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INTERNATIONAL

REVIEW OF CYTOLOGY VOLUMEI

This Page Intentionally Left Blank

INTERNATIONAL

Review of Cytology EDITED BY G. H. BOURNE

J. F. DANIELLI

London Hospital Medical College London, England

Zoology Department King's College London, England

VOLUME I

Prepared Under the Auspices of The International Society for Cell Biology

ACADEMIC PRESS INC. PUBLISHERS NEW YORK

1952

Copyright 1952, by ACADEMIC PRESS INC. 125 East 23rd Street, New York 10, N. Y All Rights Reserved NO PART OF THIS BOOE MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOW WRITTEN PERMISSION FROM THE PUBLISHERS

Library of Congress Catalog Card Number : (52-5203)

PRINTED I N T H E UNITED STATES OF AMERICA

Contributors to Volume I L. G. E. BELL,King's College, University of London, England. K . A. BISSET,University of Birmningham, Birininghm, England. L. H . BRETSCHNEIDER, University of Utrecht, Holland. R. BROWN,University of Leeds, Leeds, England. G. FANKHAUSER, Princeton University, Princeton, New Jersey. R. J . GOLDACRE, Chester Beatty Research Institute, London, Elzgland.

G. GOMORI, University of Chicago, Clzicago, Illinois. A. D. HERSHEY, Carnegie Institution of Washington, Cold Spring Harbor, New York.

ARTHURHUGHES,Strangeways Research Laboratory, Cawbridge, England.

C. LEONARD HUSKINS,University of Wisconsin, Madison, Wisconsin. GEORGE W . KIDDER, Amherst ColEege, Amherst, Massachusetts. WILLIAMMONTAGNA, Brozrm University, Providence, Rhode Island. TH. ROSENBERG, Steno Mentorial Hospital and Nordisk Insulinlaboratorium, Gentofte, Denmark.

LORDROTHSCHILD, Cambridge University, Cambridge, England. MARCUS SINGER, Harvard Medical Sclzool, Boston, Massachusetts. M. M. SWANN,Cambridge University, Cawbridge, England. W . WILBRANDT, University of Berne, Berne, Switzerland.


Foreword Much has happened in the world of Cytology since Schleiden ant1 Schwann enunciated their “cell theory” in 1838 and 1839. Beginning with the study of cell structure cytology rapidly became functional and chemical with the development of organic and biochemistry, Then with the rise of the dye industry and with the application of dyestuffs to the cell, delineation of structure became fatally facile and cytological chemistry sank more or less into obscurity. Gradually during the present century, however, the new developments in chemistry and the development of new physical and chemical microtechniques have led to an outburst of activity in the fields of cytochemistry and fine structure and their relation to cell physiology-all of which might well be incorporated in the inclusive term of “Cell Biology.” In recent years there has been a remarkable increase in the volume of published research in this field, and like so many others, it has now become too large to be adequately covered by any individual without the assistance of regular review service. Some years ago we decided to provide such a periodical review and were gratified to find that the International Society for Cell Biology welcomed this proposal and agreed that the review should be issued under its auspices. It is aimed to produce this review annually and to make it truly international. Among the authors of the present volume are seven British, five American, one Dutch, one Danish and one Swiss and in later volumes we hope to obtain a greater proportion of continental authors. It is proposed to keep the scope of the “International Review of Cytology” as wide as possible-to deal with all aspects of Cell Biology, including morphological and chemical studies of both cells and tissues. Papers presenting new theories of general interest will be welcomed. It should be pointed out that the articles contained in the present and subsequent volumes are not intended to cover completely any particular aspect of Cell Biology. The various chapters are individual and unrelated reviews of specific subjects by experts in those fields who have contributed at the invitation of the Editors. Succeeding volumes will follow the same pattern, and over a period of years the whole field of cytology and cell physiology will be covered. The policy of the Editors has been to obtain reviews which are critical discussions of data already published elsewhere or of new theoretical contributions. A certain amount of new work has been and will be admitted in some articles where it is thought that it makes for completeness or otherwise embeIlishes the review. GEOFFREY H. BOURNE Januwy 1952 JAMES F. DANIELLI


CONTENTS Some Historical Features in Cell Biology BY ARTHURHUGHES. Strangeways Research Laboratory. Cambridge. England I. Introduction ........................................................ I1. Microscopy ........................................................ I11. Nucleic Acids ..................................................... IV. Study of Living Cells .............................................. V . References .........................................................

1 1 2 5 6

Nuclear Reproduction BY C. LFQNARD HUSKINS, University of Wisconsin. Madison. Wisconsin I . Introduction ........................................................ I1. Nuclear or Cell Division ........................................... I11. Chromosome Reproduction .......................................... IV. Variation in Nuclear Size .......................................... V. Reduction in Chromosome Number .................................. VI . Conclusions ........................................................ VII . References . !.......................................................

9 10 12 15 18 21 34

Enzymic Capacities and Their Relation to Cell Nutrition in Animals

I. I1. I11. IV . V.

BY GWRGEW . KIDDER,Amherst College, Amherst. Massachusetts Introduction ........................................................ Nutritional Requirements of Tetrahymena ........................... Nutritional Requirements of Higher Animal Cells .................... Conclusion ......................................................... References .........................................................

27 28 32 32 33

T h e Application of Freezing and Drying Techniques in Cytology

I. I1. I11. I v. V.

BY L. G . E. BELL.King’s College. University of London. England Introduction ........................................................ Method ............................................................ Comparison with Histological Fixation .............................. Advantages of Freezing and Drying Techniques ...................... References .........................................................

35 36 53 57 62

Enzymatic Processes in Cell Membrane Penetration

Steno Memorial Hospital and Nordisk Insliliitlaboratorium. BY TH. ROSENBERG. Gentofte. Denmark A N D W . WILBRANDT. University of Berne. Berne. Switzerland I . Introduction ........................................................ I1. Some General Considerations ....................................... I11. Enzymes with Non-Penetrating Substrates ........................... I V. The Enzymatically Controlled Transport ............................. V . Transport of Glucose ............................................... VI . Conclusion ......................................................... V I I . References .........................................................

65 66 68 70 70 85 89

Bacterial Cytology BY K . A . BISSET.University of Birmitiglsam. Binrririghan.. Etiglawi

I. I1. I11. IV V. V I. VII VIII .

.

.

Introduction ........................................................ The Bacterial Nucleus .............................................. Growth and Cell Division in Bacteria ................................ Granular Inclusions ................................................ Bacterial Flagella .................................................. Specialized and Reproductive Methods .............................. Cytology and Systematics .......................................... References .........................................................

9.3 96 98 100 102 102 103 104

Protoplast Surface Enzymes and Absorption of Sugar BY R . BROWN.Uiiiversity of Leeds. Leeds. Eiiglaiid

I . Introduction

. I1. I11.

I V.

V.

VI

.

........................................................

Nature of Experimental Material ................................... Characteristics of Absorption Process ............................... Enzyme Systems in the External Surface ............................ Discussion ......................................................... References .........................................................

107 107 110 114 117 118

Reproduction of Bacteriophage BY A . D . HERSHEY. Carizegie Iiistitutiou of Washington. cold Spririg Harbor N e w York

I . Introduction

I1. I11. I V. V. VI V I I.

.

........................................................

Ideas about Origin ................................................. Ideas about Growth ................................................ Program and Objectives ........................................... Facts about Growth ................................................ Conclusions ........................................................ References .........................................................

119 119 120 123 126 133 133

The Folding and Unfolding of Protein Molecules as a Basis of Osmotic W o r k BY R . J . GOLDACRE. Chester Beatty Research Institute. London. England I . Introduction ......................................................... I1. The Folding and Unfolding of Protein Molecules in Living Cells ...... I11. Osmotic Work-The Accumulation of Material against a Concentration Gradient in Amoeba .............................................. I V. Osmotic Work in Other Cells ...................................... V The Inversion Tube Analogy ....................................... V I . Osmotic Work in Metazoijn Cells ................................... VII . Fungi ............................................................. VIII . General Discussion on Osmotic Work ............................... IX. Concluding Remarks ............................................... X . References .........................................................

.

135 136 141 . 147 150 151 153 153 161 163

Nucleo-Cytoplasmic Relations in Amphibian Development BY G . FANKHAUSER, Princeton University. Princeton. New Jersey I . Introduction ......................................................... I1. Quantitative Changes in Cytoplasm of Egg ...........................

165 166 168 177 179 181

I11. Quantitative Changes in the Nucleus : Polyploidy and Haploidy ....... I V . Unbalanced Chromosome Combination (Aneuploidy) ................. V. Invisible Chromosome Changes (Gene Mutations?) .................. V I . Development without Chromosomes ................................. V I I Nucleo-Cytoplasmic Relations in the Early Development of Species 183 Hybrids ......................................................... 188 VIII . Summary and Conclusions .......................................... 192 I X . References .........................................................

.

Structural Agents in Mitosis BY M. M. SWANN, Canabridge University. Cambridge. England

I. I1 I11. IV. V

.

VI

. .

Introduction ........................................................ Birefringence Changes in the Sea Urchin Egg during Mitosis ......... Further Evidence on the Release of Chemical Agents in Mitosis ...... The Nature of Chemical Agents in Mitosis .......................... Conclusion ......................................................... References .........................................................

195 197 203 208 209 210

Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes BY MARCUSSINGER. Harvard Medical School. Boston. Massachusetts I . Introduction ........................................................ 211 I1. The Influence of pH of the Staining Solution on the Interaction of Dye and Protein

......................................................

I11. The Nature of the Influence of pH on Staining

......................

I V. The Site of Dye-Binding and the Nature of the Bond between Dye and Protein .......................................................... V The Relation between the Isoelectric Point and Staining .............. V I The Ionic Strength of the Dye Solution ............................. VII . The Influence of Dye Concentration ................................. VIII . The Affinity of Dyes ............................................... I X . The Influence of Fixation and Other Modifications of Tissues on Subsequent Staining ................................................. X . The Influence of Temperature of the Staining Solution ............... X I . Some Observations on the Kinetics of Staining ...................... XI1. The Reversibility of Staining Reactions ; Equilibrium of Staining. and Other Factors Which Influence Staining ........................... XI11. References .........................................................

. .

215 221 224 230 233 236 237 212 215 246 248 250

The Behavior of Spermatozoa in the Neighborhood of Eggs BY LORDROTHSCHILD. Cambridge Uwiversity. Cambridge. England

I. I1 I11. I V. V.

.

Introduction ........................................................ The Block to Polyspermy .......................................... Chemotaxis of Spermatozoa ........................................ Conclusion ......................................................... References .........................................................

257 258 260 263 263

The Cytology of Mammalian Epidermis and Sebaceous Glands BY WILLIAMMONTAGNA. Brown UvGiverSity. Proz&fe.iEe. Rhode Island

. .

I I1 I11. IV.

Introduction ........................................................ The Epidermis .................................................... The Sebaceous Glands ............................................. References .........................................................

263 266 290 299

The Electron-Microscopic Investigation of Tissue Sections

BY L. H. BRETSCHNEIDER. University of Utrecht. Holland I. I1. I11. IV. V. VI

.

Objectives ......................................................... The History of Ultramicrotomy .................................... The Influence of Fixation upon the Electron-Optical Image ........... Primary Nuclear and Plasmatic Ultrastructures ..................... Secondary Ultrastructure of Cells .................................. References .........................................................

305 308 313 311 316 321

The Histochemistry of Eeterases BY G. GOMORI.Uiiiversity of Chicago. Chicago. lllinois

.........................................................

335

AUTHOR INDEX............................................................ SUBJECT INDEX............................................................

337 349

References

Some Historical Features in Cell Biology ARTHUR HUGHES Strangeways Research Laboratories, Cambridge, England.

CONTENTS

I. 11. 111. IV. V.

Introduction ........................................................... Microscopy ............................................................ Nucleic Acids .......................................................... Study of Living Cells .................................................. References .............................................................

Page 1 1 2 5 6

I. INTRODUCTION One of the questions which confronts the historian of science is the problem of the relations between the progress of discovery and advances in technique. How fully at each stage are the available methods and apparatus utilized? In the history of microscopical observation, it is clear that the imperfections of contemporary instruments have not exclusively determined the pace of research. The era of discovery which began in the late seventeenth century associated with the great names of Hooke, Leeuwenhoek, Grew, and Malpighi was not maintained, and although the succeeding century was not barren of microscopical researches, these tended to become more scattered as the century advanced. It is certainly true that the scope of the eighteenth century microscope with uncorrected lenses was limited. However, Sachs (1890) says, with respect to botanical microscopy : There was in fact no original phytotomic research in the first fifty or sixty years This state of decline must not be ascribed to imperfect of the last century; no one saw and described clearly even what can be seen microscopes only; with the naked eye o r with very small magnifying power; I t is not easy to discover the causes of this decline in phytotomy in the first half of the 18th century, but one of the most important appears to lie in the circumstance that botanists did not make the knowledge of structure the sole aim in their anatomical investigations, but sought it chiefly for the purpose of explaining physiological processes. D 2461

...

...

...

...

11. MICROSCOPY

The fundamental improvement in the microscope which resulted from the development of the achromatic objective was made between 1815 and 1830 (Mayall, 1886; Carpenter, 1857; Chevalier, 1839), but this great technical advance did not take effect in the several branches of microscopy at the same time. The study of plant histology was resumed very 1

2

ARTHUR HUGHES

early in the nineteenth century (Sachs, 1890, p. 256), well before improved microscopes were available and at a time when the other branches of microscopy were still dormant, for Dobell (1932) says that “from the standpoint of protozoology and bacteriology the first quarter of the last century is a blank.” (p. 381) Carpenter (1857, p. 7) tells us the same with regard to animal histology. The elements of microscopic structure can be more readily apprehended by simple methods in plants than in animals, and thus some scope in the botanical field still remained in the early nineteenth century for the uncorrected objective. Fresnel ( 1824) found that a contemporary achromatic microscope had no advantage at magnifications greater than 200 X. Indeed, we are told that Meyen, as late as 1836, still preferred an English eighteenth century instrument (Sachs, 1890, p. 258) though this may to some extent illustrate the notorious conservatism of microscopists. It is difficult at this period to trace in detail the parallel course of optical development and the resulting enhanced powers of observation. One cannot tell, for instance, from Amici’s paper of 1824 what microscope he used in following the growth of the pollen tube, here described for the first time, though it was probably his reflecting instrument. This he had developed subsequent to some early efforts in constructing an achromatic lens. The principle of the two aplanatic foci, on which the construction of the achromatic objective has since been based was described by J. J. Lister in 1830. Three years previously, however, he had constructed an object glass sufficiently corrected to enable Hodgkin to make some observations on the structure of animal cells and tissues which mark the real beginning of vertebrate histology. In a short paper (Hodgkin and Lister, 1827) are described the shape of the human erthyrocyte, the construction of muscle fibers and the fibrous network of the arterial intima. The then current view that all animal tissues were composed of uniform globules did not survive this refutation. Baker (1948, 1949) is admirably tracing this, and other aspects of the development of cellular theory. 111. NUCLEIC ACIDS

It is probable that early in the nineteenth century the technical development o€ the microscope and its use in biological research were reacting one upon another, as is indeed true also of the present day. This is not the only feature of contemporary cell biology which is reminiscent of an earlier period ; the reawakening of interest during the last twenty years

SOME HISTORICAL FEATURES I N CELL BIOLOGY

3

in the problems of the distribution and functions of the nucleic acids within the cell provides a striking parallel with the cell biology of the 1870’s. In this renaissance again, advances in technique have both facilitated and have been evoked by progress in the study of the cell. It may not be without interest to trace some of these parallel features which extend in some instances almost to the details of research. As is well known, Friedrich Miescher in the late 1860’s prepared from pus cells a substance with stronger acidic properties than any organic cell constituent then known ; this he termed “nuclein” ; it was further distinguished by a high content of phosphorous (Miescher, 1871). The first stage of this investigation has a very modern sound, for he separated the pus leucocytes into nuclear and cytoplasmic fractions. The isolated nuclei were clearly recognizable as such; within them was a nucleolus, they were merely slightly smaller than those of intact cells. Nor was Miescher alone in preparing separated nuclei ; a method for bird erthyrocytes was described at about the same time by Brunton (1870). Among the properties of nuclein which Miescher described were its solubility in alkalis and its resistance to peptic digestion. These criteria were used some ten years later by Zacharias (1881) in a cytochemical study. The use of reagents under the microscope to identify the chemical constituents of biological material is much older than this, for as Dr. Baker (1943) has shown, the subject begins with Raspail in the late 1820’s. Zacharias’ work however was probably the first example of the use of enzymes in microscopical study. H e found that the nucleus of the frog erthyrocyte, and the macronuclei of Vorticella and Paramecium all remained behind when the rest of the cell was digested with pepsin, but dissolved if soda was then added. His clearest results on plant cells were obtained with pollen mother cells in division. H e found that the “Kernplatten-elemente” of Strasburger, which Waldeyer ( 1888) later called “chromosomes,” retained their affinity for stains after treatment with pepsin. On the other hand, the spindle was digested away. Sixty years later, Mazia ( 1941) described how larval dipteran salivary chromosomes shrink when treated with pepsin, but retain their positive reaction toward the Feulgen reagent. H e concluded that the digested chromosome had lost the globulin-like protein of the matrix, but had retained the histones of the chromosome skeleton, which he found also to be resistant to pepsin. Numerous cell constituents, of course, are not attacked by this enzyme ; Zacharias (1883) gave the name of “Plastin” to cytoplasmic material which remains after peptic digestion, and it is not surprising that properties

4

ARTHUR HUGHES

common to plastin and nuclein should have led in those days to statements that nucleic acids occur in the cytoplasm. However, the first observations which we can interpret as a demonstration of this are due to van Hewerden in 1913, who then showed that the basophilia of the sea urchin egg is diminished by digestion with a preparation of what we now t e r n ribonuclease. Van Hewerden’s work has been recognized by recent investigators in this field (Catcheside and Holmes, 1947) but there still remains another type of study in microenzymology which has yet to be resumed. I n 1908 Adolf Oes published a study on the autolysis of cells, mostly in bean roots, which were incubated under toluene for periods from an hour to a day at 30-40“, and were then fixed and sectioned. The cytoplasm was relatively little affected by this treatment, but in mitotic cells from metaphase to telophase the chromosomes were digested away. Nuclei in prophase were more resistant, and those in interphase still more so. The autolysis proceeded in the presence of weak alkalis, but was inhibited by acids and by ions such as copper and magnesium. The only comparable experiment in modern times which I have come across is also due .to Mazia (1941), who found that there was no digestion of chromosomes in Drosophila salivary glands kept under toluene for seven days. Again, permanent nuclear elements resist autolysis. Another reminiscent feature of modern studies on cell nuclei is in their staining reactions. For instance, methyl green, used in Unna’s wellknown method, has recently been shown by Kurnick (1950) to react quantitatively with highly polymerized desoxyribonucleic acid. This dye was first introduced into microscopical technique by Calberla ( 1874). The most interesting early example of its use is Balbiani’s famous account of the salivary gland nucleus of the Chironomus larva (Balbiani, 1881). He described the chromosomes as a “cordon cylindrique,” the arrangement of which reminded him of an intestine. He found that only the bands took up methyl green ; other stains colored the nucleolar material, and that mixtures of the two resulted in “jolis effects de double coloration.” Not often have the staining reactions of cell constituents led to results as clear as this distinction between the nucleoli and the chromosomes. Ehrlich in 1879 realized the difference in effect of acid and basic dyes, but in later years physical explanations of staining increased in prominence ; Fischer in 1899 held that all staining was due to adsorption. The attempt was made by double staining to distinguish between free nucleic acid and nucleoprotein in the nucleus ; Lilienfeld in 1893 maintained that mitotic chromosomes and the resting nucleus differed in this respect, but two years later Heine was unable to confirm this distinction.

SOME HISTORICAL FEATURES IN CELL BIOLOGY

5

IV. STUDY OF LIVING CELLS One of the features of the cell biology of the last century is the occasional prominence of the study of living cells. Here again at the present day there is a tendency in this direction, now encouraged by the Zernicke phase microscope. The stained preparation, which has long been nearly the exclusive means of approach to the problems of cell structure did not at once acquire this predominance when dyeing technique was introduced into biology. In the 1870’s it was still customary to observe both stained and unstained material in microscopical researches. I n the elucidation of the complex events by which cells divide, the study of the living cell in the process of division played a conclusive part in that annus nzirabilis, 1879, for then three separate authors described the course of nuclear and cell division in life. Strasburger (1879) followed the process in the stamina1 hair cell of Tmdescantia while larval Amphibia were chosen by Schleicher (1879) and by Flemming (1879), who respectively studied cells in cranial cartilage and the skin. These two authors published their papers in the same volume of one journal. The fundamental importance of these studies is illustrated by a remark of Nordenokiold in referring to the editions of Strasburger’s “Zellbildung and ZelltheiIung,” which respectively preceded and followed this year : Even in the first edition of his said work (1875), Strasburger makes the nucleus of the egg cell in the plants he investigated dissolve upon fertilization and its mass disperse into the plasm of the cell; in the latter are then formed a number of concretions] which give rise to fresh nuclei. In the third edition (1880) on the other hand, it is asserted that examples of independent cell-formation can no longer be cited from the vegetable kingdom ; fresh nuclei invariably arise through the division of older ones C1928, p. 5351.

It is of much interest to compare Strasburger’s drawings with Bilai-’s photographs of the Tradescantia hair cell in division, which were published in 1929. One can recognize the parallel arrangement of the chromosomes in late prophase in Strasburger’s drawings, the significance of which he was not then aware. Attention may be drawn to two other nineteenth century studies on living cells, which have yet to be resumed in this era. In 1875 Ranvier described in his textbook of Histology how leucocytes in Amphibian lymph within a moist chamber can be seen to undergo amitotic division and says the observation is simple enough for a class exercise. He also described how fragments of elder pith inserted in the dorsaI lymph sac of the frog become infiltrated by leucocytes. In 1887, Arnold repeated

6

ARTHUR HUGHES

this work, by combining the two observations. H e mounted this infiltrated pith on a coverslip under sterile conditions, sealed the preparation, and was then able to observe the behavior of the leucocytes which wandered out of the pith fragment for a period of four or five days. Arnold confirmed Ranvier’s account of this amitotic division, and as far as I know these are the only descriptions in living cells of direct nuclear fission being followed by cytoplasmic division. It might be claimed that Ranvier and Arnold were the first parents of tissue culture, and it would surely be worth someone’s while to try their experiments again. The second example goes back still farther into the last century and relates closely to a subject of present day interest. Balbiani in 1864 described movements which occur in the nucleoli of the oocytes of spiders. He said that under the microscope this activity could be watched for several hours in the larger oocytes of an excised ovary. There was an ameboid movement of the whole nucleolus and also a continuous change in size of vacuoles within. H e was convinced that these were normal events because inspection of an ovary immediately on incision showed that all phases of activity were to be seen in individual nucleoli. Bradfield (1949) has found that the oocyte nucleolus of the spider gives a very strong positive alkaline phosphatase reaction, and is rich in ribonucleic acid. If these facts mean that this body is actively concerned in synthetic activity, as Caspersson would claim, it is possible that the nucleolar movements are related thereto, and so we may here have the possibility of watching material being synthesized in a cell under the microscope. Thr scope for futher study of such a system needs no emphasis. In conclusion, one may say that since in some ways cell biologists are on old ground working with new implements it would be as well if they recognized more fully the antiquity of their sites and looked out for thc old forgotten tracks, along which something of value might still be found A few more ideas, however ancient, would still be useful in cell biology. V. REFERENCES Amici, G. B. (1824) Ann. Sci. nat., 4 41. Arnold, J. (1887) Arch. mikr. Anat., SO, 205. Baker, J. R. (1943) J. Quekett micr. C!., Ser. 4, 1, 256. Baker, J. R. (1948) Quart. 1. micr. Sci., 89, 103. Baker, J. R. (1949) Quart. J. micr. Sci., 90, 87. Balbiani, E. G. (1864) C. R. SOC.Riol., Ser. 4, 1, 64. Balbiani, E. G. (1881) Zoo!. AM., 4, 637. Bslaf, K. (1929) 2. Zellforsch., 10, 73. Bradfield, J. R. G. (1949) Exp. Cell Res., Suppl. 1, 338

SOME HISTORICAL FEATURES I N CELL BIOLOGY

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Brunton, T. L. (1870) 1. A w t . Physiol., Ser. 2, 3, 91. Calberla, E. (1878) Morph. Jb., 3, 635. Carpenter, W. B. (1857) The Microscope and Its Revelations, 2nd ed. London. exp. Biol., 1, 2 5 . Catcheside, D. G., and Holmes, B. (1947) Synp. SOC. Chevalier, C. (1839) Des microscopes et de leur usage. Paris. Dobell, C. (1932) Antony van Leeuwenhoek and His “Little Animals.” London. Ehrlich, P. (1879) Arch. Anat. Physiol. (Physiol. Abt.), p. 571. Fischer, A. (1899) Fixierung, Farbung und Bau des Protoplasmas. Jena. Flemming, W. (1879) Arch. mikr. Anat., 16, 302. Fresnel, A. J. (1824) Ann. Sci. nat., 3, 345. Heine, E. (1895) 2. physiol. Chem., P,494. van Herwerden, M. A. (1913) Arch. Zellforsch., 10, 431. Hodgkin, T., and Lister, J. J. (1827) Phil. Mag., 2, 130. Kurnick, J. B. (1950) Ex#. Cell Res., 1, 151. Lilienfeld, L. (1893) Arch. Anat. Physiol. (Physiol. Abt.), p. 391. Lister, J. J. (1830) Phil. Trans., p. 187. Mayall, J. (1886) J . Sot. A r t s , 34, 1055. Mazia, D. (1941) CoZd Sprifig Harb. Symp. qiruiit. Biol., 9, 40. Miescher, F. (1871) Hoppe-Seyleu‘s Med-chem. Urrtersttchiricgen, 46, 441. Nordenskiold, E. (1928) The History of Biology. Translated by L. B. Eyre, New York. Tudor Publishing Co. Oes, A. (1908) Bot. Z., 16, 89. Ranvier, L. (1875) Trait4 technique h’histologie. Paris. Sachs, J. v. (1890) History of Botany. Translated by H. E. F. Garnsey, Oxford. Schleicher, W. (1879) Arch. mikr. Aptat., 16, 248. Strasburger, E. (1879) Jena. Z . 19, Sitzungsber, p. 93. Strasburger, E. (1879) J e w . 2. Nafurw., 19, Sitzungsber, p. 93. Waldeyer, W. (1888) Arch. mikr. Apia#., 32, 1. Zacharias, E. (1881) Bot. L.,39, 169. Zacharias, E. (1883) Bot. Z., 41, 209.


Nuclear Reproduction* C. LEONARD HUSKINS Department of Botany, University of Wiscoiisin, Madison, Wiscowin. CONTENTS Pllgc

I. 11. 111. IV. V. VI. VII.

Introduction ......................................................... Nuclear or Cell Division ............................................. Chromosome Reproductioii ............................................ Variation in Nuclear Size ............................................. Reduction in Chromosome Number ..................................... Conclusions .......................................................... References ...........................................................

9 10 12 15 18 21 24

I. INTRODUCTION I t is difficult to find anything new to say about morphological aspects of nuclear reproduction, but there are cytochemical and genetic data which should be correlated with the descriptive in any evaluation of the problem. It may also be useful to emphasize the distinct aspects of some of the processes that conlnlonly occur in association. Any correlated analysis or evaluation must at the present time contain many speculations, but I do not think that harmful, provided facts and ideas are plainly differentiated. Everyone knows that reproduction of the elements within the nucleus is not the same thing as reproduction of the nucleus itself, yet a number of geneticists, for example, have failed to make this distinction explicit in their consideration of problems of gene action. Usually this makes no difference to the argument, but it can do so when as in some recent papers on the possible role of heterochromatin in differentiation, conclusions as diverse as that of Caspersson (1939) and others, that heterochromatin is concerned with the division of the chromosomes is cited along, for instance, with Darlington and Thomas’ (1941) conclusion that it is responsible for supernumerary divisions of the pollen cell in Sorghum, and so on, without any indication being given that these are very different processes or that the evidence for the conclusions has been obtained from very different observational levels. Though chromosome and nuclear reproduction are both normally antecedent to reproduction of the cell, any one of the three processes can, of course, occur without either one of the others.

* Presented at the Seventh International Congress of Cell Eiology, Yal: University, September 4-8, 1950. 9

10

C. LEONARD H U S K I N S

If the geneticist sometimes errs by implying nuclear and cell division when his evidence relates only to gene or chromosome reproduction, the experimental cytologist in the past often paid too little attention to the chromosome and genes. Total disregard is not possible today, especially when once widely divergent disciplines are brought together in a congress such as this, but there is still evident need, even in the abstracts of our program, for all of us to make more extended use of each other’s data.

11. NUCLEAR OR CELLDIVISION Professor Heilbrunn will show later in this program that “cell division is not necessarily initiated by an increase in cell permeability, nor is it always accompanied by an overall increase in cell permeability,” and will present evidence on the gelatin-liquefaction cycle associated with mitosis. H e maintains that : “The colloid-chemical theory offers a logical explanation of all the known facts” of cell division. It is evident that in his argument he is including both karyokinesis and cytokinesis but not chromosome reproduction. I shall therefore concentrate chiefly on chromosomes, especially since it is the field with which our research group is predominantly concerned. As for the correlation between karyokinesis and cytokinesis, we may say that the concept of nucleoplasmic ratio determining nuclear and cell division, which was derived predominantly from morphological observations, can no longer be considered seriously in its original form, but that the data it subsumed must still be taken into consideration and that the concept itself is not wholly invalid. As for morphogenesis, while fully realizing that nothing should be regarded as unimportant in the present embryonic state of our knowledge, I shall for today also assume that we need pay little attention to nuclear or cell division or size as such in this connection. W e know, e.g., Weisz (1947), that cell mass may influence the course of differentiation and also that nuclear and cell size may have striking effects in some cases. The latter is evidenced by the differences, both morphological and physiological, sometimes found between diploids and their autopolyploids. But in some cases an increase of the chromosome number appreciably increases neither nuclear size nor any other characteristic. Correlatively, differences in the number of nuclei per cell do not appear to be causally related to differentiation. I n Acetabularia, for instance, Hammerling ( 1946) has found that nuclear divisions normally begin only after niorphogenesis is completed, However, a young nucleus will divide when transplanted into an old system, which, since the nucleus controls the differentiation, can be taken to indicate that nuclear division is ultimately regulated

NUCLEAR REPRODUCTION

11

through the mediation of its own products. Schulze (1939) dismissed the possibility that increase of chromosome material, either by polyteny or polyploidy, is involved in the increase of nuclear size which accompanies differentiation in this alga, but the material does not seem sufficiently favorable for this to be ruled out by a descriptive cytological analysis. DNA measurements should give a more decisive answer. In dikaryotic fungi a gene in one nucleus can dominate in its effect over a gene in the other, just as one allele over another in a diploid or polyploid nucleus. Heterosis also is exhibited in heterokaryonts. If we look for functional significance of the multinucleate condition it seems to be found not in anything connected with differentiation but in the occurrence of nuclear competition, whereby nuclei containing disadvantageous genes are apparently subject to adverse selection, as found in Neurospora. The reported stability of the polykaryotic Mucor species after an initial adaptive period when first placed on artificial media (Hesseltine, unpublished thesis, University of Wisconsin) could also have this explanation. Fankhauser (1948) has shown that while a polynucleate condition causes abnormal cleavage in frogs and toads, it does not do so in most Urodeles. I n these, fertilization is normally polyspermic but at the critical stage in development the principal sperm nucleus unites with the egg nucleus, and the accessory nuclei begin to degenerate. The latter may go through prophase, and hence doubtless through chromosome reproduction, but not through mitosis. Barber (1942) found that orchid pollen grains with sub-haploid nuclei could divide normally if separated by only a thin cell wall from grains with a full haploid chromosome complement. As shown by Clark (1942) even fragmentation of the nucleus has no necessary effect on development and germination of corn pollen so long as all the fragments remain in the cell. To conclude this section of our discussion on the limited significance of nuclear or cell division for differentiation and development, brief reference may be made to a few data on the time and scope of gene action relative to mitosis and cytokinesis. Berrill and Huskins (1936) stimulated discussion of this issue by proposing that “energic” replace the term “resting” nucleus. C. Stern (1938) showed that in a number of decisive cases specific genes interact with the cytoplasm during the energic state of the nucleus. He pointed out that in some cases it is possible, as in some examples of pollen dimorphism, that gene-controlled substances exert visible effects only after the breakdown of the nuclear membrane, but decisive evidence of this was, at that time lacking. I am not aware of any more definitive evidence having been advanced since then. H. Stern

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(1946) showed that there is an increased permeability to sucrose of the plasma membrane during meiosis and postmeiotic mitosis in Trillium pollen and that it begins to rise before breakdown of the nuclear membrane. Data on permeability during endomitosis are lacking and should be sought, but sugar intake need, of course, bear little or no relation to diffusion of nuclear products, Jones (1947) shows that changes in the nucleus may have visible effects in the cytoplasm and that gene-determined pigments in corn may be either cell-limited or diffusible over a considerable area of tissue. Commoner (1949), from further analyses of somatic mutations at the A locus, suggests that genetic determination of the anthocyanin content occurs before cell enlargement and that the specific action of the A gene is based on initiation of production of a precursor. The radial pattern suggests to him distribution of the gene product during formation of constituent cells ; evidence on the issue of influence during mitosis might be found here. 111. CHROMOSOME REPRODUCTION Let us turn to the problem of chromosome reproduction without nuclear division. Almost without exception, biology textbooks teach that the chromosome number is constant in all somatic cells of a multicellular organism. A brief sketch of the development of this concept may be of interest and not without value for future work and concept formulation. Weismann (1893) wrote: “With certain exceptions . . , the number of chromosomes is constant for each species.” Wilson (1900) went further : “The remarkable fact has now been established with high probability that every species of plant or animal has a fixed and characteristic number of chromosomes which regularly recurs in the division of all its cells; and in all forms arising by sexual reproduction, the number is even.” I t remained for 0. Hertwig (1918) to formulate in detail the “law of constancy of chromosome numbers”: “This law tells us that the number of chromosomes in all cdls of a plant or animal species, with the occurrence of nuclear division, is always exactly the same whether we are dealing with epidermis, cartilage, muscle, or glandular cells, etc. However, . . the egg and sperm contain one half the number of chromosomes of the somatic cells. This also is a lawful phenomenon.” One embryologist later extrapolated the law to the extent of writing: “All cells whether they continue to divide or not ultimately contain the same genetic proteins in equal quantities.” There were many factors involved in the gradual consolidation of the law of constancy of chromosome numbers. Nemec in 1904 and 1910 had

.


