may have an important influence on healing. It has been shown that during platelet aggregation and release one or several factors are released into serum, which are responsible for the proliferation of cultivated smooth muscle cells and fibroblasts (Ross et al., 1974). During the early phases of wound healing, inflammatory phenomena are constantly present. Neutrophils are numerous, and it was a common notion that marked exudation of neutrophils is a prerequisite for rapid fibroplasia (Selye, 1953). However, recent experiments showed that, in guinea pigs made neutropenic after injections of antineutrophil serum, no other cells were affected by the antiserum and the healing of a wound was normal (Simpson and Ross, 1972). It was concluded that, although neutrophils appear to be important for the phagocytosis of bacteria, they do not play any other role during wound repair. Similar results were obtained in decomplemented animals (Wahl et al., 1974). Macrophages appear in wounds shortly after neutrophils and last for a longer time (Ross, 1968a).Their precursors are blood monocytes and local histiocytes (Volkman and Gowans, 1965). The role of macro-

CONTRACTILE PHENOMENA IN WOUND HEALING

189

fibrin

APMN leukocyte\ ,

lymphocyte---

'. macrophages

fibroblasts

3~c01pl

capillaries-

2

---A'

I

'0

2

4

6

0

10

1213

Days

FIG. 1. Quantification of the elements of a healing wound in a normal guinea pig determined from 0 to 13 days. The various cellular and acellular elements are graded on a 0 to 3 basis, using the number of cells per high-power field as the basis of grading. These observations werc made on wounds prepared for routine light microscopy and stained with hematoxylin and eosin, Van Gieson stain, phosphotungstic acid-hematoxylin, and Wilder's reticulin stain. (From Ross and Benditt, 1961, p. 679.)

phages is to phagocytose and digest bacteria as well as cell debris present in the wound. When animals are made monocytopenic by means of antimonocytic antiserum or corticosteroids, wound healing is delayed, particularly if antimonocytic serum is injected locally into the wound to prevent the few monocytes present from exerting their

190


phagocytic power (Ross, 1974).The delay of wound healing, in the absence of macrophages, may be due in part to the lack of production by these cells of a factor stimulating the synthetic activity of fibroblasts. It has been shown that, after phagocytosis of silica, peritoneal macrophages produce a substance stimulating collagen formation in cultivated chick fibroblasts (Heppleston and Styles, 1967).

TISSUEFORMATION B. GRANULATION After inflammatory phenomena occur, the area of the wound is gradually invaded by fibroblasts which become the most important cellular element until healing is complete. Fibroblasts are responsible for the synthesis of new connective tissue (Ross, 1968a) and, as we shall see, for the phenomenon of wound contraction. Fibroblasts of granulation tissue differ from normal fibroblasts in many aspects. We now review the characteristics of normal and granulation tissue fibroblasts.

1. The Normal Fibroblast The fibroblast (Ross, 196813) was first identified b y means of light microscopy on the basis of its shape and its relationship with the extracellular substance. The use of electron microscopy has allowed a better definition of the cytological characteristics of fibroblasts (Fig. 2). The nucleus is generally large and contains one or more nucleoli. This is typical of active synthesis. RNA is synthesized in the nucleus and then generally transformed in the cytoplasm. The nucleolus participates in the synthesis and/or the transformation of RNA (Perry et al., 1961).The most prominent cytoplasmic organelle is rough endoplasmic reticulum which consists of a series of interconnected sacklike or tubular structures present throughout the cytoplasm (Fig. 2). Prominent rough endoplasmic reticulum is again a characteristic feature of active protein synthesis. The content of the cisternae is relatively dense and sometimes finely filamentous (Ross and Benditt, 1961; Movat and Fernando, 1962). Ribosomes form large aggregates on the membranes (Palade, 1958; Ross, 1968b). They are often arranged in double rows, taking the form of curves or spirals. Some aggregates of ribosomes are also found free within the cytoplasm. The Golgi apparatus is generally prominent and has no particular location (Ross, 1968b). It consists of stacks of flattened lamellae and vesicles. As in other types of cells, it has been related to polysaccharide synthesis (Ross, 196813; Neutra and Leblond, 1966). It has been shown that proteins synthesized in the rough endoplasmic reticulum are collected in the Golgi apparatus before excretion (Caro and Palade,


191

FIG.2. Normal fibroblast from rat subcutaneous tissue. Note mitochondria, small peripheral vesicles, and regular arrangement of the rough endoplasniic reticulnm. x 16,400.(From Gabbiani et al., 1972a.)

192


1964). This seems to be true also for collagen synthesized by the fibroblasts (Ross and Benditt, 1965).The Golgi apparatus has also been implicated in cytoplasmic membrane synthesis (Goldberg and Green, 1964). Peripheral vesicles and vacuoles may be related to the process of pinocytosis or phagocytosis, or to the excretion of cell products. Abundant mitochondria are present throughout the cytoplasm. They have regular cristae and a relatively dense matrix. A few cytoplasmic microfilaments (40-70 A in diameter) may be seen in fibroblasts of adult animals or humans, particularly, close to the plasmalemma (FittonJackson, 1968; Ross and Benditt, 1961). Microfilaments are more common in cultivated (Goldberg and Green, 1964), embryonic, and fetal fibroblasts (Greenle and Ross, 1967). Other rare components of fibroblast cytoplasm are centrioles, cilia, multivesicular bodies, and lipid droplets (particularly in old fibroblasts). In normal tissues of adult animals, there are no contacts between fibroblasts. However, contacts can be seen between cultivated (Davis and James, 1964; Goldberg and Green, 1964), embryonic, and fetal fibroblasts, as well as between fibroblasts of pewborn animals (Trelstad et al., 1970; Ross and Greenle, 1966; Greenle and Ross, 1967). These contacts most commonly take the form of tight junctions.

2. Collagen, Glycoproteins, and Proteoglycans of Normal Connective Tissue It is now accepted that fibroblasts are the source of collagen fibers (Grant and Prockop, 1972). Ribosomes synthesize all the polypeptide chains of collagen. The polypeptide chains coded for by mRNAs do not contain hydroxyproline or hydroxylysine, and they are correspondingly rich in proline and lysine. The hydroxylysine and hydroxyproline found in collagen are synthesized by the hydroxylation of lysine and proline after these amino acids have been incorporated into peptide linkages. The enzymes that synthesize hydroxyproline (Holme et al., 1970; Pankalainen et al., 1970; Rhoads and Udenfriend, 1970) and hydroxylysine (Prockop et al., 1966; Miller, 1971; Popenoe and Aronson, 1972) in collagen have been characterized. Completion of hydroxylation is not essential for the chains to be released (Juva and Prockop, 1966). The transport form of collagen (procollagen) contains extensions at the NH, terminal of the chain (Layman et al., 1971; Lenaers et al., 1971; Stark et al., 1971). The NH2 terminal extensions may facilitate the triple-helical structure of collagen, but more probably facilitate


193

the transport of the collagen molecule during the initial stages of biosynthesis by keeping it more soluble under physiological conditions (Layman et al., 1971; Stark et aZ., 1971; Dehm et al., 1972). Generally, it is agreed that the NH2 terminal extensions are cleaved off after the molecule is secreted, although there may be some exceptions (Grant and Prockop, 1972). The mechanism of collagen secretion involves the transfer of procollagen from the rough endoplasmic reticulum to the Golgi complex. The procollagen is then transported in vesicles directly to the extracellular space (Revel and Hay, 1963) by a mechanism involving microtubules (Ehrlich and Bornstein, 1972; Dehm and Prockop, 1972) and possibly microfilaments ( Bornstein and Ehrlich, 1973). Here procollagen peptidase transforms procollagen into collagen (Jimenez et d.,1971; Layman et d.,1971; Bellamy and Bornstein, 1971; Lapikre et al., 1971). Alternative possibilities are: (1) fusion of Golgi vacuoles with vacuoles containing procollagen peptidase, with intracellular transformation of procollagen into collagen (Bornstein and Ehrlich, 1973), and (2) direct intermittent communication of cisternae of endoplasmic reticulum with the extracellular space (Ross and Benditt, 1965). In the extracellular tissue, collagen has a typical periodic structure of about 640 A. Similar fibers can be reconstituted in uitro from soluble fractions (Gross et al., 1955; Jackson and Fessler, 1955). The term tropocollagen was introduced to designate the precursor of collagen fibers, characterized by electron microscopy (Gross et al., 1954). The native collagen molecule is composed of three polypeptide chains (a chains), each of approximately 100,000 molecular weight, organized in a right-handed triple helix ( Fitton-Jackson, 1968).The periodic structure arises from linear arrays of monomers in which the head end of a monomer is associated with the tail end of the next. In order to explain the banding, it has been proposed that adjacent macromolecules are displayed laterally with respect to each other at a distance of one-fourth the length of the tropocollagen monomer (Schmitt et d.,1955). This hypothesis of “quarter-stagger” has been modified successively by proposing that the length of the monomer is equivalent to 4.4 periods, and that the overlap region is only 0.4 of a period, followed by a gap of 0.6. These linear arrays would each extend over 5 periods (Hodge et d.,1965). Alternatively, it has been suggested that there are specific bonding regions, each located one-fifth of the distance along the length of the tropocollagen monomer (Grant et al., 1965; Cox et al., 1967). Such regions, by bonding in a side-to-side fashion, produce the fibrillar banding. The aggregation of a tropocollagen monomer would be essentially a random process, since a bonding zone of a monomer can cross-link with any of the bonding zones in an adjacent monomer.

194


A recent advance in the knowledge of collagen biology has been the discovery of its molecular heterogeneity (Trelstad, 1973, 1974a; Hay, 1973). Thus far, four different species of collagen have been recognized, which depend on at least five structural gene products. Type I collagen was isolated from several tissues (e.g., skin, tendon, bone) (Traub and Piez, 1971). It is composed of two different molecules called a-1 and a-2, which have a molar ratio of 2: 1. The molecule is designated [a-l(I)], a-2. Type I1 collagen was isolated from cartilage and consists of three identical a chains which elute on carboxymethyl cellulose close to a-l(I ) and have been called a-l(11) (Miller and Matukas, 1969; Miller, 1971; Trelstad et ul., 1970). Hence the molecule is designated [a-l(11)13.Type I11 collagen was isolated from embryonic skin, aorta, and leiomyomas (Chung and Miller, 1974; Epstein, 1974). It is composed of three a-I(111)chains, and the molecule is designated [a-l(III)I3.On the basis of the tissues containing type I11 collagen, it has been assumed that the producing cells are smooth muscle cells. Type IV collagen was isolated from basal laminae of different tissues (Kefalides, 1971; Trelstad, 1974b). It is composed of three identical chains of the type a-l(IV), and the molecule is designated [a-1(IV)I3. The biological significance of these different types of collagen is not known, but it might be supposed that they correspond to different specific functions. Extracellular collagen is generally insoluble. However, there are situations in which important amounts of insoluble collagen are degraded, such as during postpartum involution of the uterus. Also, onethird of newly synthetized collagen is degraded before being transformed into the insoluble fonn (Kivirikko, 1971). The mechanism of collagen degradation has not yet been clarified. Gross and Lapikre (1962) found a collagenase in the resorbing tadpole tail, and a similar enzyme has been described in several normal and pathological tissues. However, it has not been established whether this enzyme always has an important physiological role i n uiuo. Lysosomal cathepsins have also been shown to depolymerize collagen at low pH (Milsom et al., 1973). Possibly, depolymerizing enzymes cleave the fibers into short segments which are phagocytosed and further digested by lysosomic enzymes into tropocollagen. These can be further attacked b y specific collagenases and then by peptidases. Glycoproteins and proteoglycans are, with collagen and elastin, major components of the extracellular connective tissue. The specific structure and function of different connective tissues probably depends on the proportions of these components and on a particular pattern of glucosaminoglycans (Muir, 1964). Glycoproteins and proteo-


---

195

COLLAGEN TO COLLAGEN INTERACTION

I'

//

Tropocollogen

-

----rrI-nL

Approgalton

Slabilizolion

COLLAGEN TO GP 8 PG INTERACTION

Polymeric collagen

Glycoprolein

Pro1eoplyCm

FIG.3. Schematic drawing of L.ollagen-to-collagen and collagen-to-glycoprotein-toproteoglycan interactions. (From Jackson, 1974.)

glycans have similar structures (Jackson, 1974) consisting of a protein core to which are attached carbohydrate side chains. The side chains of glycoproteins are short oligosaccharides which are straight or branched. The side chains of proteoglycans are found in doublets separated by long stretches of polypeptide chains (Mathews, 1970; Jackson and Bentley, 1968; Jackson, 1972). Electron microscope studies of connective tissue, using ruthenium red or bismuth nitrate to stain proteoglycans, show a definite orientation of these substances and a close relationship with collagen with a periodicity characteristic of the collagen fibers (Jackson, 1974). It appears that most connective tissues contain glycoproteins (Bowes et ul., 1955). Some are involved in interaction with proteoglycans, and others are closely associated with collagen and are referred to as structural glycoproteins. When proteoglycans and glycoproteins are removed from tissues such as tendon and skin (Steven and Jackson, 1967), electron microscopy shows that the collagen fibers are markedly separated compared to the compact fibril arrangement found in the original tissue. Probably, the proteoglycans and/or glycoproteins represent the cement substances holding together collagen fibers (Fig. 3 ) .

3. The Fibroblusts of Grunulution Tissue (Myofibroblusts) During the evolution of granulation tissue in experimental animals and in humans, fibroblasts acquire ultrastructural, chemical, immunological, and functional characteristics that clearly distinguish them

196


FIG.4. Myofibroblast from a croton oil-induced 21-day-old granuloma pouch in a rat. A large part of the cytoplasm contains bundles of densely packed filaments with attachment sites typical of smooth muscle. Note the nuclear folds and indentations. There is still abundant endoplasmic reticulum recalling that of a normal fibroblast. Extracellular tissue consists of a few collagen fibers, microfibrils, and dense homogeneous material. x 13,000.


197

from fibroblasts of normal tissues (Gabbiani et al., 1972a; Ryan et al., 1974). We describe these changes briefly. a. Morplaology. A fibrillar system develops within the cytoplasm (Gabbiani et al., 1971), not the few fibrils seen in normal fibroblasts but bundles of parallel fibrils resembling those of smooth muscle cells (Fig. 4). Individual fibrils measure 40-80 A in diameter, more rarely 100-120 A, and are usually arranged parallel to the long axis of the cell. Many electron-opaque areas are scattered among the bundles or located beneath the plasmalemma. These are similar to the attachment sites of smooth muscle. Although these fibrillar structures often occupy a large portion of the cell, the remaining cytoplasm contains packed cisternae of rough endoplasmic reticulum typical of normal fibroblasts (Fig. 4). The nuclei consistently show multiple indentations or deep folds (Fig. 4), an appearance quite unlike that of normal fibroblasts (or other cells in the same granulation tissues such as macrophages or mast cells). There are numerous intercellular connections between granulation tissue fibroblasts. Their stnicture identifies them as tight junctions and more often gap junctions (Fig. 54. In addition, part of the cell surface is often covered by a well-defined layer of material having the structural features of a basal lamina and generally separated from the cell membrane b y a translucent layer. Where it is covered by a basal lamina, the cell often shows dense zones in the fibrillar bundles immediately beneath the surface membrane. The resulting complex is reminiscent of hemidesmosomes which bind endothelial cells, pericytes, and smooth muscle cells to their basal laminae (Fig. 5b).

FIG.5. Cell-to-cell and cell-to-stroma connections of myofibroblasts. (a) A typical gap junction between two myofibrohlasts in the granulation tissue of a human healing wound. x 107,000. (11) Basal lamina parallel to the cell membrane in a myofibroblast from an experimental wound in a rat. Note the bundles of intracytoplasmic fibrils with condensation immediately beneath the cell membrane (arrows). x 82,000.

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FIG.6. Contraction of a strip of tissue from a 21-day-old rat granuloma pouch in gm/ml). One diviresponse to successive increasing doses of 5-HT (1 x lo-' to 1 x sion on the scale represents 1 minute. (From Gabbiani et al., 1972a.)

b. Pharmacology. Strips of granulation tissue from animals or humans, placed in a pharmacological bath, behave like smooth muscle in that they are contracted or relaxed by substances that contract or relax smooth muscle (Majno et al., 1971; Ryan et al., 1973, 1974). Among the substances most active in inducing contraction are 5hydroxytryptamine (5-HT or serotonin) (Fig. 6), angiotensin, vasopressin, norepinephrine, bradykinin (Fig. 7), epinephrine, and prostaglandin Fla (Fig. 8). Among the most active relaxing agents are papaverine and prostaglandins E l and E2 (Fig. 8) (Gabbiani et d., 1972a; Ryan et al., 1974).Agents without effect are histamine, acetylcholine, tryptophan, histidine, and barium chloride. The reactivity of the tissue depends on the age of the granulation tissue. For example, in a granuloma pouch induced in the rat by croton oil, 5-HT has no clear-cut effect at 7 days. The first definite response is registered at 8 days, and the maximal response by 15-20 days. Thereafter, the reactivity stays at this plateau for 4-5 weeks. Contractions, although somewhat smaller, are still obtained with strips from SO-day-old pouches. The pharmacological response of granulation tissue strips to smooth muscle-contracting agents is influenced by several factors. Anoxia partially inhibits the response to 5-HT. Specific 5-HT antagonists (e.g.,

BRADWNIN

PAPAVERINE

FIG. 7. Granulation tissue strip from an 11-day-old rat wound. Contraction in response to bradykinin (1 x gm/ml), followed by relaxation due to papaverine gm/ml). (From Gabbiani et a l . , 1973b.) (1 x


A

199

1

1

0

2 0 3 0 Time (min)

4

0

5

0

FIG. 8. Contraction shown i n tjitro by strips of human granulation tissue in gm/ml), folto 1 x response to increasing doses of prostaglandin F l a (1 x gm/ml). (From Ryan e t lowed by relaxation in response to prostaglandin E l (1 x al., 1974.)

methysergide or cyproheptadine, which is both a n t i 9 H T and antihistamine) completely inhibit the contraction produced b y this drug but not the contraction due to angiotensin or vasopressin. Cytochalasin B causes, b y itself, a slight relaxation of the strip and inhibits contraction due to 5-HT or angiotensin. Potassium cyanide relaxes strips of granuloma pouch untreated or contracted by 5-HT. If potassium cyanide is supplied before 5-HT or vasopressin, these drugs have no effect. c. Chemistry. The yield of actomyosin obtained by extraction from a croton oil-induced granuloma pouch (4.0 mg of actomyosin per gram wet weight of pouch tissue) is comparable to that obtained with identically prepared extracts of pregnant rat uteri (3.5 mg per gram wet weight) (Majno et al., 1971).The calcium-activated adenosine triphosphatase activity of these extracts is similar, splitting approximately 10 nmoles of adenosine triphosphate per milligram of protein per minute. d. Immunology. Granulation tissue fibroblasts gradually develop intracellular neoantigens which are similar to those present in smooth muscle cells (Hirschel et ul., 1971).This was shown by the selective fixation, on myofibroblasts, of smooth muscle autoantibodies from patients with chronic aggressive hepatitis (Fig. 9). In order to precise the nature of the antigens against which these antibodies are directed, the smooth muscle autoantibodies were incubated with different contractile proteins. Their binding to smooth muscle cells and to myofibroblasts was abolished only after incubation with purified actin from platelets (thrombosthenin A) (Gabbiani et ul., 1973a), or from skeletal

200


FIG.9. Immunofluorescent staining of actin in frozen sections of granulation tissue. x 400. (a) Wall of a 14-day-old turpentine-induced granuloma in a rat, incubated first with normal huinan seniin followed by fluorescein-conjugated goat antihuman IgG. N o stain. (b) Adjacent section incubated first with AAA seruni followed by flnoresceinconjugated goat antihuman IgG. Intense labeling is localized in myofibroblasts. (c) Adjacent section incubated first with AAA previously absorbed with skeletal muscle actin followed by fluorescein-conjugated antihuman IgC. N o stain. (From Gabbiani et ol., 1976.)

muscle (Gabbiani et al., 1975a). This indicated that smooth muscle autoantibodies are antiactin autoantibodies and that myofibroblasts (contrary to normal fibroblasts) contain important amounts of actin. The presence of this contractile protein, as determined by immunofluorescence, correlates well with the development of a microfilamentous apparatus as seen on electron microscope examination (Gabbiani et al., 1971). When granulation tissue disappears after the healing of a wound, no more fixation of antiactin antibodies to fibroblasts is observed. Following the pioneer work of Carrel, it is now widely accepted that the forces producing wound contraction reside in the granulation tissue that fills the wound. The nature of these forces, however, has not been clearly defined. The development of new features in fibroblasts of granulation tissue has led to the suggestion that, at least in part, the characteristic contraction of granulation tissue depends ultimately on the contraction of these modified fibroblasts or myofibroblasts (Gabbiani et al., 1972a).It remains to be seen whether or not the morphological and functional features of myofibroblasts are compatible with their proposed histogenetic origin from fibroblasts. There has been some debate about the origin of granulation tissue fibroblasts. Conheim suggested in 1867 that cells from the blood may be transformed into fibroblasts, and this coiicept has been revived several times (Maximov, 1927; Allgower and Hulliger, 1960; Petrakis et al., 1961), in contrast to the opinion of a local origin (Grillo, 1963; Glucksmann, 1964).For healing wounds, the idea that new fibroblasts derive

CONTRACTILE PHENOMENA I N WOUND HEALING

201

from emigrated blood cells was refuted by Ross et al. (1970), who showed in parabiotic rats that blood cells labeled with tritiated thymidine do not transform into granulation tissue fibroblasts. Earlier results in which diffusion chambers filled with buffy coat cells were later found to contain fibroblasts and collagen (Petrakis et al., 1961) were probably due to contamination with a small number of connective tissue cells (picked up by during cardiac puncture or venipuncture). Ross and Lilywhite (1965) showed that, when buffy coat cells were obtained from blood collected by arterial or venous cannulation, no collagen formation occurred in the diffusion chambers. Thus it is likely that most, perhaps all, granulation tissue fibroblasts develop from local fibroblasts or possibly from less differentiated mesenchymal cells among which pericytes appear to be likely candidates. The development of new vessels in granulation tissue begins in the endothelial cells of small vessels (Schoefl and Majno, 1964). These form numerous new capillary buds which become canalized and anastomosed. The newly formed vessels start to disappear during the period of wound contraction, and finally the scar becomes slightly vascularized. Granulation tissue vessels have a high degree of p m e a b i l ity, because interendothelial junctions are generally not complete. A peculiarity of the endothelial cells of granulation tissue vessels is their phagocytic capacity which disappears as soon as the wound has . the healed (Hurley et al., 1970; Gabbiani et al., 1 9 7 2 ~ )Although majority of the cells in experimental or human granulation tissues are myofibroblasts, it may be argued that these cells have derived from smooth muscle, for example, of local blood vessels. This appears unlikely, because it implies that the commonest connective tissue cell, the fibroblast, takes little part in the formation of granulation tissue. Moreover, myofibroblasts can develop in the avascular fibrous tissue that forms around blood clots implanted in the rat peritoneal cavity (Ryan et al., 1973). It is also known that fibroblasts cultivated in vitro normally develop an extensive cytoplasmic fibrillar system (Goldberg and Green, 1964) and interconnections (Devis and James, 1964). Contractile proteins can be isolated from these cultivated cells (Bray and Thomas, 1975; Adelstein et al., 1972) or stained b y means of immunofluorescence (Fig. 10) (Gabbiani et al., 1973b; Trenchev et a1., 1974; Lazarides and Weber, 1974; Weber and Groeschel-Stewart, 1974; Peinter et al., 1975); in preliminary studies on cultivated fibroblasts obtained from normal rat dermis, we observed that the addition of 5-HT to the culture medium caused cellular contraction within 15-20 minutes, whereas tryptophan had no effect under the same conditions (Majno et al., 1971).

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FIG.10. Iinmunofluorescent staining with AAA of cultured fibroblast. (a) The cells were incubated with AAA serum followed by fluorescent antihuman IgG. Intense labeling in the form of long lines is visible in several cells. (b) The same area photographed with phase-contrast shows that the intensely fluorescent lines correspond to stress fibers. x 400.

There are some differences between the pharmacological reactivity of granulation tissue strips and that of classical smooth muscle preparations. For example, granulation tissue strips fail to react to barium chloride and acetylcholine, agents that normally cause contraction of smooth muscle. Furthermore, the pattern of response is somewhat different in that the peak of contraction is reached more slowly and maintained longer b y the granulation tissue strips. In some instances (e.g., after stimulation by 5-HT), the contractions remain stable at the peak for more than 2 hours. This “spastic” behavior suggests the presence of a contractile system similar to that of the catch muscles of invertebrates (Ruegg, 1971) and falls well into place with the biological process of wound contraction which is relatively slow but continuous. There also appear to be differences in the reactivity of granulation tissues from different sources, as granuloma pouch strips are sensitive to 5-HT, whereas wound strips do not respond to this agent under the same conditions of testing. All these data suggest that fibroblasts of granulation tissue progres-


203

sively develop intracytoplasmic “muscles” as well as cell-to-cell and cell-to-stroma connections. These myofibroblasts contract (either spontaneously or in response to endogenous mediators) at the same time that collagen is laid down (thus providing a “lock-step” system), and so the whole tissue shrinks; in other words, granulation tissue becomes a contractile organ (Fig. l l ) .This of course is of primary importance in closing a skin wound but, under other circumstances, such as postburn disfigurements and postinflammatory luminal strictures, it can be disastrous. It is also likely that the valvular deformity of chronic rheumatic heart disease is due to a similar process (Ryan e t ul,, 1973). This is a particularly interesting example, because it illustrates how apparently logical was the old belief (now outmoded) that attributed connective tissue contraction to collagen shrinkage. Normal valves and chordae consist of mature collagen and little else, and so d o chronically deformed valves and chordae. However, pathologists d o not often see the stage in between, that is, when there is involvement of these structures by active granulation tissue. Some normal fibrous tissues, such as the splenic capsule and the tunica albuginea of the testis, contract in vitro and in vivo. This property has, however, been correlated with the presence of smooth muscle cells (Bloom and Fawcett, 1962; Davis and Langford, 1970). Cells that are similar to myofibroblasts have been seen in certain other human and animal tissues: intima of chicken aorta (Moss and Benditt, 1970), rat ovary (O’Shea, 1970),interstitial cells ofnormal rat and human lungs (Kapanci et al., 1974), aortic intimal thickening (Geer, 1965), ganglia of the wrist (Ghadially and Metha, 1971), and cirrhosis of the liver (Bhathal, 1972; I r k et al., 1974). This shows that myofibroblasts may develop in different situations and participate in several physiological and pathological phenomena. Smooth muscle cells with fibroblastic features have been described in the uterus of rats treated with estrogens (Ross and Klebanoff, 1967), and in human and experimental arteriosclerotic lesions (Thomas et ul., 1963; Parker and Odland, 1966; Ross and Glomset, 1973). Furthermore, there is evidence that smooth muscle can produce collagen and elastin (Ross, 1968a).These data indicate that smooth muscle cells can assume morphological and functional characteristics of fibroblasts. The reverse process is also possible, that is, that fibroblasts can become modified into smooth-musclelike cells with a contractile capacity. Hence it appears more and more obvious that fibroblasts and smooth muscle cells are much more closely related than classic histology would have allowed one to suppose, and that either cell may be capable of modulating toward an intermediate type.

204


FIG. 11. Scheme comparing the characteristics of fibroblasts and myofibroblasts. The upper part of the figure shows a typical fibroblast with a smooth contour of the nucleus which contains a nucleolus. The cytoplasm contains abundant cistemae of rough endoplasmic reticulum, mitochondria, a Golgi apparatus, and peripheral vesicles, but only few intracytoplasmic fibrils. The extracellular tissue is mainly composed of collagen bundles. The lower part of the figure shows an area of granulation tissue. The cellular concentration is higher than in normal connective tissue. Myofibroblasts have a nucleus with numerous folds and indentations. The cytoplasm still has some cistemae

CONTRACTILE PHENOMENA I N WO U N D HEALING

205

4. Collagen, Glycoproteins, and Proteoglycuns of Granulation Tissue In inflamed tissues, collagen is synthesized more rapidly and is present in a higher concentration than in normal tissues (Madden and Peacock, 1971). When the inflammatory reaction subsides, collagen is progressively resorbed, and the repaired tissue returns to normal composition. Collagen from acutely or chronically inflamed tissue is less soluble than collagen from normal tissue. This corresponds to the presence in granulation tissue collagen of cross-links different from those present in collagen of normal skin, but similar to those present in collagen of embryonic skin (Bailey et al., 1973; Hansen, 1975). Moreover, granulation tissue induced in the rat by subcutaneous injection of turpentine oil or by subcutaneous implantation of polyvinyl sponges, contains a higher proportion of type I11 collagen than normal skin (Bailey et ul., 1975b). Myofibroblasts are present while the tissue is synthesizing type I11 collagen (Figs. 12 and 13)and disappear when normal type I collagen with different stabilizing cross-links is being synthesized (Gabbiani et al., 1976).Therefore it appears probable that inyofibroblast:, are, at least in part, responsible for the synthesis of type I11 collagen. The collagen in normal skin is almost totally of the classic type I, the fibers of which possess a typical 640-A periodicity, whereas in granulation tissue it is composed of relatively few classic collagen fibers (Fig. 3 ) , some fibers without periodicity, and a significant quantity of finely filamentous material. It may be speculated that the small filaments and the fibers without periodicity are composed mainly of type I11 collagen. These fibers are probably analogous to those generally referred to as reticulin. By using immunofluorescent techniques to localize type I and type I11 collagen, an increase in type I11 collagen has been found in hepatic fibrous septa and portal tracts of patients with hepatic cirrhosis of various etiology (Remberger et al., 1975). Further studies are, however, required to locate the type 111 fibers precisely. Whether there is a relation between the presence of such modified collagen and the contractile activity of myofibroblasts has not yet been established. However, it is worth noting that type I11 of rough endoplasinic reticulum, but its most characteristic feature is the presence of massive bundles of filaments usually arranged parallel to the long axis of the cell. Electron-dense areas are scattered among the bundles or located beneath the plasmalemma. Intercellular connections in the form of gap junctions are present between fibroblasts. In addition, part of the cell surface is often covered by a well-defined layer of material similar to a basal lamina; in such regions, the cell commonly shows a dense zone (giving a heniidesmosome complex) in the fibrillar bundles immediately beneath the surface membrane. T h e extracellular tissue contains microfibrils without periodicity, as well as mature collagen fibers. (From Gabbiani et al., 1973b.)

206

GIULIO GABBIANI AND DENYS MONTANDON a

b

c

d

e

f

FIG. 12. Analysis of pepsin-solubilized 10-day-old acute (turpentine-induced) and 5-month-old chronic (sponge implant-induced) rat granulation tissue following fractionation into type 111 (1.5Msodium chloride precipitate) and type I (2.5M sodium chloride precipitate) SDS polyacrylaniide gel electrophoresis: (a-d) without mercaptoethanol; (a) type 111 acute granuloma; (b) type I acute granuloma; (c) type 111 chronic granuloma; (d) type I chronic granuloma; (e-h) following incubation with 2% mercaptoethanol to convert 111 to a-111; (e) type 111 acute granuloma; (f) type I acute granuloma; (g) type 111 chronic granuloma (contaminated with type I on first precipitation); (h) type I chronic granuloma. (From Gabbiani et nl., 1976.)

ELUTION

VOLUME (ml)

FIG. 13. Carhoxyniethyl cellulose chromatography of type I and type 111 precipitates from 10-day-old acute rat granulation tissue. Solid line, 1.5M sodium chloride precipitate (type 111); dotted line, 2.5 M sodium chloride precipitate (type I ) . (From Gabhiani et al., 1976.)


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collagen is present in tissues that need a certain plasticity, such as embryonic skin, normal smooth muscle and granulation tissue. However, Hardwood et a1. (1974)failed to find type I11 collagen in healing skin wounds of guinea pigs. T h e content of glycoproteins and proteoglycans is different in granulation tissue compared to normal tissues (Kisher and Shetlar, 1974; Bazin et al., 1974). Soluble sialoglycoproteins, which are essentially derived from the exudate of blood proteins, are increased. Chondroitin sulfate is remarkably increased in young granulation tissues and diminishes with the progressive accumulation of collagen. The same is tnie for hyaluronic acid.

111. Epithelialization of a Wound Epithelial cells are constantly subject to a certain amount of regeneration. However, regenerative processes become essential for the integrity of the tissue, and sometimes for the survival of the organism, after the loss of large surfaces of epithelium. Repair has been studied mostly in the epidermis, but essentially similar phenomena take place in other epithelia such as that of the digestive and respiratory tracts (Ordman and Gillman, 1966; Peacock and Van Winkle, 1970; Odland and Ross, 1968). Schematically, the most important steps of skin epithelialization are:

1. Mobilization of basal cells which appear to be no longer attached to the underlying dermis. 2. Migration of epithelial cells toward the damaged area. Migrating cells usually move along the remaining basal lamina along fibrin deposits. This phenomenon has been called contact guidance (Weiss,

1959). 3. Proliferution of basul cells close to the head of the wound followed by proliferation of adjacent prickle cells and of migrating cells. 4. Differentiation of the migrated cells that have filled the gap between the wound margins and stop migration as soon as they come in contact with other cells (contact inhibition). In the skin, this consists of keratin production and of the establishment of well-developed intercellular junctions (Krawczyk and Wilgram, 1973). In linear wounds of the skin, as well as in minimal traumatisms, epithelial regenerative processes take place before any new connective tissue forms. Increased mitoses of epidermal cells are not usually observed until 1-2 days after restoration of epidermal continuity (Bul-

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FIG. 14. Staining by AAA serum of epithelial cells growing over skin wound. (a) Epithelial cells growing over center of wound are brightly fluorescent after incubating with AAA serum followed by fluorescein-con,jugated goat antihuman IgC (the detachment of epithelial cells from the underlying granulation is an artifact which occurred during sectioning). Frozen section. x 400.(b) Epithelium covering a more peripheral region ofthe wound treated as in (a).The fluorescence is brighter on the cells on the left side of the section closer to the center of the wound. Frozen section. x 400. (c) Region of normal skin close to wound treated as in Fig. 1. No intracellular fluorescence is visible. (The hair is autofluorescent.) Frozen section. x 400. (d) Section corresponding to Fig. 2 incubated with normal human serum followed by fluorescein-conjugated goat antihuman I&. No specific fluorescence. Frozen section. x 400. (From Gabbiani and Ryan, 1974.)

lough and Lawrence, 1960; Peacock and Van Winkle, 1970). In open wounds, cell mobilization and migration start at the wound edges, but they can be accompanied by mobilization and migration of cells from skin appendages, particularly hair follicles, if the full thickness of the dermis has not been removed. Migrating epithelial cells move under the blood clot which usually covers the surface of the wound and over granulation tissue. Keratin production by epithelial cells may evoke an important inflammatory response in granulation tissue. It has been shown that migration plays a most important role in epithelial repair (Bullough, 1969). Migrating epithelial cells lose many of their junctional complexes and develop a “cortical band” of filaments 40-80 A in diameter, which are different from tonofilaments (Krawczyk, 1971).


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In addition, the periphery of migrating cells selectively fixes antiactin autoantibodies (Gabbiani and Ryan, 1974; Montandon and Gabbiani, 1976),showing de novo accumulation of this protein (Fig. 14). After immunofluorescent staining, the number of positive cells and the intensity of the staining are greater toward the central part of the wound (where the cells are actively moving) and decreases gradually toward the periphery. No staining is present in areas of normal epithelium adjacent to the wound, and the staining disappears after healing in the thin layer of epithelium covering the scar. The presence of bundles of microfilaments has been associated with contraction of epidermal cells during the metamorphosis of the acsidian Amaroucium constellatum (Cloney, 1966). The presence of actin as judged by immunofluorescence correlates well with the presence of microfilaments as seen in electron microscopy (Figs. 15-18) and suggests that, during the healing of a wound, epithelial cells develop a contractile microfilamentous apparatus which probably represents the morphological basis of motile activities (Gabbiani and Ryan, 1974).

IV. Pathology of Granulation Tissue and Fibromatoses Hypertrophic scarring is a condition that may develop in a certain proportion of patients a few weeks or months after wounding or following third-degree burns. Hypertrophic scars are usually red and sometimes exhibit a certain degree of contraction even at a very late stage. Most often they appear in colored people and in children, but they can be seen in any human, especially in certain areas of the body such as the anterior chest. For unknown reasons, part of the scar heals perfectly well compared to another part. Hypertrophic scars should be differentiated from cheloids which have a different clinical evolution, although the histopathological features of these two conditions are quite similar. So far, it has not been possible to induce hypertrophic scars or cheloids in animals. The mechanism of these pathological changes is not known. However, it cannot be excluded that myofibroblasts persist longer than normally after the closure of the wound. This hypothesis has been confirmed by Baur et al. (1975), who observed the presence of myofibroblasts in human hypertrophic scars and propose that such cells are at the origin of the retraction. In addition, Bailey et (11. (1975a) showed that hypertrophic scars retain the characteristics of embryonic collagen, indicating a rapid turnover of collagen. Moreover, a high proportion of type I11 collagen is present in hypertrophic scars, similar to the situation in normally evolving young granulation tissue.

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GIULIO GABBIANI AND D E W S MONTANDON

FIG.15. Apposing cell membranes between adjacent epidermal cells of a rat. Tonofilaments are visible in association with junctional complexes of the desmosome t .y D- e and also lying more centrally in the cytoplasm. x 66,200. (From Gabbiani and Ryan,

1974.)