13

discovered tetraploid nuclei in chloralized roots, but Strasburger ( 1907) was convinced that they could not persist and therefore were of no significance. H e even explained away as obviously due to some “disturbing influences, such as wounding by small animals, etc.,” his own discovery of rows of tetraploid cells in control roots. Of this Winkler (1916) wrote : [trans.] “This last remark of Strasburger is extraordinarily characteristic. I t shows that the conviction that in normal somatic tissue only diploid cells could occur has become a dogma under the influence of which the best plant cytologist comes at once to the opinion that the occurrence of tetraploid cells found therein must be pathological, without even considering any other possibilities.” Winkler himself, after establishing with certainty the occurrence of polyploid cells and tissue in Solanum species and of the production of polyploid plants from the callus of grafts, concludes : “since the germ cells always arise directly from embryonic tissues they will always have the typical chromosome number and hand it down to the next generation. The constancy of chromosome number is safeguarded even when there is vegetative reproduction, since plants grow with their growing points which, by definition, are always embryonal. . . . W e therefore come to the view that the regular occurrence of polyploid cells in the somatic tissue of higher plants by no means refutes the laws of constancy of chromosome number but must be expected in view of the importance of the chromosome number for cell size.” Except possibly for the last clause, with its teleological flavor, this is as clear and acceptable a statement on polysomaty as could be made today. Why has it so very generally been ignored, not only by textbook writers but also by most research workers ? It must be remembered that from the time the correlation of chromosome behavior in meiosis and of Mendelian factors in segregation and recombination was first clearly enunciated it took about a quarter of a century to establish the “chromosome theory of Keredity” to the satisfaction of the overwhelming majority of biologists. As late as the 1920’s there were some who still considered the chromosome complement as a variable characteristic which was no more and no less a part of the plant phenotype than, say, the number and shapes of the leaves. The important general concepts involved in the chromosome theory of heredity were most conclusively established by showing that in exceptional organisms with chromosome numbers or arrangements of their parts that deviated from the norm of the species, such as haplo-IV Drosophila, trisomic Oenothera, tetraploid Datura and translocation stocks of Drosophila, maize, etc., the genetic behavior was altered correlatively.

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By 1937 it was realized that colchicine produces polyploid cells by inhibiting spindle action, and in view) of the importance of polyploidy in plant breeding there was immediately a wide search for other chemical agencies that would induce it. I t is not surprising, therefore, that when polyploid divisions were found in mature plant tissues that had been stimulated by “growth substances” it was assumed that the polyploidy was caused by the treatment. It seemed to me, however, that the effect of “auxins” was more probably stimulation of divisions in already polyploid nuclei. Geitler (1948) apparently reached this conclusion at about the same time and during the war he effectively established that “polysomaty” is common in the leaves and stems of a large series of plants, as he had earlier found it is in many insects. He had stimulated divisions by wounding. Unaware of his work, we had initiated complementary studies on roots with indole acetic acid in dosages that gave results quickly enough for us to determine that at least the higher polyploid nuclei had not been produced after initiation of the treatment (Huskins and Steinitz,

1948). Since polysomaty has not been considered to have any effect on genetic behavior, it was of no significance in that stage of the rapidly developing science of genetics where establishment of rules of transmission was the major goal. The question today is whether or not it is significant in developmental or physiological genetics. Let us first consider from this point of view some of the recent work, including that of members of our own group, on polysomaty. Incidentally, there is much confusion due to unsatisfactory terminology in many discussions of nuclear reproduction. I have been accused of confusing endomitosis, endodivision, polysomaty, and polyteny because I have not always in all contexts differentiated sharply between them. I risk this charge again and for the same reason as previously, namely, that I think the distinctions unimportant at the present time in discussions of possible functions. They are not unimportant in descriptive cytology, and I do not ignore them in that context. Further, since I have been so widely misunderstood on another point, despite two separate and specific warnings in my original speculative discussion on the possible significance of polysomaty ( Huskins, 1947), let me here emphasize that polysomaty as suck cannot possibly be of any great general significance in differentiation. It may be in special cases; in all cases it docs prove that chromosome, and therefore gene, reproduction continues after nuclear or cell division ceases and thereby opens the way to coilsideration of the possibility that gene action i s correlated zcuWa gene reproduction. The cyto-


15

logical evidence, now generally accepted, that the chromatid is not a transversely unitary structure, shows that Mendelian segregation involves units at a higher level of integration than those resolvable by even such a relatively crude analyzer as the light microscope. We may therefore justifiably consider the possibility that the ultimate units which may be effective in differentiation could be at a very much lower level. There is considerable evidence that, at the microscopic level, reproduction of the component strands of a chromosome is not uniform throughout its length. The possibility that reproduction of the materials making up a Mendelian gene may be differential for different genes in different tissues therefore becomes almost an a priori probability. The problem is to devise methods for testing i t ; such are, of course, appearing as soon as the problem is envisaged. IV. VARIATION IN NLTCLEAR SIZE To return to simpler levels of discussion: Huskins and Steinitz (1948) attempted to analyze the great variation in nuclear size in differentiated regions of Rhoeo roots. Evidence obtained by counting the number of heterochromatic bodies (which was the method devised by Geitler for insect tissues) and by treatment with indole acetic acid, coincided in showing the variation to be correlated with degree of polysomaty. Similar results were obtained with barley (Leonard-Bennett, unpublished). Duncan and Ross (1950) have shown that in nuclei of niaize endosperm undergoing mitosis the normal triploid number of chromosomes is usually present. However, in giant energic nuclei a high degree of polyteny is observed in regions of chromosomes marked, for observational purposes, by heterochroinatic “knobs.” They have, further, shown a different range in nuclear size in different areas of the endosperm. An apparent reduction in the size of endosperm nuclei adjacent to the embroyo as its development proceeds is of special interest and is currently being studied further. Nuclear and cell volume, chromosome and chromatid number in pith cells of Nicotium towentosu are being studied by Dr. Muriel Bradley. Her data show, iiiter ulia that in this material nuclear volume is related directly to chromatid and not to chromosome number. I t seems to make little or no difference whether 8n chromatids are present as the 4n number of ordinary two-chromatid chromosomes or as 2n chromosomes each having four chromatids. The data show also that following the halving of the total cell volume after telophase there is within each of the polyploid classes a relatively slight increase in nuclear volume which is followed by something like a doubling before the next prophase. These morphological data accord with cytochemical studies. They support indirectly the as-

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sumption made from microchemical analysis by Boivin, Vendrely and Vendrely (1948), Mirsky and Ris (1949), and others that with certain exceptions the DNA content is the same in all somatic cells. Certain variations in DNA content have been related, obviously correctly, to reproduction of the chromosomes and to polyploidy. Relating the constancy, apart from polysomaty, to the concept that the genotype is the same in all cells, it is usually concluded that DNA is an essential, and probably constant, component of the gene. The most recent published work along these lines is that of Swift (1950) who has shown, by absorption spectrophotometry following Feulgen staining, that the nuclei of ten different somatic tissues of young and adult mice all show approximately the same amount of DNA except for some of those in the liver, pancreas, thymus, blood lymphocytes, and Sertoli cells which contained two or four times the common amount. Occasional rare intermediate values were found and presumed to be associated with mitosis. Mouse spermatid nuclei had half the DNA of the common somatic nuclei. Primary spermatocytes had four times and secondary spermatocytes twice the spermatid value. Some premeiotic sex cells had the somatic amount and some twice as much. The former predominate in testes of 1- to 10day old mice and the latter are commoner at maturity. Nuclei of six tissues of adult frogs all had approximately the same amount of D N A (excepting for a few liver nuclei which had twice as much), and this was slightly less than twice that characteristic of the mouse. From studies of embryonic mouse liver and Amblystoma larvae, it was shown that DNA content “builds up in the interphase nucleus before the visible onset of prophase” and that “during the visible stages of mitosis no DNA is synthesized.” I n the Malpighian tuhule nuclei of a grasshopper four classes of DNA content with the ratio 1 :2:4:8 were found. In our laboratory (Bloch and Patau, unpublished) the relative DNA content of mouse liver nuclei has been determined, following Feulgen staining, with both an electro photometric and a visual microphotometer, the latter instrument having been designed by Dr. K. Patau. The results with liver of adult mice of pure lines and their hybrids agree with those of Swift and confirm that the step from one class to the next higher is a very accurate doubling of the DNA content accompanied by a doubling of nuclear size. In embryonic liver the DNA content ranges from 1 :2. It doubtless is reflecting the synthesis of DNA in nuclei preparing to undergo mitosis. Intermediate values between higher classes may be reflecting chromosome reproduction preparatory to either mitosis or endomitosis. Schrader and Leuchtenberger ( 1949) have stressed the

N U CLEAR REPRODUCTION

17

variation “which may be due to different degrees of polyteny” in different tissues of Tradescantia. Since oral presentation of this article, Swift (1950) has reported findings in maize and Tradescantia. In our laboratory, measurements are being made on Allium and Tradescantia tissues (Nelson, unpublished). For the roots the results are similar to those of Swift, excepting that he reports none of the lowest class in the elongation region of maize roots but several higher multiples, whereas in the same region of Allium roots, Nelson finds large numbers of the lowest and second classes, very few of the third, and none higher. Interesting additional findings by Nelson are : (1) that the doubling which precedes mitosis occurs early in the interphase period (unpublished data of Dr. Alma Howard, Radiotherapeutic Research Unit, Hammersmith, on uptake of radioactive phosphorus, are in accord) ; ( 2 ) that the guard cells of stomata have constantly the lowest amount of DNA normal for diploid cells, while other cells of the epidermis have double this amount ; (3) immediately after the first division of the microspore nucleus the resulting “vegetative” and “generative” nuclei have the same (haploid) amount of DNA but as the pollen grain “ripens” the generative nucleus doubles its content - in readiness for its division - while the content of the vegetative remains constant, contrary to the opinion of the many descriptive cytologists who have noted its fainter staining. In all of the foregoing, the findings confirm the ideas of Jacobj (1925) and many subsequent workers that increase of nuclear size may be caused by geometric increase of chromosome number, i.e., by polysomaty. It has, however, also long been clear that nuclear size can increase greatly without change in chromosome number. In some cases it is now evident that this may be correlated with increase in degree of polyteny, as shown by Duncan and Ross in maize endosperm. In yet others it may have little or no relation to the “chromatin” content. Schrader and Leuchtenberger ( 1950) have shown cytochemically that in the very different-sized nuclei which characterize different lobes of the testes of Armelitis albopunctatus, a hemipteran insect, the DNA content is approximately the same; it is the total protein and the RNA content that are correlated with nuclear, nucleolar and cytoplasmic volume. Apart from internally regulated “permanent” changes in nuclear volume, there are, of course, changes which appear to be correlated with developmental or physiological factors. Metz and his students (Buck and Roche, 1938) have shown that osmotic and mechanical pressure changes may cause reversable increase or decrease in nuclear and chromosome size as great as 25 per cent. The diverse effects of various fixatives on nuclear size are also, of course, well recognized.

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To summarize this discussion of polysomaty we may safely say: (1) The concept of genic and chromosome identity of all cells obviously needs both amplification and circumscription; (2) Many more data on its occurrence are needed before we can even decide whether or not it, as stich, is at all likely to be found of any general significance in differentiation and development. (Regular and characteristic differences in number of chromosomes in certain tissues such as Geitler and others have found in various insects and in DNA content such as Nelson is finding in plant epidermis seem to point affirmatively, whereas the difference in these regards between morphologically similar regions of maize and onion roots and many other data point negatively.) ; (3) The newer techniques if used on specially chosen materials, particularly if these permit a concomitant genetic analysis, are capable of giving us answers to problems that were unassailable by the methods of cytological study available until very recently; (4) Whatever the relationship of D N A to ultimate gene structure, it seems certain that it is precisely related to the reproduction of the chromatid, which is the subdivision of the chromosome that is significant for Mendelian heredity-so far there is no evidence of a chromosome’s being able to divide without first doubling its DNA content, but, of course, very few studies of this question have yet been made: ( 5 ) Though chromosome reproduction must “normally” precede nuclear division, either of the two processes can proceed independently of the other -the relationship is parallel to that of cytokinesis to karyokinesis.

v.

REDUCTION IN

CHROMOSOME

NUMBER

We may next consider the process of reduction of chromosome number. To the long-established account of the process in germ cells the only important item to be added from current studies with the newer techniques seems to be the discovery (Swift, Nelson, and probably many others unpublished) that the D N A content reaches the same heightened level by pachytene of the first meiotic division as that typical for early mitotic prophase. Therefore, two divisions without an intervening increase during their interkinesis are required to restore the normal relationship between number of chromosomes and nuclear D N A content since the former has been halved. W e have moved far from the concept held by many cytologists not so long ago that the first division was the reduction division, and the second an unexplained concomitant ; genetics has long shown that both divisions are essential to genetic reduction, the occurrence at first prophase of four chromatids in each bivalent indicated the same from the descriptive cytological aspect, and now we see that they are needed to restore cytochemical balance also.


19

Somatic reduction and segregation have long been known but little investigated until recently. Many textbooks list Winkler’s ( 1910) Solununz danoriniaiticm as having originated by somatic reduction, but none that I know of list his 1916 cases. I believe (see Huskins, 1948) the former to be an erroneous interpretation while the latter are clearly valid. Winkler’s 1916 remarks warrant quotation as a basis for discussion of current studies. [translation] To obtain such reversions to the diploid normal form from a tetraploid gigas type, the cells which gave rise to the atavistic tissue complex must have undergone a reduction-division. We may therefore not doubt the possibility that reductiondivisions occur in somatic cells. What stimulates them has to be left undecided. That the tetraploid condition of the nucleus by itself should have caused a tendency for the reduction of the increased chromosome number cannot be assumed, since tetraploid types exist and in general persist. It will also have to remain undecided for the time being whether the halving of the chromosome number occurs by typical reduction-divisions or otherwise. As a matter of fact it will be very difficult to find such a reduction-division since the reversions, at least so far, have occurred rarely and quite irregularly, that is in places which could not be predicted.

Two cases are reported in which somatic reduction appears to be a regular process: (1) prior to meiosis in the hermaphrodite gonad of a coccid (Hughes-Schrader, 1927) and (2) in the ileum of mosquito larvae (Berger, 1941 ; Grell, 1946). Somatic pairing and segregation are, of course, well known in the Diptera, but adequate data are lacking on reduction, though it was early reported by Bridges. Bateson (1926) insisted that there was much genetic evidence for somatic segregation in various tissues, but most such cases investigated in the past quarter century have been interpreted as somatic mutation, which he warned against as likely to obscure the issue. Recent evidence indicates that in any specific case both must be considered as possible explanations until the one is ruled out. The sporadic occurrence of chromosome pairing and/or reduction in somatic tissues has frequently been recorded. For example, Gates (1912) observed it in the nucellus of Oenotheru Zutu; Ludford (1935) and others have recorded it in tumors and tissue cultures of tumors; Metz (1942) found in a Sciara hybrid a salivary gland nucleus containing chromosomes from only one of the parents; Love (1936) found pollen mother cells that had undergone reduction prior to meiosis. East ( 1934), Nishiyama (1933), Kiellander (1941), Sparrow (1941), and Vaarama (1949) obtained plants with reduced, ancestral, chromosome numbers among the progeny of polyploid strawberries, oats, Poa, wheat, and Ribes, respectively. Brown (1947) found a reduced sector in an unbalanced poly-

20

C. LEONARD HUSKINS

ploid cotton plant. Upcott ( 1939) observed irregularly reduced chroniosome numbers in tetraploid Primnula kewensis; it is possible that by attributing these to “split spindles” she may have diverted attention from the problem as Strasburger, in Winkler’s opinion, quoted earlier lierein (1916), diverted attention from polysoniaty by assuming that it must be pathological. Following sporadic discovery of haploid cells in various plant roots, consideration of Caspersson’s ( 1939) suggestion that nucleic acid plays a role in synapsis and chromosome division led to a search for somatic reduction in preparations of Allium root tips treated by Dr. M. Kodani (1948) with sodium nucleate. Many cells with two reduced groups of chromosomes were found (Huskins, 1948). They were later found also in roots grown in solutions rich in phosphates (Galinsky, 1949). More recently it has been found (Huskins and Cheng, 1950, and unpublished) that prolonged low temperature treatment also increases the frequency with which “reductional grouping” occurs. There is also evidence that genetic factors affect the frequency with which reduced tissues or organs occur. In one strain of tetraploid Rhoeo we have obtained diploid and triploid roots and shoots with and without treatment. In another strain, treatments increase the frequency of reductional groupings, but no reduced tissues have yet been obtained. Allen, Wilson, and Powell (1950) have recently compared the sodium nucleate results with the chromosome groupings that occur after colchicine treatment. An extensive study has been made by Patau and Steinitz (1951) on the origin of reductional groupings and of reduced cells (see also, Patau, 1950). It is clear that somatic reduction is of not infrequent spontaneous occurrence, that its frequency can be increased by various treatments and “natural” conditions such as low temperatures, and therefore that polyploidy is a reversible evolutionary process. Battaglia ( 1948) has recently reported that somatic reduction occurs regularly in the basal portion of the style of Sambucus, and Christoff and Christoff (1948) report it in the integumental cells of Hieracium. If these and the cases in insects mentioned earlier are confirmed and extended, the “law of constancy of chromosome numbers” and our concepts of nuclear reproduction will have to be extended in this direction also. Besides polysomaty, polyteny, and reduction, which affect the total chromosome complement, there is also aneusomaty, i.e., the occurrence of cells with variable numbers of individual chromosonles, to be taken into account. It appears probable that aneusomaty (not to be confused with aneuploidy which refers to deviations between, not within, organisms)


21

most commonly involves chromosomes that are wholly or in large part heterochromatic. Too little carefully controlled work has yet been done on aneusomaty to warrant any conclusions on either the mechanism of its occurrence or its significance. Duncan (1945) concluded that in the root tips of an orchid the occurrence of variable numbers of chromosomes was due to a differential rate of reproduction of euchromatic and heterochromatic chromosomes. Darlington and Thomas ( 1941) attributed similar variable numbers in Sorghum to selective elimination in the roots but not in the shoots. Randolph (1941) presents numerous cytogenetic data which show that in maize the problem of B chromosome function and behavior is very complex. In Cimex, Darlington (1939) found from 0 to 12 extra X chromosomes in the males, with the average number higher in natural populations (9.0) than in mass cultures (4.3). H e correlates cycles of chromosome and centromere division with the “differential precocity” of autosomes, M chromosomes, and sex chromosomes and functionally relates the various changes observed to “adaptive balance” in sex-determining mechanisms. The mechanism of variability is related to “the state or precocity” of the centromere and the relative size of the chromosomes. These issues would carry us far beyond the scope of the present review, but they lead up (as Darlington points out in his Appendix 11) to the problem of preferential segregation, which is also beyond present scope except that it must be pointed out that special spindle mechanisms exist which provide for elimination of whole sets of chromosomes. The best analyzed of such cases is probably Sciara, for which both genetic and cytological data are available. It seems probable that such elimination is functionally related to chromatin diminution that involves only parts of chromosomes and that some form of differential reproduction may be basic to all such. Investigation with the newer techniques and with wider concepts in mind than those which guided earlier descriptive studies may lead to the establishment of some of the generalizations which at present are almost entirely speculative.

VI. CONCLUSIONS To conclude we may summarize a few of the data which, though none alone may be conclusive, together suggest a need for revision and extension of some of our more orthodox concepts on various aspects of the reproduction of the nucleus and its components and of the role of the nucleus in differentiation and development. First, against the simplest unitary concept of the gene there is, to repeat, the cytological evidence that there are, frequently at least, more microscopically separable strands in both the

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mitotic and meiotic chromosomes than there are chromatids, which are the unitary gene strings of Mendelian segregation. The concept of the Mendelian gene as made up of identical “lamellae” would fit this. (The term “lamella” must not be taken to connote an undue simplicity in either the concept or the writer ; it may at our present stage of concept-forming serve as the equivalent of the beads-on-a-string model which was useful when the linear order of the genes was the issue.) Against it are the onehit radiation data, together with the occurrence of reverse mutations. It would be easy to imagine a change in one “lamella” being transmitted to all the others if the process went only one way, but not if it goes either way with anything like the same frequency, as some few gene mutations do. However, the one-hit hypothesis is not unassailable. For recent discussions of it see Muller (1950) and Opatowski (1950). Secondly, against the concept of gene identity of all cells (leaving aside whole chromosome changes for the moment) there are observations which are taken to indicate : (1) that heterochromatic and euchromatic parts of chromosomes may reproduce at different rates (Schultz, 1941) ; (2) that the banding pattern and length and breadth is visibly different in the same giant chromosomes from different tissues (see Kosswig, 1948) ; (3) that “specific chromosome loci [produce] lateral loops” (Duryee, 1950) ; and (4) that different parts of the salivary gland nuclei give strikingly unequal phosphatase reaction (see Brachet and Jeener, 1948), which suggests the possibility of variation from one gene to another in speed of renewal of phosphorus in the DNA. This would leave open the alternatives of phosphorus renewal in DNA playing a role in synthesis of proteins concerned with growth or of differential reproduction of chromosome regions. Differential reproduction of gene lamellae would provide a mechanism for differentiation not envisaged by Goldschmidt and probably not compatible with his present ideas on the gene, but it can be related conceptually to his early theory of timed, sequential physiological activity of the genes. Thirdly, it now seems fairly certain that the DNA content of nuclei from different tissues is constant, or, more strictly speaking, that it is constant relative to the total number of chromatids per nucleus, whatever the tissue. However, the R N A and protein content are both variable in different tissues. Chargaff (1950) reports that the D N A from different species differs in chemical composition and puts out the interesting suggestion: “It would be gratifying if one could say-but this is for the moment no more than an unfounded speculation-that just as the desoxy-pentose nucleic acids of the nucleus are species-specific and con-


23

cerned with the maintenance of the species, the pentose nucleic acids of the cytoplasm are organ-specific and involved in the important task of differentiation.” Daly, Allfrey, and Mirsky (1950), however, dispute his findings. As for the possible role of the nucleus in differentiation, it is pointed out by Dunn ( 1949), Gluecksohn-Schoenheimer (1949), and others that the developmental reactions controlled by mutated genes are in some cases very like those occurring in normal differentiation, and that they are possibly “more fundamental and perhaps much closer to gene action than we suspect now.” Weiss (1950) emphasizes that the term differentiation has been very loosely used and that morphological criteria have played too large a part in the classification of cellular changes. H e classes as “modulations” those changes which are reversible and stresses the fact that many specialized cells cannot “dedifferentiate.” It is, of course, generally agreed that though permanent changes take place in many cells, the early crude concepts of differentiation always being determined by segregation of particles during early segmentation of animal eggs, or of its determination by any simple type of regulated gene segregation or mutation, are quite untenable. Further, the concept of genic identity of all cells was an essential step in the development of our understanding of Mendelian heredity and of the essential differences between asexual and sexual reproduction for which the basic mechanisms are mitosis and meiosis. Have we now reached the stage when we can profitably consider the possibility that our concept of the gene of hereditary transmission subsumes the “gene of differentiation and development” ( 1947) and that the two must now be distinguished? So long as we assume the chromosomal genes to be the same in all cells we are forced either to consider the cytoplasm as the seat of the primary differentiating materials on which the genes act or as (see Schultz, 1950) the variable member of the reciprocally interacting units-these are analytically the same, though the latter is conceptually more satisfying. The assumption that there must be units in the cytoplasm that determine differentiation has led to the very fruitful discoveries of the entities that currently are most frequently referred to as plasmagenes though the concepts this term implies cause its rejection by many (see Schultz, 1950). Plasmagenes that are found to be dependent on chromosome genes in their function, even though autonomous in reproduction (Ephrussi, 1950) do not of course conflict with the orthodox concept of gene identity of all cells. If, however, we should be forced to the conclusion maintained by Darlington (1949) that the only difference between nuclear genes and plasmagenes is that the latter “have been denied . . . the gift . . . of coordinated seg-

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regation at meiosis,” we run into logical, or at least semantic difficulties. Though nucleus and cytoplasm are probably always interacting, the Acetabularia and other evidence seem to me to indicate that the nucleus primarily controls differentiation. But if cellular differentiation should in other cases be determined primarily by fully autonomous cytogenes which arise during ontogeny, as Sonneborn (1949) and others have suggested, then such cells are by definition genetically different even though we call their new “genes” plasmagenes. These cells should show different hereditary capacities not only in vegetative reproduction but also in the sexual reproduction of plants, if such differentiated cells can ever give rise, however remotely and indirectly, to female ,gametes. This latter would demonstrate definitely that the plasmagenes really are genes in the accepted meaning of the term, but it is the concept of chromosome identity, not of genic identity, of all cells that can be saved by the concept of two sorts of genes differing only in their location. This with the data on constant DNA content appeals to those of us who picture the chromosomes, but not necessarily the genes, as characterized by DNA at least during their period of reproduction and also of “division.” W e have, however, seen the limitations of “the law of constancy of chromosome numbers” and the constancy of chromosome parts seems very likely to prove even more limited. Which brings us back to nuclear reproduction: it is a very complex process which normally comprises many subsidiary processes which for the present we can safely classify into only two, viz., chromosome reproduction and chromosome separation. Various types of separation are accomplished by mitosis, endomitosis, and meiosis, each with many variants, but in each there is an essential basic uniformity. To these processes we must add the very incompletely known mechanisms of somatic reduction and of aneusomaty and differential elimination of parts of chromosomes, whole chromosomes of special types, and of genomes or sets of chromosomes. W e do not have to consider amitosis, for most of the descriptive work on it has long ago been shown to include errors of interpretation and the evidence of genetics shows clearly that it cannot be a normal process of nuclear reproduction, if by normal we mean the production of nuclei continuing to have potentially unlimited capacity for further reproduction. VII. REFERENCES Allen, N. S., Wilson, G. B., and Powell, S. (1950) J. Hered. 41, 159. Barber, H. N. (1942) J. Getwt., 48, 97. Bateson, W. (1926) 1. Genet., 16, 201: Battaglia, Emilio (1948) Nuovo G. bot. it&., 65, I.


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Berger, C. A. (1941) Cold Spr. Harb. Symp. quant. Biol., 9, 191. Berrill, N. J., and Huskins, C. L. (1936) Amer. Nut., 70, 258. Boivin, A., Vendrely, R., and Vandrely, C. (1948) C. R. Acad. Sci., 226, 1061. Brachet, J., and Jeener, R. (1948) Biochim. Biophys. Acta, 2, 423. Bridges, C. B. (1930) Science, 72, 405. Brown, Meta S. (1947) Amer. J . Bot., !M, 384. Buck, John B., and Boche, Robert D. (1938) Coll. Net., 19. Caspersson, T. (1939) Arch. exp. Zellforsch., 22, 655. Chargaff, E. (1950) Experentia, 6, 201. Christoff, M., and Christoff, M. A., (1948) Genetics, 53, 36. Clark, F. J. (1942) Genetics, 27, 137. Commoner, B. (1949) Abst. Amer. J. Bot., 86, 822. Daly, M. M., Allfrey, V. G., and Mirsky, A. E. (1950) J . yen. Phjsiol., SS, 497. Darlington, C. D. (1939) J. Genet., 39, 101. Darlington, C. D. (1949) Hereditas Suppl., 1949, 189. Darlington, C. D., and Thomas, P. T. (1941) Proc. roy. Soc., B190, 127. Duncan, R. E. (1945) Amer. J. Bot., 32, 506. Duncan, R. E., and Ross, J. G. (1950) J . Hered., 41, 259. Dunn, L. C. (1949) Anniversary Symp., Jackson Laboratory, Ear Harbor, Maine. Duryee, William R. (1950) Ann. N . Y. Acad. Sci., 60, 920. East, E. M. (1934) Genetics, 19, 167. Ephrussi, Boris (1950) VIIth Int. Congr. Cell Biology, Yale University, p. 24. Fankhauser, G. (1948) Ann. N. Y . Acad. Sci., 49, 684. Galinsky, Irving (1949) J. Hered., 40, 289. Gates, R. R. (1912) Ann. Bot., 26, 993. Geitler, Lothar 1948) Ost. bot. Z., 9, 277. Gluecksohn-Schoenheimer, S. (1949) Growth, 9, 163. Grell, Sister Mary (1946) Gewtics, 31, 60-76, 77-94. Hammerling, J. (1946) Natzwwissenschaften, SS, 337. Heilbrunn, L. V. (1950) VIIth Int. Congr. Cell Biology, Yale University, p. 36. Hertwig, 0. (1918) Das Werden der Organismen. Fischer, Jena. Hughes-Schrader, S. (1927) 2. Zellforsch., 6, 509. Huskins, C. L. (1947) Amer. Nut., 81, 401. Huskins, C. L. (1948) J . Hered., 99, 311. Huskins, C. L. (1950) I. Hered., 41, 13. Huskins, C. L., and Cheng, K. C. (1950) J . Hered., 41, 13-18. Huskins, C. L., and Steinitz, L. M. (1948a) J. Hered., 99, 34. Huskins, C. L., and Steinitz, L. M. (1948b) J. Hered., 99, 66. Jacobj, W. (1925) Arch. EnfzuMech. Org., lU6, 124. Jones, D. F. (1947) Proc. eat. Acad. Sci., Wash., 99, 363. Kiellander, C. I. (1941) Svemk bot. Tidskr., 96, 321. Kodani, Masuo (1948) I. Hered., 83, 115. Kosswig, C. (1948) Proc. 8th Int. Congr. Genet., Stockholm. Love, R. M. (1936) Nature, Lond.,1S8, 589. Ludford, R. J. (1935) Arch. exp. Zellforsch., 17, 411. Metz, G. W. (1942) Amer. Nut., 88, 623. Mirsky, A. E., and Ris, Hans (1949) Nature, Lond., la,666. Muller, H. J. (1950) I . cell. comp. PhySioJ., 35, 9.

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Nemec, B. (1904) Jb. z k s . Bot., 89, 645. Nemec, B. (1910) Das Problem der Befruchtungsfrage. Berlin. Nishiyama, I. (1933) Jap. 1. Genet., 8, 107. Opatowski, I. (1950) Genetics, I, 56. Patau, Klaus (1950) Genetics, MI 128. Patau, Klaus, and Steinitz, L. M. (1951) (In press.) Randolph, L. F. (1941) Genetics M, 608. Schrader, F., and Leuchtenberger, Cecilie (1949) Proc. nat. Acad. Sci., Wash, 86, 464. Schrader, F., and Leuchtenberger, Cecilie (1950) Ezp. Cell Res., 1, 421. Schultz, Jack (1941) Cold Spr. Harb. Symp. qzcant. Biol., 9, 55. Schultz, Jack (1950) Science, 111, 403. Schulze, K. L. (1939) Arch. Protistertk., 92, 167. Sonneborn, T. M. (1949) AMY. Scient., 87, 33. Stern, C. (1938) Amer. Nat., 7!2, 350. Stern, Herbert (1946) Philos. Tram., 40, 141. Strasburger, E. (1907) Jb. w'ss. Bot., 44, 482. Sparrow, A. H., Huskins, C. L., and Wilson, G. B. (1941) Canud. J. Res., 19, 323. Swift, H. H, (1950a) Proc. nut. Acad. Sci., Wash., SS, 643. Swift, H. H. (1950b) Physiol. ZoGI., 2S, 169. Upcott, M. (1939) J. Genet., 89, 79. Vaarama, A. (1949) Hereditas I,136. Weismann, August (1893) The Germ Plasm. W. Scott Ltd. London. Weiss, P. A. (1950) Quart. Rev. Biol., I,177. Weisz, P. B. (1947) J . Morph., 81, 45. Wilson, E. B. (1900) The Cell. Macmillan Co., N. Y. Winkler, Hans (1910) Ber. dfsch. bot. Ges., 28, 116. Winkler, Hans (1916) 2. Bot., 8, 417.

Enzymic Capacities and Their Relation to Cell Nutrition in Animals" GEORGE W. KIDDER Biological Laboratory, Amherst College, Amherst, Massachusetts

CONTENTS Introduction .......................................................... Nutritional Requirements of Tetrahymena .............................. Nutritional Requirements of Higher Animal Cells ....................... Conclusion ............................................................ V. References ............................................................

I. 11. 111. IV.

Page 27 28 32 32 33

I. INTRODUCTION Intensive studies of mammalian and avian nutrition have been stimulated by the practical importance of these higher animals in man's economy. It is well recognized that our newer knowledge of animal nutrition has led to the production and maintenance of more and better food animals, and this knowledge has been used to tremendous advantage in bettering man's own nutritional status. In studies of animal nutrition one is almost invariably studying, however, the nutritional conditions of a community of heterogeneous organisms. Nutritional requirements of the laboratory rat, for instance, have been determined largely for the rat plus his associated microflora and microfauna. Evaluation of enzyme systems capable of synthesizing biologically important compounds are often impossible when working with the whole animal. Techniques involving the use of sulfonamids and antibiotics to reduce the population of intestinal bacteria have frequently been successful in demonstrating enzyme lacks which otherwise were obscured. There appear to be two sets of results which can be expected when dealing with a higher animal, together with the intestinal bacteria, yeasts, and molds. First, a requirement for an organic compound may not manifest itself on account of the ability of highly synthetic organisms, within the gut, to produce sufficient amounts of the compound to supply the needs of the host tissues. This certainly has been demonstrated in the case of folic acid and vitamin BIZ, where deficiences in laboratory mammals can rarely be demonstrated without antibiotics. The second possibility to be kept in mind is that gut microorganisms may rob the host *Given before the Cell Nutrition session of the Seventh International Congress of Cell Biology.