CONTRACTILE PHENOMENA I N WOUND HEALING

21 1

FIG. 16. Apposing cell membranes of two epidermal cells during regeneration. An incomplete junctional complex connects the cells. An important microfilamentous network is visible (M), particularly in the cell on the right side. More centrally in the cytoplasm, tonofilaments (T) are present. X 43,000. (From Gabbiani and Ryan, 1974.)

212


FIG.17. Epidermal cells of normal human skin. The nucleus is surrounded by a cytoplasm composed mostly of free ribosomes, mitochondria, melanin granules, and tonofilaments. The basement membrane separating epithelium from dermis is also visible. x 26,500. (From Montandon and Gabbiani, 1976.)

Wound contraction is generally beneficial, particularly when wounds are located in areas far from articulation, such as the head and neck. However, wound contraction often produces distortions in the architecture of skin submitted to a certain degree of tension. These excessive contractiors or contractures are seen frequently after thirddegree bums of the neck, elbow, wrist, and knee. Excessive contraction of granulation tissue leading to contractures is observed not only in the skin but also in various viscera. Myofibroblasts have been found in stenotic trachea following a prolonged tracheostomy (Lehmann and Gabbiani, 1975), and in esophageal stenosis after injection of caustic products or after surgical interventions with anastomoses made under tension. The utilization of silicone implants as a mammary prosthesis may lead, in some instances, to contraction of the thin capsule that normally surrounds the implanted material. This results in unesthetic and painful distortions. The presence of myofibroblasts in the retractile tissue (Ryan et al., 1974; Montandon et al., 1973) has suggested the possibility of a mechanism similar to that of granulation tissue contraction. These different types of contractures may all be explained by


213

FIG. 18. Epidemial cell regenerated over a 7-day-old wound in a human. Some basement membrane material is evident in relation to the cell. The peripheral part of the cytoplasm contains a distinct microfilamentous network (arrows). x 26,700. (From Montandon and Gabbiani, 1976.)

an excessive and prolonged myofibroblastic response after different kinds of inflammatory stimuli. Myofibroblasts also develop during various pathological conditions called fibromatoses, which are mostly characterized b y contractures and in which inflammation does not seem to play a major role. This is the case for the nodules of Dupuytren’s disease (Gabbiani and Majno, 1972), a progressive irreversible contracture of one or more digits. Dupuytren’s nodules, which are located in the palmar aponevrosis, have also been shown to contain a high proportion of type I11 collagen (S. Bazin, personal communication). Myofibroblasts have been identified in the nodules of Ledderhose’s disease (Gabbiani and Majno, 1972) (the situation corresponding to Dupuytren’s disease localized in the plantar region), in “knuckle pads” over interphalanged joints (Gabbiani et al., 1973c), and in the fibrous plaque of La Pkronie’s disease (Montandon and Tuchschmid, 1976). Madden and Carlson (1974)

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showed the presence of myofibroblasts in the lesions of several “fibrocontractive conditions,” for example, in the annular ligament during the carpal tunnel syndrome, in synovial membrane and tendinous nodules during chronic tenosynovitis, and in fibrotic tissue during progressive muscular fibrosis after ischemia of the arm or Volkman’s disease. These observations show that, in addition to the role played during wound healing, myofibroblasts may develop and multiply in regions normally relatively acellular and produce pathological contractures. The mechanism of myofibroblast development during this condition is not known, but these cells resemble morphologically, biochemically, and immunologically the myofibroblasts of granulation tissue.

V. Conclusions Several recent observations support the view that, in experimental animals and in humans, one important step in wound healing consists of the transformation of local fibroblasts and/or other less differentiated cells into myofibroblasts (Gabbiani et al., 1972a). On the one hand, these cells appear to be responsible for the mechanism of granulation tissue contraction and, on the other hand, synthesize until the completion of wound healing a type of collagen normally present in tissues that need plasticity for their functions. Many questions about the biology of myofibroblasts are as yet unanswered. For example: (1)What are the factors promoting the development of myofibroblasts? (2)What are the agents producing their contraction in vivo? (3)Under what influences do myofibroblasts disappear? Progress in these directions would not only be of importance in the understanding of the process of wound healing, but could be valuable in finding agents potentially able to influence the evolution of granulation tissue. It is of interest that both fibroblasts and epithelial cells under the stress of a wound develop a filamentous contractile apparatus which may be useful in such processes as migration and/or contraction. A relationship between intracytoplasmic microfilaments and cellular motion, development of tension, intracytoplasmic movements, and secretion has been proposed for a wide spectrum of cells, ranging from monocellular organisms to those of mammalian tissues (Wessels et a1., 1971; Pollard and Weihing, 1974, Lacy et al., 1968). The question arises whether mammalian cells other than fibroblasts and epithelial cells can respond to appropriate stimulation by developing microfilaments. This has been shown to be true for regenerating hepatocytes (Gabbiani and Ryan, 1974), hepatocytes during cholestasis or after


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treatment with certain specific poisons such as phalloidin (one of the poisons of the mushroom Arnanitu phalloides) (Gabbiani et ul., 1975c), aortic endothelial cells during the first stages of hypertension (Gabbiani et ul., 1975b), and tumoral cells from skin and mammary carcinomas (Gabbiani et ul., 1975d). Such development of a contractile filamentous apparatus probably takes place when cells of different embryological origin face situations that require the enhancement of certain characteristic functions such as the ability to move about and to contract. The study of such properties may be useful in elucidating certain aspects of the biology of wound healing and of other important pathological processes such as tumor invasion; it may also contribute to the understanding of more general phenomena such as the physiological or pathological variations of intercellular relationships.

ACKNOWLEDGMENTS

This work was partly supported by the Fonds National Suisse pour la Recherche Scientifique (grant no. 3033073).The original work described here is due to the efforts o f a team including Drs. G. Majno, G. B. Ryan, P. R. Statkov, B. Hirscliel, C. Id&,W. J. Cliff, and thc authors. We thank Miss M.-C. Clottu, Miss M. Bo~il:itid,Miss M .Flolir, Mrs. F. Calhiani, and Mrs. A. Fiarix for their technical help, and Messrs. J.-C. Huml)eli and E. Detikinger for their photograpliic work. We thank the prildishers of theJorcrnul ofBioph!/sicul Riochemicul Cytolog!/,Joitrticl ofEx))eriniental Medicine,Humrot Pat/io/ogy, Virchows Archie (B), the J o i r r t d ($Subniicrosco]~icC!/tolog{/,and the Foundation for International Cooperatioil i n the Medical Sciences, Acatlcmic Press, Iirc., and Masson et Cie for allowing the reproduction of Figs. 1, 2, 3, 6, 7, 8, 9, 11, 12, and 13.

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Potential and Limitations of Enzyme Cytochemistry: Studies of the Intracellular Digestive Apparatus of Cells in Tissue Culture M. HUNDGEN D e ~ ) a r t n i e tof ~ t Zoology, Uniuccrsity of Botin, Bonn, West Gemnany

I. Introduction . . . . . . . . . . 11. The Influence of Fixation on the Localization of Enzymes . A. The Effect of Fixation on Ultrastructure . . . . H . The Effect of Fixation on the Activity of Enzymes . . C. The Effect of Fixation on the pH Optimum of Enzymes. 111. Cytochemical Demonstration of Enzymes . . . . A. Localization of Endogenous Enzymes . . . . . . . . B. Localization of Exogenous Enzymes. C. Sequential Staining for Applied HRP and Acid Phosphatas e . . . . . . . . . . . IV. The Intracellular Digestive Apparatus . . . . . A. Light Microscope Investigations . . . . . B. Electron Microscope Investigations . . . . . C. Morphometric Analyses V. Limitations of Enzyme Cytochemistry . . . . . . . . . . A. Specificityof Lead Salt Methods B. Specificity of the Diaminobenzidine Method . . . VI. General Conclusions . . , , . , . . References. . . . . . . . . . .

28 1 282 283 287 289 290 290 292 294 295 295 299 309 314 3 14 316 317 318

I. Introduction This article presents a concrete example of the way in which the methods of enzyme cytochemistry can be applied and the results interpreted. Studies of this sort, using both the light microscope and the electron microscope, clarify cellular phenomena in terms of precise localization and quantitative measurement of certain substances or enzyme activities. Most of the enzyme cytochemical reactions used to obtain such data can be produced only in tissue that has previously been fixed. Since for each preparation there is a particular optimal fixative composition and experiniental procedure, and since the value of enzyme cytochemistry is best reflected in its application to a particular biological problem, the potential and limitations of modern enzyme cytochemistry are discussed with respect to a specific example-the intra28 1

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M. HUNDGEN

cellular digestive apparatus of cells of the chicken heart, grown in tissue culture. Model cells of this kind appear to be useful objects for such research, since heterologous nutrient media routinely induce the development of food or digestive vacuoles in these cells. Cells so cultured have the further advantage that in u i t m they grow out flat, so that their components lie nearly in a single optical plane and are readily observable under the light microscope. The preparation of sections can also be omitted with these cells. Finally, they provide an unusual opportunity for comparative measiireinents with the light and electron niicroscopes. Since morphological criteria alone do not suffice to categorize the different vacuoles of the intracellular digestive apparatus according to their function, microscopically visible indicators of both the food they contain and their specific digestive enzymes are required. Enzymes characteristic of these cells include certain hydrolases such as acid phosphatase, glucose-6-phosphatase, thiamine pyrophosphatase, and iiucleoside diphosphatase, all of which are demonstrable under both the light and the electron microscope. To follow the process of protein digestion, a tracer protein (horseradish peroxidase, HRP) is applied to the cells; its own enzymic activity is employed in a controlled reaction to reveal its location in the cells. By the use of these enzyme cytochemical procedures a reconstruction h a s been attempted of the overall process-uptake, intracellular transport, and digestion of the tracer protein-in terms of structure, spatial distribution, and time course.

11. The Influence of Fixation on the Localization of Enzymes Cells cultured in uitro may differ considerably in apparent structure, depending on the fixatives used. These differences are inevitable consequences of fixation, a process that always involves a niore-orless marked departure from the normal structural arrangement and the chemical properties of the living substrate. For present purposes it is necessary that the method of fixation preserve cell structure and enzyme activity equally well, since otherwise the products of the enzymic reactions would be difficult or impossible to localize. Unfortunately, the fixatives least disruptive of structure are not well suited for subsequent cytochemical experiments. Pilot experiments can often reveal ways to reduce fixative artifacts to a tolerable level, but this usually involves a compromise between the preservation of structure

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and the maintenance of enzyme activity. For this reason, it is necessary to begin b y investigating the effect of the various fixing media on cell structure.

A. THE EFFECT OF FIXATION O N ULTRASTRUCTURE Oxidizing reagents such as osmium tetroxide and potassium permanganate cannot be used for fixation in these experiments, because of their eiizyii~e-inactivatingeffect; the aldehydes introduced b y Sabatini et ul. (1963), however, are potentially useful fixatives. The use of cells in citro facilitates such studies, since all the stages from the living to the fixed state can be followed directly. Several changes brought about by fixation can be observed under the light microscope, and in many cases methods can be found to reduce or eliminate them. Whereas the information obtained with the light microscope tends to reflect the overall structure and activity of the cell, particular attention must be paid here to the structural state of each cell component. Therefore both light and electron microscope tests of various fixation media were made. 1. Tests o f Selected Aldehydes Following Sabatini et (11. (1963)and Gordon et al. (1963),eight different aldehydes were chosen as candidate fixatives. Each fixative solution was composed of an aldehyde and sucrose in a 0.1 M cacodylate buffer solution, a s suininarized in Table I. The optimal concentrations (0 in Table I) of the aldehyde and sucrose in each case were deterniined empirically. The aldehydes varied widely in their fixation of cytoplasm, each producing a typical appearance of the fine structure quite distinct from that due to the other aldehydes. The quality of the fixation can be evaluated by selecting preparations fixed with one of the aldehydes as a standard (glutaraldehyde seems to be the best choice given present knowledge) and comparing the effects of the other aldehydes with these. Figure 1 summarizes these results. The fixative solutions are divided into four classes, with class 4 representing the highest quality of fixation; the standard, glutaraldehyde, by definition, reaches this level with respect to five critical cell components and thus is assigned the maximum score of 20. Classes 3 , 2 , and 1 represent, in this sequence, increasing departures from the nonn. Specifically, classes 4,3, 2, and 1, respectively, connote very good, good, usable, and useless results in the morphological sense, for each cell component. The total score obtained b y addition of the scores for

284

M. HUNDGEN TABLE I COMPOSITION O F T H E FIXATIVE SOLUTIONS“

Final concentration of aldehydes

Final concentration of sucrose

Molarity (M)

Percent

Molarity (M)

Percent

Glutaraldehyde

0.20

2.00

Acrolein

0.20

1.12

Paraformaldehyde

1.33

4.00

H ydroxyadipaldeh yde

0.46

6.00

3.5 6.9 13.8 12.0 15.4 22.3 0.7 4.0 11.0 3.5 6.9 13.8

Methacrolein

0.20

1.40

0.10 0.20 0.40 0.35 0.45 0.65 0.02 0.12 0.32 0.10 0.20 0.40 0.19 0.29 0.49 0.35 0.45 0.65 0.25 0.35 0.55 0.43 0.53 0.73

Aldehydes

Crotonaldeh yde

0.55

3.90

Glyoxal

0.20

1.16

Acetaldehyde

0.45

1.96

“

6.6

10.0 16.9 12.0 15.4 22.3 8.6 12.0 18.9 14.8 18.3 25.2

Final osmolality (mosm)

380 (L)O 480 (0) 712 (H) 616 (L) 720 (0) 952 (H) 1180 (L) 1280 (0) 1496 (H) 648 (L) 760 (0) 988 ( H ) 410 (L) 520 (0) 748 (H) 1020 (L) 1130 (0) 1364 ( H ) 512 (L) 620 (0) 836 ( H ) 696 (L) 800 (0) 1032 ( H )

Hundgen et al., 1971a; Weissenfels et d., 1971. L, Low; 0, optimal; H, high.

five components gives the fixation quality of each aldehyde. In Fig. 1 the eight aldehydes are arranged in order of decreasing scores (see “Summary of Classes”). By this method of evaluation only glutaraldehyde, acrolein, and paraformaldehyde produce very good to usable results. Figure 1 also shows, however, that some of the aldehydes scoring too low for general use can be used as fixatives when only a certain cytoplasmic organelle is involved. These results should not of course obscure the fact that the structure of a cell prepared for microscopy is affected not only by the fixation but by subsequent procedures as well.

285


Glvtar .

Fixationquality Summary of

Acet-

Paraform-

Hydroxy-

Matha-

Croton-

aldehyde

Ac‘ole‘n

aldehyde

adipaldeh.

c ro l e i n

aldehyde

1 2 5 4

1 2 3 4

1 2 3 4

1

20

19

12

2

5

4

1

2

6

3

5

4

1

2

3

5

4

Glyoxal

1

2

3

4

1

aldehyde

1

2

3

b

b

classes

2. Osmolality and the Quality of Fixation Little is known about the effect of the osmolality of aldehydecontaining fixation solutions on the preservation of structure in cells. Systematic studies of this question have so far been concerned only with fixation by glutaraldehyde (Fahimi and Drochmans, 1965b; Maunsbach, 1966; Sprumont, 1967). It thus comes as no surprise that the published data vary over a wide range. Any study of the effect of aldehyde fixation on ultrastructure must take account of osmolality; examination shows that one of the factors affecting the quality of fixation is the tonicity of the fixative solution. This was varied for each aldehyde by adding different empirically determined amounts of sucrose. An “extraction effect” (Milloning, 1966) of the sugar was not discernible with the concentrations used and was detected only with extremely high concentrations of sucrose. Solutions containing a given amount of aldehyde and an optimal amount of sucrose (0 in Table I) were compared with others of the same aldehyde concentration and a sucrose content lower by 0.1 M (L in Table I) or higher by 0.2 M (H in Table I). It was necessary that the increase in sucrose be greater than the decrease, in order to obtain an appreciable departure from the result with the optimal concentration. Since both hypotonic (Fahimi and Drochmans, 1965a; Maunsbach, 1966) and hypertonic (Bone and Denton, 1971) washing solutions applied following aldehyde fixation produce a deterioration in the structural state, the osmolality of all washing media-independent of the more-or-less marked hypertonicity of the fixative used-was adjusted

286

I

M. HUNDGEN

1

Paraformaldehyde

I

Hydroryadipaldehyde

I

Melhacrolain

1

Crolonaldshyda

I

FIG.2. Th e effect of osmolality on the qriality of fixation. Final osiiiolality: L, low; 0, optimal; H, high. GP, Groundplasm; ER. endoplasmic reticulnm; PS, pcrinuclear space; M, mitochondria; D, dictyosomes. Fixation quality: 1, useless; 2, unsatisfactory but usable; 3, satisfactory; 4, excellent preservation (Hiindgen, 1973).

to the tonicity of the cultured chicken heart cells (ca. 300 mosm). The quality of fixation by glutaraldehyde and acrolein is hardly affected by errors in osmolality. At the other extreme, fixation b y glyoxal and acetaldehyde is so poor in any case that similar errors in osmolality make matters very little worse. The remaining aldehydes-paraformaldehyde, hydroxyadipaldehyde, methacrolein, and crotonaldehyde-reveal effects of osmolality on the quality of fixation, and these tend to be the same regardless of the aldehyde involved. The summary in Fig. 2 shows that both the relatively high (H) and the low (L) final osinolality produce deterioration in the structural picture when all cell components are considered (see “Summary of Classes”), though the effect is more pronounced with the lower final osmolality. These osmolar effects obscure the fixation properties characteristic of the particular aldehydes, except in the case of hydroxyadipaldehyde. A further result was that changes in the concentrations of aldehyde and cacodylate buffer amounting to ;IS much as 50% cause no deterioration in fixation quality as long as the necessary final osmolality is preserved by the addition of appropriate amounts of sucrose. It cannot at present be determined whether the artifacts caused by errors in tonicity appear during the process of fixation itself or thereafter, during subsequent procedures. The fact that well-fixed cells withstand the relatively rough treatment sometimes required in cytochemistry better than poorly fixed material indicates a positive correlation between the quality and the stability of fixation.


287

3. Other Factors Affecting Quality of Fixation: Buffer Solution, Teniperatztre, and Duration of Fixation Of the common buffer solutions, the tris and tris-maleate buffers frequently used for histochemical reactions are unsuitable here, since their amino groups react with aldehydes (Sabatini et al., 1963) and thus diminish the aldehyde concentration (Plattner, 1973). Phosphate and cacodylate buffers exhibit the smallest leaching effects. In our preparation phosphate buffer preserves structure better than cacodylate, but for obvious reasons it interferes with the demonstration of phosphatase. Hence cacodylate was the buffer of choice. I n order that autolytic processes be avoided, fixation is ordinarily done at about 4°C. In this temperature range the diffusion velocity of the fixative solution is sufficiently high (Arnold, 1968), but certain structures such as the microtubules are not revealed. For this reason, the cultured chicken heart cells were fixed at 20°C. The optimal duration of fixation depends on the nature and size of the tissue. For our preparation it is 30 minutes (Hiindgen, 1968) in the normal case, when fixation is followed by a brief postfixing with osmium tetroxide in order to enhance contrast. However, since enzyme localization must be done before structure stabilization with osmium and often at an acid pH, it is desirable to extend the period of aldehyde fixation to provide additional structural stability, as long as the enzyme activity under study is not too greatly diminished.

B. THE EFFECTO F FIXATION ON

THE

ACTIVITYOF ENZYMES

Having determined the effect of the various aldehydes on ultrastructure we examined their influence on subsequent enzyme cytochemical reactions. The results are summarized in Fig. 3. It is apparent from this diagram that none of the enzymes indicated at the head of the columns-acid phosphatase, glucose-6-phosphatase, thiamine pyrophosphatase, and nucleoside diphosphatase-can b e demonstrated in all cell components. This finding is consistent with the physiological role attributed to the cell compartments. But the activity (I to 111) of each enzyme is affected differently by the different aldehydes (1to 8), and in the extreme case the enzyme may be entirely inactivated. In conclusion, two fixatives appear potentially useful for the intended enzyme localization (of acid phosphatase, glucose-6phosphatase, thiamine pyrophosphatase, and nucleoside diphosphatase; both glutaraldehyde and acrolein provide very good structural

288

M. HUNDGEN

FIG. 3. Enzymic activity retained in cultured cells after aldehyde fixation. Enzyme activities investigated are: Acid Pase, acid phosphatase; G-6-Pase, glucose-6phosphatase; TPPuse, thiamine pyrophosphatase; NDPase, nucleoside diphosphatase. Cell organelles: ER, endoplasmic reticulum; GA, Golgi apparatus; Ly, lysosomes; N, nucleus. Fixatives: 1, glutaraldehyde; 2, acrolein; 3, paraformaldehyde, 4, hydroxyadipaldehyde; 5, methacrolein; 6, crotonaldehyde; 7, glyoxal; 8, acetaldehyde. Retained enzymic activity: I, slight; 11, moderate; 111, intense (Hundgen and Weissenfels, 1973).

preservation (see Fig. 1)and sufficient preservation of enzyme activity (see Fig. 3). It has been known for 10 years that commercially available aldehyde solutions contain impurities which can reduce structural quality and enzyme activity (Anderson, 1967; Fahimi and Drochmans, 1965a, 1968). For example, impure glutaralclehyde shows high absorption at both 235 and 280 nm, whereas purified glutaraldehyde solutions absorb only at 280 nm. The absorption at 235 nm is caused by glutaraldehyde oligomers (Robertson and Schultz, 1970), which are responsible for the diminished quality of the fixation; these can be removed by vacuum distillation (Fahimi and Drochmans, 1965a). The absorption quotient A235/A280 is used as an index of the purity of glutaraldehyde solutions. In commercially available solutions it ranges from 2.5 to 6.6, while in redistilled solutions it varies between 0.14 and 0.25. The preservation of structure that can be achieved with commercial glutaraldehyde having a purity index of 2.5 is only slightly surpassed by the use of redistilled glutaraldehyde (purity index, 0.25). This is also true with respect to the activities of acid phosphatase, glucose-6phosphatase, thiamine pyrophosphatase, and nucleoside diphosphatase (in contrast to the situation with many other enzymes). Since the effort involved in distilling glutaraldehyde seemed not to be justified for the present purposes, all fixation experiments employed glutaral-


289

dehyde solutions with purity index 2.5. The same arguments apply to acrolein.

c.

THE EFFECT OF FIXATION ON THE PH OPTIMUMOF ENZYMES It must be pointed out that the pH-dependent reaction optimum can also be displaced b y the various aldehydes, in a way not yet understood. When the fixative is changed, therefore, a new curve of activity versus pH must lie constructed for each enzymic reaction. The optimum cuives for the enzymes of interest here, after fixation with glutaraldehyde or acrolein, are shown in Fig. 4.The pH range investigated was in each case from 3.5 to 7.5. Above pH 7.5 the lead salt methods employed here can no longer lie used. Since we are not dealing with precise quantitative experiments, the pH optimum in each case is characterized not b y a single value but rather b y a moreor-less broad range of pH values. It is evident from Fig. 4 that the p H optimum of a single enzyme cannot lie generalized but, as a nile, holds only for particular reaction sites (e.g., organelles) in the cell. As the figure shows, the optimum curvc' of nucleoside diphosphatase after glutaraldehyde fixation has a shape different from that after acrolein fixation. The optimum curves for acid phosphatase, glucose-6-phosphatase, and thiamine pyrophosphatase, however, are the same with both methods of fixation. As long

FIG.4. T h e efrect of pH on the localization of enzymes in cultured cells. Enzyiile activities investigated after glutaraldehyde (+) or acrolein (+ +) fixation are: Acid Pase, acid phosphatase; G-6-Pase, glucose-6-phosphatase; TPPase, thiamine pyrophosphat a x ; NDPase, riucleoside diphosphatase. Cell organelles: ER, endoplasmic reticulum; GA, Golgi apparatris; Ly, lysosomes; N, nucleus. pH, 3.5 to 7.5. Demonstrable enzymic activity: I, slight; 11, moderate; 111, intense.

290

M. HUNDGEN

a s the effects of fixation on ultrastructure, enzyme activity, and pH op-

tiinuin are taken into account, it is possible to demonstrate these enzymes reproducibly.

111. Cytochemical Demonstration of Enzymes Since the activity of HRP, which served as a tracer protein in the following experiments, is not appreciably reduced by fixation with aldehydes, we fixed the cultured cells with glutaraldehyde or acrolein, in consideration of the results in Figs. 1-4. Crotoiialdehyde fixation was used in only one case, for the demonstration of acid phosphatase by azo linkage (Hundgen, 1968). Since this demonstration is possible only with the light microscope, the less satisfactory fixation obtained with crotonaldehyde could be accepted for the sake of better preservation of the enzymic activity.

A.

LOCALIZATION OF ENDOGENOUS ENZYMES

The endogenous enzymes studied here all belong to the hydrolase group. Acid phosphatase (EC 3.1.3.2) and glucose-6-phosphatase ( E C 3.1.3.7) are phosphomonoesterases which split ester linkages. Thiamine pyrophosphatase splits acid anhydride linkages and is probably identical to a nucleoside diphosphatase (EC 3.6.1.6) (Hiindgen, 1970).

1. Acid Phosphntase Acid phosphatase, which since the publications of de Duve (1959, 1963) and Novikoff (1961) has been considered a typical lysosomal enzyme, has been demonstrated by two different methods: the metal salt method and the axo linkage method. In the first of these, developed b y Takamatsu (1939) and Gomori (1941), the acid group liberated b y the enzyme is precipitated by Pb2+;a colorless metal salt is formed, which is directly demonstrable under the electron microscope because of its high density. If the precipitate is to be observable by light microscopy, the colorless lead salt must be converted to a black-brown lead sulfide. The substrates used have been P-glycerophosphate (Gomori, 1941) and cytidine monophosphate (Novikoff, 1963). The results of light microscopy are the same in both cases: the high-contrast lead sulfide precipitates are located in the lysosomes and in the region of the Golgi complex. Despite the long incubation period (3 hours) and the high incubation temperature (38"C), there is no sign of diffuse brown staining of the cytoplasm, which would indicate a nonspecific reaction. The second method originated with Menten et al. (1944) and was


29 1

improved by Seligiiian and Maiiheiiiier (1949) and Barka (1960).It involves the enzymic liberation of a phenol compound which is subsequently converted to a colored precipitate by a diazoniuin compound. Although this azo linkage method is more sensitive than the lead salt method, the latter must be preferred because its reaction product, a black-llrown heavy metal compound, provides considerably better contrast under the light microscope than the microcrystalline end product of azo linkage, and also appears in high contrast under the electron microscope because of its high density. Even with careful preparation for electron microscopy, occasional nonspecific precipitates in nucleus and cytoplasm are unavoidable. Improved results can be obtained h y using tris-maleate buffer instead of the inore comiiion cacodylate. Such preparations confirm the results of light microscopy, indicating that acid phosphatase is active not only in the lysosomes h i t also in the Golgi complex. Only one or two of the Golgi eisteriiae are enzyinically active, and these are filled with the reaction product only in certain regions. The Golgi vesicles, as a rule, carry iio precipitate. If the incubation temperature is reduced from 38" to 2WC, the foiination of precipitate in the nucleus and cytoplasm is considerably less. The reaction in the Golgi complex, however, also disappears. Therefore 38°C was also chosen for demonstration of the enzyme by electron microscopy. Addition of 0.01 M glutaric acid to the incubation mixture completely inhibits the reaction in the Golgi apparatus, but inhibition in the lysosomes is only partial. Iiicubation with 0.01 M sodium fluoride, 0.01 M tartaric acid, or 10% formaldehyde in the medium produces total inhibition, as does heat inactivation in buffer solution at 70°C after fixation. The precipitates in the nucleus and cytoplasm are also present in the controls, but in considerably smaller amounts.

2. GlzLco.se-fi-Phosphut(lse The first histochemical demonstration of glucose-6-phosphatase was by Chiquoine (1953); subsequelit improvements were made by Wachstein and Meisel (1956), Tice and Barrnett (1962), Teriier et 01. (1965),and Schafer and Hundgen (1971).In cultured cells the enzyme can be demonstrated b y light microscopy to occur in the region of the Golgi complex and in the perinuclear space. Electron micrographs show the reaction product primarily in the cisternae of the endoplasmic reticulum. Evidently, it occurs there in such small amounts that it is invisible under the light microscope. Even by using dark-field microscopy (Bretthauer and Hundgen, 1970), which reveals much smaller quantities of reaction product than

292

M. HUNDGEN

any other light microscope technique, it is impossible to show any reaction in the endoplasmic reticulum. Since the endoplasmic reticulum and the Golgi complex play important roles in intracellular digestion, the program was extended to include a search for thiamine pyrophosphatase and nucleoside diphosphatase.

3. Thiamine Pyrophosphatase and Nucleoside Diphosphatase Dictyosomes ordinarily show a high degree of hydrolytic activity with respect to thiamine pyrophosphate and nucleoside diphosphates (Novikoff and Goldfischer, 1961; Osinchak, 1966; Lane, 1968; Hundgen, 1970). Thiamine pyrophosphatase, which as a rule also occurs in the endoplasmic reticulum, in cultured chicken heart cells is active only in the Golgi complex; it can be best demonstrated at pH 5.5. If nucleoside diphosphate is used as the substrate rather than thiamine pyrophosphate, the same result is obtained at pH 5.5. Dictyosomes, which comprise the Golgi complex, consist of a stack of four or five cisternae, one or two of which are enzymically active on the concave side. The cisternae on the opposite side have larger lumens and contain no reaction product. There are no unspecific precipitates in the nucleoplasm or cytoplasm. The enzyme is inactivated by uranyl nitrate and acetone, but sodium fluoride and alcohol have no inhibitory action. If nucleoside diphosphate is used as a substrate and the pH is raised to 7.0, the endoplasmic reticulum, in addition to the Golgi complex, displays nucleoside diphosphatase activity. This activity is preserved in the endoplasmic reticulum particularly well after fixation with acrolein, but the activity of the Golgi complex is greatly inhibited with this fixative.

B. LOCALIZATION OF EXOGENOUS ENZYMES Straus, in 1957, was the first to use intravenously injected HRP for the study of protein uptake by cells in the tissues of organs. This peroxidase is an iron-hematin compound which in the presence of hydrogen peroxide catalyzes the oxidation of many phenols and aromatic amines. When benzidine is applied, the reaction product is colored and insoluble. This functional demonstration of the presence of HRP thus shows indirectly that the protein has been taken into the cell. The advantage of this procedure is that much smaller amounts are demonstrable than is the case with direct marking of the substance itself. It was shown that the exogenous peroxidase of certain cells is stored in heterophagosomes (Straus, 1959, 1961) and in secondary lysosomes (Straus, 1964).


293

TABLE I1 TRACER PROTEINS DEMONSTRABLE WITH 3'3-DIAMINOBENZIDINE A S OXIDABLE SURSTHATE

Enzyme

Molecular weight

Catalase M yeloperoxidase Lactoperoxidase Hemog1ol)in HRP Cytochrome c Microperoxidase

240,000 160,000 82,000 67,000 40,000 12,000 2,000

Reference Venkatachalam and Fahimi, 1969 Graham and Kamovsky, 1966b Graham and Kellermeyer, 1968 Goldfischer et al., 1970 Graham and Kamovsky, 1966b Kamovsky and Rice, 1969 Feder, 1970, 1971

This method was adapted for electron microscopy in 1966 by Graham and Karnovsky (1966a), by the introduction of 3',3diaminobenzidine which causes an osmiophilic reaction product to be formed. Since the cytochemical demonstration using diaminobenzidine is not specific for peroxidases, several other hemoproteins give a positive reaction. Consequently, it has been possible to produce a series of tracer proteins with molecular weights between 2000 and 250,000, all of which are demonstrable with diaminobenzidine (Table 11).

Horseradish Peroxiduse Of the heavy metal-containing tracers, that best suited for electron microscope studies is ferritin (M.W. 500,000), because of its intrinsically high contrast (the Fe3+content is as high as 23%).This tracer has been applied to cultured chicken heart cells by Haarmann (1970). The tracer we use in preference to ferritin, HRP, provides the following crucial advantages: (1) HRP has more physiological action than ferritin, since the latter must be applied to cultured cells in concentrations 500 times higher in order to demonstrate it intracellularly; ( 2 ) HRP can be used for both electron and light microscopy; (3) the more-or-less homogeneous reaction product used for electron microscope demonstration of introduced HRP is readily distinguishable from the lead phosphate precipitates produced for the demonstration of endogenous hydrolases. The diaminobenzidine procedure developed by Graham and Karnovsky (1966a) naturally reveals not only exogenous hemoproteins, but also endogenous hemin enzymes. If confusion between endogenous and exogenous peroxidases is to be avoided, the extent to

294

M. HUNDGEN

which these endogenous enzymes occur in cultured chicken heart cells must be established. Endogenous peroxidase (EC 1.11.1.7) has been shown to occur especially in the epithelial cells of the intestine (Venkatachalam et d., 1970) and uterus (Brokelniann, 1969), in leukocytes (Bainton and Farquhar, 1970; Miller and Herzog, 1969), in the stellate cells of the liver (Kupffer’s cells; Fahimi, 1970), and in the acinar cells of numerous glands (Herzog and Miller, 1970, 1972; Sturm et d.,1971; Essner, 1971; Nanba, 1972). It is localized in the endoplasmic reticulum, in the perinuclear space, in the Golgi complex, and in secretory vacuoles. Endogenous catalase (EC 1.11.1.6)has been found in the microbodies of liver and kidney cells (Fahimi, 1969; Essner, 1970; Goldfischer and Essner, 1970; Wood and Legg, 1970; Chang et ul., 1971), and endogenous cytochrome oxidase (EC 1.9.3.1) in the mitochondria of many types of cells (Kerpel-Fronius and Hajos, 1967; Seligman et ul., 1967, 1973; Novikoff and Goldfische, 1969; Reith and Schuler, 1972). Of these three enzymes, only cytochroine oxidase occurs in cultured chicken heart cells. In accordance with its function as an enzyme of the respiratory chain, it is active in mitochondria. Damage to the enzyme becomes more pronounced as fixation time is lengthened, so that after 1hour of glutaraldehyde fixation almost no reaction product appears. Since exogenous HRP activity is not reduced even by several hours of fixation in glutaraldehyde, prolonged fixation excludes the possibility of confusion between this peroxidase and endogenous enzymes. The procedure for demonstration of HRP leads to a reaction product (oxidized diaminobenzidine) visible under the light microscope because of its brown coloration. Contrast can be considerably enhanced by treatment with osmium. The HRP taken into the cell is found, without exception, in the vacuoles of the intracellular digestive apparatus. The electron microscope reveals the reaction product as a noncrystalline, electron-dense precipitate located both extracellularly and within the vacuoles. C. SEQUENTIALSTAININGFOR APPLIED HRP AND ACID PHOSPHATASE The lysosomal fraction of the vacuoles containing HRP was characterized b y a two-step staining procedure; the first stage demonstrated the exogenous HRP incorporated after 3 hours of exposure, and in the second the endogenous acid phosphatase was revealed. Since both


295

reactions give rise to a brown or black-brown product, light microscopy does not permit a definite discrimination between vacuoles containing HRP, acid phosphatase, or both enzymes. Under the electron microscope, however, the electron-dense, crystalline lead-containing precipitates can b e readily distinguished from the less electron-dense, homogeneous precipitates of diaminobenzidine. In order to obtain at least some light microscope evidence for the differentiation among vacuoles, a given cell was photographed first after staining for HRP and then again after the acid phosphatase reaction. The reaction products appearing in the first photograph indicated the presence of some of the incorporated HRP in those lysosonies which, in the second photograph, proved to contain the product of the subsequent acid phosphatase reaction as well. But even this procedure does not give reproducible results, since acid phosphatase activity is partially inhibited by the illumination required for microscopic examination and photography following the HRP reaction. Consequently, most of the light microscope demonstrations of HRP aiid acid phosphatase were made with different cell cultures.

IV. The Intracellular Digestive Apparatus Cells removed from their natural environment and cultured in sterile synthetic nutrient media are sensitive to external changes such as transient drops in temperature (Weissenfels, 1973b)aiid changes in the concentration of the nutrient medium (Gross and Riedel, 1969). Neither of these could be avoided during the application of HRP, and both had to be taken into account in designing the experiments and in evaluating the results. The extracellular HRP concentration optimal for the present experiments is detei-niined by two conflicting requirements: on the one hand, the HRP concentration should be as high as possible in order to permit a good staining reaction but, on the other hand, it should be as low as possible to avoid a toxic effect of this foreign protein. Pilot experiments showed that a concentration of 0.01% HRP in the nutrient medium was optimal for studies of cultured cells, both providing an adequate reaction and being sufficiently well tolerated by the cells.