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GEORGE W . KIDDER

of important nutritional materials, thereby cutting down on the efficiency of the host tissues. This may well be the explanation for the marked growth-promoting powers of aureomycin and streptomycin, as first reported by Stokstad and his associates (1950). If one is interested in the basic enzyme patterns of whole animals and wishes to study them by standard nutritional methods, then one must meet two conditions if definitive results are to be obtained. The animal in question must be freed of all associated organisms (be rendered bacteria- and fungus-free). During this process it is often necessary to use chemically crude materials to support growth and maintenance. The next step is to develop a diet composed of chemically known constituents. When these two conditions are met, one is then in a position to study the natural enzymatic capacities and limitations which are the result of gene mutations accumulated through the evolutionary history of the organism. 11. NUTRITIONAL REQUIREMENTS OF TETRAHYMENA Up to the present time, techniques have been developed for only one animal genus to meet both these conditions: free of associated organisms and maintained on a chemically defined medium. This is the animal microorganism Tetrahynuena. The discussion of enzymatic capacities and limitations in animal cells whicli follows, therefore, will deal mainly with this unicellular animal. In making biochemical and enzymatic comparisons, however, between Tetrahymena and higher animals, one must remember that the comparisons are to be made between the protozoan and whole organisms and not between the protozoan and isolated cells or tissues from the multicellular types. I would like to review briefly the nutritional requirements of Tetrah p e m and point out some similarities and differences which are to be noted between this organism’s enzyme capacities and those which have been deduced for higher forms. The basic enzyme pattern for the synthesis of specific amino acids has been modified, in animals, by loss, from that posssessed by many bacteria. Synthesizing bacteria can and do construct all the amino acids from simple N-containing compounds such as nitrates and ammonium salts. Loss of many of the enzymes responsible for these syntheses has gradually resulted in what we can call the “animal pattern.” Tetrahymena, like the rat, has lost enzymatic capacities for the synthesis of histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophane, and valine (Kidder and Dewey, 1945b, c, 1947b). Arginine can be synthesized with difficulty, indicating defective enzyme systems in regard to at

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least some portion of this configuration. This corresponds also to the rat, where mature rats can synthesize arginine at a rate which allows maintenance, but growing rats must have an exogenous supply to supplement the arginine which they make. Serine appears to be much more important in the metabolism of Tetrahymen0 than it does in the higher forms. Growth fails in Tetrahynzena in the absence of serine when relatively high levels of certain other amino acids are present in the medium (Kidder and Dewey, 1947b). For practical purposes, therefore, serine may be considered essential, but only because its rate of synthesis is low, not absent. Certain interrelationships among the amino acids may be demonstrated here, as they can be in higher forms. Thus tyrosine is synthesized by the oxidation of the p-position of the phenylalanine but reductases for this position are lacking (Kidder, 1947). Similarly cystine is formed from methionine, but methionine cannot be synthesized from cystine (Kidder, 1947). On the other hand, Tetruhynzena has lost the ability to deacetylate tryptophane (Dewey, Kidder, and Parks, 1951). N-acetyl-tryptophane is without activity, whereas it has been reported to replace tryptophane for mammals. This does not mean that Tetrahymena has lost its deacetylating enzymes, however, as N-acetyl-leucine and N-acetylmethionine were able to replace leucine and methionine respectively. In addition to the essential amino acids, which must be balanced correctly for most efficient utilization by the cell (Kidder and Dewey, 1947b ; Dewey, Parks, and Kidder, 1951) , certain non-essential amino acids prove to be somewhat stimulatory. Some growth stimulation can be demonstrated in Tetrahymenu by alanine, aspartic acid, glutamic acid, glycine, and proline (Dewey, Parks, and Kidder, 1951 ; Kidder and Dewey, 1 9 4 9 ~ ) . It would appear that this simply reflects the ability of the organism to incorporate amino acids from the medium and thereby save energy otherwise required for reactions which it is perfectly capable of performing. Investigations of the well-known relationship between arginine, citrulline, and ornithine in the urea cycle have indicated that ornithine and/or citrulline do play a role in the metabolism of Tetrulaywna, and both are synthesized via arginine (Dewey, Kidder, and Parks, 1951). Just what these roles are, however, has not been worked out, although it does not appear to have anything to do with the urea cycle. Ammonia is the nitrogen end product of amino acid metabolism here, and neither urea nor uric acid is formed. Dextrose spares amino acids in the nutrition of Tetrahymenu but no

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carbohydrate source is essential to metabolism. Likewise acetate is stimulatory and spares the vitamin protogen, to be mentioned later (Kidder, Dewey, and Parks, 1950). Inorganic requirements are difficult to determine due to traces of various minerals found in amino acids and in other supposedly pure constituents of the medium. Where high requirements exist, the detection of essential ions becomes possible. It has been shown, for instance (Kidder, Dewey, and Parks, 1951), that phosphate, magnesium, copper, iron, and potassium are required. Likewise it can be demonstrated that calcium is non-essential. In the case of calcium, removal by oxalate or citrate can be accomplished. Citrate and oxalate inhibition can be completely overcome by additions of magnesium but not by calcium. Unlike the mammal, Tetrahymena is dependent upon an exogenous source of pyrimidine and purine (Kidder and Dewey, 1945a; 1948; 1949a, b ; Kidder, Dewey, Parks, and Heinrich, 1950). Of the naturally occurring pyrimidine bases only uracil will fulfill the requirements of this organism. Cytosine deaminase is lacking as are also enzymes for the decarboxylation of orotic acid. Cytidine deaminase is present, however, as cytidine is as active, on a molar basis, as is uracil or uridine. There is good evidence to show that thymine is synthesized from non-pyrimidine sources, as neither thymine or thymidine will spare uracil. Guanine is an absolute requirement for Tetruhymena, and no other naturally occurring base can act as a substitute. Adenine and hypoxanthine spare guanine, while xanthine and uric acid are both inert. I n the purine system the sugar-base linkage does not appear to be the limiting factor, for adenosine cannot be converted to guanosine. Xanthine oxidase appears to be lacking in Tetrahymena as evidenced by two observations: first, 2-aminopurine is inert and it has been found that xanthine oxidase converts this compound to guanine (Lorz and Hitchings, 1950) ; second, hypoxanthine is as active, on a molar basis, as is adenine in sparing guanine. This indicates that all the hypoxanthine is being aminated to adenine and none oxidized to the inert xanthine. Advantage has been taken of the knowledge of the enzyme patterns of Tetruhymena in purine metabolism to pilot experiments which have revealed a qualitative biochemical difference between certain neoplastic cells and normal cells of mice (Kidder, Parks, and Woodside, 1949). When one considers the vitamins, it becomes apparent that Tetrahymenu, like higher animals, has lost enzymes for the synthesis of the major B-group (Kidder and Dewey, 1951). An exception is biotin. No requirement for biotin exists and, what is more conclusive, raw


31

egg white and crystalline avidin have no inhibitory effect. Another exception is vitamin BIZ. It can be shown by appropriate assay techniques that vitamin BIZ activity increases in a culture of Tletrahymena, originally devoid of BIZ. Like all animals so far studied, Tetrahymena is dependent upon an exogenous source of PGA (Kidder and Dewey, 1947d, 1949d). It lacks enzymes for coupling the pteridine portion of the molecule to the p-aminobenzoyl-glutamic acid portion, and also lacks the necessary enzymes for the peptide linkage between the carboxyl of pteroic acid and the amino group of glutamic acid. Like higher animals, and distinctly in contrast to bacteria, Tetrahymenu possesses conjugases which enable it to utilize conjugated PGA such as the tri- and heptaglutamates. Recently it has been shown that what has been called the citrovorum factor (Sauberlich and Baumann, 1948) is a substituted PGA. This substance was shown by Broquist, Stokstad, and Jukes (1950) and others to be far more effective in releasing the inhibitory action of 4-amino PGA or aminopterin in chicks than was PGA itself. This and many other observations make it seem probable that PGA may not be the active molecule after all but must be changed to citrovorum factor (CF), which in turn becomes a part of the coenzyme-enzyme complex leading to a vital reaction. I n as much as PGA can be utilized by animals, they must pbssess the enzyme necessary for changing it to CF. The recent work of Shive, Bardos, Bond, and Rogers (1950) has indicated that C F (which they call folinic acid) may be a formylated PGA, unsaturated beyond the condition of PGA itself. W e can say, therefore, that animals including Tetrahymenu have retained the enzymes necessary for this formylation and dehydrogenation while these enzymes have been lost in the bacterium, Leuconostoc citroaorum. Tetrahymena is dependent upon a new member of the B vitamin complex which has been called protogen (Stokstad, Hoffmann, Regan, Fordham, and Jukes, 1949). This factor appears to be identical with the socalled acetate factor and the pyruvate oxidase for certain lactic acid bacteria (Snell and Broquist, 1949). That it functions during enzymatic oxidation of pyruvate to acetate can be shown manimetrically, and minute amounts of protogen dispel the, acetate requirement in Lactobacilli I n our animal system, however, protogen is active in one or more additional vital reactions since the addition of acetate only spares, never replaces protogen (Kidder, Dewey, and Parks, 1950). It may be that protogen is more active than even vitamin BIZ, and so far conditions of protogen depletion have not been attained in higher animals. Assays of various

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tiEORGE W. KIDDER

plants and animals show it to be very widespread in nature (Keevil, 19.50), and it can be safely predicted that this factor will be found to function in more organisms than Tetralaymena and the lactic acid bacteria.

REQUIREMENTS OF HIGHER ANIMAL CELLS 111. NUTRITIONAL I would like to consider briefly the implications of what I have discussed on the nutrition of higher animal cells. I believe we should draw a sharp distinction between nutrition in a higher organism and nutrition in any of its isolated tissue cells on the basis of enzymatic capacities. W e know, for example; that the enzyme complex of cells of the liver are far more elaborate than those of most other tissues. There is, in fact, a segregation of enzymatic capacities during histogenesis so that the cells of one set of tissues develop certain enzymes while other cells develop other sets. In the intact animal, therefore, the nutritional requirements reflect the lack of synthetic ability after all the synthetic enzyme systems of the different tissues have been added together. W e know, for instance, that cells in tissue culture appear to require heat labile fractions from serum and/or embryo juice. If the embryo juice is heated, certain necessary configurations appear to be destroyed. And yet we know there is no such requirement for heat labile compounds in the intact aseptic animal. The group at Notre Dame (Reyniers, Trexler, and Ervin, 1946; Reyniers, Trexler, Wagner, Luckey, and Gordon, 1947) have shown that rats, and other animals, can be grown in the absence of other organisms on heat-sterilized foods. The biochemical and metabolic differences exhibited between cells of a higher organism when in Vivo and iiz vitro may be accounted for by the segregation of synthetic enzyme systems. Thus tissue 1 may synthesize compound A for itself and other tissues. Tissue 2 may synthesize compound B and utilize performed compound A. So in the whole economy of the organism this segregation of enzyme systems has resulted in a strict symbiosis. If this is so, then, with our present limited knowledge of the complex biologically important compounds, successful tissue cultures may be expected on chemically defined media, only when a sufficiently large number of representative tissue types are allowed to contribute one to the other. IV. CONCLUSION In spite of the apparent pessimism about the growth of animal tissues in chemically defined media, I believe that serious attempts should be continued. Drawing on our experience of many years with Tetrahymena and our experience of only a year with attempts at tissue culture, I would


33

like to suggest what I believe to be of prime importance for future trials. It would seem logical that an attempt should be made to supply the tissues with all known active compounds. In looking over the various formulas for tissue culture media, three types of compounds are most frequently missing : vitamin BIZ, protogen, and pyrimidine and purine bases. Moreover I would suggest the substitution of citrovorum factor for PGA, on the chance that your type of tissue lacks the specific formylating and dehydrogenating enzymes necessary for the conversion. I believe it would be wise to substitute pyridoxal and pyridoxamine for pyridoxine for the same reason. In addition to purine and pyrimidine bases I would suggest the use of both ribose and desoxyribose nucleosides, again to guard against the lack of enzymes for the sugar-base linkages. Finally I would suggest the use of the natural configurations of the amino acids used. It has been found for Tetrahynzena (Dewey, Kidder, and Parks, 19Sl), as has been found in other systems, that a number of the D-amino acids are inhibitory, and that this inhibition occurs even at the fifty-fifty ratio of the racemic mixture. D-histidine, D-leucine, and D-serine are strong growth inhibitors. The stakes are high, for if the various normal and abnormal tissues can be handled quantitatively in a chemically defined medium, many of the problems of animal biochemistry and metabolism will be simplified. V. REFERENCES Broquist, H. P., Stokstad, E. L. R., and Jukes, T. H. (1950) Fed. Proc., 9, 18. Dewey, V. C., Kidder, G. W., and Parks, R. E. (1951) J . biol. Chew. (In press.) Dewey, V. C., Parks, R. E., and Kidder, G. W. (1950) Arch. Biochem., 29, 281. Keevil, C. S., Jr. (1950) Unpublished Thesis, Amherst College, Massachusetts. Kidder, G. W. (1947) Ann. N. Y. Acad. Sci., 49, 99. Kidder, G. W., and Dewey, V. C. (1945a) Arch. Biochem., 8, 293. Kidder, G. W., and Dewey, V. C. (1945b) Arch. Biochem., 6, 425. Kidder, G. W., and Dewey, V. C. (1945~) PhySioZ. Zodl., 18, 137. Kidder, G. W., and Dewey, V. C. (1947a) Proc. nut. Acad. Sci., Wash., S, 95. Kidder, G. W., and Dewey, V. C. (1947b) Proc. nut. Acad. Sci., Wash., 59, 347. Kidder, G. W., and Dewey, V. C. (1948) Proc. nat. Acad. Sci., Wash., 34, 566. Kidder, G. W., and Dewey, V. C. (1949a) J. biol. Chem., 178, 383. Kidder, G. W., and Dewey, V. C. (1949b) J. biol. Chem., 179, 181. Kidder, G. W., and Dewey, V. C. (1949~) Arch. Biochem., 20, 433. Kidder, G. W., and Dewey, V. C. (1949d) Arch. Biochem., 21, 66. Kidder, G. W., and Dewey, V. C. in Biochemistry of Protozoa (1951) A. Lwoff, ed. Academic Press, N. Y. Kidder, G. W., Dewey, V. C., and Parks, R. E. (1950) Arch. Biochem., 27, 463. Kidder, G. W., Dewey, V. C., and Parks, R. E. (1951) Physiol. Zoiil., 24, 69. Kidder, G. W., Dewey, V. C., Parks, R. E., and Heinrich, M. H. (1950) Proc. nut. Acad. Sci., Wash., 86, 431.

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Kidder, G. W., Dewey, V. C., Parks, R. E., and Woodside, G. L. (1949) Scictice, log, 511. Kidder, G. W., and Fuller, R. C. (1946) Science, 104, 160. Lorz, D. C., and Hitchings, G. H. (1950) Fed. Proc., 9, 1Y7. Reyniers, J. A., Trexler, P. C., and Ervin, R. F. (1946) Lobund Repts., No. 1, Notre Dame, Indiana. Reyniers, J. A., Trexler, P. C., Ervin, R. F.,Wagner, M., Luckey, T. D., and Gordon, H. A. (1947) Lobund Repfs., No. 2, Notre Dame, Indiana. Sauberlich, H. E., and Baumann, C. A. (1948) J . b i d . Chem., 176, 165. Shive, W., Bardos, T. J., Bond, T. J., and Rogers, L. L. (1950) J. Amer. Chem. SOC.,72, 2817 Snell, E. E., and Broquist, H. P. (1949) Arch. Biochem., 23, 326. Stokstad, E. L. R., Hoffmann, C. E., Regan, M., Fordham, D., and Jukes, T. H. (1949) Arch. Biochcm., 20, 75. Stokstad, E. L. R., and Jukes, T. H. (1950) Proc. SOC.exp. B i d . Med., 73, 523.

The Application of Freezing and Drying Techniques in Cytology L. G. E. BELL Department of Zoology, King’s College, University of London

CONTENTS Page I. Introduction .......................................................... 35 11. Method .............................................................. 36 1. Quenching ...................................................... 36 2. Drying ............................ ......... ...... 41 3. Microtomy . . . . . . . . . ............... 50 111. Comparison with Histologica ............... 53 IV. Advantages of Freezing and Drying Techniques .......................... 57 V. References ...............................................

I. INTRODUCTION The method of dehydrating a material by a process of vacuum desiccation at a low temperature is of great scientific and industrial importance. The biochemist and pharmacist use the method on a large scale in the preparation of proteins, hormones, and other labile products. The bacteriologist uses the method extensively in the preservation of cultures. These applications are discussed by Flosdorf (1949). More recently physiologists have been using the method in an attempt to preserve life in animal cells (Smith and Polge, 1950). The cytological use of the method seems to have been among the earliest applications and to have developed independently of the other and later uses. Leeuwenhoek was among the first workers to dry tissues for microscopical examination, but it was Altmann (1890) who originated the method in use today. His procedure was to freeze small pieces of tissue and to keep them over sulfuric acid in vacuo at a temperature of -20°C for some days. The tissue was then infiltrated with xylene or paraffin wax in wacuo. The sections from such material were then floated on to fixative solutions to study the action of fixation. Altmann recommends as low a drying temperature as possible. H e remarks that the method is difficult and time consuming. Early workers in cytochemistry were well aware of the criteria for accurate, work and the artifacts of chemical fixation. Among these was Mann (1902) who used Altmann’s method and recommends it for cytochemical work. H e introduced cooling the tissue in alcohol cooled by a mixture of solid carbon dioxide and alcohol, but largely negatived this

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L. G. E. BELL

improvement by quenching whole frogs and afterwards breaking up the animal into small pieces for drying. H e used a drying temperature of -30°C. Bayliss (1915) dried tissue at -35°C to be below the eutectic temperature of the tissue salts. H e was discouraged from using the method as his sections disintegrated when brought into contact with water. Other authors mention the method (Romeis, 1932) , but little was done until Bensley and his co-workers elaborated and improved the method (Gersh, 1932). Since the publication of Gersh’s paper the method has been used extensively in the United States (Scott, 1933; Goodspeed and Uber, 1934; Hoerr, 1936 ; Simpson, 1941a. I n other countries development has been slower. Lison (1936) mentions the method but says it is too specialized for general use. Scandinavian workers have used the technique (HydCn, 1943 ; Sjostrand, 1944). Recently, Reed and Udall (19SO), quoting unpublished experiments, refer to the results of freeze drying as disappointing, but from the few details given in their paper, it is clear that they are not acquainted with recent work. The increasing availability of the necessary physical equipment is an important factor in the wider use of the method. 11. METHOD The procedure of cytological freezing and drying is conveniently divided into three stages: The initial sampling and cooling of the tissue, the vacuum desiccation, and the preparation of the dried tissue for microscopical examination. These steps will be considered separately.

1. Quenching This involves taking a sample of tissue and immersing it in a cold bath. By this means, all chemical reactions and diffusion of substances in the tissue are slowed down to a minimum. Material such as protein and salt which is dissolved in the water of the tissue is precipitated from solution at the crystal boundaries of the ice crystals formed from the free water of the tissue. This precipitation of material means that the more minute the ice crystals formed in the tissue, the more faithfully will the structure of the frozen tissue reflect that of the original sample. The larger the ice crystals, the larger the distance dissolved material will be moved from its original site to be precipitated at the crystal boundaries. In a fixed volume of a solidifying liquid the number of crystals formed is directly proportional to the number of nuclei of crystallization and inversely proportional to the rate of growth in size of the crystals. The larger the number of crystals in a given volume, the smaller will each

FREEZING AND DRYING TECHNIQUES I N CYTOLOGY

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crystal be. This means that a high rate of nucleation and a low rate of growth is desirable to obtain small crystals. Luyet (1938) has shown that the rate of growth of ice crystals in gelatin-water mixtures is reduced by twice for 1 per cent gelatin to three hundred and fifty times slower than pure water for 3 per cent gelatin. It is to be expected that the protein material in tissues will be able to slow the rate of growth of ice crystals in a similar way. The growth-retarding action of material in solution depends on its concentration and its structure. At certain concentrations, many substances have such a retarding action that crystal growth is entirely inhibited, and a solid with a subcrystalline or vitrified structure is obtained on cooling. The mechanism of retardation involves the binding of water by hydration of the dissolved molecules (Luyet, 1938), the adsorption of the precipitated material at the growing crystal boundaries (Adam, 1941) and the dissolved molecules hindering diffusion of water from the liquid phase to the growing crystal nuclei. Though the growth-retarding action of tissue substances is not sufficient to prevent the formation of ice crystals by itself, it is probable that it influences their final size and, as will be discussed later, the growth involved in recrystallization. The most important factor under the control of the experimenter is the rate of nucleation. It is first necessary to consider the extent to which water can be cooled without crystallization. Kistler (1936) has shown that small droplets of water can be cooled to about -35°C before crystallization begins. Similar critical temperatures are found by Cwilong (1945) and Schaeffer (1946). Using this value of -35"C, Fisher, Holloman, and Turnbull (1949) have shown that the rate of nucleation of water is very small up to the critical temperature and then increases very rapidly. Between -33°C and -43°C the rate of formation of new crystal nuclei is increased by a factor of lo1*. These considerations show that the more quickly the tissue can be cooled to well below the critical temperature, the finer the ice crystal structure. If a system can be cooled quickly enough to a temperature where the rate of nucleation is very large or infinite, a non-crystalline or vitrified structure is formed, even in the absence of dissolved material. If the concentration of dissolved substances is insufficient by itself to inhibit crystallization entirely, it is clear that it may slow crystal growth enough to enable the temperature to be reached where the rate of nucleation is very great, before the solution has solidified. This is confirmed by the work of Luyet, who shows that it is very difficult to vitrify pure water, but much easier to vitrify solutions of sugars and glycerine.

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L. G. E. BELL

Factors which govern the rate of cooling and hence the rate of nucleation are the thermal conductivity of the cooling bath and the temperature difference between the tissue and the bath. A high conductivity and a large temperature difference are desirable. These criteria have been emphasized by Scott (1933), Hoerr (1936), and Simpson (1941a). The earlier work by Gersh and others used liquid air directly as the cooling bath. This was satisfactory in establishing a large temperature gradient, but had a very poor conductivity because of the formation of a film of vaporized air around the specimen. Gersh (1932) used large pieces of tissue, 0.5 by 0.5 by 1 cm., in which case the conductivity of the tissue becomes a limiting factor. The results obtained with such pieces do not seem to have been very good as Bensley and Gersh (1933) say “The quality of fixation by this method is much better than might be expected and can be compared favourably in some cases with that obtained by ordinary histological fixatives.” Hoerr ( 1936) mentions that he obtained improved results in quenching by wrapping the piece of tissue in liver or spinal cord. Simpson (1941a) confirms this and further shows that if a large enough piece of tissue is used, the crystal artifacts form three zones, an outer zone with a well-preserved structure, an intermediate zone very badly preserved, and an indifferently preserved inner zone. Simpson is inclined to ascribe the very bad intermediate zone to the effect of the pressure exerted by the outer hard shell of frozen tissue, It is possible, however, that this intermediate zone reflects the conditions where the solidification temperature is too high for rapid nucleation, but the rate of crystal growth is great, while the temperature in the inner zone is too high for rapid nucleation and also too high for the rate of crystal growth which gives the large artifacts of the intermediate zone. These circumstances would give the observed large ice crystal artifacts of the intermediate zone and the smaller artifacts of the inner zone. Evidence for this interpretation comes from the demonstration by Simpson that the extent of the three zones could be varied by altering the temperature of the cooling bath. With selected temperatures, both the outer and intermediate zones could be eliminated. Using a bath at -90°C only the inner zone was obtained. As Simpson points out, for cytological work it is best to take such a size of sample and to use such a cooling bath as will give only the outer zone type of structure with artifacts of minimal size. In order to improve the conductivity of the cooling bath, various liquids have been used. Scott (1933) used ethyl alcohol at -100°C. H e says better results were obtained than with liquid air alone. The disadvantages of this liquid are that it solidifies near -115°C and that near its melting


39

point is a viscous liquid, which means that stirring is inefficient. Hoerr (1936) uses pentane (m.p. -131"C), and impure isopentane, which remains liquid until -190"C, cooled with liquid nitrogen. He says that the best results were obtained with a bath at -190°C. Simpson (1941a) confirms this. Emmel (1946) lowers the melting point of pure isopentane by dissolving dry butane in it. The author has found that the addition of three volumes of propane to one volume of isopentane gives a bath that is still very fluid at -190°C; even better is propane by itself, with a melting point near -185°C. By quenching a small piece of tissue in these low temperature baths it is possible to obtain a structure which does not show ice crystal artifacts with the light microscope. Simpson (1941a) recommends that the bath temperature should be at least -165"C, the melting point of pure isopentane, and preferably as near -190°C as possible. H e says that above these temperatures the next best result was obtained by using a bath a t --6o"C when the quality of fixation was that of his inner zone. Pease and Baker (1949), quenching small pieces of muscle in liquid air, found no advantage in using isopentane. Hoerr (1936) emphasized that the cooling of such inflammable liquids as isopentane by liquid air was attended by great hazard and said it was much safer to use liquid nitrogen. If liquid nitrogen is not obtainable, by using only metal Dewar flasks and metal tubes, with extreme care it is possible that liquid air could be safely used with these liquids. The arrangement for a cooling bath can be very simple, consisting of a wide test tube containing the isopentane supported in a Dewar flask containing liquid nitrogen. A simple stirrer is needed for the isopentane. %hen using pure isopentane an indication of the bath temperature is given by the presence of solid isopentane at the bottom of the tube. With mixed baths it is necessary to use a thermo-couple to measure the bath temperature, if it needs to be known accurately. The piece of tissue can be dropped in the bath or preferably plunged in on a piece of metal gauze or strip (Gersh, 1932) or cardboard (Simpson, 1941a). Long narrow forceps make a convenient tool for submersing the specimens. The different thermal contractions of metal and tissue release the specimen once it is in the bath. Some authors use metal baskets for the specimens (Pease and Baker, 1949; Sjostrand, 1944), but the use of a container must hinder the rapidity of the quenching. Once the specimens are quenched they are allowed to stay in the bath until transference to the drying apparatus, or stored a t solid carbon dioxide and alcohol temperatures (Mendelow and Hamilton, 1950). It is essential that the specimens do not warm up beyond the selected drying temperature.

40

L. G . E. BELL

The most perfect quenching can only preserve the structures present in the tissue the instant before it enters the bath. I t is therefore necessary to obtain the sample as rapidly as possible to minimize post-sampling artifacts. Bartelmez ( 1940), in an investigation using both chemical and freezing drying fixation on uterine epithelium demonstrated that cellular material shows aggregation and vacuolation within thirty seconds after removal from the animal. This emphasizes the need for rapid sampling of fresh material if the advantages of the freeze drying method are not to be wasted. Some authors (Hoerr, 1936; Simpson, 1941a) have sought to improve the quality of the fixation by withholding water from the experimental animal, supposing that the partially dehydrated tissues, having a lesser amount of water than usual, would quench more satisfactorily. This may be so, but it raises the problem of evaluating changes in the material induced by this procedure, and the method cannot be recommended as a general procedure. Most animal tissues can be quenched so as to give negligible ice crystal artifacts. The least satisfactory results are obtained with loose connective tissue, nervous tissue, testis, and bone marrow (Simpson 1941a). With such tissues it is important to use as small a sample as possible and to use a cooling bath near -190°C. The author has obtained the best results with testis by making a thick smear, about one tubule thick, on thin celloidin and quenching strips of this material in propane at -185°C. Goodspeed and Uber (1934) quenched anthers and root tips in liquid air. They reported a coarse reticulate structure at the exterior of the specimens with a finer structure inside. It seems that their material had the characteristics of the intermediate zone and inner zone of Simpson. It is probable that the cellulose cell walls of plant material act as thermal insulators and make efficient quenching more difficult. It is possible to obtain good results, using small samples and an isopentane bath. Quenched specimens are transferred to the drying apparatus in gauze baskets, on small trays or by a spatula, all of which must first be cooled to the temperature of the quenching bath. It is not necessary to drain off all the adhering isopentane from the specimens, as most pumping systems can deal with a small amount, but it is better not to carry over large amounts on the specimens. The transference must be done rapidly into an apparatus at or below the selected drying temperature. Hoerr (1936) recommends that the specimens should be warmed up rather slowly to the drying temperature and says this gives better results. Simpson (1941a) used a procedure of freezing and substitution to dis-


41

tinguish artifacts of quenching from those of later stages. This procedure consists of placing the quenched specimen in water-soluble organic solvents at temperatures of from -40°C to -78°C. Methyl cellusolve and ethyl alcohol were among those used. These solvents dissolve the ice out of the specimen, and on warming up to room temperature the specimen retains those distortions originally caused by the quenching. It is not certain at what stage this substitution of alcohol for water occurs. It may well take place during the warming up stage. This means diffusion artifacts are possible and that the fixation is essentially chemical. For morphological studies it can be used as a second best to complete freeze drying. The author has found it useful as a quick method of checking the quality of quenching of difficult material. 2. Drying

A critical factor in drying is the temperature. Altmann dried his material at -20°C and suggests -30°C would be better. Mann (1902) dried at -30°C; Bayliss (1915) selected a temperature of -35°C with the intention of operating below the eutectic temperature of the tissue salts. Gersh (1932) used -2O"C, Goodspeed and Uber (1934) -3O"C, as have many later workers. Scott and Hoerr (1950) recommend temperatures below -30°C as giving better results. There are not sufficient quantitative physical data concerning the various factors which influence drying temperature to enable a drying temperature to be selected theoretically. It is, however, instructive to consider the qualitative aspects of this problem as they must be taken into account in evaluating the results of this method. The structure of the specimen immediately after efficient quenching will be that of a vitrified or a microcrystalline solid. It is well known, and the fact is used. extensively in metallurgy, that such a quenched structure is very unstable. The changes that tend to take place are: devitrification, recrystallization to relieve the mechanical stresses of the sudden contraction in quenching, and crystal growth. The magnitude of such changes depends on the increase in temperature of the specimen and also on the amount of material present other than the ice. Luyet (1938) says that a vitrified 1 molar solution of sucrose devitrifies in 10 seconds at -26"C, 60 seconds at -3O"C, and is stable at -35°C. H e further says that with ten or less molecules of water to one of sucrose there is no devitrification, but with higher concentrations of water devitrification starts at -50°C. The same type of result is obtained with other solutions. The action of the dissolved substances is the same as in quench-

42

L. G. E. BELL

ing, namely to hinder the growth of ice crystals. It seems likely that recrystallization and increase in size of ice crystals will not become appreciable until the specimen warms to the devitrification temperature. The extent of such recrystallization which may involve the movement of material at the boundaries of the old crystals will depend therefore on the temperature of the specimen and the length of time it is kept at that temperature. The devitrifying temperature of protoplasm is not accurately known. Burton and Oliver (1935) give the devitrifying temperature of pure water as -8O"C, so it may be taken that that of protoplasm is above this temperature. Recent work which has a bearing on this problem is that of Hazel, Parker, and Schipper (1949). This work concerns the behavior of quenched silica sols. When allowed to thaw rapidly from the quenched state to room temperature, the colloidal system remains stable, but holding the quenched specimen at any temperature above -55°C produces a coagulation of the sol. This is explained by the authors as being due to a recrystallization to a more stable crystal lattice at -55°C. It seems likely that some of the coagulation results from the concentration of the colloidal particles at the crystal boundaries. The authors say that the extent of coagulation is modified by the presence of salts in the solution. It is certain that the structure of quenched protoplasm will be influenced by similar changes. It is not yet possible to evaluate these changes quantitatively, but a drying temperature near -55°C seems to be indicated by these considerations. Another factor influencing the drying temperature first mentioned by Bayliss is the melting point of the eutectic mixtures in the tissue. Scott and Hoerr (1950) point out that the eutectic temperatures for such a complex mixture as protoplasm are very difficult to arrive at. The binary eutectic which may be present in the tissue, with the lowest melting point, is that of calcium chloride and water, with a melting point of -54.9"C. The tissue eutectics will be more complex tertiary and quaternary systems, which generally have lower eutectic temperatures than simple binary systems. Workers studying salt distribution in tissues have generally dried below -55°C in order to minimize diffusion of material which might occur if liquid eutectic was formed. Scott has shown that tissue dehydrated below -30°C shows a different ash distribution from that dehydrated a t -2WC. Why -30°C should be a critical temperature is not clear, if only euctectic mixtures are considered, but if another factor-recrystallization-becomes appreciable above this temperature, this would help to explain the result. Scott (1943) uses a drying temperature of -63°Cin his micro-incineration studies. It is not likely that

FREEZING AND DRYING TECHNIQUES IN CYTOLOGY

43

eutectics will be present in quenched tissue as they represent an equilibrium condition, and the constituents of the tissue do not have time to reach equilibrium during the very rapid quenching process. Much of the salt content of protoplasm is likely to be closely associated with protein, and the amount of free salt available for eutectic formation may be quite small. This consideration may explain why Pease (1947) was unable to find free salt on electron microscope examination of frozen and dried myosin from potassium chloride solutions. His explanation of the lack of free salt deposits is that during the dehydration at -72°C free ions become exposed and evaporate. It is true that at low temperatures the ionization of substances is depressed and the attractive forces may be sufficiently modified to enable evaporation to take place, but further experimental evidence is needed before the explanation of Pease can be accepted. H e suggests that experiments with radioactive salts should settle the problem. Most workers who are concerned with the larger molecules in tissues and the preservation of cytological structure seem to agree that drying at or below 4 ° C gives superior results to drying at higher temperatures, but that still lower temperatures prolong the drying time with no great improvement in the quality of the preparation. Much published work is based on material dried at -30°C. The work previously cited of Hoerr, Simpson, and others shows that the results obtained on material dried at -20°C or above are not of good quality. It is important to remember that due to the poor conductivity of the tissue and the use of high vacua in drying that it will be difficult to maintain a specimen accurately at any drying temperature. Evaporation of the ice during drying will cause the surface of the specimen to be cooler than the bulk of the tissue. However, it is possible to ensure that a specimen is not exposed to a higher temperature than the selected drying temperature during dehydration, even if, in fact, its actual temperature is lower. Details of the drying apparatus used by earlier workers are not available. The task of the drying apparatus is to remove the water from a previously quenched specimen held at the selected drying temperatiure. A drying apparatus must have provision for maintaining the drying temperature and for removing water vapor from the gas space of the apparatus. The most useful range of drying temperatures is from -30°C to -70°C. Temperatures down to -40°C can be attained by mechanical refrigerators, but the refrigerator needs to be of exceptionally good design and workmanship to give trouble-free operation. Goodspeed and Uber (1934) use a refrigerator at -30°C, Sylvdn (1950) has a re-

44

L. G. E. BELL

frigerated dryer running at - 4 7 " C , a commercial dryer, Aloe Co. (1947), uses a refrigerating unit. Gersh (1932) uses a method of boiling a volatile liquid under a controlled pressure. H e uses liquid ammonia boiling at 1304 mm. Hg, which gives -20°C. Harris, Sloane, and King (1950) use methyl chloride boiling at 350 mm. Hg, to give a temperature of -50°C. These liquids are contained in a jacket around the drying chamber. The disadvantages of this method seem to be mainly those of ventilation and the complication of the ancillary apparatus. A method first used in freezing drying by Mann (1902) is the use of a mixture of ethyl alcohol and solid carbon dioxide. This mixture gives a temperature of approximately -78°C. It is a stable mixture when contained in a Dewar flask and will remain cold as long as there is solid carbon dioxide present. While there is still carbon dioxide in the mixture it is non-inflammable. The temperature of the mixture is too low for most freeze drying, and the apparatus must include a method of warming the drying chamber or specimens to the drying temperature. Packer and Scott (1942) use a vacuum jacket around the drying chamber which reduces the heat conduction between the bath and the chamber. With a pressure in the 0.5-inch gas space of 1 mm. Hg, they obtain a temperature at - W C in the drying chamber. Their apparatus is so designed that an electric heater in the vacuum jacket could be used to raise the drying chamber temperature if necessary. An apparatus manufactured by W. Edwards and Co. uses an electric heater inside the drying chamber controlled by a bimetallic strip thermostat mounted on the heater support. An ingenious method of controlling the temperature of the drying chamber is to use the melting point of a low melting point liquid. Mendelow and Hamilton (1950) use a paste of solid and liquid ethyl oxalate which maintains a temperature of 4 C so long as there is solid present. These authors say that an occasional addition of a small quantity of solid carbon dioxide is sufficient to maintain some solid ethyl oxalate in the mixture. The method is obviously applicable to other temperatures with suitable liquids, e.g., nonane m.p. --53"C, isopropyl ether m.p. -60°C. This method is certainly the simplest and most economical to apply. Solid carbon dioxide is a fairly cheap and easily available material in all countries where ice cream is manufactured. As mentioned previously, the most that can be expected of a temperature control under the conditions in a freeze dryer is that the specimen is not allowed to warm up above the drying temperature. Its actual temperature may well be lower than the temperature of the heater.


45

The desiccation of the specimen depends on the fact that solids have a vapor pressure of their own molecules in equilibrium with the solid. If the vapor pressure of a substance in the atmosphere over a solid of that substance is greater than the equilibrium pressure at that temperature, then material will pass from the vapor to the solid. This process is condensation. If the vapor pressure is less than the equilibrium vapor pressure, then some solid will pass to the vapor. This is evaporation. Material is actually always evaporating, but at equilibrium the same amount is also condensing, so the net change is zero. If the vapor pressure above a solid is maintained by some means below the. equilibrium pressure for any temperature, then the solid will evaporate completely. Packer and Scott ( 1942), using simple kinetic theory for evaporation rates, work on the assumption that the desiccating action involves pumping water vapor from the specimen. This leads them to conclude that high capacity oil diffusion pumps are necessary. The diffusion pump on their analysis is needed to reduce the pressure in the system below the vapor pressure of the specimen so that negligible recondensation of water takes place. The high capacity is needed because they consider pumping the water vapor from the specimens expanded to the pressure of the system. This gives very large volumes of water vapor to be handled by the pumps. They calculate the volume of water vapor evaporating from a square centimeter of ice at 4 0 ° C and at the pressure in their apparatus would be 1600 liters per second. This is more than a hundred times the capacity of the diffusion pumps used, as the authors point out. They ascribe the ability of their apparatus to handle the water vapor to unknown factors slowing the diffusion of water vapor from the specimen. They regard the traps of phosphorus pentoxide and of carbon dioxide alcohol as protection for the pumps. It seems, however, that it is these traps which actually do the drying by removing the water vapor from the system. It appears to the author that the above analysis neglects several factors, the main one being that the essential point in drying specimens is to have in the system a trap which continuously removes water from the gas space of the system. This trap may be either chemical, such as phosphorus pentoxide, which combines chemically with the water, or a surface held at such a low temperature that water condensing on it has an extremely low vapor pressure so that re-evaporation is negligible. Such temperatures are obtained by using liquid air or carbon dioxide alcohol mixtures. The other main factor to consider is that at the low pressures employed the distance that a molecule in the gas space can travel before hitting another molecule, known as the mean free path, becomes com-

L. G . E. BELL

46

parable with the dimensions of the apparatus. If the trap is farther away from the specimens than the mean free path, the rate of drying will be governed by diffusion of water vapor from the specimens to the trap. The greater this distance the longer the process will take, and in some dryers the distance is very great. It is clear that if the trap is brought within the distance of the mean free path from the specimen, most water molecules evaporating from the specimen at the vapor pressure of the specimen will pass directly to the trap without suffering any deflecting collisions. There can be no quicker process of drying than this, in fact it is molecular distillation. The limiting factor then becomes the vapor pressure of the specimens, and this is controlled by the temperature of the specimens. Many workers have designed dryers on the basis of the work of Packer and Scott, using oil diffusion pumps to give very low pressures. The traps are often placed a long distance from the drying chamber and though possibly within the mean free path at the pressures used, the connecting tubing usually has at least two bends in it. This means that a molecule will suffer at least two collisions before reaching the trap, and the drying time will be correspondingly increased. The basic principles of a freeze drier can be best appreciated by considering Fig. 1, which is a cross section of an idea1 dryer. The bottom Top plate

Trap a t -78.C.

G=

-

Bottom plate

To vacuum pump

Specimen at -4O'C.

FIG.1.

is assumed to be kept at the drying temperature and the top at -78°C to act as the trap. As the distance between top and bottom is increased, so must the vacuum be raised to increase the mean free path of the molecules. If the distance between top and bottom plates is 0.5 cm. the vacuum need only be 1W2mm. H g for the water molecules to.leave the specimen and pass directly to the trap. If the distance is increased to 4.5 cm. the vacuum must be lowered to 10-* mm. H g for water molecules to pass directly to the trap. Lowering the vacuum further will not increase the rate of drying which is governed by the vapor pressure of the specimen. If the drying temperature is lowered to --6O"C, the drying time will be correspondingly longer, and it can be calculated from the kinetic equa-


47

tions used by Packer and Scott that ice will evaporate ten times more slowly at -60°C than at -40°C. It is necessary that the trap should have such a small vapor pressure of water that the evaporation from it is negligible compared with the evaporation from the specimen. A trap at -78°C has a vapor pressure of water approximately two hundred times lower than a specimen of ice at -40°C. Details of dryers designed as pumps have been published by Gersh (1932), Packer and Scott (1942), Scott and Hoerr (1950), Harris, Sloane, and King (1950), Sylven (1950), and Pease and Baker (1949). These all employ diffusion pumps. Goodspeed and Uber (1934) and Sjostrand (1944) have built dryers using only rotary oil pumps. The dimensions of their apparatus are much larger than the mean free paths at the operating pressures. Sjostrand dries at a pressure of from to mm. Hg, giving a mean free path of from 0.5 to 5 cm. The trap is at least 50 cm. away from the specimen and is connected at right angles to the drying chamber by a narrower tube. It is not surprising that this apparatus takes from one to two weeks to dry at 4 ° C . The apparatus of Goodspeed and Uber suffers from similar disadvantages and takes two weeks to dry at -32°C. The author has found that an apparatus manufactured commercially (Edwards and Co., 1950) can be used as a short path condensation drier. The quenched specimens are held on trays inside an annular electric heater. The heater is suspended in a glass tube immersed in a carbon dioxide alcohol bath. Using a two-stage rotary pump operating at approximately 5 X lWS mm. Hg, the water vapor leaves the specimens at, say, -40°C and condenses at the bottom of the tube at -78°C opposite the end of the heater, or on a charge of phosphorus pentoxide placed in the bottom. The water does not react with the phosphorus pentoxide at such low temperatures, but when the dryer is allowed to warm up to room temperature when drying is finished the water is absorbed and cannot pass back to the specimens. The drying times with this apparatus are of the order of two or three days at -40°C. This apparatus operates on a short condensation path, but the design does not take into account the full possibilities of molecular distillation. The author feels that evidence supporting the molecular distillation analysis of freeze drying is provided by the recent paper of Mendelow and Hamilton (1950). These authors, using an all metal apparatus, maintain the specimens at -40°C a short distance from a liquid nitrogen cold trap inserted directly into the gas space above the specimens. Using

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L. G. E. BELL

an oil diffusion pump to give a vacuum of 5 X lCP4 mni. H g they are able to dry their specimens in seven hours, which is a very great decrease in drying time compared with any other published data. The authors, however, do not seem to have appreciated the reasons why their design is so efficient. They follow the reasoning of Packer and Scott and assume it is essential to lower the pressure in the system below the vapor pressure of ice at the temperature of the specimen. They state that in their opinion the short drying time is due to the use of liquid nitrogen as a trap. The author suggests that the reason for the great efficiency of Mendelow and Hamilton’s apparatus is that the vacuum they use of 5 X 1 V mm. H g is more than sufficient to increase the mean free path of the water molecules to the dimensions of the apparatus. This means that most of the water molecules will pass directly from the specimens to the trap without suffering collisions with any other molecule. Further, a surface at the temperature of liquid nitrogen will hold a molecule of water at the first impact without a rebound. The drying times given in the literature vary greatly. Scott (1937) gives three days at -32°C; Emmel (1946) gives one to two weeks at - W C to -78°C; Scott and Hoerr (1950) say several days to weeks at 4 C ; Harris et al. (1950) says two days at -50°C. Factors apart from the apparatus which will influence the drying time are the size and shape of the specimen, and whether many specimens are loaded in one chamber in such a way as to obstruct each other. Hoerr (1936) points out that a limiting factor may be the diffusion of water vapor out of the specimen but says that it is quite rapid. Packer and Scott (1942) emphasize that little is known about this process and suggest that it slows evaporation considerably. They also point out that evaporation produces surface cooling and so slows the process. Gaseous diffusion may not be the controlling factor; as is well known, molecules migrate easily along solid surfaces. Volmer and Estermann (1921) give data I t seems concerning the phenomena with mercury crystals at -63°C. possible that such a migration of water molecules could occur in the solidified tissue giving the result that evaporation occurred mainly from the surface of the block. However, there is as yet no experimental evidence which can help to evaluate the extent to which either process controls the rate of drying. To obtain the best quenching for cytological purposes it is necessary to have a thin specimen. This kind of specimen will also have the best dirhensions for rapid drying. Hoerr (1936) points out that normally all the water is not removed from frozen dried tissue even by running the dryer for four or five days

FREEZING A N D DRYING TECHNIQUES I N CYTOLOGY

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after completion. H e finds that frozen dried tissue loses from to 1 per cent of its original weight after drying to constant weight in an incubator. Mendelow and Hamilton (1950) have investigated the dryness of a variety of frozen dried tissues. They obtain the loss in weight of the dried specimens after heating to constant weight at 50°C in a vacuum oven. They find that most frozen dried tissues lose between 0.2 and 1 per cent of their original wet weight after heating to constant weight. The most probable source of error in their method is the loss of volatile constituents other than water from the tissue during the heating at 50°C. This error, if present, would make the results obtained for residual water content maximum figures. The completeness of drying will depend finally on the attainment of equilibrium of vapor pressures between the trap and the specimen. When the vapor pressure of the specimen equals the vapor pressure of the trap, no more water will be removed from the tissue by the trap. It is probable that the water left in the dried tissue represents water bound by protein or by salts and will have a very low vapor pressure. For this reason it is best to use a trap having the lowest possible vapor pressure. This consideration means that liquid nitrogen and liquid air traps will dry more completely than any others. However, as the majority of freeze dryers use either phosphorus pentoxide or carbon dioxide alcohol mixtures, it seems that for most practical purposes the degree of desiccation obtained by their use as traps is adequate. The problem of deciding when tissue is dried has been mentioned by numerous workers. The normal vacuum gauges such as the McCleod and Pirani gauges are accurate instruments for true gases but cannot be used to measure water vapor pressures. For this reason they are not usefully employed on freeze driers except as leak detectors. Packer and Scott (1942) applied the method of measuring the pressure differences between two ionization gauges sealed into the apparatus. They assume that when both gauges register the same pressure no more water vapor is being pumped along the apparatus, as there is no pressure gradient. It appears to the author that for this method to be useful the electrodes of the two gauges should be sealed into the gas space of a straight tube for accurate measurements of a pressure gradient. The diagram given by Packer and Scott shows one gauge connected to a wide tube containing the phosphorus pentoxide trap, while the other is connected to a narrower tube leading off the wider tube at right angles. Packer and Scott say that the results of this method are sometimes erratic. In an apparatus with the optimum short distance between the trap and

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the specimens, gauges are of little use for determining the dryness of the specimens. Gersh (1947) sums up the situation very well in saying that the time of drying varies considerably with temperature, size, and shape of specimen and that the operator learns empirically when drying should be complete. Most dryers have been constructed of glass. Scott and Hoerr (1950) list the advantages of this material as cheapness, ease of finding and repairing leaks, and reduced adsorption of gases compared with metal tubing. The main disadvantage of this material is fragility, and where an apparatus is to be used by many workers, perhaps inexperienced in handling glass, there is no doubt this outweighs the advantages. The author has found a drier constructed almost entirely of metal very satisfactory in use. Glass is very useful where one end of a tube needs to be kept colder than the other, as metal tubing has too high a thermal conductivity for such a task. The apparatus of Mendelow and Hamilton is constructed entirely of metal except for a glass liquid nitrogen trap.

3. Microtomy The dried material can be treated in several ways. The most widely used method is to inflltrate with wax in vacuo. Many driers can be loaded (Scott, SylvCn, Harris) with degassed wax which is melted when the sample is dry. The sample is then infiltrated directly. There is little doubt that this is the most desirable method. It avoids exposure of the sample to the atmosphere and removes the risk of absorption of moisture or of oxidation. However, it is quite practicable to allow the sample to warm up to room temperature when dry and then to remove it quickly from the dryer to an embedding apparatus, which need consist only of a tube containing degassed wax connected to a vacuum line. The apparatus is evacuated, and the wax is melted by heating the tube in hot water. If the sample is dry, it will sink in the melted wax, and any moisture present will cause vigorous bubbling. Some authors transfer the dried tissues directly to melted paraffin, with or without first warming to room temperature. (Gersh, 1947). This seems more drastic than is desirable, as dropping cold tissue directly into hot paraffin will cause a very rapid rise in temperature. Simpson (1941a) infiltrates the tissue first with ethyl alcohol for a week or more to remove the last traces of moisture not removed by the drying, and to reduce shrinkage in paraffin embedding, Emmel (196) uses four hours in alcohol. Simpson recommends celloidin embedding without the use of heat for the best preservation of general histological detail. The author has found in-


51

filtration with methyl benzoate or methyl benzoate celloidin very satisfactory as a preliminary to the use of some of the ester wax mixtures available. Goodspeed and Uber (1934) remark on the difficulty of infiltrating frozen and dried plant tissue with wax directly. This has also been found by the author who has confirmed that the pretreatment of the dried tissue with butyl alcohol recommended by Goodspeed and Uber is beneficial. I t is not clear why infiltration with wax should be difficult in this case. The surfaces left exposed by the removal of water may be hydrophilic and not wetted by the non-polar wax, or the cellulose cell walls may offer mechanical obstruction to the entry of liquids. Harris et ad. (1950) caution against the use of a high vacuum in degassing the embedding wax. They suggest volatile constituents may be lost, giving a brittle wax which cuts badly. Hoerr (1936) cuts freehand sections of unembedded material. H e shows that his sections cleared in liquid paraffin contain many irregular vacuoles, probably due to ice crystals. Similar sections after thirty seconds exposure to aqueous toluidine blue show no vacuoles. This experiment illustrates the hygroscopic nature of dried tissue and the ability of frozen dried protein to become rehydrated. The rehydration could be prevented by first exposing the material to alcohol. In frozen and dried tissue unaltered by subsequent treatment, the tissue proteins retain a large part of their original solubility. Hoerr noted that this solubility might vary and suggested that the variation was due to the action of the hot paraffin in embedding or the use of petroleum ether to remove the wax from the section. H e therefore used' freehand sections for solubility studies and showed that the solubility decreased after standing at room temperature or paraffin embedding, and more slowly in the refrigerator. (Bensley and Hoerr, 1934). Hoerr has also stated that it is possible to reduce the solubility of tissue protein by the action of sunlight on frozen dried tissue in a vacuum desiccator. It is not certain how far this is an oxidizing or a thermal action, and it does not seem desirable to use it for cytochemical or enzyme studies. Heat also makes frozen dried tissue insoluble, and it is likely that the heat generated at the cutting edge of the microtome knife will have a similar action. Although such treatment may be undesirable for critical solubility studies, the tissue obtained in paraffin sections is still extremely soluble and able to absorb water. Hoerr (1936) shows that when freehand sections of nervous tissue are placed in aqueous fixatives, myelin takes up water rapidly with the formation of myelin figures. Most cytochemical or cytological techniques require the use of aqueous solutions, and the tissue substances must be made in-

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soluble before expasure to such solutions. Frozen dried sections disintegrate when flattened on warm water in the usual way. Such sections are best flattened on a warm clean or albumenized slide by gentle pressure of a clean finger or a fine brush. If the ribbon of sections is attached first at one end to the slide and the ribbon is supported at the other end by forceps, it can be lowered on the slide a small portion at a time, starting from the attached end. The ribbon expands just as it comes down on the warm slide and gentle stretching by a fine brush will remove most of the creases from the section. The sections are then consolidated on the slide by gentle pressure. It is possible to flatten paraffin wax sections on a clean warm mercury surface and to remove the sections on a warm glass slide lowered onto the mercury surface, being careful not to trap air between the slide and the sections. Alcohol and diacetin can also be used, but are not so effective. After sections have been flattened by any of these methods, it is best to use gentle finger pressure and a few hours in an incubator to fix the sections to the slide, especially after mercury flattening, and if albumen has not been used. Mendelow and Hamilton (1950) overcome the difficulty of cutting thin sections without creases by cutting single sections and coating the block with paraffin before cutting the next. This gives a greater total thickness of wax which is not easily crumpled. Sections thus mounted are treated in the usual way by removal of wax in a non-polar solvent and passing through ethyl alcohol to water. It is usual to let the sections remain in absolute ethyl alcohol at least fifteen minutes to make the sections reBistant to water. Some authors (Gersh and Catchpole, 1949) keep sections overnight in absolute alcohol. If the tissue has been kept in alcohol before embedding, as in Simpson’s method, the sections may be floated on water. It is not clear what is the action of absolute alcohol on the dried tissue, and it is not known what materials may be removed from the section by this solvent. The action is not the same as that of an alcoholic fixative which involves precipitation and aggregation of material and the movement of substances by diffusion currents. Mancini (1948) avoids the action of alcohol by treating his sections after the removal of wax by the vapor of iodine or a non-polar solution of iodine to reveal glycogen. Unfortunately there are few cytochemical reactions which can be used in this way. It is possible to float the sections onto the fixatives usually used in cytology or cytochemistry, as was originally done by Altmann. The author has used 80 per cent alcohol in this procedure and finds the results with this fixative not so satisfactory as the dry flattening methods. Sec-


53

tions in the water-soluble waxes with the lipoid preserved can be floated on aqueous fixatives such as neutral formalin, which flattens the sections and dissolves the wax at the same time. The use of ultraviolet absorption and phase contrast microscopy enables observations to be made on sections that have had the wax removed by liquid paraffin and are mounted in the same liquid. This material will be freer than most sections from artifacts caused by the action of solvents. This kind of technique should be useful in studying cell structures such as Golgi complexes, which have been criticized as being artifacts, due to the action of some solvents (Palade and Claude, 1949). For ultraviolet absorption microscopy, in order to diminish absorption of ultraviolet light due to scatter at retractive index boundaries, it has been usual to mount frozen dried sections in glycerine. The protein imbibes glycerine and the sharp boundaries of different refractive indices are diminished (Caspersson, 1950). Fulham and Gessler (1946) use a eutectic mixture of camphor and naphthalene as an embedding medium for use in a highspeed microtome and sublime off the embedding medium without the use of solvents. Harris et al. (1950) mount frozen dried sections onto photographic emulsions without the use of solvents and emphasize that only in this way can it be possible to preserve all the radioactive material in the tissue. 111. COMPARISON WITH HISTOLOGICAL FIXATION

Frozen dried tissue, though free from most of the artifacts of chemical fixation, has a structure which reflects the treatment it has undergone. In the initial quenching there may be up to a 2 per cent volume contraction due to the cooling. This contraction can give rise to cracks, especially in large pieces of tissue. These cracks are always easily recognizable and are not likely to be confused with natural spaces in the tissue. The solid elements in the tissue will have been deposited around the boundaries of ice crystals. This means that structure will only be resolvable down to the size of the ice crystals. Any resolution below this size will only reveal the ice crystal artifact. I t is possible to prepare most tissues by freezing drying so that they do not show ice crystal artifacts in the light microscope. It is not known how much orientation the precipitated material will undergo a t the growing crystal face, but it is well known that crystal faces influence the orientation of material deposited on them. In this connection it is interesting to recall that Pease and Baker (1949) state that the birefringence of froien dried striped muscle was much greater

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than that of chemically fixed muscle. Luyet (1938) showed that gelatinwater mixtures when quenched in liquid air showed a laminated structure. It is often possible to detect a similar laminated structure in frozen dried tissue with the higher powers of the microscope. It is especially obvious in fluid filled tissue spaces and in the ground substance of connective tissue. The better the quenching, the less obvious is this artifact. It is probably always present, but may be below the size resolved by the microscope. This laminated structure probably owes its origin to the same mechanism which gives a solid eutectic a laminated structure. This involves alternate supercooling and precipitation of material over short distances. For many cytochemical techniques the best cytological preservation is not essential. As long as the artifacts are not resolved at the power of the microscope used, they will not influence the interpretation. The chemical fixation of tissues has always been the object of investigation by histologists who have tried to determine what happens when a piece of living tissue is placed in solutions of protein denaturants and precipitants. Altmann developed the freeze drying technique to investigate this problem. Baker (1945, 1950) gives a review of the classical work of Fisher, Hardy, and others on this problem. Baker emphasizes that to elucidate the action of complex mixed fixatives is very difficult and for this reason advocates the use of simple fixatives where there is a possibility of understanding the mode of action. Cytochemists need fixatives that do not react with the chemical groups or substances they wish to identify. Fixatives such as 80 per cent alcohol and neutral formalin are popular for this reason. Unfortunately cytochemical fixatives often do not preserve structure very well, apart from this disadvantage it is important to realize the existence of others. The object of cytochemistry is to estimate qualitatively and quantitatively, in their natural cytological positions, the various chemical substances occurring in cells. Most cytochemistry is done on dead fixed cells. Ultraviolet microscopy of surviving cells has its own special problems and will not be considered here (Faraday Society Symposium, 1950). Cytochemistry does not require the use of large pieces of tissue. Pieces 0.5 mm. thick or less are usually adequate. Even so, as fixatives penetrate slowly (Bolles Lee, 1950) there is ample time for post mortem changes to occur before fixation is complete. That these post mortem changes may be serious has been shown by Bartelmez (1940). The penetration of fixatives sets up diffusion currents in the tissue. These sweep cell substances into fictitious localizations and concentrations. The


55

case of glycogen is a well-known example. One of the first applications of freeze drying (Gersh, 1932) was to confirm Fisher’s work on the even distribution of glycogen in the liver cell. Mancini (1948)in a comprehensive investigation of glycogen in animal tissues, clearly showed, by comparing frozen dried material with Zenker or Bouin fixed material, that glycogen may even be displaced extracellularly. He showed that frozen dried muscle contains glycogen only inside the fibers, while workers using chemical fixatives (Dempsey, Wislocki, and Singer, 1946) have described intra- and extra- fibrillar glycogen. Mancini further shows the presence of glycogen in skin and fatty tissue, while it could not be detected in similar tissue after chemical fixation. An important aspect of Mancini’s work is his use of non-polar solvents to carry out his glycogen reaction. He is thus able to reduce diffusion artifacts in the test to a minimum. Reactions which can be carried out in non-polar solvents or in strong alcoholic solutions are especially useful in reducing the possibility of diffusion, or removal of water-soluble materials from the specimen during a cytochemical reaction. The reaction of Hotchkiss for carbohydrate can be modified so that all the reagents are in 60 per cent alcohol (Staple, 1949). This reduces the possible diffusion of glycogen and muco protein in a section. Several workers have shown by comparing frozen dried tissue with chemically fixed tissues that materials may be seriously translocated in fixation. Gersh (1933-34)in an investigation of rabbit kidney describes differences in the distribution of uric acid between frozen dried and alcohol fixed tissue. H e finds that frozen dried material shows uric acid in Bowman’s space and only in the lumen of the tubules which all contain it. Alcohol fixed material shows no uric acid in Bowman’s space, little in the lumen of the tubules, and some in the cytoplasm and nuclei of the tubular cells. This work clearly shows the very bad diffusion which may occur with molecules the size of uric acid. With small inorganic ions, diffusion may be even more serious. By dehydrating at a low temperature (-6O’C.) it is possible to prevent the diffusion of inorganic ions up to the time that sections are cut. Scott and his co-workers have worked extensively on the distribution of calcium and magnesium in frozen dried tissue. Scott (1943) has reviewed this work. H e uses the method of micro-incineration and by taking a motion picture of the process shows that the picture remains static during the incineration. H e points out that this means any distortion which occurs is not observable at a magnification of 800x and therefore will not influence the interpretation of the results. Efforts by Gersh to establish unequivocally the site of chloride

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and phosphate have been criticized by Scott and Packer (1939) on the grounds that although diffusion is prevented up to mounting the section on the slide, diffusion very likely occurs during the cytochemical reaction. The problem of localizing chloride does not yet seem to have been solved. The non-destructive nature of freeze drying means that enzymes retain their activity very much better after this process than by chemical fixation. Hoerr (1936) showed that ground up frozen dried liver suspended in saline retains its glycolytic activity, glycogen disappearing from the tissue with the fqrmation of glucose. Several workers have published comparisons of alkaline phosphatase activity and distribution in frozen dried tissue. Emmel (1946), comparing frozen dried and cold acetone fixed kidney and intestine, uses Gomori's (1939) phosphatase method with an incubation time of three hours. He finds substantially the same distribution with the two methods of fixation. H e remarks on the excellent preservation of cytological structure. It appears, however, that using such long incubation times may introduce diffusion artifacts and that a more critical comparison, both of activity and distribution, would be obtained by the use of very much shorter incubation times. Bevelander and Johnson (1950) make extensive use of frozen dried embryo pig heads in an investigation of the development of membrane bone by histochemical methods. Frozen dried material has been used for cytological investigations. Mitochondria were studied by Bensley and Gersh (1933). They found them insoluble in alkalis, acetic acid, and organic solvents. Using Bensley's Millon reaction for tyrosine it is shown that a large part is protein. The results of this work and that of Bensley and Hoerr (1934) on the solubility of frozen dried liver protein led to the work of Bensley and others in isolating cellular constituents from fresh tissue. This work has been reviewed by Hoerr (1943).Nuclear structures have been studied by Bensley (1933) and Goodspeed, Uber, and Avery (1935). Bensley, using Amblystoma and rodent tissues, demonstrates that the material staining with hematoxylin and giving a positive Feulgen reaction in interkinetic nuclei is concentrated on the inner surface of the nuclear membrane and also as an investment around the nucleolus. H e also reports that nuclear ground substance gave a slight positive Feulgen reaction. Goodspeed et d. investigated the dividing nuclei of root tips of Lilium, which had been quenched in liquid air and dried at -32°C. The cytological preservation does not seem to have been of the highest quality, but they were able to demonstrate the multi-strand nature of chromosomes. They report four chromonemata at anaphase and eight at metaphase.

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57

One of the most striking differences seen between frozen dried and cheniically fixed material is the almost complete absence of shrinkage in the former. This is well demonstrated in kidney. Chemically fixed kidney shows the usual capsular space between the glomerulus and Bowman’s capsule, frozen dried kidney shows the glomerulus filling Bowman’s capsule completely, leaving no capsular space. This absence of shrinkage may be a disadvantage to the cytologist making chromosome analyses, for which purpose the shrunken chromosomes obtained by chemical fixation are easier to observe. Simpson ( 1941b) investigated the Golgi apparatus in various frozen dried tissues of Cavia. He used freehand sections of unstained as well as stained material. He was not successful in obtaining an osmium impregnation of the Golgi apparatus, but obtained an even impregnation through the tissue. This was possibly due to the preservation of reducing groups such as sulfhydryl, normally destroyed to a large extent especially by fixatives containing oxidizing agents. Gersh (1949) has studied a component in the Golgi apparatus which gives a positive reaction with the periodate and leuco-fuchsin test for vicinal hydroxyl groups (Hotchkiss, 1948).

IV. ADVANTAGES OF FREEZING AND DRYINGTECHNIQUES The advantages of frozen dried material for quantitative cytochemistry have been emphasized by Caspersson and his school. This is now the standard method adopted by these workers for preparing material for ultraviolet absorption measurements. HydCn ( 1943) compares carnoy fixed mrve cells with frozen dried material and points out the clumped and precipitated appearance of the carnoy nuclei compared with the frozen dried material. He states that chemical fixatives such as acetic alcohol and formalin precipitate protein in clumps and cause shrinkage and that these artifacts will cause false absorption of ultraviolet light due to diffuse reflection and scattering of light at the surfaces. His photographs of frozen dried ganglion cells photographed at 2570A show diffuse dark masses of absorbing material in the cytoplasm, while carnoy fixed cells show sharply defined, more deeply absorbing areas. The author has compared the action of some cytochemical fixatives with frozen dried material. In rat kidney, frozen and dried, phase contrast and ultraviolet photographs show large evenly absorbing nuclei with no thick nuclear membrane. The cytoplasm is homogenous and contains well-preserved mitochondria. Absorbing material is preserved in the lumen of the proximal tubules. Intertubular structures are preserved.

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59

FIG.2. Rat kidney, frozen dried, 4-micron section mounted in nonane. Visual phase contrast. FIG.3. Rat kidney, carnoy fixation, 4-micron section mounted in nonane. Visual phase contrast. FIG.4. Same section as Fig. 2, 2570A, Quartz Objective. NA 1.25. Condenser NA 0.4. FIG. 5. Rat kidney, carnoy fixation, photographed as Fig. 4. FIG.6. Rat walker tumor frozen dried, 4-micron section mounted in glycerine, photographed as Fig. 4. FIG.7. Rat walker tumor, carnoy fixation, 4-micron section mounted in glycerine, photographed as Fig. 4. FIGS.2-7 are from original photographs by R. J. King and E. M. F. Roe. FIG.8. Rat kidney, fixed by freeze drying, 5~ section reacted with tetrazo dianisidine and K acid. FIG.9. Rat kidney, carnoy fixed, rest as Fig. 8. FIG.10. Rat kidney, acetic formol fixed, rest as Fig. 8.

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61

Rat kidney fixed in carnoy shows smaller nuclei with a clumped appearance and heavy nuclear membrane. The nucleolus appears larger than in frozen dried sections. The cytoplasm shows clear spaces and a clumped appearance. Mitochondria are not preserved. There is no absorbing material in the lumen of the tubules. These points are illustrated in Figs. 2-5. I n similarly treated material from the Walker rat tumor, frozen dried sections show evenly absorbing nuclei and cytoplasm with a fine nuclear membrane. Carnoy material shows shrunken nuclei with a clumped appearance and a heavy nuclear membrane (Figs. 6-7). In the frozen dried material it is seen that there is even absorption in the cytoplasm of the metaphase cell, while the carnoy fixed metaphase cell shows a transparent area around the metaphase plate. This means the chemical fixation has removed absorbing material from its original site, either completely from the cell or possibly onto the chromosomes. Using the tetrazo-benzidine reagent for tyrosine, histidine, and tryptophan end groups (Danielli, 1947) the author has found that in rat kidney, frozen dried nuclei are large and evenly staining with a more densely staining nucleolus. Nuclei from acid fixatives such as acetic alcohol or 10 per cent formol plus 5 per cent acetic acid, show a more clumped structure with a heavy nuclear membrane. These points are illustrated by Figs. 8-10. Nuclei from neutral formalin material show an even absorption which is apparently much heavier than in the other fixations, but this may be due to the greater shrinkage with this fixative after paraffin embedding. Only in the frozen dried material are the mitochondria preserved, and they stain more deeply than the cytoplasm. The cytoplasm with the other fixatives mentioned shows a heterogenous appearance, the brush borders in acetic formol material appearing darker than the cytoplasm. Frozen dried material shows staining material in the lumen of the proximal tubules. There is little left in the lumen of the chemically fixed tubules. Sjostrand (1944) shows photographs of frozen dried kidney stained with iron hematoxylin with material in the lumen of the proximal tubules. What the chemical nature is of the substances translocated by chemical fixation is not yet known. It is clear, due to their content of protein end groups, that they are of a protein nature, and therefore presumably of a molecular weight, approaching that of at least the smaller protein molecules. This underlines the necessity of interpreting the cytochemical results on chemically fixed tissue with extreme caution. Even in frozen dried material, though the rapid quenching reduces diffusion artifacts to a minimum, the action of the various solvents used to treat the sections is not completely understood. The extent to which material is removed from

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sections by alcohols and other reagents needs investigation. When quantitative measurements are to be made, especially on protein end groups the degree of denaturation or unfolding of the protein molecules will influence the results. It is not known to what extent the end groups of frozen dried protein are available and how their availability may be influenced by various reagents and the length of time elapsing between drying and carrying out the test. The observations of Hoerr (1936) on the solubility of frozen dried tissues has a bearing on this point. It seems clear, however, that even allowing for these uncertainties frozen dried material should be used wherever possible for cytochemical work in preference to chemical fixation. The rapid-quenching of freezing drying should be useful in studying rapidly changing structures such as cilia, or the state of capillaries. Such studies have not yet been very extensive. V. REFERENCES Adam, N. K. (1941) The Physics and Chemistry of Surfaces, 3rd ed., Oxford. Oxford University Press. Aloe Co. (1947) Catalogue U. S. A. Altmann, R. (1890) Die elementarorganism und ihre Beziehungen zu den Zellen. Leipzig. Baker, J. R. (1945 and 1950) Cytological Technique, 1st and 2nd ed. London. Bartelmez, G. W. (1940) Anat. Rec., 77, 509. Bayliss, W. M. (1915) Principles of General Physiology, p. 17. Longmans, Green & Co. London. Bensley, R. R. (1933) Anat. Rec., 58, 1. Bensley, R. R., and Gersh, I. (1933) Anat. Rec., 57, 205. Bensley, R. R., and Hoerr, N. L. (1934) Anat. Rec., SO, 251. Bevelander, G., and Johnson, P. L. (1950) Anat. Rec., 108, 1950. Bolles Lee, A. (1950) Microtomists Vade Mecum, 11th ed. J. & A. Churchill. London. Burton, E. F., and Oliver, W. F. (1935) Proc. TOY.Soc., 158, 166. Caspersson, T. (1950) Cell Growth and Cell Function. Norton & Co. New York. Cwilong, B. M. (1945) Nature, Lond., Iw,361. Danielli, J. F. (1947) S. E. B. Symfiosia, 1, 101. Dempsey, E. W., Wislockie, C. B., and Singer, N. (1916) Atid. Rec., 96, 221. Edwards & Co. (1950) Catalogue, London. Emmel, V. M. (1946) Anat. Rec., 06, 159. Faraday Society ( 1950) Symposium on Microspectography. Fisher, J. C., Holloman, J. H., and Turnbull, D. (1949) Scierrce, 109, 168. Flosdorf, E. W. (1949) Freeze Drying. Rheinhold Publishing Co., N. Y. Fullam, E. F., and Gessler, A. E. (1946) Rev. sci. Insfriwieir., 17, 23. Gersh, I. (1932) h u t . Rec., 88, 309. Gersh, I. (1933-4) Anat. Rec., 68, 369. Gersh, I. (1947) The Aloe Co. Cutalogwc.

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Gersh, I. (1949) Arch. Path., 47, 99. 457. Gersh, I., and Catchpole, H. R. (1949) Ailat. Rec., I, Gomori, G. (1939) Proc. SOC.exp. Biol. Med., 9, 23. Goodspeed, T. H., and Uber, F. M. (1934) Proc. mt. Acad. Sci., Wash., a0, 495. Goodspeed, T. H., Uber, F. M., and Avery, P. (1935) Univ. Calif. Pub. Bot., 18, 33-44. Harris, J. E., Sloane, J. F., King, D. T. (1950) ‘Nature, Lond., 166, 25. Hazel, F., Parker, J. A,, and Schipper, E. (1949) Scielzce, 110, 160. Hoerr, N. L. (1936) Anat. Rec., 63, 293. Hoerr, N. L. (1943) Bid. Symposia, 10, 185. Hotchkiss, R. D. (1948) Arch. Biochem., 16, 131. HydCn, H. (1943) Acta physiol. scad., Suppl. VII, 6. Kistler, S. S. (1936) J. Amer. C k m . Soc., 68, 901. Lison, L. (1936) Histochemie animale. Paris. Gauthier-Villars. Luyet, (1938) Biodynumica, 2. Mancini, R E. (1948) Anat. Rec., 101,149. Mann, G. (1902) Physiological Histology. Oxford, pp. 139, 142. Oxford University Press. Mendelow, H., and Hamilton, J. €3. (1950) Anat. Rec., 101, 443. Packer, 0. M., and Scott, S. H. (1942) J. Tech. Methods No. 22, 85. Palade, G. E., and Claude, A. (1949) I . Morph., 88, 35. Pease, D. C. (1947) Science, 108, 543. Pease, D .C., and Baker, R. F. (1949) Amer. J. Afraf.,84. Reed, R., and Udall, K. M. (1950) J . roy. micr. SOC.London, 70, 92. Romeis, B. (1932) Taschenbuch der mikroscopischen Technik. Berlin, p. 93. Schaeffer, V. J. (1946) Science, 104, 457. Scott, G. H. (1933) Protoplamia, a0, 133. Scott, G. H. (1937) McClung’s Microscopical Technique, 2nd ed. Hoeber, New York. 643. Scott, G. H. (1943) B i d . Symjosia, 10, 277. Scott, G. H., and Hoerr, N. L. (1950) Medical Physics, 2, 293. Chicago. Year Book Publishers. Scott, G. H., and Packer, D. M. (1939) Awat. Rec., 74, 31. Simpson, W. L. (1941a) Anat. Rec., 80, 173. Simpson, W. L. (1941b) d4nat. Rrc., 80, 329. Sjostrand, F. (1944) Acta. Anat., Suppl. 1. Smith, A. V., and Polge, C. (1950) Naftrre, Lorrd.. 166, 668. Staple, P. H. (1949) Private communication. SylvCn, B (1950) Private communication. Voliner, M . and Estermann, I. (1921) Z. Pltys., I, 1. 13.


Enzymatic Processes in Cell Membrane Penetration TH. ROSENBERG Biochemistry Deportment, Strno Memorial Hospital and Nordisk Insdinlab., Denmark

W. WILBRANDT Department of Pharmacology, University of Beme, Berrce, Switserlarrd CONTENTS Page 65 66

I. Introduction ........................ ..................... 11. Some General Considerations . . . . . . . . . . . . . . ...................... 111. Enzymes with Non-Penetrating Substrat:s . . . ............. 1. Invertase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.......................................