A. LIGHT MICROSCOPE INVESTIGATIONS In order to obtain evidence of all stages of the process-the uptake of the tracer protein, its transport, and its breakdown by the intracellular digestive apparatus-HRP was included in the nutrient medium

296

M. HUNDGEN

for various periods of time. This was done by introducing the protein 0 , 2 , 4 , 8 , 16,32, and 64 hours prior to the end of the 64-hour culture period; thus all the cultures, regardless of the time of exposure to the enzyme, were 64 hours old at the time of fixation. According to the studies of Neubert-Kirfel (1970), cultured chicken heart cells show a strong positive correlation between the interphase age and the number of lysosomes. Young cells, characterized by the small size of both cell and nucleus, as a rule have fewer lysosomes than older interphase stages. The size, as well as the number, of individual lysosomes increases during the interphase. The total area of lysosomes seen in microscope preparations increases linearly in time until it has doubled. In order to distinguish experimentally produced changes from size differences related to age, we used phase-contrast to select the cells with nuclei of approximately the same size, that is, cells of nearly the same interphase age. With a mean interphase age for the culture of ca. 17 hours, the interphase age of the selected cells was 6.5-8 hours. Figures 5 and 6 show cells exposed to HRP for 0 , 2 , 4 , 8 , 16,32, and 64 hours. Following the exposure, some cells were stained for exogenous HRP (Fig. 5), and cells in parallel cultures were stained for endogenous acid phosphatase (Fig. 6). At the top of each column of photographs (0 in Figs. 5 and 6) is a control. These cells, like those in the micrographs below them (2 in Figs. 5 and 6), received added nutrient medium 2 hours before fixation, but in the case of the controls this medium contained no HRP. It is necessary to treat the controls in this way, since the addition of even very small amounts of fresh nutrient medium-in fact, even agitation of the fluid in the culture-constitutes a stimulus to pinocytosis and thus leads to an increased uptake of substances by the cells. Since for present purposes only the influence of HRP is of interest, but such undesirable effects cannot be excluded, the controls are essential. In Fig. 5 the incorporated HRP, represented by oxidized diaminobenzidine, appears clearly. It is located in vacuoles which may be either heterophagosomes or secondary lysosomes; the present experiments do not distinguish between them. After 2 hours a considerable number of vacuoles varying in size and containing reaction product appears in the cells. The reaction product stains the vacuoles only lightly, indicating that it is present in small amounts. After 4 hours the food vacuoles are more deeply stained, and their number has increased by about 20%. Further extension of the exposure time to 8,16,32, and 64 hours causes no further increase in the number of vacuoles containing HRP, but there is a considerable in-


297

crease in their average size. As the number of large vacuoles increases, the proportion of small vacuoles is reduced. After 64 hours only a few small vacuoles remain. Hence the incorporation of HRP has almost entirely ceased. The increase in total area of vacuoles containing HRP, which is clearly associated with increased duration of exposure, is thus d u e less to an increase in number rather than to an increase in size of the vacuoles. Figure 6 shows the effect of uptake of HRP on the vacuoles exhibiting acid phosphatase activity (= lysosomes). After 2 hours of exposure to HRP the area occupied by the lysosomes is greater than in the controls, chiefly as the result of an increase in their number. Longer exposure, for 4 or 8 hours, causes almost no appreciable increase in lysosomes. From the sixteenth hour on, however, there is again a clear increase, with the number of lysosomes-large ones in particularrising steadily. Comparison of Figs. 5 and 6 reveals that in both cases there is a positive correlation between duration of exposure and area occupied by the vacuoles containing HRP and acid phosphatase, respectively. Staining of the tracer proteins reveals that after only 4 hours of exposure the number of vacuoles containing HRP is already maximal. Longer exposures then produce enlargement of these vacuoles. Since cells contain a well-developed intracellular digestive apparatus regardless of exposure to HRP, even the controls display vacuoles with acid phosphatase activity. The mean total lysosome area increases relatively uniformly with time, from the control level to the full 64-hour exposure, with growth in both the number and the size of the lysosomes. It is striking that a considerable proportion of small lysosomes is always present, whereas small HRP-containing vacuoles are no longer observed as exposure time is increased. Thus, for both the vacuoles containing HRP and those containing acid phosphatase, there is a definite dependence of total volume on duration of exposure. In order to obtain some idea of the time cultured chicken heart cells require for the HRP they have taken up to be broken down to a degree such that it can no longer be demonstrated histochemically, a method of “pulsed” exposure was adopted. The cells were precultured in normal nutrient medium, exposed to HRP for 4 hours, washed in physiological saline at 38”C, and kept for a further 8 or 24 hours in nutrient medium free of HRP. The cells were fixed at the e n d of this final postculture period. The preculture times were adjusted so that all the cultures were 64 hours old at the time of fixation. After 8 hours of postculture, in the absence of HRP, the cells

298

M. HUNDGEN

FIG.5.

FIG.6.


299

showed only a slight decrease in HRP activity as compared with the control, but after 24 hours there was a clear reduction. It is not practical to continue postculture until the activity of the enzyme has entirely vanished, since the interphase of cultured chicken heart cells lasts only about 20 hours. Then the next mitosis occurs, in the course of which all the vacuoles-and thus the quantity of tracer protein they have taken up--are divided about equally among the daughter cells. Each mitosis halves the amount of intracellular HRP, quite apart from any intracellular digestive processes. There is thus no point in using a post-culture period longer than that of one interphase. Since the present cells break down only part ofthe tracer protein in this time, the time span between uptake of HRP and its digestion must exceed 24 hours. The results of pulsed exposure, then, were not quite as had been hoped.

B. ELECTRONMICROSCOPE INVESTIGATIONS The results of light microscopy showed that the amounts of HRP cultured cells can take up vary depending on the duration of exposure (2, 4, 8, 16, 32, or 64 hours’ exposure to 0.01% HRP). The electron microscope studies showed that the protein quantities taken up in each case are disposed of by different mechanisms. 1. Uptake, Trcmqmrt, untl Digestion of S m a l l Amounts of H R P Only a short time after application of HRP, the tracer protein begins to be adsorbed onto the glycocalyx which overlies the plasmalemma and which, in cultured chicken heart cells as in numerous other cell types, consists of acid niucopolysaccharides (Haarmann, 1970). But this process is not a general one, since cells are also known that cannot adsorl) soluble proteins (Steinman and Silver, 1972; Steinman and Cohn, 1972). Cultured chicken heart cells adsorb so strongly that soon after the addition of HRP the surfwe ofthe cell is completely covered with the protein. The adsorbed protein cannot be removed by washing in physiological saline. In the course of the following day the adsorbed HRP moves into the cell, but little or no further protein is adsorbed in its place. In time therefore the cell surface becomes “clean” again. FIGS.5 and 6. Cultured chicken heart cells after exposure for 0, 2, 4, 8, 16, 32, and 64 hours to 0.1 mg/ml HHP. FIG. 5. Cells incubated in a diaminobenzidine-hydrogen peroxide medium to demonstrate HHP activity. FIG. 6. Cells stained for acid phosphatase to demonstrate lysosomes. 1 0 0 0 ~ .

300

M. HUNDGEN

FIG.7. Diagram siimmarizing the transport of HRP from the membrane through the digestive apparatus. Left, after adsorption of small amounts of' HRP. Right, after adsorption of large amounts of HRP. Dotted areas, HRP; N, nucleus; ER, endoplasmic reticulum; D, dictyosome. For description see text.

After its adsorption on the cell surface, the HRP enters the cultured cells b y micropinocytosis (arrow in Fig. 8). In cultured chicken heart cells, as in amebas (Wohlfahrth-Bottermann and Stockem, 1966) and HeLa cells (Riedel and Gross, 1968), pinocytosis proceeds steadily during interphase and to a slight extent also during mitosis. The rate of pinocytosis depends primarily on the concentration of the protein component of the nutrient solution (Cohn and Parks, 1967; Steinman and Silver, 1972). Only coated vesicles participate in the process of HRP endocytosis (Friend, 1969; Zacks and Saito, 1969; Gervin and Holtzman, 1972); these vesicles are thought to serve in the selective uptake of certain groups of substances, proteins in particular. The coated vesicles that contain HRP following endocytosis (1 in Fig. 7; arrow in Fig. 8) fuse either with one another (2 in Fig. 7) or with preexisting vacuoles (3 in Fig. 7; open arrow in Fig. 8), losing their membrane coat in the process. Such membrane fusion processes have been studied in detail by Palade and Bruns (1968), Cornell


301

(1970), and Haarmann (1970).A striking feature is that the preexisting vacuoles with which the coated vesicles fuse are always heterophagosomes (HPh in Fig. 8) and never lysosomes (Kessel, 1970). The HRP-containing heterophagosomes can attain a considerable size by fusion with micropinocytosis vesicles (4a in Fig. 7). These vacuoles are considered to function as storage elements. If the vacuoles remain small (4b in Fig. 7), in some cases they assume a crescent shape (4c in Fig. 6; Fig. 9). These smaller heterophagosomes can participate in autophagic processes (4b and 4c in Fig. 7 ) ,as discussed extensively below. In parallel with endocytotic food uptake, lysosomal digestive enzymes are synthesized at the ribosomes of the endoplasmic reticulum (ER in Fig. 7 )and thence move to the cisternae of the endoplasmic reticulum. There are many indications that in cultured chicken heart cells, in analogy with the pathway of secretions in gland cells, the Golgi complex (D in Fig. 7) participates in the transport of hydrolases from the endoplasmic reticulum to the food vacuoles. The Golgi cisternae contain, among other things, the readily demonstrable digestive enzyme acid phosphatase (Friend, 1969; Hausmann and Stockem, 1973; Weissenfels, 1973a). From the Golgi complex coated Golgi vesicles (=primary lysosomes; 5a in Fig. 7; arrow in Fig. 10) carry the digestive enzymes, all of them acid hydrolases, to the heterophagosomes (Shannon and Graham, 1971; Gordon, 1973). The latter fuse with primary lysosomes (arrow in Fig. 11) to become heterolysosomes (6 in Fig. 7). The transport of enzymes from the Golgi complex to the heterophagosomes cannot be definitely documented for cultured chicken heart cells by enzyme cytochemistry, since their Golgi vesicles give a negative reaction to the test for acid phosphatase. This fact can be explained by the presence of inadequate amounts of enzyme, particularly since the larger stages of fusion of primary lysosomes, which still bear the membrane coat (Fig. 12),show a positive reaction. A factor in favor of enzyme transport from the Golgi complex to the heterophagosomes is the close proximity of these two cytoplasm components. The path of the Golgi vesicles, identifiable by their membrane coat, can be reconstructed morphologically. Since these vesicles measure only about 450 x 450 x 700 A, they can be clearly distinguished from the larger (650 x 650 x 1000 A) pinocytotic vesicles. Coated vesicles also break off directly from the endoplasmic reticulum (5b in Fig. 7 ; arrow in Fig. 13). Morphologically, they are indistinguishable from the Golgi vesicles. No conclusions can be drawn from the present results about the extent to which these endoplasmic

302

M. HUNDGEN


303

reticulum vesicles participate in the transport of hydrolases. We regard such participation a s a possibility, since in certain protozoans 1963; Elliott, 1965),as well as in nerve cells, it has (Goldfischer et d., been shown that digestive enzymes are transported directly from the endoplasmic reticulum to the digestive vacuoles (Lane, 1968; Holtzman and Peterson, 1968). The content of heterolysosomes often comprises only the reaction products ofthe test for HRP or for acid phosphatase; but on occasion it can also include vesicles and smaller vacuoles (6 in Fig. 7). These vesicles arise by constriction of the lysosome membrane (arrow in Fig. 14); they have been found in many other preparations (Friend and Farquhar, 1967; Fedorko et nl., 1973). In shape and size they resemble Golgi vesicles, and for this reason have often been considered identical to them (de Duve and Wattiaux, 1966; Riedel and Gross, 1969). The vacuoles appearing in heterolysosome sections usually prove to lie membrane invaginations when serial sections are examined (arrow in Fig. 15), which comes as no surprise in view of the plasticity of lysosoines (Pfeifer, 1971). The HRP reaction products in the heterolysosomes decrease in quantity with time. This indicates digestion of the HRP. But it must be noted that this is an indirect conclusion; the method demonstrates strictly only the loss of HRP activity, and not the decomposition ofthe protein molecule itself. Among the contents of the lysosornes are all the enzymes that serve to split peptide bonds. Since the lysosome membrane is permeable to FIGS.8-17. Cttlturetl chicken heart cells after exposure to 0.1 mg/ml HRP. FIG.8. Uptake of HRP (b)b y foriiiation of coated pinocytosis vesicles (+) and their fusion

(3)with hcterophagosoines. 25,000x.

FIG.9. Invaginated Iieterophagosonie. 30,000x. FIG. 10. Coated vesicle (+) which is in continuity with a Golgi saccule. 85,000x. FIG. 11. Fusion of a small coated Golgi vesicle (+) and a vacuole (V). 6 5 , 0 0 0 ~ . FIG. 12. Large coated Golgi vesicle with dense deposits of acid phosphatase reaction product. 6 5 , 0 0 0 ~ . FIG. 13. Coated vesicle (4which ) is in continuity with endoplasmic rcticrtlum (ER). 85,000~. FIG.14. Bidding of ii vesicle from a vacuole membrane invagination (+), reminiscent of :I Golgi vesicle hut without a memlirnne coat. 65,000~. FIC:.15. Drcp invagination of ii vacuole membrane. 50,000x. FIG. 16. The heteropliagosome (HPli)-containing reaction product of HRP is surrounded by endoplasmic reticulum (ER). 25,000x. FIG. 17. Two autophagic vacuoles bounded b y ii single (AV,) or b y two (AV,) membranes. The space lwtween the two enveloping memhranes is filled with deposits of nucleoside-diphosphatase reaction product (+). 2 0 , 0 0 0 ~ .

304

M. HUNDGEN

molecules with a molecular weight below 230 to 200 (Ehrenreich and Cohn, 1969; Lloyd, 1971), the amino acids and dipeptides thus formed can escape from the lysosomes. The dipeptides are thought to be split into free amino acids eventually, in the cytoplasm (Gordon, 1973), and then to be available for the synthesis of endogenous proteins. The ability to break down exogenous proteins is particularly important for cells in tissue culture, since in uitro they must meet most of their amino acid requirements by this means (e.g., by the decomposition of fetal calf serum) (Riedel and Gross, 1969); in this respect they differ fundamentally from cells in uiuo. During the process of digestion the quantity of active HRP, and both the number and size of the vesicles and vacuoles in the heterolysosomes, decrease (7 in Fig. 7). These secondary lysosomes, together with the smaller heterophagosomes (4b and 4c in Fig. 7), become autophagic (8 in Fig. 7; Fig. 16).Production of the autophagosomes involves parts of the endoplasmic reticulum (ER in Fig. 16) (de Duve and Wattiaux, 1966; Ericsson, 1969; Schafer, 1972),which enclose the older heterolysosomes in particular, but also cell organelles surrounded b y cytoplasm (8 in Fig. 7). As a result, the autophagosomes are bounded b y two membranes (9 in Fig. 7). Between these two membranes, both the endoplasmic reticulum enzyme nucleoside diphosphatase (arrow in Fig. 17) and glucose-6-phosphatase (Schafer, 1972) can be demonstrated. In addition to the morphological data, the results of enzyme cytochemistry argue in favor of participation of the endoplasmic reticulum in the production of autophagosomes. It is not clear how the autophagosomes are supplied with digestive enzymes, for no current hypothesis gives a satisfactory explanation of the origin of the digestive enzymes that can be shown to occur in the autolysosomes. Older autolysosomes break down the inner enveloping membrane (10 in Fig. 7) and later also lose HRP and acid phosphatase activity. These older organelles are to be regarded as “postlysosomes,” and as such they can release their contents by exocytosis (Fig. 18).The exocytotic vacuoles of vertebrate cells in uitro are free of acid phosphatase and HRP (Steinman and Cohn, 1972; Walker et al., 1972). This ability of cultured chicken heart cells to defecate is exceptional, since most netazoan cells are unable to dispose of secondary heterolysosomes by exocytosis. As a result, residual bodies are frequently formed, which are regarded as a sign of aging (de Duve and Wattiaux, 1966). The exocytoses known to occur in vertebrate cells function chiefly in secretion and less commonly in excretion, or serve in the transcellular transport of substances (cytopempsis). Cultured chicken heart cells become capable of defecation through exocytosis


305

only b y the roundabout method of autophagosome formation (Gordon, 1973). Under normal conditions, however, exocytoses are rare. After unphysiological treatment-for example, after radiation with sublethal or lethal doses of x rays-the rate of autolysosome formation and of exocytosis is very high (Schafer, 1969, 1972; Neubert-Kirfel,

1970). 2. Uptake, Trunsport, und Digestion of Intermediate Amounts of HRP If larger amounts of adsorbed HRP accumulate, the cultured chicken heart cells make use of additional mechanisms of transport and digestion, for in contrast to other cell types they cannot make repeated use of the hydrolases in older secondary lysosomes b y fusion with new heterophagosomes. Consequently, digestive enzymes must be newly synthesized as needed and supplied to the heterophagosomes via primary lysosomes. Since synthesis de n o w of digestive enzymes cannot at first keep pace with the uptake of HRP (Straus, 1971) and/or the enzymes do not arrive at the heterophagosomes rapidly enough, the number of HRP-containing heterophagosomes steadily increases. But this increase proceeds more slowly than would be expected, because of a special mode of autophagy in the course of which the number of heterophagosomes is reduced. The uptake of large quantities of HRP by coated micropinocytotic vesicles (1 in Fig. 7) and their fusion with other pinocytotic vesicles (2 in Fig. 7) or preexisting vacuoles to form larger HRP-containing heterophagosomes (3 in Fig. 7) proceed as with the uptake and transport of smaller quantities of the enzyme (see 1 to 3 in Fig. 7). The special formation of autophagosomes, which takes place particularly often after 8 and 16 hours of exposure to the protein, begins with an indentation of the heterophagosomes, which gives them a crescent shape (4c in Fig. 7; Fig. 9).This process continues until the former vacuole is reduced almost to a shell consisting of two membranes (V in Fig. 7; AV2 in Fig. 19). These invaginated structures are usually situated near the Golgi complex. The HRP content of the sickle-shaped lumen of the vacuole identifies these structures as heterophagosomes. Other heterophagosomes, and probably primary lysosomes as well, enter this cavity (VI in Fig. 7). Eventually, the edges of the invagination meet and the membrane closes over, separating the cavity and its contents entirely from the ground substance. The autophagosome or autolysosome thus formed is therefore bounded by a wall formed of two membranes, with regions that show HRP activity (VII in Fig. 7). The autophagic vacuoles formed by the endoplasmic reticulum are also en-

306

M. HUNDGEN


307

closed by a two-membrane layer (AV, in Fig. 19). In this case, however, the space between the two membranes is always free of HRP. In time the inner membrane degenerates (10 in Fig. 7), and the autolysosome ages and eventually expels its contents by exocytosis. The origin of auto1ysosoines with only one enveloping nienibrane is unidentifiable, whether from heterophagosomes or from parts of the endoplasmic reticulnni.