IV. V.

VI. VII.

3. Trehalase . . . . . . . . . . . . . . . . . . . . .................... 4. Amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5. Phosphatases .................................................... 6. Phosphorylating enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Enzymatically Controll ............................... Transport of Glucose . . . . . ............................... 1. Muscle . . . . . . . . . . . . . ............................... 2. Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Erythrocyte . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Kidney and Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion ......................................................... References . . . . . . . . . . . . . . . . . . . ................

68 68 68

68 69 69 69 70 70 71 73 75 79

85 89

I. INTRODUCTION

Es ist anzunehmen, dass der physiologische Import und Export ein komplizierter, unanalysierter, an die Lebenstatigkeit der Zelle gebundener Vorgang in der Zelloberflache, der Plasmahaut ist. Dieser Vorgang setzt meist unter bestimmten Bedingungen ein; diese sind uns noch nicht geniigend bekannt. Es ist nicht anders denkbar, als dass fur solche Aktion der Plasmahaut eine komplizierte Organisation erforderlich ist. [R, HOBER,Physikalische Chemie der Zellen und Gewebe, 1911.1 The cell membrane has been widely considered as a passive structure allowing solutes to penetrate according to the properties of their molecules such as size, membrane solubility, and electric charge. More recently evidence has begun to accumulate indicating that, in addition, the membrane is a part of the cell machinery which contains special enzymes participating both in control of cell metabolism and transport of substances. In the present review an attempt is made to show some general principles and possible mechanisms underlying this activity rather than to give a complete representation of the pertinent published data. The reviewers are aware that this implies the introduction of concepts and 65

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suggestions some of which may appear rather hypothetical. They feel, however, that at the present stage an outline and discussion of some possible fundamental elements may be more useful than a compilation of observed facts. It was decided therefore to focus the interest on the role of surface enzymes in glucose transport which so far appears to have been studied most extensively,

11. SOMEGENERALCONSIDERATIONS Considering the submicroscopic dimension of the cell membrane, the evidence for the membrane localization of enzymes is necessarily more indirect than direct. The methods of histochemistry introduced the possibility of localizing enzyme activity with surprising accuracy. Their resolution power remains insufficient so far to enable decision to be made as to whether the enzyme is attached to the cell membrane. Therefore, for this purpose, histochemical results can be used only in combination with evidence derived from other observations. So far this has been possible mainly with regard to alkaline phosphatase, which in kidney, intestine, and other organs has given very clear and interesting pictures. (For a review see Bradfield, 1950.) In the indirect evidence available the most conclusive is based on impermeability of the membrane for substrates or enzyme effectors (inhibitors or activators). If a cell shows enzymatic action on a non-penetrating substrate or if the activity of a cellular enzyme is affected directly by non-penetrating substances, the site of the enzyme involved must be the outer surface of the cell membrane. The localization of an enzyme on the inner surface of the membrane (which should by no means be regarded as less frequent or less important), cannot, of course, be similarly demonstrated with equal ease and conclusiveness. A special group of enzymes with non-penetrating substrates are those whose action resembles those involved in digestion in the alimentary canal. These act by splitting larger, non-penetrating molecules of a substrate into smaller units which can be metabolized in the cell interior. They have adequately been termed “digestive enzymes” by Rothstein (1950). Wider biological importance appears to be attached to those processes which in the present review will be termed “enzymatically controlled transport.” Some introductory remarks concerning the meaning of this notion seem adequate. The essential feature of an enzymatically controlled transport is the

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combined action of two surface enzymes separated by the cell membrane whereby the substrate is temporarily changed to a membrane-soluble transport form capable of penetrating the membrane. The existence of this transport form will thus be confined essentially to the membrane proper. It may be assumed to consist of the penetrating substrate and some other molecule or molecules which may be regarded as “membrane carriers.” The transport direction of this substrate-carrier complex will be determined by the natural diffusion tendencies of both substrate and carrier. If these tendencies are opposite and if the resulting transport follows the tendency of the carrier rather than of the substrate, the latter will move from lower to higher chemical or electrochemical potential. This case will be termed “thermodynamically active transport.” It should be pointed out that the thermodynamically active transport is a possible, but by no means a necessary consequence of the participation of enzymes in membrane penetration; in other words, in general it represents a special case of enzymatically controlled transport. The essential point for a continuous thermodynamically active transport appears to be the maintenance of the carrier gradient which usually will require both enzymatic formation and elimination of the carrier on the two sides of the membrane. A more detailed discussion has been given by Rosenberg (1948). The identification of a transport as enzymatically controlled meets with greater difficulties if it is not thermodynamically active. Indirect criteria then have to be used. They include: 1. Non-conformity with the laws of diffusion, particularly a constant penetration rate independent of the concentration difference. Minor deviations may possibly be ascribed to variations of activity coefficients in the membrane or to a change of the membrane structure by high concentrations of the penetrating solute in the membrane. 2. Competition of simultaneously penetrating substrates. The limitations of this criterion coincide to a certain degree with those of the foregoing in so far as the principle of independent diffusion streams holds strictly only for dilute solutions. 3. High structural specificity, particularly stereospecificity. Although the possible existence of stereospecific pore structures cannot be denied in principle such “keyholes” have never been demonstrated and would appear to have no reasonable degree of probability. 4. Action of enzyme effectors (inhibitors or activators) on the rate of transport, provided that their effect cannot be ascribed to a changed concentration difference of the penetrating substances.

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As to other criteria, occasionally an unusually high temperature coefficient of the penetration rate has been taken as an indication for chemical transport mechanisms. Danielli and Davson (1934) have shown, however, that high energy barriers in the, path of diffusion may lead to temperature coefficients of considerable magnitude (“activated diffusion”), without participation of chemical reactions. None of the above criteria alone will be conclusive and it will depend on the case under consideration, how much weight can be attributed to any of them. 111. ENZYMES WITH NON-PENETRATING SUBSTRATES

Disregarding the possibility of an enzyme action over long distances (through the cell membrane), the outside localization of enzymes acting on non-penetrating substrates seems to be conclusive. A number of the enzymes belonging to this group clearly are of the digestive type, particularly carbohydrases of microorganisms. Also certain phosphatases have been assigned to this group. 1. Invertase. Wilkes and Palmer (1933) studied the pH-activity curves of yeast invertase in free solution and in the living cells and found them practically identical. They concluded that the locus of the enzyme in the cell must have free exchange with the external medium as regards [HI+ ions and that the enzyme would be supposed to function on the surface of the cells. 2. Lactuse. Essentially the same behavior was later found concerning lactase in yeast by Myrback and Vasseur (1943), who arrived at the same conclusion as regards the localization of the enzyme. 3. Trehulase. Through a different approach the conclusion of outside localization was reached in the case of trehalase. Myrback and Oertenblad (1936) found that yeast cells contain trehalose which is not metabolized although the cells contain an enzyme trehalase which splits trehalose added to the suspension. Thus apparently the naturally occurring enzyme and substrate are separated by the cell membrane, and the enzyme must be sited on the outer surface of the membrane. Accordingly the pH-fermentation curve for added trehalose was found by Myrback and Vasseur (1943) to differ substantially from that for glucose, showing a narrow optimum about pH 5 , whereas glucose fermentation has a wide range of practically constant activity without a sharp maximum, extending from about pH 2-3 to beyond p H 7. The pH optimum of trehalose fermentation agreed well with that found earlier by Myrback and Oertenblad (1937) for yeast trehalase in solution ( p H 5-6), indicating that the rate of hydrolysis on the outside of the cell was the limiting factor.

69

ENZYMATIC PROCESSES 1.N CELL MEMBRANE PENETRATION

4. Amylace. According to Rahn and Leet ( 1949) Streptococcus bovis forms adaptive amylase in the presence of insoluble starch in the medium. In this case both the adaptive enzyme and the enzyme involved in the process of adaptation appear to be situated on the outside of the membrane. 5 . Phosphatases. Rothstein and Meier ( 1948) described surface phosphatases in yeast cells acting among others on adenosinetriphosphate. The outside localization in this case was established by quantitative recovery of the added nitrogen, pentose ester, phosphate, and total phosphate in the outside medium by experiments with ATP containing PS2in which no P3*entered the cells. I n later experiments Rothstein showed that yeast cells utilize phosphorylated sugars only after enzymatic dephosphorylation, the inorganic phosphate remaining outside. Inhibition of the surface phosphatases by molybdate deprived the cells of the ability to metabolize hexose phosphates. Vishniac ( 1950) described hydrolysis of tripolyphosphate, which also may be considered as non-penetrating into yeast cells. 6, Phosphorylating enzymes. Synthetic enzymes likewise must in some cases be localized on the surface of cells. Lindberg (1948, 1950) working with 32Pphosphate on sea urchin eggs studied the equilibration of the external inorganic phosphate with both inorganic phosphate and energy-rich phosphate of the cell. In the case of the unfertilized egg his experiments showed an increase of specific activity first in the inorganic phosphate and somewhat later in the energy-rich phosphate of the cell which, however, did not rise above 0.03 per cent of the specific activity of the external inorganic phosphate and remained constant at this level. His conclusion that on the outer surface of the eggs energy-rich phosphate was formed from inorganic phosphate, both fractions being unable to penetrate the membrane, appears to be reasonable although the striking fact that the equilibrium specific activities of the two fractions were found identical does not clearly follow from this assumption. Since according to prevailing views formation of energy-rich phosphate from inorganic phosphate involves coupling to other processes, a further consequence would appear to be that the total reaction on the surface is still more complex. In the case of the fertilized eggs equilibrium was not reached within the time of the experiment (135 minutes), indicating slow penetratibn. The continuous rise of the activity, however, was considerably steeper and reached much higher values in the energy-rich fraction, indicating that the formation of the energy-rich bonds in this case likewise must have taken place on the outer surface of the cell. It seems possible that r

r

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the presence of energy-rich phosphate bonds on the outer surface of a cell as shown in these experiments may be of a more general significance for enzymatically controlled transports. IV. THE ENZYMATICALLY CONTROLLED TRANSPORT The number of enzymatically controlled transports used by the organism is probably greater than we know at present. In many cases no compelling criteria are available, and sometimes the enzymatic nature of a transport can only be supposed. There is mainly the alternative possibility of a .

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plasm is demonstrated by the arrest of development at the gastrula stage (e.g., Bufo 9 x Rana 3 ) . An early cessation of development of the hybrid, following normal fertilization phenomena, as in the last example, has been found in a number of hybrid combinations and subjected to detailed analysis.

2. Analysis of Nucleo-Cytoplasmic Incompatibility in Diploid and Androgenetic Haploid Hybrids A detailed study of the factors involved in the early arrest of development in such cases is of obvious importance since the block to the progress of development must be caused by the incompatibility between the foreign chromosomes and the cytoplasm (and chromosomes) sf the egg. The situation is especially favorable for experimental treatment since the analysis can be extended by combining hybridization with androgenesis and testing directly the developmental possibilities of the combination of cytoplasm of species A with a haploid set of chromosomes of species B, following removal of the chromosomes of A. Since the problems of early lethality are similar in both types of experiments, the evidence gained from diploid hybrids and from androgenetic, haploid hybrids will be reviewed together, reserving the application of hybrid androgenesis to the study of the development of species characteristics for the following section. For detailed references to the literature the reader is referred to the recent reviews by Baltzer ( 1940, 1949, 1950). For convenience, androgenetic hybrid combinations will be designated by placing the name of the species furnishing the egg cytoplasm but no chromosomes in parentheses. The final stage to which development proceeds varies considerably with different hybrid combinations. In the androgenetic hybrid Triton (pdinatus) 9 x Salamandra inaculosa d development is arrested before gastrulation ; in the corresponding diploid hybrid gastrulation is not completed. In several androgenetic hybrids, such as Triton (palmatus) 9 x T. cristatus 3, Tritoi, (dpestris) 9 x T . palmatus 8 , Rana palzcstris 9 x R. pipiens 3, development proceeds through neurulation or to the formation of the eye vesicles ; in the combination Triton (taeniatus) x T . palinatus the lethal effect is not expressed until an advanced embryonic stage with branching gills and pigment. The lethal effect is more often general, affecting all regions of the embryo. In the androgenetic hybrid Triton palinatus or taeniutus x T . crisfatus, however, the effect is strictly localized in the head mesodet-1x1. Pycnosis and degeneration of the cells are not preceded by visible mitotic disturbances, with the exception of the diploid and androgenetic hybrids

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Triton palmatus x Salunaandra (and, possibly, Rana esculenta x R. fusca), where failure of the chromosomes to separate normally during anaphase is a striking phenomenon in the middle blastula stage. The causes of the early arrest of development have been investigated in a number of ways. Valuable information has been obtained by physiological and biochemical methods which define more accurately the nature of the block to development. In the arrested hybrid gastrulae Rana pipiens x sylvatica there is a marked reduction in respiration and glycolysis (Barth and Jaeger, 1947 ; Gregg, 1948). In the lethal combination Rana esculenta x R. fusca the synthesis of ribosenucleoprotein is reduced (Brachet, 1944). The foreign chromosomes are not only unable to take part in basic metabolic processes but also seem to interfere with the activity of the maternal chromosomes. Moore (1947) has suggested that the foreign genes compete with the maternal ones for a substrate present in limited amount and form an analog that cannot take the place of the substance formed by the normal cells. More important still was the discovery that the development of hybrid tissue may be resumed when parts of hybrid gastrulae are grafted to normal embryos where they survive and may differentiate normally. In one combination only has transplantation failed to improve the performance of the cells ; in the androgenetic hybrid (Triton palmatus) ? x Salamandra d, all grafts so far have failed to survive. In some cases transplants from all regions of the hybrid gastrula differentiate normally in their new site and survive until the hosts have developed into free-swimming larvae ; such full viability is shown by grafts from the diploid hybrid Triton palmatus x Salanzandra, and from the androgenetic hybrid Triton (dpestris) x T . palwtatus. In the hybrid Ram pipiens x R. sylvatica, differentiation of the grafts is limited ; ectudermal grafts show a reduced competence in response to inductive stimuli of normal embryos, while grafts from the dorsal lip have a limited power to induce secondary structures in competent ectoderm of a normal host (Moore, 1947, 1948). In the most thoroughly analyzed combination, the androgenetic hybrid Triton. (palmatus) or (tueniatits) x T.cristatzts, the behavior of the grafts depends both on their prospective significance and on their relations to the host tissues. If a series of relatively small grafts are made to normal embryos, the different organ territories of the androgenetic hybrid gastrula show a clear-cut gradation in their response: (1) head mesoderm, which is the focus of degeneration in the intact androgenetic hybrid, degenerates ; (2) muscle segments and notochord both contain some pycnotic nuclei ;

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but muscle fibrils differentiate normally while the notochord cells begin to differentiate but do not become vacuolated; (3) neural tube and epidermis differentiate normally to tlie condition reached in young larval stages (Hadorn, 1932). When similar pieces of androgenetic hybrid gastrulae are explanted in vitro in salt solution, the epidermis and notochord alone show normal differentiation ; neural tissue and somites survive but fail to differentiate, demonstrating the importance of the host environment in furthering their differentiation when grafted (Hadorn, 1934). This dependence is more clearly shown by the development of chimaeras, consisting of the anterior half of a normal palnzatus gastrula and tlie posterior half of an androgenetic hybrid gastrula. Epidermis and notochord differentiate well, but the neural tissue develops poorly, except in the zone of contact with normal host tissues where histological differentiation is normal ; this stimulating effect decreases rapidly with increasing distance from the boundary (“histogenetic stimulation,” Hadorn, 1935, 1937). I n his most recent theoretical discussions of the beneficial effects of transplantation Baltzer ( 1949, 1950) offers three possible interpretations. (1) The grafts are able to survive and differentiate because they have been removed from the harmful or lethal influence of the disintegrating head mesoderm ; the differentiation of tlie graft cells is autonomous, i.e., independent of any stimulation by the surrounding host tissues. This would mean that the palmatus nucleus may be effectively replaced by the cristatus nucleus in its functions during embryonic development and differentiation of the tissues under consideration. (2) The differentiation of the grafts is made possible entirely by the proximity of normal host tissues and is dependent on the transfer of substances from the host to the hybrid tissues which the latter are unable to elaborate. (3) The cells of the graft differentiate autonomously, not under the “guidance” of the cristatus chromosomes cooperating with the palmatus cytoplasm, but because of a much earlier “predetermination” of the characteristics of these tissues in the cytoplasm of the oocyte, before fertilization, under the control of the maternal gene complex. Which of these interpretations, alone or in combination, is the correct one cannot be decided without additional evidence, although the “histogenetic stimulation” of hybrid cells by host tissue seems to be well established by the observations on chimaeras which ‘emphasize the complications introduced by the graft-host relationships.

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3. Nztclco-Cytoplasnaic Relatiom in the Developnzetit of Species Characteristics Experiments on hybrid androgenesis, combining cytoplasm and nucleus belonging to two different species, were undertaken originally to determine the relative role of these two components in the development of species characteristics. Unfortunately, androgenetic hybrids of any of the combinations tried could not be raised to a stage where species characteristics first appear. I t was only through the use of transplantation that this critical period could be approached. So far, three experiments of this type have been at least partially successful. (1) A large piece of ectoderm from a gastrula of the combination Triton (palmatus) x T. cristatus was grafted to a normal embryo of T . alpestris where it differentiated into normal skin and survived metamorphosis of the host animal (Hadorn, 1936). At metamorphosis, a specific difference between T . palmatus and T. cristatus becomes apparent in the surface structure of the epidermis; in T . cristatus the surface is smooth; in T. palmatus the epidermis forms small protuberances consisting of short columns of cornified cells. The epidermis developed froin the graft, with cytoplasm of T. palmatiis and nuclei of T . cristatzls, showed the typical palmatus protuberances. The cristatus chromosomes apparently had no influence on this characteristic which followed the maternal species entirely ; probably, this trait, although it appears late in development, is “determined” very early in the cytoplasm of the egg before fertilization. However, the interpretation of the result is complicated by the fact that the epidermis of alpestris, serving as host, also forms protuberances. (2) The species of California newts can be recognized in early larval stages by distinctive pigment patterns. In Triturus torosus, the melanophores form two compact, dorsal bands, while in T . rivularis the individual pigment cells are scattered. Hybrids between the two species show an intermediate condition which also develops when neural crest from hybrid embryos is grafted to embryos of either one of the pure species (Twitty, 1936). The basic difference determined by the genetic constitution of the two species appears to be a difference in the rate of differentiation of the melanophores and in the ultimate degree of differentiation and melanization attained (Twitty, 1945, 1949) . Androgenetic hybrids with rivzrlaris cytoplasm and torosus chromosomes do not live to the stage of pigment formation, but their neural crest may be transplanted to normal toroszis hosts where it produces a pigment pattern that is essentially like torosus. However, an increase in the number of pigment cells on the flank and the degree of dispersion in this region show a definite influence of the rizrziluris cytoplasm (Dalton, 1946).

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(3) The third experiment involves androgenetic hybrids between two races of the axolotl, rather than between two species. The recessive white race shows a limited number of melanophores in a very restricted distribution, compared with the black race. It seems that this difference is caused by the absence of a substance in the “white” epidermis which is necessary for melanin formation in the propigment cells below ; a graft of skin from a white donor prevents the melanization of the pigment cells of black hosts in the region under the graft (Dushane, 1935). Baltzer (1947) produced androgenetic hybrids with cytoplasm of the black race and a haploid nucleus of the white. A piece of ectoderm of this hybrid grafted to a black neurula formed a large area of epidermis and prevented melanization of the pigment cells of the black host. If the mechanism of melanin formation is correctly interpreted (cf. the recent work of Dalton, 195Oa, b) , the result of this experiment demonstrates that the peculiarity of the white epidermis is determined by a chromosomal factor acting early in development and not by “predetermination” by maternal genes in the cytoplasm of the oocyte before fertilization. Altogether, the limited evidence available points to the importance of both nucleus and cytoplasm in the development of early species characteristics. The constitution of the cytoplasm of the egg emerges as more important than had been anticipated, since it may control characteristics that appear relatively late in development. Such long-range cytoplasmic effects are no longer too difficult to visualize if particles with various degrees of genetic continuity and dependence on nuclear genes exist in the cytoplasm of the egg. A N D CONCLUSIONS VIII. SUMMARY The experimental evidence reviewed in this paper, though incomplete in many respects, demonstrates clearly the existence during development of nucleo-cytoplasmic relations at various levels. ( 1 ) Within the nucleus, a delicate balance exists between the genes of the whole complex. Whole sets of genes may be added without disturbing development; addition of one or more single chromosomes to the diploid complement, however, almost invariably results in abnormal development. Not alone viability and growth, but differentiation as well are affected, particularly the development of circulation. The fact that certain groups of genes are present in triplicate, while the others are in the normal duplicate condition, disturbs the activity of the gene complex. The mechanism of this effect of a numerical imbalance among the genes remains obscure ; it may involve competition for various raw materials in the cytoplasm. (2) The study of unviable hybrid embryos deals more directly with

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the relations between the chromosomes and their cytoplastnic environment. In the diploid hybrid which fails to develop beyond the gastrula or neurula stage, there remains the possibility of a conflict between the two sets of chromosomes within the nucleus. Even here, however, it is perhaps itlore logical to transfer the site of conflict into the cytoplasm of the cells if one follows Moore’s (1947) suggestion that the foreign genes compete with the maternal ones for cytoplasmic substrates and form related analogs that cannot be used properly by the cells of the developing embryo. The depression in respiratory activity and in the synthesis of ribonucleic acid, which was demonstrated in the arrested hybrid gastrulae of frogs, shows that the foreign genes fail to operate properly in the control of basic biochemical processes, possibly because of specific differences in the structure of proteins. To what extent the genetically “inert,” heterochromatic sections of the chromosomes, rather than the euchromatic gene-carrying portions, may be involved in such biochemical activity cannot yet be discussed profitably because of the lack of critical evidence. In androgenetic hybrids, the incompatibility exists obviously between the foreign chromosomes and the maternal cytoplasm. In those combinations where the corresponding diploid hybrid is completely viable, we meet the problem of the nature of the relations between the two chromosome sets within the diploid nucleus which are not antagonistic as in the lethal combinations. With the maternal chromosomes present and taking the lead, the paternal ones are able to cooperate properly, although they are incapable of doing so when left alone in the strange cytoplasm. One of the most interesting and at the same time puzzling features of the analysis of hybrid lethality is the beneficial effect of transplantation which has been observed in the majority of combinations. I n a normal host environment, all or most of the grafted tissues remain alive and differentiate normally. It has not been possible as yet to decide between two interpretations of this improved performance : (a) The hybrid cells may be able to develop normally, under their own power, once they have passed the critical stage and have been removed from the lethal environment of the degenerating hybrid embryo ; this would in turn presuppose that the nuclei of the two species are interchangeable as far as their activity in the differentiation of these particular tissues is concerned, unless we assume a “predetermination” of these characteristics before fertilization, under the effect of the maternal gene complex. ( b ) The hybrid cells are “vitalized” by the intimate relations with the normal host tissues from which substances essential for differentiation

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diffuse into the graft cells that are themselves still unable to manufacture them. That such “histogenetic stimulation” (Hadorn, 1937) by the host exists is shown negatively by the generally poor differentiation of explants in zdro and, positively, by the normal differentiation of hybrid tissues in the boundary zone of chimaeras, where they are in contact with the tissues of the other coniponent derived from a normal embryo. The possibility that faulty gene-cytoplasm relations may be corrected by bringing in substances (gene products ?) from neighboring healthy cells opens a very interesting perspective of gene action in development. (3) Further light is thrown on the nucleo-cytoplasmic relations during cleavage of the egg by the observation that cells without chromosomes can survive and divide repeatedly until the blastula stage is reached. During this period of rapid cell division there seems to be little time for synthetic activities that require nuclear control. The fact that non-nucleated cells fail to survive through gastrulation and to differentiate when grafted to normal embryos demonstrates participation of the nucleus at this stage and can be correlated with biochemical evidence of increased enzymatic activity beginning at the end of cleavage. The longer survival of cells with remnants of sperm chromatin that had been heavily damaged by x-irradiation, as compared with completely achroniosomal cells, may indicate that some components of the damaged chromosomes (heterochromatin?) are still active in a limited way. (4) The graded increase in nuclear and cell size in amphibian embryos with various degrees of polyploidy reaffirms the existence of a definite nucleo-cytoplasmic ratio. Just how and why the regulation of cell size is brought about remains unknown. In haploid embryos developing from whole eggs, an additional mitosis at the end of cleavage would, theoretically, reduce the cell size to one-half of the diploid value and thus restore the normal nucleo-plasmic ratio. Conversely, omission of the last cleavage mitosis in a tetraploid blastula would produce large tetraploid cells with the same nucleo-cytoplasmic ratio as exists in the diploid cells. This interpretation becomes inadequate when one considers the fact that the same ratio is obtained in diploid and haploid embryos regardless of the size of the original egg cell, whether normal, tliree-fourths, or onehalf normal. There remains also to be explained the regulation in triploid embryos which involves an increase by 50 per cent over the diploid cell size. Briggs (1947, pp. 257-58) has suggested that the adjustment takes place, at least in part, when cell growth begins at the end of the period of rapid cell division during cleavage. In triploid frog embryos the cells are definitely larger than in diploids in the gastrula stage which suggests

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that a regulation in triploid mitotic rate and cell size occurs during gastrulation or late blastula stages. At this time the cells acquire new synthetic activities, in which nucleo-cytoplasmic interactions are important. If the nucleus becomes active in protein synthesis at this stage, it might also acquire the ability to regulate cell size, if the synthesis of cytoplasmic substances were proportional to the chromosome number. A suitable prolongation of the first “growth interphase” would regulate cell size and cell number in one step; from this time on, the mitotic rate in both diploid and triploid embryos would again be equal, as actual observations show it to be. (5) One of the outstanding features of polyploid development in amphibians is the normal character of morphogenesis at the triploid, tetraploid, and pentaploid levels. Embryonic differentiation is to a high degree independent of cell size. The upper limit of polyploidy compatible with normal morphogenesis has not been established ‘with certainty. The fact that the two heptaploid embryos discovered so far were both abnormal may well indicate that, with cells of approximately three and one-half times the normal size, some of the morphogenetic processes are meeting with serious obstacles. While early development of tetraploid and pentaploid embryos is normal, their later growth and viability are often adversely affected, although with considerable individual variations. The most plausible explanation would seem to be that the large polyploid cells do not function at maximum efficiency. The interactions between the nucleus and the cytoplasm, and between the latter and the environment, must be influenced by the increase in cellular dimensions. The distance between the surface of the cell and that of the nucleus may be a critical factor, as well as the relations between cell surface and cell volume. Until more definite information is available, another more strictly genetical interpretation must also be considered. The multiplication of identical sets of genes beyond a certain point may have a harmful effect, perhaps because some genes when present in multiple dose increase their activity more than others and thus disrupt the harmony of the genetic system. (6) In spite of these limitations the adaptability of embryonic development to differences in cell size remains truly remarkable. Increases in the dimensions of the individual cells are balanced by a regulation of cell number and, in thin-layered organs, also by an adjustment of cell shape, so that the size and structure of the organs and the size of the body remain essentially normal. One could not wish for a more forceful reminder that

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the subdivision of the embryo into cells is in some respects incidental and subject to the regulating influences of the embryo as a unit. The mechanisms that determine the normal limits of growth and thus the final body size remain unknown. All we have learned is that growth, within wide limits, is independent of the initial mass of cytoplasm of the egg and of the size and number of the individual cells, since changes in cell size invariably induce compensatory changes in cell number. It appears as if the total mass of living material that is produced during developnient were fixed by the genetic constitution of the species. It does not matter how much or how little the embryo starts with, or how the material is divided up into individual units. This subordination of the cells to the developing embryo as a whole, since it in turn must be under the control of the genes within the nucleus, emphasizes the complexity of nucleo-cytoplasmic relations in amphibian development. IX. REFERENCES Baltzer, F. (1922) Verh. schweie. nalurf. Ges., 248. Baltzer, F. (1940) Naturwissenschaften, S,177, 1%. Baltzer, F. (1947) Rev. mime Zool., 54, 260. Baltzer, F. (1949) XI11 Congr Int. Zool.,234. Baltzer, F. (1950) Rev. suisse Zool.,57, suppl. No. 1, 93. Barth, L. G., and Jaeger, L. (1947) Physiol. Zool., 20, 117. Brachet, J. (1944) Embryologie Chimiqw. Masson, Paris. Briggs, R. (1946) Growth, 10, 45. Briggs, R. (1947) J. exp. Zool., 106, 237. Briggs, R. (1949) J . exp. Zool., 111, 255. Briggs, R.,Green, E. U., and King, T. J. (1951) J . exp. 2001.. 116, 455. Dalton, H. C. (1946) J . ezp. Zool., 103, 169. Dalton, H. C. (195Oa) J. exp. Zool., 116, 17. Dalton, H. C. (1950b) J. exp. Zool., 116, 157. Dalton, H. C. (1950~) Auat. Rec., 108, No. 3, 30. Dushane, G. P. (1935) J. exp. Zool., 72, 1. Fankhauser, G. (1929) Rev. suisse Zool., 86, 179. Fankhauser, G. (1934a) J . ex$. Zool., 67, 349. Fankhauser, G. (1934b) J. exp. Zool., 08, 1. Fankhauser, G. (1937) J. Hered., 28, 1. Fankhauser, G. (1938) J. Morph., Ba, 393. Fankhauser, G. (1941) J. Morph., 66, 161. Fankhauser, G. (1945a) Quart. Rev. Biol., 20, 20. Fankhauser, G. (1945b) J. exp. Zool., lw, 445. Fankhauser, G. (1948) A m N. Y. Acad. Sci., 48, 684. Fankhauser, G., and Godwin, D. (1948) Proc. nat. Acad. Sci.,LVuslt.. 34, 544. Fankhauser, G., and Griffiths, R. B. (1939) Proc. nat. Acad. Sci., Wash., 26, 233. Fankhauser. G., and Humphrey, R. R. (1950) J. cxp. Zool., 116,207.

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Fankhauser, G., and Moore, C. (1941) J. Morph., 68, 387. Fankhauser, G., and Watson, R. C. (1942) Proc. nat. Acad. Sci., Wash., 28, 436. Fischberg, M. (1947) Genetica, 24, 1. Gallien, L.,and Muguard, H. (1950) C. R. SOC.B i d , Paris, 144, 657. Goldschmidt, R. (1937) Amer. Not., 71, 83. Gregg, J. R. (1948) J . exp. Zool., 109, 119. Griffiths, R. B. (1941) Getietics, 26, 69. Hadorn, E. (1932) Arch. EiifwMCch. Org., l26, 495. Hadorn, E. (1934) Arch. EiztwMech. Org., 131, 238. Hadorn, E. (1935) Rev. d s s e Zool., 49, 417. Hadorn, E. (1936) Verh. dtsclz. zool. Ges., 97. Hadorn, E. (1937) Arch. ElttwMecR. Org., 136, 400. Humphrey, R. R. (1948) J. Hered., 89, 255. Jollos, V., and Peterfi, T. (1923) Biol. Zbl., 43, 286. Kaylor, C. T. (1941) Biol. Bull., 81, 402. Mangold, O., and Seidel, F. (1927) Arch. EntwAlech. Org., 111, 593. Moore, J. A. (1946) Genetics, 31, 304. Moore, J. A. (1947) J . exp. Zool., lM, 349. Moore, J. A. (1948) J. exp. Zool., 108, 127. Penners, A. (1935) 2. w'ss. Zool., 146, 463. Randolph, L. F., and Hand, D. B. (1940) J: agric. Res., 60, 51. Spemann, H., and Falkenberg, H. (1919) Arch. EnfwMrrh. Org., 45, 371. Stauffer, E. (1945) Rev. suisse Zool., 62, 231. Tchou-Su (1931) Archs. Attat. uiicr., 27, 1. Twitty, V. C. (1936) J. ezp. Zool., 74, 239. Twitty, V. C. (1945) J. exp. Zool., 100, 141. Twitty. V. C. (1949) Growth Symfiosiiini, 9, 133.


Structural Agents in Mitosis* M. M. SWANN D<par-tiiient of Zoology, Cambridge Uaiversity, Cambridge, Eikglatkd

CONTENTS

Page

I. Introduction ......................................................... 195 11. Birefringence Changes in the Sea Urchin Egg during Mitosis . . . . . . . . . . . 197

111. Further Evidence on the Release of Chemical Agents in Mitosis . . . . . . . . . . 203

Iv. The Nature of Chemical Agents in Mitosis ............................ V. Conclusion ........................................................... VI. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

208 209 210

I. INTRODUCTION In the course of mitosis, the physical properties of cells undergo a series of changes. Cytoplasmic viscosity, membrane tension and permeability, birefringence and light scattering all rise and fall in a definite pattern. Most of these changes are concerned in some way with the formation and disappearance of the mitotic figure or with the surface activity involved in cleavage; but so little is known of the mechanisms involved, that the significance of the various changes is far from clear. The multiplicity of theories about mitosis and cleavage is evidence enough of the state of confusion that exists. A full understanding of some of these physical changes would probably lead to the solution of a great many cytological problems. If, for instance, the decrease in birefringence in the spindle at anaphase could be interpreted for certain in molecular terms, the cause of chromosome movement might become clear. As it is, the physical data to be obtained from studies of living cells are too scanty for drawing any but the most tentative conclusions. Sometimes, however, it is possible to reach beyond difficulties of this kind, by investigating phenomena at a different level. The study of nervous conduction is an interesting case in point. Many aspects of conduction, such as its self-propagating nature and the rise and fall of the action potential were understood in outline many years ago; the physico-chemical mechanisms responsible for the action potential, on the other hand, are only now beginning to be understood. To reach beyond the difficulties inherent in interpreting scanty physical data implies, in the case of mitosis, a study of what may be called the behavior of the physical changes. Much work has, of course, already been

* A ,short version of this paper was delivered at the Seventh International Cytological Congress, held at Yale University in September, 1950. 195

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done on these lines, particularly with regard to the timing of the various changes in the mitotic cycle. There are, for example, the observations of Heilbrunn (1943) on cytoplasmic viscosity, VlCs (1921) on refractive index, Herlant (1920) on permeability, and Raven (1948) on membrane tension, to mention only a few. But curiously enough, not much attention has been paid to the spatial patterns of these changes. No doubt the limitations of the experiniental methods are responsible for this ; it is scarcely possible for instance, to measure refractive index, membrane tension, or permeability in different regions of the cell. Viscosity can be measured at different points, though with difficulty, and the observations of Carlson (1946) are a valuable beginning in this direction. But for some time to come, practical difficulties are likely to limit the study of spatial and temporal variations in the physical properties of dividing cells, to methods involving direct microscopical observation. Since there is so little work on the variation of physical properties in space as well as time, it is not surprising that we know little of what causes such changes. This is not a deficiency peculiar to mitosis; it is true of almost all the activities of the cell that involve extensive changes of protoplasmic structure : muscular contraction, ameboid and ciliary movement, protoplasmic streaming and rnorphogenetic movements, to mention only the more important ones. All structural changes involve, ultimately, changes in the forces acting between molecules and molecular aggregates. A considerable amount is known about the effect of various physical and chemical factors on such forces in protein and other model systems (Frey-Wyssling, 1948). Temperature, for instance, affects the cohesion of lipophilic groups ; the presence of ions affects the amount of bound water, and hence the amount of hydrogen bonding; p H affects salt linkages] and r H probably affects various other forms of valency bond. But the extent to which the living cell employs any of these mechanisms is uncertain. Though Heilbrunn (1943) attaches great importance to changes in calcium concentration, the evidence on the whole suggests that the internal environment of the cell is surprisingly constant. On the other hand, it is possible that some of these factors, including no doubt calcium ions, may be employed locally as intermediary mechanisms ; but if so, they must presumably be controlled by other more specific means. There are, in any case, certain general reasons for supposing that the structure of the living cell is controlled by highly specific substances with an essentially catalytic effect. Hormones, for example, are usually effective in very great dilution; so are various inhibitors such as colchicine which appear to act on the

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mechanisms controlling structure. But whether the ultimate controlling agents act directly, or whether they operate through some of the mechanisms discussed above, is not known. If we look at the specific mechanisms that exert an effect on protoplasmic structure in living systems, we find essentially two different types : selfpropagating action potentials, and hormones or comparable chemical agents. The self-propagating type of mechanism is most highly developed in nerve, but is found also in muscle and a few other cells. Being primarily electrical it appears to be limited to the surface of cylindrical cells.* Whether this form of transmission can affect the internal structure of cells directly is uncertain. The fact that nerve endings liberate various types of hormone, which can themselves stimulate muscles and other organs, suggests that chemical intermediaries are usually necessary. The chemical type of control is widely distributed in living systems. Acetyl choline, adrenaline, and histamine are particularly well known for their effects on muscle, though it is not clear whether they act directly on the contractile mechanism or on the conduction mechanism of the muscle fiber. The effect of these and other hormones on the expansion and contraction of chromatophores, however, is presumably a direct one. Auxins appear to have a direct effect on protoplasmic streaming in plants, and in muscle it seems that adenosine triphosphate is directly concerned in altering protoplasmic structure. It is possible that self-propagating mechanisms are important at some stage in the control of the more rapid cell functions, such as ciliary and flagellar movement. Ameboid movement, mitosis, and protoplasmic streaming, on the other hand, are so slow that action potentials must be ruled out. Only further research, however, will show whether the control in these cases is by chemical agents. CHANCES 11. BIREFRINGENCE

I N THE SEA URCHIN

EGGDURING

MITOSIS

The first detailed study of birefringence in mitotic figures was that of Schmidt (1937, 1939) ; references to other work are given by Swann (1951a). Schmidt found that the spindle of the sea urchin egg was positively birefringent with respect to its length, and the asters positive with respect to their radii. H e also found that the birefringence, which was strongest at metaphase, disappeared almost completely during anaphase. Since protein fibers are positively birefringent with respect to their length, he supposed the mitotic figure to consist of such fibers, and ~

* Though the only self-propagating mechanisms so far demonstrated are primarily electrical, it is possible that there may also be purely chemical ones.

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since muscle and protozoan myonemes lose their birefringence to a greater or lesser extent when they contract, he supposed further that the decrease in birefringence during anapliase was due to contraction. His ideas have lent considerable support to the traction fiber hypothesis of chromosome separation. Beyond measuring the maximum birefringence of the mitotic figure, Schmidt did not make a quantitative study, nor did he examine the changes during anaphase in any detail. Using ordinary methods of observation, indeed, this is scarcely possible. Recently, however, Swann and Mitchison (1950) have made certain refinements in polarised light technique, which open the way to a more detailed examination of weakly birefringent structures in living cells. They describe various means of obtaining increased sensitivity, and show how, by taking photographs and measuring them with a densitometer, it is possible to construct a series of curves of retardation for structures whose birefringence varies from point to point and moment to moment. The various problems involved in applying this technique to sea urchin eggs are dealt with by Swann (1951a). In the same paper he examines in some detail the birefringence of the living spindle and asters of Psamwchinus miliaris at metaphase (Plate I ) . From curves of retardation it is possible to calculate curves for coefficient of birefringence, exactly in the case of the asters, and approximately in*the case of the spindle. Both spindle and aster curves show essentially the same features (Figs. 1 and 2) ; coefficient of birefringence is nil in the centrosomes, rises to a maximum at about 5 microns from the center, and then falls again to a minimum at the periphery of the asters, and at th'e equator of the spindle. The rise in coefficient of birefringence to a maximum at 5 microns represents, of course, the build-up of orientation round the centrosomes. The fall beyond 5 microns is not so easily accounted for. Since the total amount of material in the mitotic figure does not vary significantly, it must be due either to changes in molecular and micellar structure, or to a decreasing proportion of the material of the mitotic figure being oriented. A structure of submicroscopic fibrils radiating from the centrosomes like the spokes of a wheel, for instance, would give an inverse square law fall in coefficient of birefringence, if the material between the fibrils were not oriented. In fact, the observed fall in coefficient of birefringence corresponds fairly closely to an inverse square, from which it is tentatively assumed that the structure of the spindle and asters may be one in which submicroscopic fibrils radiate from the centrosomes. A certain amount of other evidence points in the same direction. On the

STRUCTURAL AGENTS IN MITOSIS

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PLATEI. Photographs from a time-lapse film, taken in polarized light, of an egg of the sea urchin Psammechinus miliaris. 450 X. Compensated. Fertilization membrane removed. (1) 52 minutes after fertilization, prophase ; (2) 56 minutes, metaphase; (3) 58 minutes, early anaphase; (4) 60 minutes, late anaphase; (5) 62 minutes, telophase; (6) 64 minutes, starting to cleave.

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fiistance along the spindle axis,* microns from the equator

FIG.1. Coefficient of birefringence of a spindle at metaphase.

i j

Distance from the aster center, microns

FIG.2. Coefficient of birefringence of an aster at metaphase.

other hand it is always possible that the mitotic figure is really a homogeneous body, in which all the material present is oriented; if so, the fall in coefficient of birefringence with distance from the centers, must be due to molecular and micellar changes. Further analysis is not possible without evidence about form and intrinsic birefringence. A detailed interpreta-


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tion of structure is not necessary, however, for an examination of the changes in the spindle and asters during anaphase. These are described by Swann (1951b). By using time-lapse photography and constructing curves for retardation and coefficient of birefringence, it is possible to compare the birefringence of living spindles or asters at successive stages in anaphase. The results show that the decrease in birefringence noticed by Schmidt is not uniform, but proceeds on a definite, though somewhat complicated, pattern. The birefringence of the spindle is at a maximum in nietaphase; it first decreases in anaphase at the equator, though without any change elsewhere. In the course of a minute or two, quite a large region in the center of the spindle is affected. But as time goes on, it becomes apparent that there are in reality two separate regions in which the birefringence is decreasing more rapidly than elsewhere ; these are centered somewhere between the equator and either pole. Later still, the birefringence decreases mainly in the region close to either pole, the equator remaining more or less constant. In short, the decrease of birefringence starts at the equator and then moves towards either pole. The region affected by the decrease at any particular moment extends over a number of microns and is by no means sharply defined. There is an interesting correspondence between the movements of these regions of decreasing birefringence and the familiar pattern of movement of the chromosomes. They also start at the equator in metaphase and move apart in two groups towards either pole. I n the sea urchin egg, unfortunately, the chromosomes are very small and invisible in the living cell. But by following single eggs under polarized light and then fixing and staining them, it is possible to show that there is not merely a general similarity between the patterns of movement of the chromosomes and the regions of decreasing birefringence, but that there is a precise correspondence. The chromosome groups actually lie within the region of maximum birefringence decrease. Since the chromosome groups are small and compact, and since the chromosomes themselves appear not to be birefringent, it seems likely that the decrease must be due, not to any opposite sign of birefringence in the chromosomes, but to an indirect effect exerted by them on the structure of the spindle. The changes in the asters during cleavage lend further weight to this idea. For the first few minutes of anaphase they show no decrease in birefringence at all; but at the time when the chromosomes reach the poles of the spindle, the asters also begin to show a decrease. This begins near the centrosomes, and, in the course of the next few minutes, moves steadily outwards at the rate of about 5 or 6 microns per minute.

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Returning to the two mechanisms for producing structural changes, it will be seen that we have here to decide between a self-propagating process, initiated by the chromosomes, and the release of a chemical agent from the chromosomes. Fortunately, there is one clear-cut difference between mechanisms of these two types which enables us to make a decision with some certainty. Once started, self-propagating mechanisms are necessarily independent of the subsequent behavior of the initiating stimulus. The nerve action potential for instance, is not affected by the removal or repetition of the original stimulus. A diffusion mechanism, on the other hand, is inevitably affected by the subsequent behavior of the source. In the case of the birefringence changes it is evident that the movements of the chromosomes do affect the pattern of change. Provisionally therefore, we may accept the hypothesis that the changes in spindle and aster structure are brought about by the release of a chemical agent from the chromosomes, rather than by any self-propggating mechanism. The decrease of birefringence is not the only change in the mitotic figure that takes place in anaphase. From the curves of spindle and aster birefringence, it is evident that there is a marked growth of the whole mitotic figure during this period (see Plate I). The spindle lengthens, and the asters increase in radius. I t might be supposed that such growth is simply part of a general growth going on throughout mitosis; but this does not appear to be the case. Curves from birefringence data and from fixed material, show that both spindle and asters reach a certain size quite early in prophase and remain more or less constant for the rest of prophase and metaphase. In anaphase, however, there is a sharp increase in size. The same effect was noticed in the chick spindle by Hughes and Swann (1948) and is discussed further by Swann ( 1 9 5 1 ~ ) . It is evident that, once formed, the whole mitotic figure remains in a more or less static condition until the beginning of anaphase, when it suddenly beconies able to incorporate and orient fresh material. In the case of the asters this leads simply to an increase in radius, but in the spindle it leads mainly to an increase in length. The cause of this sudden change in the power of aggregation of the cytoplasm is not clear. Since it appears to affect the whole mitotic figure more or less simultaneously it is not possible to decide between a self-propagating and a diffusion mechanism; nor is there any reason to connect the change with the chromosomes. But since the growth clearly represents a second type of structural change, which starts at the same moment as the first type discussed earlier, it is perhaps not unreasonable to regard it as being due to a similar mechanism, that is to say, to the release of a second type of chemical agent.


203

There is considerable evidence that outside the mitotic figure, the cytoplasm of the sea urchin egg at metaphase is in a fluid condition (Heilbrunn, 1943; Chambers, 1924). Seen in ordinary light it is not fibrous, nor is it birefringent; there is, in fact, every reason for supposing it to be unoriented and structureless. At this stage therefore, it is possible to distinguish two different protoplasmic configurations : one, in the spindle and asters, which is fibrous in ordinary light, and strongly birefringent ; the other, outside the spindle and asters, which is structureless. At anaphase however, both these configurations undergo a change. The spindle and asters show a marked fall in birefringence, to between a third and a sixth of their metaphase value. The structureless outer cytoplasm on the other hand, having previously been isotropic, shows a slight rise in birefringence, since it becomes incorporated into the growing mitotic figure. But although it becomes fibrous in appearance, it never becomes strongly birefringent like the original metaphase spindle and asters. The final result is two large asters, which virtually fill the cell, but are only weakly birefringent . Let the structureless configuration in the cytoplasm be called A, the weakly birefringent one B, and the strongly birefringent one C. The changes that occur in anaphase are then A to B and C to B. The meaning of these changes is not yet clear. The decrease in coefficient of birefringence involved in the change from C to B, however, may well be the result of randomization or folding up of protein chains, which is usually associated with contraction. This is the interpretation originally put forward by Schmidt, and there is no reason to think that it is not substantially correct. If so, it would seem that the chromosomes must be responsible for initiating their own movement. This may be of some significance in view of the instances cited by Schrader (1944) where chromosomes appear to behave autonomously.

111. FURTHER EVIDENCE ON

RELEASEOF CHEMICALAGENTSI N MITOSIS

THE

Further evidence on the release of chemical agents from the chromosomes comes from a study of the cell surface using various optical methods, (Mitchison and Swann, 1952). A number of changes are visible, which fall into two groups, one at the beginning of anaphase, the other at cleavage. From shortly after fertilization until the end of metaphase, the cortex of the sea urchin egg scatters little or no light under a particular form of dark-ground microscopy known as vertical illumination. At the beginning of anaphase, however, at the same time as the structureless

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regions of the cytoplasm begin to be incorporated into the mitotic figure, the cortex suddenly develops a marked color. At the same time, the hyaline layer outside the cortex, which is only faintly birefringent from fertilization onwards, becomes more strongly birefringent. These two changes are clearly correlated in time with the A to B structural change in the cytoplasm, and may therefore be caused by the same chemical agent. More striking changes are apparent at the surface during cleavage. During anaphase, the light-scattering visible under vertical illumination is apparent all round the egg. Just before cleavage begins, this scatter decreases at either pole of the aell. As cleavage progresses, the subpolar and later the equatorial regions of the surface also decrease (Plate 11, 5-8.) Corresponding changes can be seen in the cortex by polarized light, though there appear to be no marked changes of birefringence in the hyaline layer, which must be removed with calcium free sea water if the changes in the cortex proper are to be seen. Before cleavage the cortex is slightly birefringent, being negative in a tangential direction. As cleavage begins, there is a decrease in birefringence, which starts at either pole of the cell, and later spreads round to the subpolar and equatorial regions. Similar changes in birefringence are mentioned by Monroy ( 1945). Two points about the changes at cleavage are important. The first is their general timing. It was mentioned earlier that the decrease of birefringence in the aster ( C to B), spreads outwards at about 5 or 6 microns per minute. Since, at the end of anaphase, the chromosome groups lie at the poles of the spindle, which are then about 30 microns from the surface, the change should take some 5 or 6 minutes to reach the cortex. It is significant that cleavage normally begins at about this time after the decrease of birefringence first becomes visible in the asters. Secondly, since the chromosome groups lie at either pole of the spindle, they are considerably nearer to the poles of the cell than the equator. A chemical agent released from the chromosomes will therefore reach the poles of the cell before it reaches other parts of the surface. The observed pattern of decrease in birefringence and light scattering in the cortex in fact, is exactly what would be expected, if the changes were to be caused by the same chemical agent as causes the decrease in birefringence in the spindle and asters. It is interesting that this pattern of optical changes corresponds precisely to the pattern of cortical expansion found in another species of sea urchin egg by Dan, Yanagita, and Sugiyama (1937). The significance of these various changes from the point of view of the mechanism of cleavage, is discussed by Mitchison (1952) and Swann (1952). Evidence of a different kind in favor of the release of chemical agents


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PLATE11. Fertilization and cleavage in the egg of the sea urchin. Figs. 1-1 ?dia?%. Figs. 5-8 PU?-aCf?ntrOtUslividus. 14. Photographs from a time-lapse film, taken by ordinary dark-ground illumination. 300 X . (1) unfertilized egg; (2) sperm has just entered at about 8 o'clock; (3) a few seconds after 2, the brightening of the cortex has spread about halfway round the egg; ( 4 ) 20 seconds after the entry of the sperm, the brightening has spread all round the egg. 5-8. Photographs of an egg in cleavage, taken by vertical illumination. 300 X. The effect of four quadrants is the result of using a polarizing system to cut out light reflected from the coverslip; the whole egg surface does in fact scatter light. (5) 60 minutes after fertilization, egg in telophase; (6) 61.5 minutes; (7) 62.5 minutes ; (8) 64.5 minutes. PSOJJlJJlCChinUS

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comes from a study of the fertilization reaction in the sea urchin egg, using ordinary dark-ground illumination. From time to time, various authors have noticed that a wave spreads round the surface of the egg at fertilization, without, however, describing it in detail. Recently Rothschild and Swann (1949) have taken time-lapse films of the phenomenon. They find that the surface of the unfertilized egg scatters very little light but that shortly after the attachment of the spermatozoon, a bright patch appears round the point of entry. This brightening gradually spreads round the egg, taking about 20 seconds in all (Plate 11, 1-4). They believe the change to be associated with the block to polyspermy (Rothschild and Swann, 1950, 1951a, b). They also plotted the time course of the wave moving round the egg. Initially it spreads at a more or less constant rate ; it then slows down slightly at the equator, and finally speeds up considerably. For a number of reasons they conclude that the change is not likely to be due to a self-propagating mechanism of an action potential type, and they suggest that the time relations are of the right order for diffusion. Rothschild (1949) has calculated the type of curve to be expected, assuming the change to be due to the release by the sperm head, of an active substance which then diffuses through the body of the egg. The theoretical and observed curves bear a fairly close resemblance to each other. The particular shape of the observed curve suggests a molecular weight for the diffusing substance of some tens of thousands, but this conclusion must be treated with considerable reserve. Corresponding changes in the cell surface at fertilization can be observed by vertical illumination. Here the position is the reverse of that under ordinary dark ground ; the unfertilized egg shows a strong scatter which disappears on fertilization (Mitchison and Swann, 1952). The cortex of the unfertilized egg, like the cortex before cleavage, shows a slight birefringence, which also disappears at fertilization ( Monroy, 1935). Both vertical illumination and polarized light appear to show the same spread of a change round the surface of the egg as does ordinary dark ground, but for practical reasons the time course is more difficult to follow. Not only do the changes at fertilization suggest that a chemical agent is released from the sperm head, but it will be noticed that the changes visible by vertical illumination and polarized light are almost identical with those to be seen at cleavage. Since the sperm head consists largely of nuclear material, it would not be surprising if it were to release the same chemical agent as the chromosomes. There is yet another interesting parallel between events at fertilization and at anaphase. Immediately after fertilization, the sperm aster starts


207

to form round the sperm head. The cytoplasm of the unfertilized egg has a low viscosity, and appears to be structureless in ordinary and polarized light. But unlike the spindle and asters of prophase and metaphase, the sperm aster built up from this structureless cytoplasm, though strongly fibrillar, is only weakly birefringent. In fact, it closely resembles the asters of late anaphase, which, it will be remembered, are built up in their outer regions from the structureless cytoplasm of metaphase. It is interesting too, that the sperm aster grows in radius at about the same rate as the asters in anaphase, namely about 2 to 3 microns per minute. It was suggested earlier that the growth of the asters in anaphase may be the result of the release of a second chemical agent. I t seems possible at least, that this same agent is also released by the sperm head and causes the growth of the sperm aster. Beyond these observations on the sea urchin egg, there is little evidence for the release of chemical agents from the chromosomes or the sperm head, though there are a number of points in the literature which may be relevant to the idea. One of the most interesting suggestions is that of Metz (1933) on the curious type of anaphase found in the first spermatocyte division of Sciara. H e argues that the movement of the chromosomes must be due to some activity of their own, and that “this activity operates by bringing about a progressive alteration in the physical state of the protoplasm adjacent to the chromosome.” Somewhat similar observations were made by Chalkley (1935) on mitosis in the ameba. He claimed that protoplasmic viscosity was lowered in the neighborhood of the telophase nucleus and put forward a theory of cell division based on the idea that the presence of daughter nuclei at either side of the cell caused ameboid movement in opposite directions, with the result that the cell pulled itself in two. Similarly, Chambers (1938) noticed that the well-known “bubbling” of tissue culture cells began near the daughter nuclei at either pole of the dividing cell, and only later reached the equator. The changes involved in the maturation of the sea urchin egg are another interesting problem. Whereas the cytoplasm of the mature egg is capable of forming asters, that of the immature egg is not. Fry (1925) reviewing the evidence, attributes this to the release of substances from the germinal vesicle when it breaks down, There seems to be little direct evidence on whether nuclear substances can affect protoplasmic structure, though the existence of proteolytic enzymes in spermatozoa has been reported by a number of workers (e.g., Lundblad, 1949).

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IV. THE NATUREOF

THE

CHEMICAL AGENTSI N MITOSIS

The evidence for the existence of chemical agents which are released from the chromosomes o r the sperm head is only indirect. The isolation of nuclear substances with an effect on protoplasmic configurations in the living cell is clearly necessary to establish the hypothesis, and it is as well to make it clear that, as yet, there is no direct evidence of this sort whatsoever. There is, however, a certain amount of more indirect evidence of some interest. The ratio of total chromosome volume to cell volume in the sea urchin egg is only about 1:104. The ratio of the volume of the sperm head to the whole cell is even less than this. It follows that any chemical agents in the chromosomes or the sperm head can only be present in very small quantities, and must be catalytic in nature. The non-committal term “structural agent” would perhaps be suitable for them. Cytochemical tests on sea urchin eggs (Swann, unpublished) have not shown that any of the recognizable substances are released from the chromosomes into the cytoplasm during anaphase. The only evidence on the nature of the structural agents comes from the work described earlier on the change in light scattering at fertilization. Diffusion considerations suggest a molecular weight of some tens of thousands, though this figure may well be seriously in error. But whereas the cortical changes at fertilization take only about 20 seconds, the changes in the mitotic figure at anaphase take several minutes. The rate of movement of the decrease of birefringence in the spindle is so slow in fact, that it would seem to imply a molecule of at least virus-like dimensions. This seems unlikely, though it is not, presumably, out of the question. There are two other possible explanations. In the case of the unfertilized egg, the structural agents released from the sperm head diffuse through a structureless medium of low viscosity, whereas in mitosis, they have to diffuse through the oriented structure of the mitotic figure. This might possibly account for the slowing down, since it is known that when the size of a molecule approaches the pore size of the gel in which it is moving, the rate of diffusion is greatly reduced. An alternative explanation is that the structural agents do not move freely, but are subject to trapped diffusion; that is to say, they are bound by the protoplasmic structure, so that the diffusion front does not move on until all the traps in its way are filled. Under these circumstances the apparent rate of diffusion will be governed in reality by the rate of release of the substance in question. If this is the case, it becomes necessary to suppose that at fertilization the structural agents are released very rapidly, whereas at


209

anaphase one of them is only released slowly. It will be remembered that the asters start growing at the very beginning of anaphase, so that the second structural agent, associated with this growth, must either be released rapidly, or must diffuse in the normal way. The release of substances from the chromosomes during anaphase has a certain parallel in the apparent disappearance of nucleic acids from the chromosomes in the later stages of mitosis. It has been supposed by Brachet (1947) that nucleic acids are released directly into the cytoplasni at the end of mitosis, after conversion to the ribose form, and reabsorbed again on to the chromosomes in prophase. Such a scheme would explain why the cytoplasm of many cells is basophilic in interphase, but loses its staining capacity at metaphase, when the nucleic acid charge on the chromosomes is at a maximum. But it is now thought unlikely that DNA and R N A are directly interconvertible (Villee et d.,1949). The extensive studies of the Caspersson school, however, seem to support the view that some nucleic acid, at any rate, is lost from the nucleus in the later stages of mitosis (Caspersson, 1950). Ris (1947) finds that the DNA content of grasshopper chromosomes decreases in anaphase and telophase, while Jacobson and Webb (1950) claim that nucleic acids are discharged onto the spindle at anaphase in chick tissue culture cells. On the other hand, Lison and Pasteels (1951) find that the DNA content of sea urchin nuclei is somewhat lower in metaphase than interphase. Although there is a possible parallel between the behavior of nucleic acids and the postulated behavior of the structural agents, there is no direct evidence to connect the two. The idea is nevertheless an interesting one that is not altogether out of keeping with what is known of nucleic acids. It is well established that they are connected in some way with protein synthesis, both in the nucleus and the cytoplasm, and it is known that nucleotides are constituents of certain coenzymes, while one particular nucleotide, adenosine triphosphate has an effect on the structure of muscle proteins.

V. CONCLUSION The idea that chemical agents are responsible for the structural changes of the cell, has a number of points to commend i t : it fits in with what is known of biological controlling mechanisms in general ; it links together mitosis and cleavage and relates them to fertilization; and it allows of the possibility that individual chromosomes may behave to some extent independently. It is easy to see also, how it might give rise to a cyclical controlling mechanism, for if chemical agents are released from the chro-

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mosomes at one stage, they must presumably recondense there at some other stage, or else be broken down in the cytoplasm, so that a freshly synthesized batch may be released. But there may be other explanations for the observed patterns of structural change during mitosis, a i d the idea of structural agents must stand or fall in the end on whether it is possible to identify or isolate nuclear substances with a catalytic effect on the structure of living protoplasm. This is not likely to be an easy problem. VI. REFFXENCES Brachet, J. (1947) Embryologie Chimique. Masson, Paris. Carlson, J. G. (1946) Biol. Bull., 90, 109. Caspersson, T. (1950) Cell Growth and Cell Function. New York. Chalkley, H. W . (1935) Protoplasma, 24, 607. Chambers, R. (1925) Chapter in Cowdry’s General Cytology. Chicago. Dan, K., Yanagita, T., and Sugiyama, M. (1937) Protoplasma, !28, 66. Frey-Wyssling, A. (1948) Submicrascopic Morphology of Protoplasm and Its Derivatives. Elsevier, New York. Fry, H. J. (1925) J. exp. Zool., 43, 49. Heilbrunn, L. V. (1943) Outline of General Physiology. Philadelphia. Herlant, M. (1920) Arch. Biol., Paris, So, 517. Hughes, A. F., and Swann, M. M. (1948) I . exp. Riol., 26, 45. Jacobson, W., and Webb, M. (1950) J . Physiol., ll2, 2 P. Lison, L., and Pasteels, J. (1951) Arch. Uiol., Paris, 6!2, 1. Lundblad, G. (1949) Nature, Lord., 169, 643. Metz, C. W. (1933) Biol. Bull., 64, 333. Mitchison, J. M. (1952) Symp. SOC.ex+. Biol., 6. ( I n press.) Mitchison, J. M., and Swann, M. M. (1952) ( I n preparation). Monroy, A. (1945) Experientia, 1, 335. Raven, Chr. P. (1948) Biol. Rev., aS, 333. Ris, H . (1947) Cold. Spr. Harb. Symp. q w n t . Biol., 12, 158. Rothschild, Lord (1949) J. exp. Biol., 26, 177. Rothschild, Lord, and Swann, M. M. (1949) J . exp. Biol., 26, 164. Rothschild, Lord, and Swann, M. M. (1950) J . exp. Biol., !27, 40. Rothschild, Lord, and Swann, M. M. (1951a) Exp. Cell Rcs., 2, 137. Rothschild, Lord, and Swann, M. M. (1951b) J. exp. Biol., 28, 403. Schmidt, W. J. (1937) Die Doppelbrechung von Karyoplasma, Zytoplasnia und Metaplasma. Berlin. Schmidt, W. J. (1939) Chronzosoma, 1, 253. Schrader, F. (1944) Mitosis. New York. Swann, M. M. (1951a) J. exp. Biol., 28, 417. Swann, M. M. (1951b) J . exp. Biol., 28, 434. Swann, M. M. (19.51~) Chapter in The Mitotic Cycle, by A. F. Hughes. London. Swann, M. M. (1952) Symp. SOC.exp. Biol., 6. (In press.) Swann, M. M., and Mitchison, J. M. (1950) J . ex). Biol., 27, 226. Villee, C. A., Lowens, M., Gordon, M., Leonard, E., and Rich, A. (1949) J. cell. comb. Physiol., 83, 93. VKs, F. (1921) C. R. SOC.Biol., 86, 494.

Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes* MARCUS SINGER? Department of Anatomy, Harvard Mcdical School, Bostotc, Massachusetts CONTENTS I. Introduction ....................................................... 11. The Influence of pH of the Staining Solution on the Interaction of Dye and Protein ................................................. 111. The Nature of the Influence of pH on Staining ....................... IV. The Site of Dye Binding and the Nature of the Bond between Dye and Protein ................................. V. The Relation between the Isoelectric Point and Staining . . . . VI. The Ionic Strength of the Dye Solution ............................. VII. The Influence of Dye Concentration ................ VIII. The Afinity of Dyes ............................................. IX. The Influence of Fixation and Other Modifications of Tissues on Subsequent Staining ............................................ X. The Influence of Temperature of the Staining Solution . . . . . . . . . . . . . . . XI. Some Observations on the Kinetics of Staining ....................... XII. The Reversibility of Staining Reactions ; Equilibrium of Staining and Other Factors which Influence Staining .................. ... .............................. ... XIII. References .............

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242 245 246

248 250

I. INTRODUCTION Acid and basic dyes are employed in histology mainly to reveal the morphology of cells and tissues. Yet, these stains may also be used in the histochemical study of cellular and tissue proteins, an application which was appreciated in a number of early works on the staining reaction (for example, Miescher, 1874 ; Ehrlich, 1879a, b ; Lilienfeld, 1893 ; Michaelis, 1900, 1901, 1911 ; Pappenheim, 1901, 1917; Heidenhain, 1902, 1903; Mann, 1902; Magnus, 1903; Bethe, 1905). Mann (1902) in his remarkable book on histology noted: “It is not enough to regard dyes as simply acids or bases, as oxidizers or reducers, but we must aim at microchemical methods, and endeavour to know the composition of the dye and the tissue, to apply tests in a purposive way. Not till then will progress be made in the most difficult of all branches of Physiology, namely inicrochemistry.’’ But relative to the total literature on staining, interest in the

* This work was supported in part by funds received from the Eugene Higgins Trust. t Present address: Cornell University, Ithaca, N. Y . 21 1

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use of acid and basic dyes as histochemical tools has always been a limited one. A major reason for this has been the purely morphological demands which such growing fields as genetics, embryology, experimental inorphology, and endocrinology have hitherto made on the use of dyes and staining techniques. Another and perhaps more important reason for the limited histochemical application of staining procedures has been the absence of adequate information on the nature of the staining reaction. A prerequisite of a histochemical analysis of tissue sections with dye is, of course, a working knowledge of the mechanism of the staining reaction. The present review hopes to define the more important factors which govern interactions of acid and basic dyes with proteins and is preliminary to a second article, now in preparation, which applies this information to histochemical study of proteins of cells and tissues. Many facts of interest have appeared in the literature on staining of proteins, and it is one of the purposes of the present work to review the most salient of these and, whenever possible and necessary, to reinterpret them in the light of modern concepts. The review of literature is not confined to histological sources but also considers pertinent works in the field of protein, dye and textile chemistry. Much work has been done in the latter field on the protein-dye interaction, but these works have been largely ignored by histologists except for the detailed references and the comparison of textile and tissue staining of Pappenheim (1901; see also Baker, 1945) and for casual notations by other workers. The textile literature is particularly instructive since, as in the case of tissues, the substrate is also in a solid, insoluble state. Indeed, even studies of dyeing of cellulose derivatives and polyamide fibers are instructive for tissue staining. In contrast to that of textile materials, however, the stainable substance of cells and tissues is much more complex and heterogeneous and shows considerable variation from one region to the next in the kind, the interrelations, and the arrangement of substances. Much of the early literature on staining was devoted to the controversy, then extant, on whether the staining process is physical or chemical in nature. These speculations are adequately covered in one sense or another in many works such as those of Hofmeister (1891), Fischer (1899), Pappenheim (1901), Heidenhain (1902, 1903), Mann (1902), Dreaper (1906), Schwalbe (19O7), Zacharias ( 1908), Michaelis ( 1911), Gee and Harrison (1910), Pelet-Jolivet (1910), Harrison (1911), Bancroft (1914a, b, c, 1915 a, b), Briggs and Bull (1922), von Mollendorff and Krebs (1923), Unna (1928), Conn and Holmes (1928), Holmes

STAINING OF

TISSUE SECTIONS

213

(1929), Stearn and Stearn (1929, 1930), Zeiger (1938), Conn (1940), Baker (1945), Bourne (1951), and therefore no attempt will be made to review and evaluate these works systematically. However, a few comments taken from the literature may be pertinent in placing this controversy in proper perspective for the ensuing discussion of the forces which influence staining. In the light of modern information on intermolecular forces “the different theories are merely different ways of looking at the same phenomenon” (Vickerstaff, 1950). Mann (1902) wrote in similar vein fifty years ago when, in describing his views on the nature of staining, he noted : “Before discussing whether chemical or physical factors are at work in staining, the question must be asked, Is there any difference between chemistry and physics? There is not. Frankland used to teach that chemistry is but a branch of physics, and the whole recent development of chemistry points to the importance of studying chemical interaction apart from those conditions where substances join in definite molecular proportions. Even adsorption, which is perhaps the best example of physical action, has certain resemblances to chemical action. This matter has been gone into to make the reader realize that hard and fast lines cannot be drawn between chemical and physical action.” These sentiments have since been reemphasized in views of others. For example, the working assumption of Rawlins and Schmidt (1930) in analyzing the mode of combination between certain dyes and gelatin granules (see also Goldstein, 1949) was the fundamental statement of Langmuir ( 1916, 1917) that “there is no present justification for dividing interatomic (or intermolecular) forces into physical and chemical forces. It is much more profitable to consider all such forces as strictly chemical in nature. Evaporation, condensation, solution, crystallization, adsorption, surface tension, etc., should all be regarded as typical chemical phenomena.” Michaelis (1920) in a theoretical work on staining pointed out that practically all phenomena of staining with the exception, perhaps, of dye uptake of charcoal, could be described in chemical terms. He later noted ( 1926) that processes of adsorption, generally considered by physical theorists to be the mechanism of interaction between dye and substrate, “do not represent a fundamental contrast to chemical union.” This view was stressed by Stearn and Stearn (1930). Similar thoughts and sentiments concerning the significance of this controversy have been expressed by Dubos (1945), Baker (1945), Dempsey and Wislocki (1946), Rideal (1950), Vickerstaff (1950), Bourne (1951), and others. Studies of the interaction of dyes and proteins, although originally directed toward elucidating the physical or chemical nature of staining, un-

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covered many facts which are of greater interest here than the controversy which originally stimulated them. These facts will be focused upon in some detail. The experiments were varied and considered one or another aspect of the staining process. Some were concerned especially with the structure of dyes ; others with the nature of the dye reaction ; and still others dealt primarily with the physical-chemical nature of the protein interactant. In analyzing the staining of protein, many substances were employed as “models,” and along with tissue sections, subjected to a variety of dyes and conditions of staining. The model substances included, among others, gelatin, glue, wool, charcoal, celloidin, fibrin film, filter paper, and soluble proteins (e.g., casein, albumin). From these many and varied investigations the obvious fact emerges that there are numerous and diverse factors which profoundly influence the dye uptake of proteins of cells and tissues. The more salient of these factors are considered in detail in this review since they throw light upon the nature of the staining mechanism. The variable which affects dyeing most profoundly is pH, and consequently most extensive treatment is given it. Other factors of importance are ionic strength of the dye medium, concentration of dye, nature and affinity of the dye, fixation and other chemical modification of the protein, temperature, diffusion, rate of staining, and the chemical and physical characteristics of the protein. With few exceptions, the discussion is confined to aqueous solutions of molecularly dispersed acid and basic dyes. Staining under conditions other than these is frequently done in the histological laboratory, and it is appreciated here that the forces which operate under such conditions cannot be equated with those described here and are worthy of separate consideration at another time. Finally, as for the chemistry and constitution of acid and basic dyes, no attempt will be made here to discuss this subject in detail. Instead, the reader is referred to appropriate works. A brief summary of features of these dyes important for the following discussion is, however, pertinent here. Works which treat this subject in proper detail are the concise and illuminating descriptions by Conn ( 1940), Baker ( 1945), and Bourne (1951), and the more detailed ones by Rowe (Colour Index, 1924), Mayer ( 1934), Pratt ( 1947), Fierz-David and Blangey ( 1949). Acid (anionic) and basic (cationic) dyes are aromatic compounds of various complexities containing a water-solubilizing group, which in the case of acid dyes is frequently a sulfonic radical (--SOsH) but which also may be a carboxyl or hydroxyl group. In the latter event a nitroso, nitro, or another hydroxyl group is present. The solubilizing group of the basic

215

STAINING O F TISSUE SECTIONS

dye is an amino group or one of its derivatives. These acid and basic groups are also in large part the groups responsible for the reaction of the dye with the protein, for which activity they have been termed auxochronzes. The acid dyes are generally used as the sodium salt of the dye acid ; basic dyes are prepared as chlorides, sometimes as acetates, sulfates, or as still other salts. There may be both acid and basic groups in a dye molecule, in which instance the overall acid or basic tendency is determined by the relative strength of these groups. Certain unsaturated groups of the dyes are chiefly responsible for their color, although the auxochrome may influence the quality and intensity of the color. Groups which impart the color are called chronzophorcs and include among others the carboxyl group >C = 0 ; the azo group -N = N- ; the nitroso group -N = 0 ; the nitro group -N Go ; the quinoid group 0 = 0 = = N ; and the ethylene group -CH = CH -. 1 0