3. Uptake, Trcirisport, iiiitl Digestion of Unphysiologiccilly L u g e Aniozcrits o f H R P Immediately following application of HRP doses beyond the physiological range (0.1% in the nutrient medium), the cell surface adsorbs large quantities of the tracer protein. Since the cells cannot reverse the process of adsorption, the entire amount must be disposed of by endocytosis. Consequently, as little a s 1 hour after application the cells contain more vacuoles holding HRP than after 32 or 64 hours of exposure to the protein at a concentration of 0.01% in the nutrient in edi urn. After a 2-hoar exposure to HRP, pathological changes appear in the dictyosomes. The Golgi complex region is still recognizable, but the cistemae 25 grains) in the germinal centers may be attributed to a more rapid rate of turnover of the bursal cell population that migrates to splenic germinal centers. Intrabursal labeling of 18-day embryos, neonatal chicks, and 6-week-old chickens revealed labeled cells in the thymus 24-48 hours after Td€b3H injection (Hemmingsson and Linna, 1972; Hemmingsson, 1972a). The labeled cells first appeared in the medulla and then in the cortex. No transport of labeled cells to the thymus or spleen was observed following intrabursal labeling of 14-week-old White Leghorns (Hemmingsson and Linna, 1972), even though the specific activity (counts per minute per milligram of DNA) of the bursa was quite high. At this age the bursa of White Leghorns is in the late stage of regression (Glick, 1956; Beach et al., 1934), which is a period characterized by increased metabolic activity (Kulkarni et al., 1971, 1972) and extensive loss of lymphocytes. Also, the chicken may retain a bursal epithelium throughout its life, and the persistence of this epithelium may be associated with its improved survival (McConnachie and Ruth, 1974). These data demonstrate that the period of bursa regression is an active period and should not be considered an event without biological significance. While thymic cells have not been found to migrate to the bursa (Linna et al., 1969; Hemmingsson 1972b), the thymus’ influence on the cellular makeup of the bursa cannot be ignored (Jankovii. and Isakovik, 1964) (see Section V,B,2 for more detail). Thymic cells labeled exogenously with a d e n ~ s i n e - ~(Durkinet H al., 1972), or in situ with t h ~ m i d i n e - ~(Hemmingsson, H 1972b), localized in splenic red and white pulp but not in germinal centers. Unlike the bursa, which appeared to cease exporting lymphocytes at 14 weeks of age, the thymus continued to send lymphocytes to the spleen at this age (Hemmingsson, 1972b). In situ labeling (TdR-3H)of the bursa of 18-day embryos, neonatal chicks, and 6- and 14-week-old chickens failed to reveal labeled cells in the bone marrow (Hemmingsson 1972a; Hemmingsson and Linna,

THE BURSA AND IMMUNOGLOBULIN

357

BONE MARROW

t

WOODS AND LINNA, 1965

- - - - _ _ _ _',_ _HEMMINGSSON AND LINNA.1972 I

L------

MOOREHEAD et al., 1974

FIG. 7. The traffic of lymphoid cells among the tissues of the lymphomyeloid complex. Chromosome marker studies have revealed a migration of bursa1 cells to the bone marrow (Weber, 197213).-, Cells labeled in situ;all others were exogenous injections of cells.

1972; Back and Linna, 1973). However, labeled cells appeared in the bone marrow of 9-day-old chicks following an intrathymic injection of TdK3H (Hemmingsson, 1972b) and in 6-week-old antigen-stimulated chickens (Back and Linna, 1973). These data suggest that bursal cells do not traffic to the bone marrow (Fig. 7); yet, as we will see in Sections IV,B and V,D, the bone marrow contains a population of cells considered to be B or bursal cells. The question may now be asked, Are all B cells bursa-dependent or are some bursa-independent? This question is central to the discussion of Sections IV,B and V,D. Intravenously injected aden~sine-~H-labeled bone marrow cells from 66-day-old donors trafficked to the follicular and periarteriolar lymphatic tissues of the spleen of 8-day-old recipients, but did not enter the bursa (deKruyff et al., 1975). Chromosomally marked 19-day embryonic bone marrow cells injected IV into 4-day-old chicks populated the marrow and thymus (Weber, 1975). Furthermore, birds made chimeric by IV injection of 45 x lo6 chromosomally marked bone marrow cells challenged with Brucella abortus and sampled over a 3.5week period failed to exhibit migration of the donor bone marrow cells to the bursa. Once again a conflict is found when we compare data from in situ labeling with data from the injection of labeled cells. Back et al. (1973)failed to note germinal center labeling of the spleen following an intra-bone-marrow injection of TdK3H, but reported labeled cells in the spleen and bursa (Back, 1972). Also, they found heavily labeled cells in the thymus. The research that identifies labeled bursal, thymic,

358

BRUCE GLICK

and bone marrow cells in the spleen is significant, for it demonstrates that thymic and bursal cells intermingle in the classic areas of the spleen, thus offering visual evidence of their interaction in the immune response.

111. Bursa Kinetics A. GROWTH The literature through 1952 was in agreement that maximum bursa growth is attained by 4 months of age, followed by regression (Jolly, 1913, 1915; Schauder, 1923; Taibel, 1941; see Glick, 1960a, for further discussion). At that time we initiated experiments which emphasized the study of bursa growth. Our data revealed that bursa regression occurred earlier than 4 months of age and was influenced b y the breed and strain of chicken (Glick, 1956, 1960a; Landreth and Glick, 1973). The most characteristic period of bursa growth we have observed occurred between hatch and 3 weeks of age (Glick, 1956, 1960a; Landreth and Glick, 1973). While occasionally the absolute weight of the bursa exhibited a marked increase beyond the third week, that is, between 3 and 4 weeks and 4 and 5 weeks of age, the relative bursal values (bursdbody) markedly increased from hatch to 3 weeks of age and then either remained constant for several weeks or declined steadily. Other workers have confirmed a rapid growth period for the bursa occurring up to the third or fifth week after hatching (Dieter and Breitenbach, 1968; Vriend et al., 1975; Wolfe et al., 1962). The growth of the bursa during this period may not be due to lipid storage, but may be a result of protein storage (Stefoni et al., 1971). It should be emphasized that the observation of a restricted rapid growth period for the bursa was critical to our implicating the bursa in the control of antibody production, since other workers investigating bursa function bursectomized after this period (Glick, 1955). Our early bursectomy experiments were always conducted within the period of rapid bursa growth. The rapid growth period of the bursa may be followed by immediate regression (Glick, 1956, 1960a; Landreth and Glick, 1973), or a period of continued absolute growth with regression occurring well before 4 months of age (Glick, 1956,1960a; Hoffmann-Fezer and Lade, 1972; Vriend et al., 1975; Wolfe et al., 1962; Dieter and Breitenbach, 1968). This plateau period is not always predictable, since it depends on the strain of chicken and on environmental conditions. This is best illustrated with data from White Leghorn and Nigerian strains raised in Nigeria (Aire, 1973,1974).The Nigerian strain’s bursa


359

peaked at 14 weeks and then regressed both on an absolute and relative scale. However, the White Leghorn’s bursa continued to grow up to 12 weeks (1.1gm) and then regressed until 20 weeks, at which time it increased to 2.8 gm and attained a peak weight of 3.5 gm by 28 weeks. The apparent regeneration of bursa growth in the White Leghorn at 20 weeks of age could reflect a regeneration of lymphoid development. If such is the case, one may expect to detect increased numbers of B cells migrating to the thymus between 20 and 28 weeks of age. If this is true, then, discovering what triggered the regeneration of the bursa may lead to a possible clue concerning how one could rejuvenate humoral immunity. Our observations have been that, while the bursa may experience slight weight gains up to 2 months of age, its cytoarchitecture at this time reveals loss of medullary lymphocytes, suggesting bursa regression. Therefore studies depicting several periods of bursa growth should be accompanied by histological data for the bursa to ascertain the true cellular picture at this time. CONTROL OF BURSAGROWTH B. EXPERIMENTAL Bursa growth has been reported to be inversely related to growth of the testes (Riddle, 1928; Jolly, 1913; Glick, 1956; Dieter and Breitenbach, 1968).This suggested regressive influence of the testes on bursa growth was confirmed after recording larger bursae in birds following caponization (Jolly and Pezard, 1928; Glick, 1957a; Wolfe et al., 1962). The bursa of young birds is known to be compromised in the presence of exogenous injections of male hormone (Kirkpatrick and Andrews, 1944; Glick, 1957a, 1970b; Panda, 1961; Zarrow et al., 1961; Dieter and Breitenbach, 1970, 1971; Schomberg et al., 1964). Meyer’s group (1959; Aspinall et al., 1961; Rao et al., 1962) evaluated sex steroid influence on the embryonic bursa. In their initial article 0.63 mg of 19-nortestosterone, injected into the pointed end of an egg on the fifth day of incubation, was reported to abrogate normal development of the bursa (Meyer et al., 1959). Other androgens like androsterone, androstane-3,17-dione, methylandrostene diol, and dehydrotestosterone were highly effective in inhibiting embryonic development of the bursa (Aspinall et al., 1961). Administering 19nortestosterone between the fifth and seventeenth day of embryonic development arrested further differentiation of the bursa and prevented lymphoid development if injections took place prior to the eleventh day (Rao et al., 1962). It was concluded that the androgens interfered with mitosis, since DNA levels in the bursa were reduced in the presence of the androgens. Meyer’s group reported that the

360

BRUCE GLICK

thymus and spleen were not significantly influenced by the injection of androgens. An alternative method for introducing steroids into the environment of the embryo was patented b y Seltzer (1956). The steroid is dissolved in a solution containing a surface-active agent or ethyl alcohol (EA), and the egg is then dipped into this solution. He claimed functional sex reversals were produced by this method. In an attempt to duplicate his results we dipped the pointed end of an egg (2.5 cm) into EA solutions containing varying concentrations of testosterone propionate (TP)and diethylstilbesterol (DES). While we were unable to confirm Seltzer’s claim for functional sex reversals, we did observe the elimination of the bursa in chicks hatched from eggs dipped for 5 seconds on the third day of incubation in 2 gm% solutions (18OC) of TP (Glick and Sadler, 1961; Wilson and Glick, 1966a). Thymic size was not significantly modified (Glick, 1961). In the injection method one can be certain of the amount injected, but not necessarily of the amount absorbed (Meyer et al., 1959). How much TP enters the egg in the dipping (TPD) method? Since the chick’s comb is accepted as the ideal tissue for assaying androgenic potencies of unknowns, we inuncted it with albumin removed from eggs dipped in varying concentrations of T P solutions. In three of five bioassays 54 pg of TP was found in the albumin 20 minutes after dipping the eggs in 0.64 gm% TP. The mean for five experiments was 81 p g (Wilson and Glick, 1966b). Measuring the volume of EA removed per egg suggested that between 74 and 111p g of TP entered the egg after dipping in EA solutions containing 570-840 mg TP/100 ml EA (Sato and Glick, 1964b). In an experiment utilizing testosterone-4-14C(T-4-14C), we reported that 52 pg of the radioactive testosterone was localized in the albumin 20 minutes after its application to the shell surface (Wilson et al., 1971). More significant was the increase in T-4-14C found in the albumin with time, suggesting that testosterone applied to the shell did not pass directly into the albumin but was released slowly from some site, for example, the shell membrane. A variety of factors such as muscular fatigue (Garren and Shaffner, 1954a,b), infection with typhoid (Garren and Barber, 1955), cold (Garren and Shaffner, 1956), and restraint of chicks (Newcomer and Connally, 1960) produced an enlarged adrenal and a reduction in the mean weight of the bursa. The inverse relationship between the bursa and the adrenal gland suggested by the aforementioned references was revealed in a statistical study (Glick, 1960a) which reemphasized previous work and data presented by later workers all demonstrating bursa regression in the presence of adrenal extracts (Selye, 1943),cortisone and corticosterone (Glick, 1957a,b, 1959, 1960c, 1967, 1972;


36 1

Zarrow et al., 1961; Bellamy and Leonard, 1964; Dieter and Breitenbach, 1970, 1971; Sato and Glick, 1970), and ACTH (Siegel, 1961; Breitenbach, 1962; Sato and Glick, 1964a). However, the dynamic nature of the bursa-adrenal relationship may best be illustrated b y a series of selection experiments initiated in our laboratory in 1957. We developed by family selection two lines of chickens which differed significantly in bursa size at hatch and at 1,3, and 5 weeks of age (Glick and Dreesen, 1967). The only parameter for our selection was bursa size. A comparison of adrenal size between the large-bursa (LB) line and the small-bursa (SB) line revealed a significantly larger adrenal in the SB line. Our selection experiments magnify the interrelationship of an endocrine, the adrenal, and a member of the lymphomyeloid complex, the bursa. One may suspect from previous experience that the smaller bursa of the SB line resulted from a more active adrenal gland. Tissue culture experiments revealed that bursal cell replication of SB line embryos or chicks was improved in the presence of foreign or LBline serum; whereas bursal cell replication from the LB line was significantly limited b y SB-line serum (Kulkami et al., 1971). Therefore the serum of SB-line chickens contained a substance that inhibited bursal replication. Since corticosteroids are regressive to bursa growth and since the adrenal of SB-line chicks is larger than that of LB-line chicks, one may conclude that the inhibitory substance in the serum of SB-line chicks was a corticosteroid. Furthermore, cell replication of the bursal lymphocytes from the LB-line chicks cultured in Basal Medium Eagle with or without calf serum exceeded that of SB-line bursal cells. Therefore the greater growth potential of LB-line bursal cells demonstrates an intrinsic growth difference between the bursa of the two lines.

IV. Characterizing Bursa1 Lymphocytes A. METABOLICACTIVITY Having characterized bursa growth into three stages, rapid, plateau, and regression, and its modification by steroids (see Section III,B), our laboratory became interested in the metabolic activity of bursal and thymic lymphocytes during these three growth periods. Changes in the metabolic activity of bursal lymphocytes in part reflect the bursa’s response to its internal environment. The objective of this section will be to compare the bursa’s metabolism with that of the thymus during the well-characterized periods of bursal development.

362

BRUCE GLICK 100

8 f

-

90 -

f

8070-

10 HotchI

2

3

4

5

AGE

6

7

8

9

10

II

12

13

14

15

IN W E K S

FIG. 8. A comparison of the oxygen consumption between bursal (solid line) and thymic (broken line) cells during the periods of rapid bursa growth (0-4 weeks), plateau (4-10 weeks), and regression (after 11 weeks). (Modified from Kulkarni et al., 1971, 1972, by courtesy of Academic Press and Marcel Dekker Journals.)

1. A Comparison of Bursal and Thymic Lymphocytes Bursal and thymic lymphocyte suspensions were obtained b y gentle disruption of tissue fragments (Mueller et al., 1971). The supernatant was discarded, and the packed cells were resuspended in Ringer's solution. The cell suspensions were transferred to a YSI Model 53 biological oxygen monitor (37°C) in which microliters of oxygen consumed per hour was determined. The sample contained 2 ml of cell suspension (100 x lo6 cells/ml), 0.3 ml of chicken serum, and 0.1 ml of glucose (30 p M ) . The oxygen consumption of bursal cells increased significantly between 1 and 4 weeks of age (7-30 p1 of oxygen consumed per hour), the period of rapid bursa growth, remained stable between 4 and 10 weeks (30-27 p1 of oxygen consumed per hour), and then significantly increased at the time of regression, between 11 and 15 weeks (27-83 pl of oxygen consumed per hour) (Fig. 8). A significantly greater metabolic activity of bursal lymphocytes than of thymic lymphocytes was apparent after 1 week of age (Kulkarni et al., 1971, 1972; Kulkarni and Glick, 1972) (Fig. 8). We had anticipated such an oxygen consumption curve for the bursa during its rapid growth period. However, the accelerated consumption of oxygen by the bursa during regression was unexpected. The latter might reflect a catabolic process with, for example, the disruption of lymphocytes and exposure of mitochondria to our system and an anabolic response to changes in the environment followed b y a final recruitment (in


363

vivo) of virgin bursal cells for the immunoglobulin (Ig) system. The significantly greater cell volume reported for the bursa over the thymus (Peterson and Good, 1965; Sherman and Auerbach, 1966) is consistent with the higher metabolic rate of the bursa. The modal cell volume differences are especially apparent during embryonic development (Sherman and Auerbach, 1966), at a time when the oxygen consumption of the bursa is two to three times greater than that of the thymus (Kulkarni et al., 1971, 1972). Our metabolic studies of the bursa and thymus confirmed and extended Warner’s (1965)autoradiographic data indicating that bursal cells incorporated in vitro two times more TdR-3H than thymic cells. Within 48 hours after single intravenous injections of TdR-3H were given to 1-day-old, 9-day-old, and 6- and 14-week-old chickens, scintillation counts revealed more TdR3H uptake by bursal tissue than by thymic tissue (Hemmingsson and Linna, 1972; Hemmingsson, 1972b; Back, 1972,1973). In 1973 we initiated a study to ascertain the optimum route of application of TdR-3H to the embryo (Glick and Schwarz, 1975).The application of 200 pCi of TdFb3H to the air cell (AC) or allantoic cavity of 12-day embryos revealed significantly more TdFb3H in lymphoid tissue 1-4 days after the AC application. Peak concentrations occurred in the 14-day embryo or 2 days after TdFb3H administration. Spleens incorporated significantly more TdR-3H at all ages than the thymus or bursa, while bursal labeling exceeded thymic labeling at all ages. Our data agree with those of Hemmingsson and Alm (1973), who injected TdR-3H intravenously into 16-day embryos and revealed 1 day later by autoradiography more grains over bursal than over thymic cells. In our study (Glick and Schwarz, 1975), in vitro incubation for 15 minutes of approximately lo7 bursal and thymic cells with 50 pCi of TdR-3H revealed three times as many labeled bursal cells as thymic cells (Table I). It is apparent that the thymus possesses relatively more small lymphocytes than the bursa; yet, at this time approximately 44% of the bursal lymphocytes are small. The presence of T cells in the thymus and B cells in the BF of chickens offers the investigator a unique opportunity to compare the turnover rate of pure populations of T and B cells without having to resort to i n vitro separations or in vivo manipulations. While numerous experiments have described the function of thymic and bursal lymphocytes, few have dealt with the development of these cells within their individual primary lymphoid tissue. There are no published reports on the existence of long-lived and rapidly turning over (shortlived) lymphocytes within the thymic, bursal, and bone marrow environments. Preliminary data collected in our laboratory have re-

364

BRUCE GLICK TABLE I BUHSALAND THYMICCELLS FHOM ~-DAY-OLD CHICKS AFTER A 15-MINUTE INCUBATION IN 50 p c I O F TDR-3H"*b

GRAINCOUNTS

OVER

Lymphocyte Small Bursa Total percent labeled = 43.8% Thymus Total percent labeled = 15.4%

" Reproduced from

Medium

Large

Dead

20/220 (9.09%) 157/215 (73.0%) 27/40 (67.5%) 15/15

30/385 (7.79%)

40/115 (34.7%)

010

3/3

Glick and Schwarz, 1975, by courtesy of Marcel Dekker, Inc.

* Values shown refer to: number labeled/total cells counted. Percent of cells labeled is given in parentheses. All slides were exposed for 14 days, and bursa1 and thymic cells were labeled with 25 or more grains.

TABLE I1 GRAINSOVER SMALL(S), MEDIUM (M), AND LARGE(L) LYMPHOCYTES FROM THYMUS AND BONE MARROW AFTER A SINGLE APPLICATION OF 200 pCI TDR-3H (SP. ACT. 6.7 CI/MMOLE TO THE AC OF AN 18-DAY EMBRYO After 3 hours Number of grains Thymus 0-4 5-9 10-19 20-29 30-39 Above 40 Bone marrow 0-4 5-9 10-19 20-29 30-39 Above 40

L

M

S

After 24 hours

After 48 hours

After 72 hours

L

L

L

M

M

0 15 0 0 0 1 3 0 1 1 0 0 3 59

385 2 1 0

0 0 0 0

0

0

4

0 0 0 0 0 0

1

4 3 0 0 0 7

6 3 0 0 0 2

S

M

S

S 12 0 0

0

0 41 0 0 1 0 0 0 0 0 9 449

0 0 0 0 0 0

0 27 0 0 0 5 0 3 0 0 15 450

0 0 0 0 0 0

0 0 0 0 0 0

488

0 0 0 0 0 0

3 8 0 0 0 1 1 0 1 8 0 4 14 7

0 0 0 0 0 0

0 6 0 0 0 0 0 3 0 0 9 40 6

0 0 0 0 0 0

0 0 1 6 4 39

10 0 4 1 4 35

0 0


365

vealed that the lymphocyte pool of the thymus, bursa, spleen, and bone marrow may be saturated in time following a single application of Td€b3H (200 pCi) to the AC. A sample of these unpublished results is presented in Table 11. These data reveal the presence of small lymphocytes in the bone marrow as early as 18 days of embryonic development, an observation not made previously (Lukii: et al., 1973). B. RECEPTORS The metabolic studies that appear to differentiate between bursal and thymic lymphocytes do not allow the investigator to identify visually the lymphocytes originating from these specific compartments. Forget et al. (1970) were the first to demonstrate that cells originating from the thymus and bursa could be identified by antisera developed against their respective lymphocytes. Antibursal (anti-B) serum identified B cells in peripheral lymphoid tissue and was capable of inhibiting hemolytic plaque formation (Potworowski et al., 1971a). Antithymus (anti-T) serum identified peripheral white blood cells (Potworowski et d.,1971b; Ivanyi and Lydard, 1972; Rouse and Warner, 1972a; Wick et al., 1975). Also, receptors for a specific antibody distinguish between B and T cells, for example, B cells and not T cells possess receptors for antipolymerized flagellin (Basten et al., 1972). Hudson and Roitt (1973) produced anti-T and anti-B sera in rabbits and absorbed the antisera with liver membrane and erythrocytes. This was followed by absorption of anti-T serum with B cells and anti-B serum with IgG and thymic cells. Since most of the activity of the anti-B serum toward B cells was removed by thymic absorption, it was concluded that the thymic cell preparation possessed B cells. The presence of B cells in the thymus was demonstrated by incubating thymic lymphocytes with anti-B serum and then adding fluoresceinconjugated goat antirabbit immunoglobulin. Also, anti-B serum, made in turkeys (Isakovib et al., 1975) and properly absorbed, identified between 2 and 8% B cells in the thymus of 2- to 3-week-old chickens by indirect immunofluorescence (IF) (Wick et al., 1973; Albini and Wick, 1974).These reports confirmed earlier work on the presence in the thymus of B cells, and that thymic lymphocytes may contain an antigen common to both thymic and bursal lymphocytes (Potworowski et al., 1971a; Potworowski, 1972; McArthur et al., 1971; Malchow et al., 1972; Jankovii: et al., 1970, 1975a). The percentage of embryonic thymic cells binding antibursal serum peaks at 13 days (ca. 50%) and then rapidly declines to less than 10% by day 21 (Albini and Wick,

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BRUCE CLICK

1975). One must ask, What are the specific characteristics of the embryonic thymus cells in question and where do these cells migrate? Chicken T antigen is found not only associated with thymocytes but also with the reticuloepithelial cells of the thymus, as evidenced by an absorption of anti-T serum with a lymphocyte-free thymus preparation, which results in the loss of anti-T activity (Potworowski et aZ., 1973). Perhaps the cell stroma of the bursa also possesses the B-cell antigen. Also, it has been reported that a soluble thymic factor from the thymic stroma converts null cells in the bursa to T cells which are capable of eliciting splenomegaly in White Leghorn embryos (Teodorczyk and Potworowski, 1975). The anti-T and anti-B sera stained 80-100% of their respective lymphocytes (Hudson and Roitt, 1973;Wick et aZ., 1973; Albini and Wick, 1974). It was also revealed that more than 50% of the lymphocytes in the spleen are T cells. The possibility that lymphocytes exist in chickens that are neither T nor B cells does not receive encouragement from the data of Hudson and Roitt (1973),who revealed 58%and 41%T and B cells, respectively, in the spleen, and 62-64% T cells and 28-35% B cells in the peripheral blood (PB). However, Wick et al. (1973) and Albini and Wick (1974) reported that approximately 13%of splenic lymphocytes did not bind to anti-T or anti-B serum. These data suggest the presence of null lymphocytes or a reduced density of determinants on the T and B cells of peripheral lymphoid tissue. The suggestion that immunoglobulin-specific determinants (ISD) are present in thymus-independent lymphocytes (Rabellino et aZ., 1970; Pernis et al., 1972) was tested in chickens by exposing PB lymphocytes to fluorescein-labeled antibodies specific for heavy-chain determinants of IgM or IgG (Kincade et al., 1971). Normal chickens had 13-22% labeled PB lymphocytes, while no positive PB lymphocytes were detected in chickens made agammaglobulinemic by embryonic injection of anti-p serum followed by surgical bursectomy at hatching. Fluoresceinated antichicken Ig serum revealed ISD on scattered bursa1 cells of 14-day embryos (Kincade and Cooper, 1971; Albini and Wick, 1973).The frequency of ISD-positive cells increases thereafter and peaks at 3 weeks of age. This correlates well with the period of most rapid bursa growth (Glick, 1956,1960a).The number of ISD-positive cells is less in subsequent weeks, ranging between 60 and 80% (Albini and Wick, 1973; Rabellino and Grey, 1971). ISDpositive cells appeared initially in the thymus of 17-day embryos, where they represented less than 5% of the lymphocyte population. At 14 and 26 weeks of age the ISD-positive cells in the thymus increased to approximately 10 and 20%, respectively. Our autoradio-


367

graphic studies (Glick et d., 1975) complement the IF studies and extend them to include the identification of different-sized lymphocytes. Rabbit antichicken Ig was labeled with "'I and incubated with bursal, thymic, splenic, and bone marrow lymphocytes. There were 1000 lymphocytes counted in each tissue, with the exception of the bone marrow where 100 lymphocytes were counted. The frequency of lZ51-labeledlarge, medium, and small lymphocytes in the bursa of 4week-old chickens was 94.5, 78.5, and 58.8, respectively, while the frequency for these cells in the thymus was 2.7,0.89, and 0.14, respectively. In older birds, 21 weeks of age, the frequency of lZ5I-labeled lymphocytes in either the bursa or thymus was similar to that in the 4week data (B. Glick, W. D. Perkins, D. S. V. SubbaRao, and F. C. McDuffie, unpublished data, 1976). However, the frequency of lZ5Ilabeled medium and small lymphocytes in bone marrow increased with age (Glick et ul., 1975): at 4 weeks, 4.2 and 1.7, respectively; at 8 weeks, 17.1 and 23.6, respectively; and at 21 weeks, 73.8 and 19.4, respectively. The last-mentioned are unpublished data (Glick et al., 1976). Our bone marrow percentage for "'I-labeled cells agrees with the I F study of Hudson and Roitt (1973)who recorded that, while 27% of bone marrow cells from a 5-week-old chicken were positive following exposure to an anti-B serum, only 2% of the bone marrow cells bound an anti-light-chain serum. Albini and Wick (1973) reported (1) the presence of cells in the bone marrow of 14- to 16-day embryos that stained with fluorescein-conjugated antichicken Ig, and (2) a marked increase in ISD-positive cells (10-25%) by 1 week of age. While these cells may have been medium-sized lymphocytes, it is doubtful that the positive cells in the bone marrow of the embryo were small lymphocytes. Lukii: et nZ. (1973)failed to identify small lymphocytes in the embryo, and we have not found small lymphocytes in embryo bone marrow prior to 17 days ofembryonic development (B. Glick, unpublished data). Also, the high percentage of ISD-positive cells in the bone marrow after hatch may include cells other than lymphocytes. The cells with ISD increase rapidly after hatching in the spleen but represent less than 50% of the total lymphocytes (Albini and Wick, 1973; Rabellino and Grey, 1971). Incubating spleen cells with lZ51-labeled anti-light chain, /I chain, and y chain produced 22.0, 24.0, and 11.8% labeled lymphocytes, respectively (Bankhurst et a1 ., 1972). Our autoradiographic data for spleens of 4-week-old birds were slightly lower in that the percent of labeled large, medium, and small lymphocytes ranged between 9 and lo%, but increased to about 16% by 21 weeks of age. While ISD are useful in identifying B cells, the T cell of the chicken does possess light chains (Theis and Thorbecke, 1972;

368

BRUCE GLICK

Szenberg et al., 1974), p chains, and a 40,000-dalton molecule (Szenberg et al., 1974). There are suggestions that the T-cell Ig of peripheral blood is bursa-dependent and therefore is a B-cell product (Hudson et al., 1975). Cells with ISD increase rapidly after hatching in the cecal tonsil and gland of Harder, peaking at 14 weeks in the cecal tonsil (70%positive cells), and at 6 weeks in the gland of Harder (90%positive cells) (Albini and Wick, 1973). Interestingly, the ISD-positive cells in the cecal tonsil and gland of Harder declined after 14 weeks, at a time when the bursa was regressing. The gland of Harder revealed (1)a larger cell and a higher percent of heavily labeled cells (ISD-positive) than other tissues, and (2)a more asymmetric distribution of label on cell surfaces. The suggestion by Albini and Wick that the labeled cells are plasmacytoid has been verified by our autoradiographic studies. Only an occasional lymphocyte was labeled with 1251-labeledantichicken Ig, while more than 80% of the plasma cells in the gland of Harder were heavily labeled (B. Glick, W. D. Perkins, D. S. V. SubbaRao, and F. C. McDuffies, unpublished data, 1976). Plasma cells in the gland of Harder may be distinctive cell types, since mammalian plasma cells do not generally reveal ISD (Pernis et al., 1972). However, in our experience plasma cells in chicken bone marrow also exhibit ISD, which suggests either a population of plasma cells only recently removed from their B-cell progenitor or that the plasma cell of chickens is not identical to the cell type found in mammals. The former possibility is an unlikely explanation in accounting for the large number of plasma cells in the gland of Harder, since only an occasional gland of Harder lymphocyte bound the lZ5I-labeledantisera. An alternative explanation, that B cells fail to migrate to the gland of Harder, would then beg the question, From what cell does the plasma celI in the gland of Harder originate? Our thesis that the bursa is not a sine qua non for IgM production suggested an extra bursal site for Ig production. The conditions within our TPD birds might support a proliferation of plasma cells in a restricted area of the lymphomyeloid complex, namely, the gland of Harder, where classic B cells do not appear to reside, thus suggesting a uniqueness of either the cell type or the environment within the gland of Harder. Thymic and bursal lymphocytes bound fluorescein-labeled concanavalin A (FITC-Con A), but 80%of the thymic cells experienced capping of the FITC-Con A while less than 5% of the bursal cells capped (Sallstrom and Alm, 1972).These data suggested a cell membrane difference between T and B cells. Bona and Anteunis (1973) reported that thymic and bursal lymphocytes were negative and positive, respectively, following staining with collodium lanthanum. However,


369

thymic cell membranes but not bursal cell membranes were positive for phototungstic acid (PTA). These data revealed the presence of pglucosidase-sensitive glycoprotein (lanthanum-positive) and a deficiency of sialoproteins (PTA-negative) on the bursa membrane, while T-cell membranes were positive for sialoproteins. Taking advantage of charge differences on the lymphocyte membrane, Droege and coworkers (Droege, 1971; Droege et al., 1972a,b, 1974; Zucker et al., 1973) revealed the existence of subpopulations of lymphocytes. The electrophoretic mobility (EM) of lymphocyte populations (98-99% pure preparations) from PB and spleen reflected a single pool of cells within each population, while the PB lymphocytes migrated slightly faster than the spleen population (Droege et al., 1972a). Differential centrifugation of thymic cells (4-month-old chickens) yielded a major fraction A (300g) and a minor fraction B (750 g). Fraction A contained 10 times more cells than fraction B and exhibited two populations of cells on the basis of EM, slow and fast. The slow fraction A was similar to fraction B, and both were absent in surgically bursectomized (BSX) irradiated birds (Droege et al., 1972a). These data demonstrated that one can differentiate between T and B cells within the thymic environment. However, the EM values for PB and splenic lymphocytes were greater than for the fastest moving cell population within the thymus, thus demonstrating that this technique cannot differentiate between T and B cells in peripheral lymphoid tissue (Droege et al., 1972a) and that these cells may change their physical-chemical characteristics once they migrate to the periphery. The latter thesis is entertained by others (e.g., Weber, 1973; Toivanen et d., 1972a,b). Neuraminidase reduced the E M in all lymphocyte populations, suggesting the involvement of sialic acid (Droege et al., 1972a).These results contrast with the staining studies of Bona and Anteunis (1973) and require further work on the chemistry of the membrane coat. Bursa1 lymphocytes labeled with W r and injected into 10-week-old White Leghorns accumulated in the spleen at a low frequency of 3, but the thymus subpopulation (300-750 g ) , which has an EM similar to that of bursal lymphocytes, accumulated in the spleen at the highest frequency, 18 (Droege et al., 1972b). However, bursectomy plus irradiation equalized accumulation of the 25-300 g and 300-750 g thymic fractions in the spleen. These data revealed elimination of bursal lymphocytes from the 300-750 g fraction and an overlap of bursaindependent lymphocytes in this fraction. Also, the absence of a bursa-dependent subpopulation from the spleen of 6-month-old chickens revealed its dependency on the bursa which has involuted by this age (Droege et al., 197213). The density gradient separation experiments of Tamminen et al. (1973) reaffirm the migration of B

370

BRUCE GLICK

cells to the thymus and suggest a maturation of T cells within the thymic environment. Finally, functional differences in tlie receptor response between T and B cells have been revealed by employing lectins and lipopolysaccharides (LPS). The initial report on the chicken disclosed that the addition ofphytohemagglutinin (PHA)to bursal, thymic, and splenic cells stimulated the uptake of TdR-3H in only the last-mentioned two cell populations over a 48-hour period (Weber, 1966). Surgical thymectomy but not surgical bursectomy (SBSX) or hormonal bursectomyinjection of TP into eggs on the sixth or seventh day of incubationreduced blast formation and thymidine uptake of PB lymphocytes in the presence of PHA (Greaves et al., 1968). Similar results have been reported with Con A (Toivanen and Toivanen, 1973a). These reports and others (Kirchner and Blaese, 1973) allow one to differentiate between chicken T and B cells on the basis of their response to PHA and Con A. However, they do not rule out the possibility that the responding lymphocyte is not a B cell or has not been conditioned by a B cell. One should verify that the test bird is agammaglobulinemic (Mancini test) and lacks B cells (fluorescent assay or 9 - l a b e l e d antiIg). Even these criteria are not sufficient, since timing is most important, that is, one must eliminate the possibility of a B- and T-cell interaction. LPS may be employed to differentiate between T and B cells in peripheral lymphoid tissue but not within the thymic or bursal environment. Chromosomally marked B cells from 19- to 20-day embryos were transferred to agammaglobulinemic birds and harvested 4 and 12 weeks later from the spleen. These B cells responded to LPS from Escherichia coli, but B cells in the bursa failed to respond (Weber, 1973). Similar results were reported by Tufieson, and Alm (1975a) who concluded, like Weber, that a maturational change may be necessary for bursal cells to respond to LPS. This contrasts with thymic cells which respond to Con A while in the thymic or splenic environment (Weber, 1973; Tufveson and Alm, 1975a). Perhaps Lydard and Ivanyi’s (1975) observation that an intravenous injection of LPS impairs bursal follicular development in embryos may be explained on the basis of this apparent maturational change in the B cell. V. Bursa1 Regulation of Immunoglobulin (Antibody) Production A.

ANTIBODY SUPPRESSION

1. The Early Experiment In 1954 T. S. Chang obtained several 6-month-old pullets from the present author for the purpose of injecting them with Salmonella-type


371

0 antigen in order to obtain serum with a high antibody titer for a class demonstration. Several of the pullets died subsequent to the immunization, and none of the surviving birds produced antibody. A check of the wing-band numbers revealed that the dead pullets and those failing to produce antibody had been BSX during the period of rapid bursa growth. We decided that the bursa was responsible for the results, since their normal penmates reacted to the inoculations by producing normal antibody titers (Glick, 1955). We then designed two experiments to further evaluate our observation. Equal numbers of male and female White Leghorns were BSX at 12 days of age and injected six times at intervals of4 days with Salmonella typhimurium 0 antigen. At 7 weeks of age, 7 of 10 BSX birds and 2 of 10 controls failed to produce antibody (Glick, 1955). These data were reinforced by a second experiment employing larger numbers and two different breeds of chickens. In the second experiment 89.3% of the BSX birds failed to produce antibody, while only 13.7% of the controls failed to do so (Chang et al., 1955; Glick et al., 1956). Confirmation of our observation that the bursa plays an important role in the development of circulating antibody was recorded with a variety of antigens in numerous laboratories throughout the world [e.g., Mueller et al., 1960, 1961, bovine serum albumin (BSA); Isakovii. et al., 1963, human 0 erythrocytes; Graetzer et al., 1963b, natural hemagglutinins to rabbit erythrocytes; Kemenes and Pethes, 1963; Pethes and Kemenes, 1967, Leptospira icterohaemorhagiae; and Edwards et al., 1968, T, coliphage].