=

OF p H OF THE STAINING SOLUTION ON 11. THE INFLUENCE INTERACTION OF DYE A N D PROTEIN

0 or

THE

Control of pH, although reached on a highly empirical basis, is implicit in many staining techniques where acid or alkali is used to enhance or depress staining. Instances of such control are abundant among the methods listed in books of histological technique (e.g., see Baker, 1945, Bourne, 1951). For example, among the various methylene blue solutions recommended by Mallory (1944) for staining of basophilic substances are Loeffler’s solution which includes sodium hydroxide, Kiinne’s with phenol, Gabbett’s with sulfuric acid, Unna’s with potassium carbonate, Goodpasture’s with potassium carbonate and acetic acid, and Sahli’s with borax. Examples of substances which are used to control staining with acid dye may be found in the work of Maneval (1941) on bacteria. References in early histological literature to acidic or basic substances which enhance dye uptake (“accentuators”) or which otherwise control the intensity of staining were frequent. The mechanism of action of these substances was not agreed upon, but it was evident to many (see Mann, 1902) that alkaline substances accentuate basic dye uptake and acid substances favor the acid one. Examples of basic accentuators listed by Mann (1902) are bicarbonate, soap, pyridine, sodium borate, aniline, and potassium or sodium hydroxide. Some acid ones are phenol and sulfuric, acetic, and oxalic acids. There are many other examples in the literattire where the effective mechanism of enhancing or otherwise controlling staining is the use of acid or alkali. The influence of acid

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and base on staining has also been appreciated by wool and silk dyers who have employed various acids as well as salts to enhance and control dyeing of textiles (see e.g., Brown, 1901b), an effect which today is still the subject of considerable interest and research. A number of early studies were devoted to the experimental analysis of the effect of acid and base on staining. Mathews (1898) observed that proteins could be precipitated from solution as the salt of acid dyes such as acid fuchsin, erythrosin, orange red, and methyl blue if a few drops of dilute acetic acid were added to the dye-albumin mixture. Basic stains did not give this reaction under such conditions, but when brought into protein solutions made alkaline with sodium carbonate a precipitate was formed of the protein in combination with the dye. H e stated, in summary, “The acid stains will combine with albumoses only in acid solutions and the basic stains will combine with the albumoses only in alkaline solution, when they form insoluble colored compounds.” That these reactions held true for solid, coagulated protein as well as protein in solution was also demonstrated by Mathews. “If two pieces of coagulated egg albumin be brought the one into slightly acid and the other into alkaline solutions of thionin, the stain poured off after a few seconds, and the albumin washed in water, the piece that has been in the alkaline solution will be an intense purple, the other barely tinged with color.” H e observed similar results when he extended his observations to tissue sections of liver, kidney, and muscle of the frog. “I find that sections of the above-mentioned tissue, if immersed for an instant in one-tenth per cent sodium carbonate solution before staining or if stained in solutions of the basic stains made slightly alkaline with sodium carhomte show the cytoplasm deeply stained, as well as the chromatin. These reactions, which are identical with those of the albumoses, show that in alkaline solution many of the basic dyes will combine with the albumin molecule whether in cytoplasm or nucleus.” This work of Mathews which was written at a time when little was known about the dissociation and reaction of proteins was one of the earliest important contributions to our understanding of acid-base relations in staining ; yet, except for the detailed treatment which Mann (1902) gave it, little reference has been made to it. Mann (1902) was also impressed by the effect of acidity or alkalinity of the staining solution on the degree of dye-binding. H e believed that in cases where staining was favored the acid or alkali prepared the protein to receive the dye radical. At that time proteins were described as pseudoacids and pseudobases whose acidic or basic nature could be brought out by various substances or conditions. An alternative explanation ad-

STAINING OF TISSUE SECTIONS

217

vanced by some textile chemists at this time was that the accentuators acted upon the dye ; thus, in the case of the accentuating action of acid in dyeing of wool with acid dyes, the acid accentuator caused the liberation of the dye acid which then reacted with the basic groups of the protein to form a stable salt. Bethe (1905) observed that the degree of staining of tissue sections varied with the acidity of the staining solution. He stained sections of spinal cord, sublingual gland, mammary gland, and kidney in toluidine blue solutions of constant dye concentration but containing different amounts of acid or alkali. Alkali favored and acid inhibited the uptake of the basic dye. This was true for all structures of cells and tissues, but the degree of staining at any given level of acid or base varied from one structure to another. A e described the staining of given structures by curves which depicted the intensity of staining as a function of the amount of acid or alkali. The curves showed that in highly alkaline solution all structures stained with the basic dye ; with decreasing alkalinity the tissue substances gradually lost their affinity for toluidine blue. The order of loss varied, however, with the tissue structure. For example, in weakly alkaline solution tracts and glia of the central nervous system no longer stained, whereas solutions of approximate neutrality were required before fibrous connective tissue and colostrum of mammary gland failed to stain. Cartilage and mucus continued to stain even in very acid solutions. Other early workers who stressed the importance of acid and alkali in dyeing were Spiro (1897), Brown (1901a), Heidenhain (1902, 1903), Halphen and Riche ( 1904, 1905), Pelet- Jolivet ( 1910), and Harrison (1911). As information was accumulated on the dissociation of protein in aqueous solution interest in the effect of acid and alkali increased greatly. Michaelis (1920) in a theoretical work on the nature of the staining reaction was among the first to emphasize the relation between the binding of acid and basic dye at different concentrations of hydrogen ion and the amphoteric nature of the protein. Loeb (1922, 1924) showed that the binding of acid or basic dye by gelatin followed the pH of the dye bath, high p H favored basic dye uptake, and low p H enhanced staining with acid dye. Staining with either dye appeared to be minimal at the isoelectric point, but some interaction with acid dye occurred above and with basic dye below the isoelectric point. Staining of collagen powder also varied with p H (Thomas and Kelly, 1922). Briggs and Bull (1922) concluded from their studies of dyeing of wool that the “hydrogen ion concentration of the dye-bath is the most important single factor affecting the process of dyeing” and that “many of the assistants or re-

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strainers, used in dyeing, produce an appreciable and often a great change in the hydrogen ion concentration of the dye-bath and their action in many cases is due more to this change than to any other specific action.” They observed that high pH favored binding of the basic dye and low pH that of the acid one. Hobbins (1923, 1924, 1926) studied the influence of acidity on the staining of plant tissues and reported that potato tuber tissue, Elodea leaves, and the mycelium of fungi responded as amphoteric colloids being stained with either acid or basic dye according to the p H of the solution. A number of important works bearing upon the influence of p H in staining bacteria were published by Stearn and Stearn (1924, 1925, 1928a, b). In solutions of varying p H the bacteria stained in a fashion similar to the protein structures discussed above; at low pH, acid fuchsin (acid dye) was taken up, but at higher ,pH, gentian violet (basic dye) was bound. When the intensity of staining was plotted as a function of pH, curves decreased with increasing p H for the acid dye, but were the reverse for basic dyes. Furthermore, such staining curves were displaced along the pH axis for different bacterial species (see also McCalla and Clark, 1941). Stearn and Stearn (1928a, b, c) also investigated the action of various chemical decolorizers on the dye fastness of stained bacteria and observed that decolorizers which were especially effective were acidic and basic ones, The acidic ones, for example phenol aldehyde, selectively removed basic dye, and basic decolorizers removed acid dyes. Pischinger (1926) , a student of Bethe, reexamined the influence of pH on staining and applied the method of p H control for the histochemical analysis of tissue structure. He studied quantitatively the binding of acid and basic dye by gelatin, egg white, thymus, and cartilage over a range of pH. The amount of bound dye was determined colorimetrically after extraction from the stained product. In each instance dye uptake followed the p H relations described above, namely that basic dye binding was inhibited as the pH of the staining solution was lowered until finally, at a given pH and below, no dye was bound; and staining with acid dye occurred most readily at low p H but dropped progressively and eventually ceased with gradual elevation of pH. Differences were observed among these protein substances in the degree of staining at any given p H or in the critical pH which marked the boundary between loss of staining capacity and increasing dye uptake. The proteins of tissue sections when subjected to p H variations responded in a manner similar to that of the model substances. As in Bethe’s earlier work, Pischinger plotted somewhat quantitatively the intensity of staining of given tissue structures


219

against pH. The curves obtained thereby were different for each tissue structure, a fact which led Pischinger and others to introduce pH staining control as a method of histochemical characterization and study of tissue proteins. The importance of p H in staining of tissue sections and in histochemical study of tissue proteins was independently recognized and reported by other workers at about the time of Pischinger’s publications. A series of publications, in which dyes were used in the study of tissue proteins emanated from the laboratories of the University of Missouri (Robbins, 1923, 1924, 1926; Naylor, 1926; Stearn, 1931, 1933; Stearn and Stearn, 1924, 1925, 1928a, b, c, 1%9, 1930; and Levine, 1940). These studies sought to extend to the proteins of cells and tissues the information on protein reactions published by Loeb (1922). Robbins (1923, 1924) applied the staining reaction at different p H to study of the isoelectric point of the proteins of fungi. Naylor (1926) showed that the staining of plant tissues followed the p H relations reported by Loeb for gelatin staining. In addition to studying dye binding from solutions of an individual dye, he analyzed staining from solutions containing both an acid (eosin) and basic (methylene blue) dye. At high p H only methylene blue was bound, but as the acidity of the staining solution was increased, the sections at first became purplish because of the partial binding of eosin as well and, then, at a low p H the sections were stained exclusively with eosin. The use of an eosin-methylene blue combination over a p H range was also applied with similar results to staining of tissue, particularly blood cells, and of bacteria by Tolstoouhov (1927, 1928, 1929), who also evaluated independently the use of dyes in the study of proteins of cells and tissues. Other contributions at this time to the study of the relations between p H and dye uptake of tissue and cellular proteins were made by Pulcher (1927) and Haynes (1928). The control of acidity has a longer history in textile dyeing, and it is evident in reviewing the literature that some of the experiences of textile colorists have been drawn upon by histologists. The history of pH control in dyeing of textile fibers has been summarized recently by Seymour, Agnew, Crumley, and Kelly (1948). The influence of pH and other factors on the dyeing of wool and various textile fibers is reviewed in a number of articles, important among which are works by Rose (1942), Abbot, Crook, and Townend (1947), and a journal-sponsored review article in the American Dyestuff Reporter (1948). Wool is dyed with acid dyes which are generally applied in acid solution between p H 2 and 5,

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depending on the type of dye employed. Sulfuric acid is used to establish the lower p H and acetic acid the higher. Finally, studies have been reported recently on the influence of pH and other factors on the binding of dye in a relatively pure protein system (Singer and Morrison, 1948). Films of fibrin of known protein content, purity, and thickness prepared from products of fractionation of human blood (Cohn, 1945; Ferry and Morrison, 1946, 1947) were used as a model substance. This model protein was of particular value because as initially prepared it was native and undenatured. Moreover, it was in a solid and insoluble form and thus was similar in condition to cellular and tissue proteins of histological sections. Some films were modified by chemical or physical denaturants such as are used in fixing and then subjected to progressive staining under a variety of conditions until equilibrium was reached. The dye uptake was measured quantitatively by photometric means and expressed in terms of the amount of fibrin. Many factors influenced staining of fibrin, but primary among these was pH. The relations observed with pH variation are exemplified in the curves of Fig. 1. The curves resemble those for the titration of protein with CAST GREEN

MmFNNE U E

vn

FIG.1. The influence of pH on the staining of fibrin film with acid (fast green) and basic dye (methylene blue). Film fixed in formaldehyde (10 per cent Tor 10 hours) dye concentration, 5 X 10-6 M.;ionic strength, 0.02. (Singer and Weiss, unpublished.)

acid and base. The importance of pH for staining is emphasized particularly by the fact that there are p H levels where the protein shows no affinity for a dye even though immersed for very long periods in a solution of high concentration. The results of these studies were applied to the histochemical characterization and identification of proteins of cells and tissues (Dempsey and Singer, 1946; Dempsey, Wislocki, aiid Singer, 1946; Dempsey, Bunting, Singer, and Wislocki, 1947 ; Wislocki, Weather-

22 1


ford, and Singer, 1947; Singer and Wislocki, 1948; Wislocki, Singer, and Waldo, 1948 ; Singer, 1949 ; Dempsey, Singer, and Wislocki, 1950).

111. THENATUREOF

THE

INFLUENCE OF pH

ON

STAINING

The profound influence which p H exerts on staining reflects in large part the sensitivity of the dissociation characteristics of proteins to alterations in the solution environment. Before proceeding to a description of the dissociation of proteins under various conditions of p H it is well to summarize at first some chemical information about proteins pertinent to the discussion. A characteristic feature of proteins in solution is that they are amphoteric, that is, they contain at the same time both basic and acidic groups which by their dissociation give rise respectively to positive and negative charges on the protein molecule. These acidic and basic groups comprise the free side groups of certain amino acids, the terminal amino and carboxyl groups of the protein molecule, and, finally, the charged substances which niay be conjugated to the protein. Free basic side groups (substituted ammonium : NH3+) are found in the amino acids-lysine, histidine, and arginine ; and acidic ones (carboxyl : COO-) in glutamic, hydroxyglutamic, and aspartic amino acids. Another acid group in addition to free carboxylic acid is the hydroxyl one of certain amino acids (e.g., of tyrosine, serine) . Many amino acids have no dissociating group other than their single amino and carboxyl ones which are used in the peptide linkage and, therefore, do not impart a charge to the protein unless they are located terminally in the polypeptide chain. Of special interest among the conjugated proteins are those which contain acid groups such as nucleoprotein (phosphoric acid) and mucoprotein (uronic and sulfuric acids). Proteins differ according to the nature and number of their constituent amino acids and their conjugated substances. For example, free basic groups may be relatively more abundant in one protein and acidic ones in another. -kcording to the relative number of acidic and basic groups and their degree of dissociation, the net or overall charge on the protein mole-’ cule at a particular time will be positive (excess basic dissociation), negative (excess acidic dissociation), or zero (isoelectric point). Positive and negative charges exist in the protein molecule even at the isoelectric point (defined as the p H of a solution in which the protein does not migrate in an electrical field and therefore in which it is electrically neutral), although the net charge is zero. Thus, the electrically neutral form of the protein molecule is a dipolar ion (Zwittem’on or amphion) and its where R structure may be represented in general as H,+N-R-COO-,

z 2

MARCUS SINGER

represents the polypeptide chain and NHs', COO- the ionized basic and acidic side and free terminal groups of the constituent amino acids. The degree of ionization of the free acidic or basic groups of proteins depends on the pH of the solution. When acid is added to the solution, the dissociation of the free carboxylic acid group (as well as any conjugated acid group, e.g., phosphoric acid) is decreased and that of the free amino is increased, and the protein becomes less negatively charged. The reverse tendency in ionization and charge follows upon addition of alkali to the solution. The responses of protein to changes in p H may be indicated according to the following formulations : Isoelectric condition

A

B

NHs+-R-COONH3+-R-COO-

acid alkali

NHs+-R-COOH NHs -R-COO-

If the pH of the solution is below the isoelectric point the protein tends to respond increasingly in the direction described by formula A and above the isoelectric point according to B. It is presumed that at extremes of p H (approximately 2 and 11) complete ionization of the substituted ammonium or carboxylic groups is attained, and in these regions the respective net positive or net negative charge on the protein is maximal. At p H levels intermediate between these extremes and the isoelectric point, the net charge on the protein falls somewhere between the zero of the isoelectric point and the maximum, being positive below the isoelectric point and negative above. The ability of proteins to take up acid or basic dye according to the acidity of the environment is an expression of these amphoteric properties and of the charge on the dye ion. Basic dyes are generally chloride salts of a dye base, whereas acid dyes are in general the sodium salts of a dye acid. In the following discussion these salts are assumed to be completely ionized. Actually a small amount of the dye exists in solutions of ordinary pH as the undissociated dye acid or base whose concentration is probably influenced by alterations in p H of the solution. In those cases where dye ion association is prevalent at the p H of staining it is important to stress that the degree of dissociation of the dye would play an important part in the extent of staining. But, with few exceptions, the dissociation constants of these dye acids or bases fall in the extremes of p H and consequently ordinary levels of pH, such as those considered here, will hardly influence the degree of dye dissociation. Indeed, the variations in dissociation of the dye with alteration of p H are evidently insignificant when compared with that of the protein. Consequently, variations in ioniza-


223

tion of the dye may be ignored in most cases in the influence of p H on staining. The uptake of dye over a range of p H (Fig. 1) follows fairly closely the alterations in electrostatic charge on the protein. Basic dye, being cationic, may be expected to be attracted to protein with an excess negative charge. And, the staining conditions which favor increase in the net negative charge, such as low acidity (high p H ) of the staining solution, should also favor basic dye uptake-a relation borne out by previously described studies of staining as a function of pH. On the other hand, increase in the net positive charge, as the p H is lowered, favors the increased attraction of acid (anionic) dye. As dissociation proceeds and the groups are uncovered, the amount of dye bound increases until at the extremes of p H complete dissociation occurs and the maximum dye binding is attained. The above description of the effect of electrostatic alterations of the protein molecule on staining is based on the experiences and ideas of many workers. The early works of Brown (1901a), Mann (1902), Gee and Harrison (1910), and Harrison ( 1911) explained staining as an electrostatic attraction between dye and protein. These early interpretations were further elaborated as more detailed information on the nature of the bond between organic substances was obtained (Michaelis, 1920 ; Loeb, 1922; Stearn and Stearn, 1928a; Tolstoouhov, 1928; Craig and Wilson, 1937; Kelley, 1939b; Zeiger, 1938; Conn, 1940; Levine, 1940; McCalla, 1941 ; Rose, 1942 ; Neale, 1947 ; Gerstner, 1949 ; Vickerstaff, 1950). It will be shown later that there are other forces besides electrostatic ones which operate in the interaction of dye and protein. Yet, the electrostatic ones explain most effectively the observed variation of staining with pH and cannot be ignored even if it is finally shown that other forces provide the bond which combines the dye and protein. In the latter event, the electrostatic forces would play the important role of attracting the dye and protein within the range of the binding forces (see below). Finally, it should also be noted here that alteration in p H of the dye liquor also affects staining in another, albeit less important way. Swelling of the protein framework may occur as the p H is shifted beyond the isoelectric point. The increased separation of the micellae attendant upon swelling facilitates penetration of dye ions, particularly aggregates, which otherwise might be excluded from more central binding sites (Gerstner, 1949). Further thoughts on swelling phenomena as they relate to dyeing are noted below.

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MARCUS SINGER

IV. THESITEOF DYEBINDING AND BETWEEN

THE

NATURE OF

THE

BOND

DYEAND PROTEIN

Electrostatic forces operate over relatively long distances and, consequently, are particularly effective in attracting the dye ion to the protein. The actual site on the protein to which the dye is bound and the nature of the bond have been frequently speculated upon. The most prevalent and firmly established view holds that the site of binding is the charged group of opposite sign which has attracted the dye ion and that the bond is an electrostatic one (also called salt linkage or primary zdence, coulombic, or ionic bond). Accordingly, the actual combination may be described by ordinary laws of chemical combination as a salt formation between dye and protein (Loeb, 1922). The idea that the dye-protein reaction is a salt formation was advanced by textile chemists more than a hundred years ago and is also prevalent in early histological literature (see reviews of Pappenheim, 1901; Mann, 1902 ; Heidenhain, 1902; Pelet-Jolivet, 1910). Mann ( 1902) repeatedly described staining phenomena as salt linkages ; for example, he stated that “Proteid, changed in this manner, can readily interact with the ions derived from the salt which we employ as a dye, and in consequence chemical union between the kat-ions of the tissue and the an-ions of the dye (or the an-ions of the tissue with the kations of the dye) can readily take place, provided the tissue and the dye are brought into contact with one another in a common solvent, i e . , in a fluid which allows of electrical dissociation of both the proteid and the dye.” And elsewhere he said, “The conception I have formed of precipitated proteids is that each component molecule, in addition to adhering probably only physically to its neighbour, is still endowed with a number of side chains, which are unsatisfied after the removal of the fixing reagent, and which, under suitable conditions, may attract toward themselves color radicals of the opposite electrical sign. Thus, for example, a tissue side-chain may be a sufficiently strong kat-ion to combine with the an-ion radical of-picric acid, and the unsatisfied nucleic acid radical in nucleins may withdraw the kat-ion on methylene-blue from its an-ion chlorine. Thus dyes and tissue-molecules can adhere chemically to one another by their side-chains.” Salt-like combinations between dye and protein were also emphasized by Mathews (1898), and, Nietzke ( 1901) observed in wool dyeing that “Certain facts speak for the view that the unions of dyes with fibres are nothing but salt-like unions, in which the fibre, analogously to an amido acid, plays in the one case the part of an acid, in the other case that of a base.” Weber (1894) concluded that “the amido group of the wool combines with the sulphonic group of the dye, while the


225

carboxyl group of the wool remains unaffected.” H e believed, moreover, that basic dye reacted with the carboxyl group, leaving the amino group unaffected. In the early literature on the affinity of chromatin for basic dye a number of works appeared which described the interaction between nucleic acid and basic dye as salt formation (see for example Miescher, 1874; Lilienfeld, 1893 ; Mathews, 1898). Description of dye binding as a salt linkage involving primary valence forces has been repeatedly advanced for reaction between various dyes and soluble proteins such as casein and albumin, or solid ones like gelatin and wool (some references are : Michaelis, 1920, 1947; Loeb, 1922, 1924 ; Chapman, Greenburg, and Schmidt, 1927 ; Hewitt, 1927 ; Rakusin, 1928; Rawlins and Schmidt, 1929, 1930; Stearn and Stearn, 1929, 1930; Stearn, 1931 ; Craig and Wilson, 1937; Ender and Miiller, 1937; Fraenkcl-Conrat and Cooper, 1944; Peters, 1945; Schmidt, 1945; Abbot, Crook, and Townend, 1947 ; Sokolova, 1948; Veller, 1948). The charged groups which woul_d form salts with acid dye are the free basic groups of the amino acids lysine, histidine, and arginine. Those to which basic dye would be bound are the free carboxyl groups of aspartic, glutamic, and hydroxyglumatic acids, the hydroxy groups of certain other amino acids, and the free acidic groups of phosphoproteins and mucoproteins. If a salt linkage is formed between dye and protein, one would expect that the amount of dye bound corresponds to the number of free acid or basic groups on the protein molecule. Such stoichiometric proportions between the quantity of dye and the number of binding .sites in the protein molecule have been looked for by many workers to support the view of salt linkages by primary valence bonds. The total number of acidic or basic groups available for interaction with dye may be calculated from the amino acid content of the protein or may be determined by titration of the protein with acid or alkali. In determining the number of dyeing sites for comparison with the calculated number of available sites, staining is done at extremes of pH where there is presumably maximum dissociation of acidic and basic groups of the protein and, consequently, where maximum uncovering of the binding sites occurs. The extremes of p H at which these groups are maximally dissociated are respectively 2 and 11 for the free amino and carboxyl groups. If the reaction is a stoichiometric one, there should presumably be a one-to-one combination between dissociating groups of the protein and dye ion at these extremes of pH. Such a result obtained in many of the works cited above where it was demonstrated that the amount of dye bound was equivalent to the calculated number of basic or acid groups.

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MARCUS SINGER

Other methods have been used for determining the stoichionietry of dye binding by protein. A unique one was employed by Stearn (1931) in his conductometric titrations of sodium gelatinate and sodium nucleinate with basic dye. H e observed alterations in electrical conductivity of the solution as the basic dye displaced hydrogen ion and calculated therefrom the amount of dye bound. His results supported those obtained by direct measurement of the dye taken up by protein. McCalla (1941) described the stoichiometry of the reaction of dye and protein in bacteria treated with MgS04 by observing the amount of Mg++displaced by the basic dye, methylene blue. The stoichiometry of staining was further studied in bacteria by observing the amount of hydrogen ion ( p H ) released in the course of staining with methylene blue (McCalla, 1941; McCalla and Clark, 1941). If combination of acid dye is determined by basic groups of the protein, then destruction of these groups should be followed by loss in capacity to bind acid dye. Experiments directed toward this end have been reported for wool in which the fiber was deaminated with nitrous acid (Speakman and Stott, 1934; Speakman and Elliott, 1943). But, deamination though extensive was never complete, and a small amount of acid dye was still taken up by the fiber. Further loss in acid dye binding was obtained by acetylation of the wool after deamination. Early experiments along these lines were done by Gelmo and Suida (1905), who looked for various groups in the fiber by first neutralizing them in various ways and then observing the alteration in dyeing. While a large body of information has been accumulated to support the view of dye binding in stoichiometric proportions by primary valence forces, there are many instances, however, where the amount of dye bound does not reflect the number of dissociating groups on the protein. Because of some of these results, as well as still other information to be presented below, the possibility has been advanced that forces other than coulombic ones may also operate in staining, though to various degrees depending on the substrate and the dye. In studies of dye binding of the solid protein, fibrin, much less dye appeared to be bound at extremes of pH than the number of groups available in the fibrin molecule (Ferry, Singer, et al., 1947; Singer and Morrison, 1948). Indeed, the quantity of dye bound at these levels of p H varied according to conditions of staining other than pH, such as ionic strength and dye concentration (see also studies with safranine 0 of Fraenkel-Conrat and Cooper, 1944). Another situation in which the number of dye equivalents probably differs from the number of binding sites obtains whenever an aggregate of dye ions rather than a single one is bound. Aggregation is quite common in solutions of dye,

STAINING O F TISSUE SECTIOXS

227

and dye is frequently taken up by the substrate in the form of aggregates ranging from dimers to colloidal particles. In the staining of agar and other sulfated substances the basic dye is probably bound as an aggregaterather than a molecular unit-to the charged side chains of the substrate, a mode of binding which would explain the metachromasia of the stained product (Michaelis and Granick, 1945 ; Michaelis, 1947). Another example, where stoichiometric proportions with the primary charged groups do not obtain, appeared in studies of the interaction of various inorganic and organic acids and wool at low p H where more acid was bound than expected from the basic amino acid content; and the conclusion was drawn that either feeble basic groups, such as the amide groups of glutamine and asparagine and of peptide nitrogen or an entirely different mechanism of binding was responsible (Steinhardt and Harris, 1940; Peters, 1945 ; Carlene, Fern, and Vickerstaff, 1947 ; Abbot, Crook, and Townend, 1947; Vickerstaff, 1950). The amide groups may become charged at very low pH and thus become sites for ionic interaction with acid dye, as is shown by a rise in binding of certain acid dyes between p H 1 and 2 (Abbot, Crook, and Townend, 1947). Strong support for binding by amide nitrogen has been obtained in the study of nylon dyeing. Vickerstaff (1950) described interaction of dye and amide groups by way of hydrogen bonding (see below) and believed that amide combination is the source of main affinity of acid dyes and protein. According to him, combination with the amide group would occur particularly in neutral solution. He described two sites of amide binding; one which is adjacent to positively charged basic sites and the other not. In solutions of low p H the former sites are more effective and have the higher affinity for acid dye by virtue of the electrostatic attraction of dye ions to these regions. In this way he explained the stoichiotnetric correspondence often reported between amount of dye bound and the number of basic sites. Interest in amide groups as sites of dye attachment has been emphasized in recent years since the advent of polyamide fibers. Nylon contains some free amino and carboxyl groups, but various lines of evidence suggest that the amide groups with which this fiber abounds are particularly active as dye-binding sites. Another possible exception to stoichiornetric proportions and salt linkages is the observation, frequently made, that protein can bind acid dye, albeit minimally, above the isoelectric region where dye and protein have a similar charge, and basic dye below the isoelectric region where the interactants are both positive (Atkin and Douglas, 1924; Loeb, 1924; Grollman, 1925 ; Robbins, 1926; Chapman, Greenherg, and Schmidt, 1927 ;

228

MARCUS SINGER

Kelley, 1939a; Schmidt, 1945 ; Skinner and Vickerstaff, 1945 ; Klotz, 1946; Neale, 1946, 1947; Singer and Morrison, 1948; and others). These relations are evident in Fig. 1 where both cationic and anionic dye are bound in the pH range above and below the point at which the curves cross. It can be argued that the limited binding of dye at a p H where staining is opposed by electrostatic repulsion may be due to interaction of dye ions with occasional and isolated protein groups of opposite charge. Yet other possibilities have been advanced to explain dye uptake under circumstances in which dye and substrate have the same electrical sign and, indeed, under ordinary conditions of staining, too. Dye ions which reach the protein surface may be anchored there by specific and powerful short range forces such as van der Waals (Neale, 1947; see discussion below of cellulose dyeing in section on dye affinity). If such bonds are important in binding of dye, then the electrostatic forces would operate in assisting or opposing diffusion of dye to the binding sites. In their analysis of the interaction of wool and dye, Gilbert and Rideal, (1944) also suggested that other forces besides primary valence bonds may be effective, such as resonance bonds, van der Waals forces, and coordinate links (covalent bonds), perhaps by way of the chromophore and auxochrome groups of the dye. The fact that large molecules have a greater affinity for wool fibers than small ones, would be expected if forces in addition to ionic ones, as for example van der Waals forces, obtain in the binding of the dye (Steinhardt, Fugitt, and-Harris, 1941a, 1942). Moreover, it is possible that forces operating between protein and dye vary according to the dyestuff, being a simple salt linkage in the case of some, such as small ions, but including additional forces in the case of others such as those mentioned above (Skinner and Vickerstaff, 1945). The important hydrogen bond, already mentioned above, may be particularly effective in the binding of certain dyes (Vickerstaff, 1950). In hydrogen bonding, hydrogen acts as a bond between two atoms, especially highly electronegative ones such as oxygen and nitrogen. Hydrogen bond formation has been postulated between the amide group of the protein and the hydroxyl, amine, or azo group of the dye (Vickerstaff, 1950). Additional discussion of this method of dye binding is given below (see section on affinity of dyes). Another possible method of linking dye and protein emerged from study of shifts in the spectral absorption curves of azo dyes during certain reactions (Gerstner, 1949) ; in addition to ordinary ionic reactions the carboxyl groups of protein may bind dye by linkage with the azo group (also refer to Gillet, 1889, 1890). There are, conceivably, still other ways by which dye and protein may be


229

combined than those listed above. And, indeed, the forces involved in dye binding may vary as staining progresses, since there is some evidence that dye ions which are first bound influence the binding of subsequent ones in various ways, for example by repelling oncoming ones electrostatically (Klotz, Walker, and Pivan, 1946). The nature of the binding site and the forces involved may vary according to the characteristics and state of the protein. In the study of the stoichiometry of staining, soluble proteins were mainly used. Yet, there is good evidence to show that binding in a solid system, such as is encountered in histological sections, is different, and consequently the mechanism of staining of soluble proteins cannot be equated precisely with that for solid ones. Major differences are evident between the titration curves of dissolved and solid proteins (Speakman and Hirst, 1933; Lloyd and Bidder 1934 ; Speakman and Stott, 1934, 1935 ; Steinhardt and Harris, 1940; Steinhardt, Fugitt, and Harris, 1941b). The p H levels over which the titration curve shows little change with added acid is much greater than for soluble proteins. Moreover, whereas the pK values of soluble proteins are very similar to the corresponding amino acids, they are shifted to higher and lower p H levels for the basic and acidic groups respectively of the solid protein (Vickerstaff, 1950). These differences between insoluble and soluble proteins have been attributed to structural differences, to the effect of a two-phase system on the reaction as explained by the Donnan equilibrium, or to other means. It is quite evident that the conditions under which dye is bound in a three-dimensional solid protein are quite different from those for protein free in solution. I n the former instance Donnan effects operate just as effectively as though the protein were in solution and separated from the staining solution by a semipermeable membrane (Eliid, 1933), and, consequently, the solution within the protein meshwork differs from that without in terms of p H as well as ionic distribution. These differences have been emphasized and quantitated by Speakman and Peters (1949), who applied the Donnan theory of membrane equilibrium to titration of wool with acids and observed that the internal p H of wool differed from that of the external solution. They were able to calculate the internal p H but could not measure it directly (see also Vickerstaff, 1950; Gilbert and Rideal, 1944; Kitchener and Alexander, 1949 ; Peters and Speakman, 1949). There is further evidence to complicate and question the view that salt linkages and stoichionietric proportions are alone characteristic of staining. This other evidence is best considered in relation to the nature of the affinity of dyes, which is discussed below. Particularly instructive is the

230

MARCUS SINGER

information on cellulose derivatives where forces other than primary valence bonds explain dyeing most logically.