2. Antibody Response of Surgically and Chemical1y Bursectomized Poultry It was evident from the previously cited reports, as well as other early articles (Chang et al., 1957; Glick, 1958b, 1960b),that SBSX did not abrogate the antibody response to cellular antigens. The importance of the rapid growth period of the bursa (Glick, 1956, 1960a; see Section II1,A) in the control of antibody production was suggested by the early experiments of Chang and Glick, and clearly defined by Chang et al. (1957) when they revealed that SBSX at 2 weeks was more effective in suppressing antibody production than SBSX at 5 or 10 weeks of age. The observations of Chang were confirmed with the soluble antigen BSA (Muelleret al., 1960; Graetzeret al., 1963b). Failure of SBSX to eliminate all antibody production might make one suspicious concerning the totipotent control of the bursa over antibody production or suggest the existence of a brief period in embryonic development during which the bursa could be functional. Actually, it

372

BRUCE CLICK

was the investigation of the latter possibility that led us to formulate our current scheme (see Section V,D,2). The first experiments to evaluate the existence of a functional embryonic period for the bursa (Meyer et al., 1959)took advantage of the known regressive influence of androgens on the posthatched bursa (Kirkpatrick and Andrews, 1944; Glick, 1957a).The injection of 19-nortestosterone into the incubating egg impaired bursa1 development (see Section II1,B for more detail). Subsequent injection of BSA into chicks hatched from eggs injected on day 5 of incubation revealed complete elimination of precipitins, while chicks from eggs injected with the hormone on the twelfth or thirteenth day of incubation possessed significantly reduced precipitin levels (Mueller et a1 ., 1960, 1962). As might be expected, systemic anaphylaxis was abrogated in BSX birds (Sato and Glick, 1965a).The bursa was generally absent in 19-day embryos that had received T P prior to the eighth day of embryonic development (Warner and Burnet, 1961). The injection of 2.5 mg of TP into the allantoic cavity of 12-day embryos eliminated the antibody response in hatched chicks to BSA, human gamma globulin, Brucella, and an endotoxin from Salmonella adelaide (Warner et al., 1962). Approximately 10% of the TP-treated chicks revealed degeneration of the thymic cortex and medulla (Szenberg and Warner, 1962). Section II1,B deals with an alternate method for introducing steroids into the environment of the embryo, in which fertile eggs are dipped into EA solutions containing TP. The TP dipping eliminated the bursa from more than 50% of the hatched chicks and prevented antibody production to Salmonella pulloruni in 83% of 5- to 6-week-old chickens (Glick and Sadler, 1961).Also, no change in thymic size was noted (Glick, 1961). Increasing the concentration of TP from 80 to 1280 mg% also increased the regressive influence of the bursa (May and Glick, 1964). Levels of TP less than 320 mg% failed to influence significantly the response to BSA. At first, these experiments seemed to support our earlier evidence that a relationship exists between the size of the bursa and antibody production (Sadler and Glick, 1962). Families of White Leghorns from the same sirain were classified on the basis of bursa size at hatching (Sadler and Glick, 1962). Chicks from large-bursa families produced significantly greater antibody titers to Vibriofoetus than chicks from families characterized by small bursa size. However, in later experiments we found no differences in antibody production between chicks from White Leghorn families classified on the basis of large or small bursa size (Sato, 1964). Also, Jaffe and Jaap (1966) failed to find differences in disease resistance or antibody titers in two different breeds of chickens which differed sig-


373

nificantly in bursa size. May and Glick (1964), on the basis of their results and data from an article by Sato and Glick (1964b),concluded that “the implication is that it is not the size of the bursa per se but the presence of active bursal follicles that determines future antibody potential.” If bursa size and/or activity of bursal follicles are factors in enhancing antibody response, then lines selected for large bursa (LB) and small bursa (SB) size (see Section II1,B) may be expected to reflect difrerences in antibody potential (Glick and Dreesen, 1967). While antibody differences were not detected between the SB and LB lines (SubbaRao, 1969), it was noted that the bursa of hatching SB chicks, unlike that of LB chicks, was nearly devoid of follicular development (Landreth and Glick, 1973). The lack of bursal follicles in the SB-line bursa at hatch suggested that the SB-line bursa develops slower than the bursa of LB-line chicks. Theoretically, the LB line should possess a more active bursa embryonically by virtue of its greater number of cellular units (Kulkarni et al., 1971) and would be more mature at hatching. The difference in maturity would be difficult to measure after several weeks, since the SB chicks would have had enough time to develop and seed the minimal number of cellular or humoral units necessary to prepare the bird for an antibody response. Therefore, to compare the immunocompetence of the LB and SB chicks, they were BSX at hatch and at sequential ages after hatching (Landreth and Glick, 1973). SB chicks BSX at hatch failed to respond to sheep red blood cells (SRBC), while at hatch bursectomy of LB chicks did not interfere with agglutinin production. Agglutinin titers were low for SB-line chicks BSX at 1 week but were unaffected by SBSX at 3 weeks of age. Thus it is not bursa size per se that influences future antibody production, but the presence of a minimal number of functional cells expressed as lymphocytes within bursal follicles (Landreth and Glick, 1973). The antibody response of the White Pekin duck, like that of our LB line, was not influenced by SBSX at hatching (Glick, 1963).The longer incubation period of the duck may be an important factor in the duck’s antibody response. The duck‘s bursa was eliminated b y dipping fertile eggs into 2 gm% TP. These TP ducks failed to respond to S. pullorurn (Glick, 1963). The White Carneau pigeon, like the White Pekin duck, produced normal titers of antibody (to Brucella abortus) subsequent to SBSX at hatching (Thompson and Linna, 1975). Failure to register a depressed antibody response in the BSX pigeon may suggest a brief period of embryonic function for the pigeon’s bursa, as we have proposed for the chicken. White Gelinan Rhine geese and the Rajna breed, unlike the White Pekin duck and pigeon, experienced a marked reduction in the primary

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BRUCE GLICK

immune response (Pethes and Losonczy, 1971) and the production of natural agglutinins (Pethes et al., 1972) following SBSX at hatch. In ozlo bursectomies have been performed. The significance of these experiments is discussed in Section V,D. B. DISSOCIATION OF THYMUS AND BURSAFUNCTION In retrospect it now seems quite elementary that after the identification of bursa1 involvement in the immune system one should have immediately suspected thymic involvement, both because of similarities in cell populations and response to steroids. The thymus was considered to be involved in the mammalian immune system by Karl Fichtelius of Uppsala and R. A. Good of Minnesota, but unfortunately in the early 1950s we were not familiar with their work. It should be mentioned in passing that several members of the Poultry Department at Ohio State University did thymectomize some chickens (1954-1955) with the intent of determining the thymus’ role in antibody production. The concept of the dissociation of immunological responsiveness originated with investigations from the Walter and Eliza Hall Institute of Medical Research (Warner and Szenberg, 1962; Warner et al., 1962; Szenberg and Warner, 1962). In these studies T P was injected into the allantoic cavity of 12-day fertile eggs. The chicks from this injection (TPI) failed to produce antibody to five different antigens but were competent in rejecting a homograft. Severe impairment of the thymic cortex occurred in approximately 10%)of the TPI chicks. These chicks exhibited a delay in homograft rejection. From these data and others, Warner and co-workers suggested a dissociation of the immune response with humoral immunity under the control of the bursa and with the thymus primarily involved in recognizing histocompatibility antigens and rejecting the invasion of foreign cells. This concept was disturbed slightly by reports that chickens previously treated with steroids in ozlo showed an impaired response to injections of foreign lymphocytes (Papermaster et al., 1962; Warner and Szenberg, 1963). Yet, SBSX did not interfere with the birds’ rejection response to foreign leukocytes (Jaffe, 1965; Marvanova, 1969). The employment of different techniques and the change in cellularity produced b y steroids may account for these different results (Warner and Szenberg, 1963; Marvanova, 1969).It is now clear that thymic or T cells initiate the graft-versus-host (GVH) response (Cain et al., 1968; Potworowski et al., 1971b; Weber, 1974). Wisconsin (Aspinall et al., 1963; Meyer et al., 1964) and Yugoslavian workers (Isakovii: et al., 1963) independently demonstrated the normal rejection of homografts by BSX


375

birds and impairment of the homograft response following thymectomy. The control of delayed hypersensitivity (Szenberg and Warner, 1962) by the thymus was clarified by Jankovik et al. (1963) and Jankovib and Isvaneski (1963), when they demonstrated that experimental allergic encephalomyelitis was not impaired by bursectomy but was reduced in intensity following thymectomy. These data were extended in experiments employing the wattle test for sensitivity to diphtheria toxoid (DT) and purified protein derivative (PPD). Wattles from thymectomized (ThyX) irradiated birds previously sensitized with DT were less responsive to an intradermal wattle injection of DT than were BSX irradiated birds or control birds (Morita and Soekawa, 1972). Similar results were reported using PPD (Okuymma, 1965a,b) and polymerized flagellin of S. adelaide, BSA, dinitrophenylated chicken serum albumin or mouse serum albumin, and PPD (Warner et al., 1971). Also, Morita and Soekawa (1972) reported splenic cells from sensitized ThyX irradiated chickens migrated farther in the presence of a sensitizing agent (DT) than splenic cells from BSX irradiated or control chickens. Further clarification of the control of thymus in delayed hypersensitivity came from work with chickens made agammaglobulinemic by TP injection into 12-day fertile eggs followed by four consecutive days of cyclophosphamide (Cy) injections beginning at hatch (Theis and Thorbecke, 1972).These chickens were capable of elaborating a normal delayed hypersensitivity response. The latter response, however, was disrupted b y the injection of rabbit antichicken IgG and IgM sera, suggesting that the interference of the T cells was a result of the light (L) chain receptors on T cells, since anti-L chains are common to both antisera. The bursa was further removed from involvement in the homograft response when first- and second-set wattle homografts occurred normally in agammaglobulinemic chickens (Perey et al., 1967). Credit should be given to Isakovii: and Jankovik (1964) for their initial description of a marked depletion of splenic plasma cells in BSX birds and a reduction in small lymphocytes surrounding arteries and Schweigger-Seidel sheaths in ThyX birds. These observations were confirmed and extended to include the dependence of splenic and gut-associated germinal centers on the bursa (Papermaster and Good, 1962; Carey and Warner, 1964; Cooper et al., 1965, 1966a; Pierce and Long, 1965; Jankovik and Mitrovib, 1966; Leancu et al., 1968; Rodak, 1970, 1971; Nieuwenhuis, 1970). Within the white pulp dendritic reticular cells did not localize antigen in BSX irradiated birds (White et al., 1975). This, along with the lack of B cells that can aggregate with dendritic cells, may be responsible for the absence of germinal centers in BSX birds.

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BRUCE GLICK

The investigations by Cooper et al. (1965, 1966a) gave the greatest impetus to the dissociation concept. Their definitive reports clearly separated the functions of the two lymphoid tissues. Birds BSX or ThyX at hatch were irradiated 24 hours later with 650 r. The BSX irradiated birds were void of Ig, did not produce antibody to BSA or Brucella, and lacked splenic germinal centers and plasma cells, but were capable of a normal homograft response, elicited a GVH response, and possessed normal numbers of circulating small lymphocytes. Splenic cells from BSX irradicated birds synthesized or secreted little or no Ig (Alm and Peterson, 1969). However, the ThyX irradiated birds possessed Ig and splenic germinal centers and plasma cells, exhibited a suppressed antibody response to BSA and Brucella, maintained a homograft for a prolonged period, did not elicit a GVH response, and contained reduced numbers of circulating small lymphocytes (Cooper et al., 1966a). A reduction in the number of circulating small lymphocytes in ThyX birds had been reported previously (Warner and Szenberg, 1962; Isakovib and Jankovib, 1964). Our research has suggested that, while the bursa may not influence the number of circulating small lymphocytes, the absolute number of lymphocytes may be affected (Glick and Sato, 1964). Birds were BSX prior to 3 days of age, and at 3 weeks of age received a single intramuscular injection of ACTH. Leukocyte counts were determined 4,6, and 12 hours later. BSX birds receiving ACTH exhibited a significantly lower absolute number of lymphocytes than comparable controls. These results suggested that the bursa is necessary for an optimum level of circulating lymphocytes. In light of our data presented in Section IV,A and several published reports (Lukib et al., 1973; Jankovib et al., 1975b; Glick and Rosse, 1976), which reveal a paucity of lymphocytes in the bone marrow, it is not surprising that, under conditions that stimulate the pituitary-adrenal axis, a major repository of lymphocytes like the bursa should be called on to help maintain cellular homeostasis. We know that the level of absolute lymphocytes increases moderately during the first 2 months after hatching (Glick, 1958a).However, the absolute lymphocyte profile beyond this period has not been exhaustively studied. In yearlings, in which the bursa is absent, one might expect to find the thymus nonfunctional, the birds possibly more vulnerable to their environment, and a marked reduction in the number of circulating lymphocytes. The reduced antibody response reported b y Cooper et al. (1966a) for ThyX irradiated birds had been observed previously in ThyX birds (Graetzer et al., 1963a) and has been extended (Rouse and Warner,


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1972b) to include a necessary synergism between T and B cells leading to a normal immune response (Ivanyi and Salerno, 1971; Jacobs et al., 1972), specifically illustrated by cooperation of carriersensitized T cells with B cells to produce a hapten-antibody response (McArthur et al., 1972; Weinbaum et al., 1973). 1. Cytotoxicity and Lymphokines Cytotoxic effector lymphocytes (CEL) may be activated in the spleen, bone marrow, and thymus, but not the bursa, by PHA, pokeweed mitogen (PWM), and Con A (Kirchner and Blaese, 1973). CEL activated by PHA reacted against chicken red blood cells (CRBC), burrow red blood cells (BRC), and human red cells (HRC), and PWM and Con A activated CEL against only HRC and BRC, respectively. Bone marrow lymphocytes of agammaglobulinemic birds that appeared to lack B cells were activated to become CEL by PHA but, unlike the spleen lymphocytes, did not show proliferation in the presence of the mitogen. Perhaps there are at least two subpopulations of T cells in the bone marrow. It was of interest to note that the evidence suggested preexisting effector cells which become cytotoxic after linkage of target cells by the mitogen (Kirchner and Blaese, 1973). Confirmation of the dependence of cytotoxic cell production on the presence of T cells and not B cells comes from experiments in which cytotoxicity was not influenced subsequent to embryonic or hormonal bursectomy (Calder et al., 1974; Granlund et al., 1974; Granlund and Loan, 1974)and from experiments involving Rous sarcoma virus (RSV).SBSX prior to 2 days after hatching did not significantly influence the development of tumors in RSV-infected birds (Radzichovskaja, 1967a). While RSV inoculation increased the incidence of tumors in ThyX chickens (Radzichovskaja, 1967b), regression of primary tumors produced by the Schmidt-Ruppin strain of RSV (SR-RSV)was rarely observed in ThyX quail but occurred normally in BSX quail (Yamanouchi et al., 1971). Further confirmation of the dissociation of bursal lymphocytes from CEL was seen in reports that bursal cells of quail whose sarcoma (to SR-RSV) had regressed were not cytotoxic to cultivated SR-RSV, while spleen and thymus cells were highly cytotoxic to SR-RSV (Hayami et al., 1972). Seven-week-old Rhode Island Red chickens were sensitized with Mycobacterium tuberculosis, and 4 weeks later their buffy coat reacted with PPD (Oates et al., 1972). The supernatant from previously sensitized spleen cells, but not normal spleen cells, increased uptake of TdFG3H,suggesting the production and release of a lymphocyte mitogenic factor by chicken lymphocytes. The stimulatory or mitogenic

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factor was less apparent in the buffy coat from BSX irradiated and ThyX irradiated birds, leading the investigators to conclude that the stimulatory factor in the chicken may be heterogeneous. The plasma from wattle-grafted BSX irradiated birds adoptively transferred the wattle homograft immunity (Perey et al., 1970). These results revealed the presence in the plasma of a mediator substance which was probably released by a T cell. Another example of a lymphokine produced by T and not B cells is mononuclear chemotactic factor (MNL CTX) (Leonard and Kirchner, 1972; Altman and Kirchner, 1972; Kirchner et al., 1974). Chickens were made agammaglobulinemic by 3 days of Cy injections (6 mg per day). Between 6 and 10 weeks of age spleens were removed and cultured in the presence of Con A and/or phytomitogen. The supernatants from both normal and agammaglobulinemic spleens produced MNL CTX factor. The evidence indicated that MNL CTX factor comes from T cells but does not entirely rule out B cells as producers. The stimulation of B cells from the bursa (B,) or from the spleen (BJ with LPS or a previously sensitized agent would be helpful in collecting direct evidence for or against the production of MNL CTX by B, or B2 cells. 2. Rosette Forming Cells (RFC) Guinea pig red blood (GPRB) cells were found to form rosettes spontaneously with bone marrow (3350 RFC/106 cells), spleen (2650/106),bursa (320/106),and thymic (50/106)lymphocytes from 8week-old chickens (Isakovik et al., 1974). SRBC spontaneously form rosettes with bursal lymphocytes beginning at 15 days of embryonic development and continue linearly through embryonic life and after hatching (Tufveson and Alm, 1975b). Anti-Ig serum inhibited completely GPRBC RFC of bursal and thymic cells but was less effective with bone marrow and spleen cells. This is further evidence for Ig receptors on both T and B cells and suggests maturation of these cell types in peripheral lymphoid tissue, with a possible loss of Ig receptors of lymphocytes which emigrated from the bursa and thymus (see Section IV,B for more detail on receptors). Rabbit red blood cells (RRBC) are high in spontaneous rosette-forming ability with bursal cells from 15-day embryos. The rosette-forming ability of RRBC declines in late embryos and rises again after hatch. The frequency of RRBC RFC was the same in thymus, spleen, yolk, and bursa in 15-day embryos. This is interpreted to suggest that the RFC in thymus, spleen, and yolk do not come from the bursa, since the latter is a poor exporter of cells until later embryonic ages (Tufveson and Alm, 1975b). If RFC are progenitors of antibody-producing cells, as has


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been suggested (Van Alten and Meuwissen, 1972), immunoglobulin cells are not restricted in their origin to the bursa (Lerner et al., 1971; Morgan and Glick, 1972; Sato and Glick, 1972; Glick and McDuffie, 1975; Glick et ul., 1976; see Section V,D). Unlike RRBC, cells binding the monomeric flagellin antigen of S . adelaide are first present in the bursa of 14-day embryos and only later appear in other tissues (Dwyer and Warner, 1971). If antigen-binding cells precede antibodyproducing cells, their absence in 13-day embryos suggests that the identification of an immunocompetent bursal cell must wait until 14 days of embryonic development (Decker et al., 1974). The appearance of cells in the embryonic and posthatch bursa (Van Alten and Meuwissen, 1972) reacting with cellular components demonstrates the presence of specific receptor-bearing bursal lymphocytes. The specificity of the receptors was evident when bursal lymphocytes were incubated with SRBC or RRBC and only separate populations of pure SRBC RFC and RRBC RFC occurred (Tufveson et d . , 1974). The above-mentioned experiments with GPRBC, SRBC, and RRBC involved spontaneous RFC. Others have revealed RFC following immunization of the chicken (Theis et al., 1973; Hemmingsson and Alm, 1972; Kiszkiss et d . , 1972). SBSX significantly reduced spontaneous RFC in the spleen and eliminated the number of RFC in the thymus (Isakovik et al., 1974). Thymectomy significantly reduced RFC in the spleen and reduced the number of RFC in the bursa by one-third. The latter may reinforce the thesis that the thymus influences the development of the bursa (Jankovii, and Isakovik, 1964). Perhaps it does so by the release of soluble thymic factor (STF) (Teodorczyk and Potworowski, 1975). The presence of T cells in the bursa may be inferred from the failure to label 100% of the bursal lymphocytes with 1251-labeledanti-Ig (see Section IV,B), and accepted by the demonstration that a small number (

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