BETWEEN T H E ISOELECTRIC POINTAND STAININC; V. THERELATION The dyeing of protein in the isoelectric condition was studied by Loeb (1922) who emphasized that in this condition gelatin was relatively inert ; but, he later reported (1924) that gelatin could bind acid dye, albeit minimally, above or basic dye below, the isoelectric point. Nevertheless, the close relation between the isoelectric point and the relative absence of protein reactivity appeared to offer a means of defining the isoelectric point by staining procedures. Many attempts were subsequently made to apply such determinations of isoelectric points to the protein complexes of cells and tissues. Robbins (1923, 1924, 1926) stained the mycelium of fungi and tissue of the potato tuber with acid or basic dye and then washed them in buffer solutions of various p H and observed the degree of dye loss in each solution. The p H region of minimum retention of dye was considered the apparent isoelectric point of the protein complex. Because of the relation between dissociation of the protein at dieerent pH and combination with dye Stearn and Stearn (1924) concluded that the isoelectric point of bacteria could be determined from curves of staining with acid and basic dye. They reported that the crossing point of staining curves of acid and basic dye reflects more accurately the isoelectric condition than the region of minimal anion or cation retention. Therefore, they defined the isoelectric point ( 1928c) as “the hydrogen-ion concentration at which there is equal retention of cation and of anion.” Tolstoouhov ( 1929) , using a mixture of eosin and methylene blue buffered to various pH, defined the isoelectric point of blood cells and other tissue proteins as that p H where staining was an approximately equal combination (purple color) of the red and blue dye components. The isoelectric point of structures of plant cells was described by Naylor (1926) and Robbins (1926) , following observations on the pH of minimal acid and basic dye uptake, as a p H range rather than a specific point. The isoelectric point of hemoglobin in hemoglobin-containing cells was observed in fixed and unfixed specimens with acid and basic dye (Kindred, 1932, 1935). The crossing point of p H curves of acid and basic dye binding, or the region of minimal basic or acidic dye uptake was used as the criterion of the isoelectric condition by Pischinger (1926, 1927a) in histochemical studies of the proteins of cells and tissues. A whole series of studies of ‘%oelectric points” on a variety of tissue and cellular components was initiated by Pischinger’s work (Mommsen, 1927; Pulcher, 1927a ;


231

Schwarz-Karsten, 1927 ; Ochs, 1928 ; Pfeiffer, 1929 ; Seki, 1933c, 1934 ; Yasuzumi, 1933a, 1933b, 1934 ; Nishimura, 1934 ; Achard, 1935 ; Ikeda, 1935, 1936a, b ; Sturm, 1935; Fautrez, 1936; Yasuzumi and Matsumoto, 1936; for review of these and other works see Zeiger, 1938 and Levine, 1940). More recently the isoelectric point of structures of skin were defined using fluorescent dyes over a pH staining range (Bejdl, 1950; Stockinger, 1950). The accuracy of isoelectric determinations of proteins by staining procedures has been questioned. The close relation between the isoelectric point and the minimum of acid and basic dye uptake observed by Loeb for gelatin did not obtain for powdered hide collagen (Thomas and Kelly, 1922). When isoelectric point determinations were attempted from titration data or from study of the interaction of the protein with various substances, a number of difficulties appeared (Speakman and Stott, 1934). Between p H 5 and 7 there was no significant binding of alkali or acid by wool, and consequently the isoelectric point of wool could not be exactly defined but rather the values 5 to 7 were considered an isoelectric range. Comparison of the staining of extracted nucleoprotein with that of tissue nuclei showed that the isoelectric point of the nucleoprotein of the cell could not be determined by staining with the basic dye, toluidine blue (Kelley, 1939a, b). According to Kelley, staining depended on the amount of nucleic acid in the nucleoprotein and not on the isoelectric point. H e observed that toluidine blue was bound to nucleoprotein below its cataphoretic isoelectric point. Levine ( 1940) reexamined the concept of isoelectric point‘ determination of tissue proteins with dyes and concluded that the relatively qualitative curves of staining with p H are inadequate sources of such information. H e found that the crossing points and other characteristics of acid and basic dye curves upon which “isoelectric point” determinations were based varied with such factors as the nature and concentration of dye and buffer salts of the staining solution. Isoelectric point determinations varied by as much as 2 p H units when different dye pairs were used. Pfeiffer (1929, 1931) also questioned the belief that the point of‘ intersection of the dye curves represents the isoelectric point. Some pertinent observations concerning isoelectric determinations by staining procedures may be drawn from studies of fibrin film, an isolated and relatively pure protein system. The isoelectric point of powdered samples of fibrin film was determined electrocataphoretically and the molar binding of orange G and methylene blue analysed quantitatively over a range of p H (Singer and Morrison, 1948). The cataphoretic iso-

232

MARCUS SINGER

electric point of freshly prepared fibrin was 6.0,but the p H of crossing of orange G and methylene blue curves was 6.5 (Fig. 5, see Fig. 3A of Singer and Morrison, 1948). When denatured by heating (20 minutes at lZO”C), the apparent isoelectric point was 5.5 but the crossing point of acid and basic dyes was 6.3;for films heated for lesser times (1 minute at 100°C)the values were 5.7and 6.4respectively (refer to same figures). The disparity between the two values, although thoroughly apparent, is not great so that the p H of crossing diverges between one-half and one p H unit from the cataphoretic isoelectric point. If such quantitative procedures could be applied as readily to the microscopic and, yet, highly heterogeneous protein systems of tissue sections, it would be possible to approximate the isoelectric conditions of tissue elements by determining the pH at which there is equivalent binding of acid and basic dye. It must be stressed that such determinations would be only approximations. The divergence between the isoelectric point and the region of crossing of the curves may be due to binding of dye by forces other than coulombic ones. These additional forces do not determine the cataphoretic isoelectric point and, consequently, binding by them should only increase the disparity between the values. The deviation between the cataphoretic isoelectric point of fibrin and the pH a t which the acid and basic dye curves intersect one another varies with the dye pairs used (unpublished results). The pH of equimolar dyeing of formalin-fixed fibrin (cataphoretic isoelectric point of 5.2) with methylene blue and the following acid dyes were: orange 11, 5.0;picric acid, 5.1; orange G, 5.2; ethyl orange, 5.3; fast green, 5.5 ; sodium 2, 4-dihydroxyazobenzene-4-sulfonate, 5.6; light green, 5.7. These results are in agreement with the more qualitative observations of Levine ( 1940), who demonstrated variation of the intersection point for dye pairs in tissue staining. There are other factors as well which influence the crossing point. It is evident, therefore, from these observations that caution must be exercised in defining the isoelectric condition of proteins by their staining characteristics, particularly in tissue sections where the conditions of binding are not easily controlled and where adequate procedures for determining the ’quantity of bound dye are not yet developed. At best, perhaps, a broad isoelectric range may be described in which the actual isoelectric point would probably fall (“isoelectric range” of Naylor, 1926;“isoelectric zone” of Stearn, 1933 ; Speakman and Stott, 1934;see also discussion by Dubos, 1945,p. 68). Perhaps too much emphasis has been placed upon the relation between the isoelectric point and dye uptake. There are characteristics about the pH curves of staining which can be drawn upon in analyzing the protein


233

without any particular reference to the isoelectric point. For example, the region of the curve showing greatest rate of change with alteration in pH reflects a most sensitive and characteristic response of the protein to alterations in its electrostatic environment. This region can be defined by that p H at which a tangent along the steep part of the curve intercepts the pH axis; or it can be described as the p H which bisects the steepest part of the curve. If dye pairs are used then the pH at which curves cross may be used as a characterization point of the protein structure. There are still other ways of characterizing proteins according to the dye curves which they yield. Although the isoelectric point of the protein cannot be precisely defined by p H staining characteristics, yet it is possible to compare curves of different proteins and to draw conclusions therefrom on the relative position of the isoelectric point of these substances. From previous considerations of dissociation of proteins in relation to staining, it would follow that staining curves displaced to regions of higher p H reflect higher isoelectric proteins and those which lie lower on the p H axis represent lower isoelectric proteins. It is also possible to determine the direction of shift of the isoelectric point of a protein or protein complex following a physiological modification by comparing staining curves of the substance before and after modification (Levine, 1940; Singer and Morrison, 1948 ; Singer and Wislocki, 1948; Singer, 1949). A point which requires some emphasis here is that studies with dyes of the “isoelectric point” or other characteristics of proteins invariably and by necessity have been made on fixed proteins. Such chemical modification undoubtedly affects the protein, and the characteristics of dye binding does not reflect those of the native undenaturecl system, as will be evident in the discussion of the effect of fixation on staining (see below). The isoelectric point may be influenced profoundly by such chemica1 modification. Pischinger ( 1926, 1927a) and many others (see e.g., Zeiger, 1930b ; Seki, 1933b) have assumed in their studies that the isoelectric point of tissue proteins is not altered by alcohol fixation, a conclusion which has been criticized by Yasuzumi (1933a). VI. THE IONICSTRENGTH OF

THE

DYE SOLUTION

The foregoing discussion of the influence of p H on staining has emphasized that the protein amphion is sensitive to alterations in the pH of its environment and reflects these alterations by a change in charge. This being the case, it may be expected that factors other than acid or alkali which alter the electrolytic environment should also affect the electro-

234

MARCUS SINGER

static condition of the ampholyte and, thereby, its interaction with dye. The amount of dissolved salt represented either as neutral or buffer salts is such a factor. The activity of salt ions in solution is best expressed by the ionic strength (see Cohn and Edsall, 1943) rather than by other concentration expressions. Salt solutions of equal concentration measured by the latter means may have different ionic activities by virtue of a difference in number and valence of dissociable ionic groups. The ionic strength ( u ) of a solution of electrolyte is defined according to Lewis and Randall (1923) as half the sum of the concentration (molality) of each ion multiplied by the square of its valence: u = 8 mi Zf Increasing ionic strength of the dye solution decreases staining with both acid and basic dye, a result which is exemplified in Fig. 2 for the stainORANGE G

0.4

k

g a3 W

n J

a

-0

oa 0.1

s a 10-5 W.

-.\.

+:, ,.'

o -\ . 0 . 0

0

3

9@Y

4

1.2

Ionic strength

' 0

\e

0

g

METHYLENE BLUE

I I 10-sY.

%,bc@ /O s

6

0 8 0

0.01 0.04 0.15

I.o

3.8 36

.d

0.4

/

01

I

7

E

9

P"

FIG.2. The influence of ionic strength on staining of fibrin film with acid (orange G ) and basic dye (methylene blue). Taken from Singer and Morrison, J. B i d . Chem., 176, 1948. ing of fibrin with methylene blue and orange G. Steinhardt, Fugitt, and Harris (1941a) described such a depressing effect on the titration of soluble and fibrous proteins with various anions. Levine (1940) observed that the staining of tissue sections decreased with increasing concentration of buffer salt. A number of reasons have been advanced for this effect of the electrolyte on staining, among which are the remarks of Singer and Morrison (1948) on the dye binding of fibrin and the more detailed discussion of Elod (1933) in his work on the effect of neutral salt on the binding of dye anions by wool at low pH. According to Donnan's theory of membrane equilibrium, salt ions may influence the staining of solid proteins by altering the distribution of dye ions between the external solution and the solution within the protein itself. For this reason, the effective


235

staining concentration, namely the concentration of dye in the solution between and within the fibrillae of the protein probably differs from that of the dye bath. In solutions of high ionic strength the internal dye concentration in equilibrium with the stained micellae is evidently much less than in solutions where little or no salt is present. A quantitative interpretation of the effect of different ionic srengths on the interaction of solid protein and various acids according to Donnan’s theory has been elaborated by Speakman and Peters (1949) in studies of the binding by wool of sulfuric acid and hydrochloric acid singly or in the presence of different concentrations of KCl. The analysis of dyeing equilibria according to the Donnan theory has been summarized and evaluated by Vickerstaff (1950). In addition to the Donnan effect it is also possible that the salt ion competes with the color ion for the binding site on the protein molecule and thereby limits the quantity of dye bound (Pelet-Jolivet, 1910; Briggs and Bull, 1922; Elod, 1933; Speakman and Clegg, 1934; Skinner and Vickerstaff, 1945). I n describing such competition Elod (1933) showed that in dyeing of wool the small salt ion penetrates the fiber most rapidly by virtue of its greater diffusibility and is bound, but then secondarily is replaced by the larger dye ion which shows less tendency to dissociate after binding (see discussion below on dye affinity). When the concentration of salt ion is increased, less of the dye ion in competition with it is bound. Neale (1946, 1947) concluded that salt ions serve to suppress the electrostatic forces on the protein, thus decreasing the attraction to acid dye below the isoelectric point and basic above. It is interesting, as Neale and others have emphasized, that higher ionic strengths have the reverse effect on dye binding when the sign of the charge on the dye and the protein is the same (above the isoelectric point for acid dyes and below for basic ones). In these instances the positive effect of the salt on staining results from suppression of forces which would normally repel the dye ion. Since he attributed dye binding, in part, to the more powerful but short-range forces, such as hydrogen bonds and covalent links, the effect of the decreased potential of the protein surface is to allow more dye ions to come within the range of action of these forces (see discussion on dye affinity). I n this way the activation energy of the dyeing process is reduced and dyeing increased with added salt (Vickerstaff, 1950). Electrolyte also plays an important role in the dyeing of cellulose fibers, but in a manner quite different from that for ordinary dyeing of wool. Cellulose derivatives are negatively charged and yet are generally stained

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MARCUS SINGER

with acid dyes. The affinity of the color anion for cellulose is quite low in the absence of electrolytes. As electrolytes are added to the color bath, dyeing increases markedly despite the similarity in charge of the color anion and the plant fiber. The effect of the salt ions is to dampen the mutual repulsion and allow dye to approach the cellobiose chains closely enough to be bound by hydrogen linkages. The effect of electrolyte on the staining of cellulose resembles its effect on dyeing of proteins above the isoelectric point with acid dye and below this point with basic dye. In both instances salt serves to facilitate dyeing. Finally, it is also possible that salt influences staining by acting on the dye itself. Increased salt causes the dye to form colloidal aggregates and even eventually to precipitate from solution (Michaelis, 1947). Large aggregates of the dye by virtue of their size find less ready access to intermicellar regions of the protein than single ions or dimers. Consequently, the effective concentration of dye in solution is lowered thereby, and the staining decreased.

VII. THEINFLUENCE OF DYE CONCENTRATION Variation in the amount of dye taken up by tissue sections according to the concentration of the dye bath is a matter of common knowledge to histologists. Greater amounts of dye are bound with increasing concentration as shown in Fig. 3 for both acid and basic dye. The amount of dye which is bound with increasing dye concentration is limited by the number of available binding sites (Knecht, 1889, 1904; Hofmeister, 1891, p. 224; ORANGE G

44.

METHYLENE BLUE Dye concentration

2.

o

- 1.2 -

I r ~ ~ M.- 5 0 SXIO'~ M. 0 2.5 ~ 1 0 M. . ~

x

-

0.8

a6

-.0.4

- 02 2

3

4

5

6

P"

7

8

9

FIG.3. The influence of dye concentration on staining of fibrin film with acid (orange G ) and basic dye (methylene blue). Taken from Singer and Morrison, J. Biol. Chem., 176, 1948.


237

Steinhardt, Fugitt, Harris, 1941a; Skinner and Vickerstaff, 1945). Except for the activity of other forces which may bind dye such as hydrogen bonds and van der Waals forces, once coulombic forces are satisfied little or no additional dye is bound. Limitation in the amount of dye taken up as staining proceeds is due, however, not merely to occupation of the dyeing sites by dye molecules but also to the effect of bound dye on the oncoming dye molecules. Klotz, Walker, and Pivan (1946) have pointed out in their studies of the adsorption of dye by serum albumin that steric hindrance or electrical effects on approaching dye molecules may result from already bound ones. The change in the rate of increase of bound dye with increase in dye concentration has been explained by Elod (1933) in terms of the Donnan equilibrium. Other experiments on the influence of dye concentration on staining are those of Craig and Wilson (1937).

VIII. THEAFFINITYO F DYES By ufinity is generally meant the tendency of a dye to combine with a given tissue structure. However, the term is userl quite loosely by histologists and may have a variety of implications. Often it implies a specificity between the substrate and a particular dye not shared by other dyes. Yet, a given protein may be stained by any one of a number of acid or basic dyes provided conditions of staining, particularly such as pH, are adequate. And, consequently, from this viewpoint, specific affinities are not the rule and molecularly dispersed dyes of quite different character are taken up by the same protein. For example, the basic dye methyl green which is ordinarily considered very specific for desoxyribonucleoprotein will interact with proteins of cells and tissues in general in aqueous solution, provided the appropriate p H conditions are used (compare Michaelis, 1947). And other basic dyes will stain the same nucleoprotein quite well. The acid stain, aniline blue, will also stain cells and tissues widely when applied under conditions of low pH, although as employed in triple staining methods it is considered fairly specific for collagen. Mathews (1898) appreciated this similarity in staining capacity of dyes at a time when dfferences or “specific affinities” were especially highlighted. Although protein shows little tendency to bind a particular acid or basic dye exclusively and reject others, nevertheless the degree of binding varies from one dye to the next. Some dyes are bound in greater amount than others. If the equivalent of acid dye or of other acid substance taken up by the protein is plotted against pH, notable differences in the number of equivalents of each substance are apparent, even though the conditions of staining are the same. Steinhardt, Fugitt, and Harris (1941a) recorded

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MARCUS SINGER

differences between a variety of substances which at the extremes reached 2 units on the p H coordinate between the curves for HC1 and flavianic acid when the p H values were compared a t which half the maximum amount of acid was bound. Such differences in the curves of staining of various dyes were also recorded by Levine (1940) and Elod (1933) and are exemplified in Fig. 4 for staining of fibrin film.

PH

FIG. 4. Differences in the affinity of two acid dyes (light green and picric acid) revealed by their pH staining curves. Note the differences in the crossing points of these curves with the curve of methylene blue (see text on discussion of isoelectric points). Formaldehyde-fixed film (10 per cent for 10 hours) ; dye concentration, 5 X 10-6 M . ; ionic strength, 0.02. (Singer and Weiss, unpublished.)

The differences in affinity of dyes for solid proteins evidently depends on a number of factors (see also review of the factors which govern the affinity of soluble proteins for dyes and other interactants by Goldstein, 1949). As mentioned previously, it is conceivable that other groups on the protein molecule than amino or carboxyl ones may be involved in the reaction with different dyes. Some forces may be more available for combination with one dye than with another. For example, combination of protein with simple acids may involve primary valence bonds with substituted ammonium groups, whereas with other substances forces such as covalent links or hydrogen bonds may be involved simultaneously or alternatively (Klotz, Walker, and Pivan, 1946; Neale, 1946, 1947; Klotz and Walker, 1947). Neale (1947) in his study of affinity and its meaning in terms of electrochemistry of staining (see also Harrison, 1948) concluded that short-range forces such as are present in covalent links or hydrogen bonds are responsible for the specific affinity of a dye for a particular fiber. The long-range electrostatic forces oppose or assist these more powerful short-range forces. But, even with electrostatic repulsion of the dye (e.g., below the isoelectric point with basic dye or above with


239

acid dye) some dye is bound, nevertheless, by the short-range forces, although work evidently must be done (thermal agitation) to bring the dye ion to the surface of the fiber. Another explanation of differences in affinity is based on studies of the degree of dissociation of various protein-anion combinations ( Steinhardt, 1940; Steinhardt and Harris, 1940; Steinhardt, Fugitt, and Harris, 194la, b, 1942). When the dissociation was great, as occurred after reaction with chloride ion, the affinity as recorded in the titration curve was much less than when the dissociation was slight as occurred with protein-dye combinations (compare Chapman, Greenberg, and Schmidt, 1927). The dissociation constants, calculated for various combinations of protein and anion, showed wide variations (also Steinhardt, 1942). Therefore, affinity of the dye was directly related to the degree of association of the color ion with the binding site of the protein. The differences in affinity were correlated with differences in size of the anion, and it was noted that with few exceptions increasing order of affinity followed increase in molecular weight and was higher in aromatic than in aliphatic ions of the same size (compare Klotz and Walker, 1947; Klotz, Triwush, and Walker, 1948). In the exceptions, considerations of shape of the molecule and its relation to steric hindrance were offered as an alternative possible explanation of differences in affinity (cf. Goldstein, 1949, p. 146). The difference in combining capacity of various acids with protein was great. When chloride ion was taken as unity, then the combining capacity of picric acid was 758 and that of Orange I1 over 23400 (Rose, 1942). It has also been stated by textile chemists as a general rule that affinity increases with molecular weight and, moreover, with complexity of the dye ion and with the introduction of additional polar groups (Abbot, Crook, and Townend, 1947; Lemin and Vickerstaff, 1947). In connection with size differences of dye molecules, Speakman and Clegg (1934) and Speakman and Smith (1936) believed that in the case of wool the cystine (-S-S-) and salt linkages (-COO-, -NH3+) offer resistance to the penetration of large dye molecules, but at low pH, salt linkages are broken since the carboxyl groups are displaced from combination and the freed amino groups combine with the added acid. As a result of lowered cohesion of the micelles the structure swells with water and is now accessible to large dye molecules which are then trapped in the protein fiber. The tendency of dye ions to aggregate in solution and to stain as aggregates will also affect the penetration and therewith the affinity of the dye for the protein. There are evidently other possibilities involving the number and kind

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MARCUS SINGER

of reacting groups on the dye molecule (Speakman and Clegg, 1934; Townend and Simpson, 1946 ; Gerstner, 1949) and the physical structure of the protein as well as that of the dye (see also Seki, 1933a; Zeiger, 1938). For each dye with more than one binding site in its molecule it would be important to inquire how many of these sites are involved in the actual attachment of the dye molecule to the protein. For example, a dye molecule with two charges may be bound in equivalent or molecular fashion (Loeb, 1922; Elod, 1933; Speakman and Stott, 1935), and its mode of attachment may vary according to the protein. Abbot, Crook, and Townend (1947) have described this effect for the dyeing of nylon. When the number of acid groups of the dye molecule is increased from one to two, the affinity of the dye decreases since it is now spread over two sites rather than one. Or, dye with three or even four charged groups has been reported to occupy an equivalent number of oppositely charged sites. Because of the probable disparity in spatial arrangement of the charges on the dye and those on the protein, it is possible that an equivalent number of charged sites are neutralized rather than occupied by the dye ion (Vickerstaff, 1950). However, if only one binding group of the dye molecule is involved, the remaining groups may influence adversely the binding of oncoming ions by electrical effects or steric hindrance (Klotz and Walker, 1947; Vickerstaff, 1950). I n these instances the affinity of dye for protein must be weighed not merely in terms of its combining capacity but also, once bound, in relation to its effect on the approach of another dye ion to an adjacent site. Therefore, as staining proceeds, the available sites are no longer equivalent to the initial ones and the dyeing mechanism may be profoundly influenced and altered. In connection with these thoughts it may be well to note that there is no need to assume that all dyeing sites are equivalent, even at the onset of dyeing (Vickerstaff, 1950). Some may not be accessible to certain dye molecules, but readily available to others. Some correlation has been drawn between the shape of the dye molecule and the affinity for protein in that planar molecules are believed to have more affinity for wool than three-dimensional or linear ones (Steinhardt and Harris, 1940) ; but in the case of protein staining in general little information is available relating shape and affinity (Vickerstaff, 1950). The relation between the structure of the substrate and the shape and constitution of the dye has been studied in most detail in direct dyeing of cellulose fibers. Cellulose is dyed by color ions (anions) having the same charge (negative) as the fiber, and consequently, other forces than


241

electrostatic ones must be invoked to explain the uptake and fixing of the dye. Indeed, the electrostatic forces serve in this case to repel the dye ion rather than attract it, as in the case of ordinary protein staining. Dyes which combine directly with cellulose are generally sodium salts of sulfonated aromatic azo dyes. Once the dye anion is brought to the surface of the fiber (as described in preceding pages, factors of thermal agitation and addition of large quantities of electrolytes to the dye bath are important in this movement) and penetrates the water swollen regions between the cellobiose chains, the dye molecule is bound by short-range forces to the cellulose molecules (Neale, 1947 ; Vickerstaff, 1950). The nature of these forces and the problems involved in binding dyes of various structure and configuration have been speculated upon. Among the theories is that of hydrogen bonding (Rose, 1935) in which hydrogen of certain groupings of the fiber or of the dye acts as the electron acceptor. According to Rose at least two hydrogen links are required to bind a colored ion. Such linkages between the color ion and the linearly arranged cellobiose chains should be more readily formed with dye ions of certain shape and structure. Indeed, dye molecules used in direct dyeing of cellulose are in general long and chain-like and thus can contact the cellulose micellae more closely than non-linear ones (Meyer, 1928). Coplanarity of the various ring nuclei (benzene and naphthalene) also determines affinity of the dye (Hodgson, 1933) ; dyeing is favored when the rings lie in one plane. Finally, a high number of double bonds in the dye molecule seems also to be important (Schirm, 1935). This description of some of the theories of cellulose dyeing emphasizes that dye interaction depends to some degree upon the shape, size, and constitution of the dye molecule. The problem of the affinity of dye is undoubtedly complicated by still other factors than those described above. In non-aqueous solutions or under conditions of staining quite different from those considered in the present review, the extent of interaction may be still further influenced; and the reaction between a particular substrate and a dye may be specifically favored. Relatively little attention has been paid to the influence of special media, such as an alcoholic or phenolic one, as is employed, for example in methyl green-pyronin staining of nucleoprotein. Perhaps the function of the special media or other peculiar conditions of staining is specifically to enhance or to facilitate forces which may favor a particular dye and protein combination.

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MARCUS SINGER

IX. THEINFLUENCE OF FIXATION AND OTHERMODIFICATIONS OF TISSUES ON SUBSEQUENT STAINING The obvious and important effect of fixation in histology is to denature and thereby render insoluble both the solid and dissolved proteins. In addition, fixation and other chemical or physical modification influence profoundly the subsequent binding of dye. In general, the living cell shows a selective affinity for some dyes which it concentrates and stores. This selective affinity is an expression of vital activities of living cells and bears little relation to forces which control the binding of dye in fixed and histologically prepared cells and tissues. After death, dyes of various character readily penetrate the cell, but the capacity to bind them is slight. However, when the cell is subjected to physical or chemical denaturing agents there is an immediate and pronounced increase in staining. The extracellular protein matrix of tissues also shows a limited affinity for dye until it is fixed. The precise nature of the effect of fixatives on proteins is not known. Elucidation of the alterations suffered by the protein during fixation is a problem of major importance in the study of the physical chemistry of tissue proteins since it is the modified and not the native protein which is studied under the microscope. Yet, relatively little work is being or has been done in recent years on fixation. Early workers devoted a considerable time to the elaboration of different fixatives designed to preserve the morphology of the cell and tissue with little change and yet to favor the staining of one or another morphological component. A considerable number of procedures was elaborated empirically or on the basis of certain chemical information. The procedures were reviewed admirably by Mann (1902), who also discussed, according to the information then available, the chemical and physical significance of the techniques. A more recent evaluation of fixation is given by Zeiger (193Oa, b, 1938). References to various methods of fixation and their general application may be obtained in technical works on histological procedures (for example Baker, 1945 ; McClung’s Handbook, Jones, 1950; Lee’s Vade-Mecum, Gatenby and Beams, 1950 ; Bourne, 1951). No attempt will be made to cover these works here. Instead, some general statements will be made about fixation based especially upon chemical information on denaturation of proteins and related information that is available in the histological literature, The manner in which fixation influences the subsequent staining is exemplified in experiments recently reported on films of fibrin (Singer and Morrison, 1948). These films are particularly suited for studies of

243


fixation, since the constituent fibrin of freshly prepared films may be considered native (Ferry, Singer, et al., 1947). Moreover, the protein is solid and insoluble having the three-dimensional relations typical of the structural proteins of cells and tissues. Consequently, the factors which obtain in fixation of fibrin film resemble somewhat those for cells and tissues. The interaction with dye of native fibrin and fibrin modified by various procedures is compared in Fig. 5. Freshly prepared fibrin has

i

I 2

10

08

06

0.4

0. I 09.

t

3

6

4

9

PH

FIG.5. The influence of different fixatives on subsequent staining of fibrin film. Note how the acid (orange G ) and basic (methylene blue) dye-binding curves shift according to the fixative (see text on discussion of isoelectric points and of fixation). Taken from Singer and Morrison, J. Biol. Chem., 176, 1948. little capacity to bind dye and resembles unfixed tissue protein in this way. In general, a notable increase in stainability with both acid and basic dye occurs after fixation, whether such fixation be p%sical (heat) or chemical in nature. However, the increase in acid and basic dye-binding capacity is not an equal one and in most cases varies according to the treatment. For example, there is a relatively greater increase in basic than in acid dye uptake following formaldehyde fixation. O n the other hand, the reverse is true after fixation with HgClz or some other salt of a heavy metal (cf. Kelley, 1939a). Fixatives appear to have two major effects on protein as reflected in the dye reaction. There is an initial effect of increase in affinity for both classes of dye attributable presumably to a physical reorganization of the protein, whereby charged and other groups to which the dye ion may attach are rendered more available to the dye (Singer and Morrison, 1918). This effect is in general shared by all fixatives. An increase in

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MARCUS SINGER

availability of reacting groups resulting from denaturation of proteins has been frequently described in the chemical literature (see reviews of Neurath, Greenstein, Putnam, and Erickson, 1944 ; and Anson, 1946). Alteration in physical structure of the protein implies also an alteration in the permeability of the protein meshwork for dye molecules. The second effect is peculiar to the particular fixative and results in an alteration in the relative uptake of acid and basic dye. This effect may be attributed to a specific influence of the fixative on groups which bind the dye, or on other groups whose proximity or mere presence influences the binding of dye to adjacent sites. The fixative, depending on its nature, may introduce other ionizing and, therefore, dye-binding sites in the protein. It may cover specifically certain groups or in some other manner prevent dye interaction with these sites. The two effects of the fixative are well exemplified in heat denaturation of fibrin film (Ferry, Singer, et al., 1947). As a result of brief heating the acid and basic dye-binding capacity increased remarkably (see also Herrmann, Nicholas, and Boricious, 1950), an effect which corresponds to the first postulate. Upon prolonged heating a second effect appeared and gradually became pronounced ; the affinity for basic dye increased whereas that for acid dye declined. Associated with the second change there was a gradual drop in the isoelectric point of the protein (Singer and Morrison, 1948). Prolonged heating probably causes a gradual and progressive deamination of the protein and thereby a decrease in the number of positive groups available for acid dye. A similar drop in the isoelectric point and a similar alteration in staining is observed following denaturation with formaldehyde. But, in this instance, the secondary effect is accomplished by combination and therefore covering of the amino groups. A secondary effect in which metal ions combine with carboxyl groups presumably occurs with HgCl? and other heavy metal fixatives. In this way the basic dye affinity is relatively diminished and the acid one is increased. Other alterations, such as mordanting, which are secondarily imposed upon the primary fixation will further influence the staining of proteins by modifying the dyeing sites or so changing the physical characteristics of the protein structure (such as degree of swelling) as to alter the extent of penetration by the dye ion. Staining intensity varies with changes in the protein and, consequently, is a sensitive criterion of the modifications to which the protein was previously subjected. Indeed, slight differences in heat treatment of fibrin film were detected through alterations in dye uptake (Ferry, Singer, et al., 1947). Another study of staining of modifietl proteins was that of Fraenkel-Conrat (1944) ; and the effect of various chemical modifications

S T A I N I N G OF TISSUE SECTIONS

215

on the dye binding of wool was reviewed by Kienle, Royer and McCleary (1945), Lemin, Vickers and Vickerstaff (1946) ; Vickerstaff ( 1950). A number of interesting studies of the variation of staining with fixation have been reported in histological literature (see reviews by Zeiger, 193Oa, 1938). Tolstoouhov (1928) recorded the staining of blood cells in mixtures of eosin and methylene blue of various pH and following various fixations. The p H of approximately equal binding of these two dyes depended upon previous fixation. After fixation in solutions of salts of heavy metals, the cells had much less affinity for basic dye and the p H of equal binding rose. The reverse occurred after formalin fixation, whereas fixation with ethyl alcohol yielded stain affinities of an intermediate character. Zeiger (1930) studied alcohol and formalin fixation and believed that there was less shift in the isoelectric point of tissue proteins after alcohol fixation than with other common fixatives. Yasuzumi ( 1933) studied the effect of alcohol fixation on the isoelectric point of red blood cells. An extensive series of experiments has been done with various fixatives on egg albumen and tissue sections (Seki, 1933b) and on extracted nucleoprotein (Kelley and Miller, 1935 ; Kelley, 193913).

X. THEINFLUENCE OF TEMPERATURE OF THE STAINING SOLUTIOK The temperature of the staining solution may influence staining in a number of ways. Probably the most pronounced effect is on the rate of diffusion or movement of the dye within the protein and, therefore, the rate of staining. The rate of dyeing is increased progressively with increased temperature so that equilibrium staining is reached much faster at higher temperatures than at lower ones. The notable effect of temperature on equilibrium staining is illustrated well in the example which Vickerstaff (1950) gives for the dyeing of wool fiber. Five months at 20°C would be required for equilibrium dyeing of wool which can be dyed in 1 hour at 100°C. This effect of temperature on the diffusivity of the dye has been treated quantitatively by determining the activation energy of dyeing (Vickerstaff, 1950). Dyes of poor diffusivity are affected most by temperature changes (Abbot, Crook, and Townend, 1947). Temperature of the dye bath is believed to have other influences besides alteration in the rate of diffusion of the dye. It may influence the affinity of the dye-protein interactants since the amount of dye bound at equilibrium decreases with increasing temperature (Boulton, Delph, Fothergill, Morton, 1933 ; Neale, 1933 ; Vickerstaff, 1950). At low temperatures equilibrium may be so slow in attainment that the erroneous impression is thereby given that less dye is bound at low temperatures (Vickerstaff,

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MARCUS SINGER

1950). Steinhardt ( 1940) and Steinhardt, Fugitt, and Harris ( 1940b, c, 1912) studied the effect of three temperatures (0,25, and 50°C) on the affinity of various anions and wool. They observed that the p H association curves of these anions showed different degrees of sensitivity to temperature change. Larger, more tightly bound anions have greater heats of dissociation, and thus their affinity was altered less by increase in temperature than smaller more readily dissociated anions. Altered temperature may be expected to produce essential responses and chemical changes in the protein itself (Elod, 1933). Heating may cause a swelling of the fiber due to loosening of the micellar structure as has been reported by Speakman and Smith (1936) for wool dyeing. Penetration of the protein by the color ion is thereby facilitated. The effect would be particularly pronounced for large dye ions or for colloidal aggregates (Gerstner, 1949). The separation of the micellar sheets can occur by loosening of the covalent bonds between them but also by destruction of the disulfide linkages. At elevated temperature there may also be a decomposition of protein (see previous discussion of heat modification of fibrin). In the case of colloidally dispersed dyes, the effect of temperature is very marked since swelling of the protein which results from increased temperature allows greater penetration of dye into the protein. Moreover, the dye is more finely dispersed at higher temperature and aggregation tendencies are diminished (Speakman and Smith, 1936; Goodall, 1938, 1947). For most successful dyeing, dyes of low dispersivity require higher temperatures. This information explains the importance in tissue staining of elevated temperatures with colloidal . solutions of dye (for example, azocarmine in triple acid staining techniques). Other works of interest for further references and description of temperature effects in dyeing are those of Brown (1901a, b), Sheppard, Houck, and Ditmar (1942) ; and Royer, Zimmerman, Walter and Robinson (1948). The latter workers described the effect of extremely elevated temperatures (200"and 300°F) on dye uptake of textiles. In the histological literature there is relatively little other than empirical studies on temperature effects on staining. Some references may be had in the work of Ochs (1928), who also described temperature alterations of the staining of blood cells and gelatin.

XI. SOMEOBSERVATIONS ON THE KINETICS OF STAINING In preceding pages attention was focused primarily on the forces which bind molecularly dispersed dye to solid protein and on the various factors


247

which influence these forces or otherwise alter the dyeing process. Another important aspect of the staining process concerns forces involved in the movement of dye ions to the surface of the protein and their distribution to the binding sites. Histological staining is invariably done in concentrated solutions with a great excess of dye so that there is essentially no decrease in concentration as staining proceeds. Consequently, diffusion of dye ions to the protein surfaces must be extremely rapid and the movement must continue at a high level particularly when the solution or tissue is agitated during staining. Moreover, coulombic forces acting between dye and protein would serve to hasten the movement. Under conditions where the dye bath is gradually exhausted during the course of dyeing as is done in textile coloring, the problem of diffusion of dye in solution to the surface of the fiber is of greater import. Diffusion of the dye ion from the surface of the protein into the internal meshwork and thence into the intermolecular spaces of tissue structures to more deeply placed dyeing sites, constitutes, however, an important factor which evidently exerts a profound control on the rate of staining. An early and particularly lucid discussion of diffusion factors is given by Pappenheim (1901 ) . The importance of factors of diffusion in staining was stressed particularly in early works which supported the physical mechanism of staining (Gierke, 1885 ; Fischer, 1899; Knoevenagel, 1911 ; Pappenheim, 1917; von Mollendorff, 1923; von Mollendorff and Krebs, 1923; von Mollendorff and von Mollendorff, 1924; excellently reviewed by Zeiger, 1938). The importance of dye particle size and pore size in the protein was speculated upon as a mechanism of staining (e.g., the “Durchtrankungsfarbung” of von Mollendorff, 1923). Problems of diffusion of dye to the binding sites are also of importance in textile dyeing since the fiber and fibril sizes to be penetrated by the dye ion are large and the micellar network dense (Vickerstaff, 1949, 1950). Once the dye reaches the surface of the tissue section it is free to react with sites available at that position. Penetration of other dye ions to deeper staining sites must occur through this layer in which staining forces are already at least partly satisfied. Since the interaction of the dye with the surface sites is probably immediate, the rate of staining is determined in large part by the time for the dye ions to reach more central regions of the protein (Vickerstaff, 1950). The movement of dye ion to deeply placed sites is much slower than to the surface because of mechanical obstruction of the protein micellae and because of other forces acting between dye and protein and, indeed, between bound and free dye. The protein structure may provide an effective barrier to some dye ions and not others. Large dye ions may

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MARCUS SINGER

penetrate with difficulty or not at all and dye aggregates may penetrate the interstices of certain meshworks but be excluded from others. The speed of penetration may vary from one region of the tissue to another according to the density of the charge in these regions. It may change as dyeing proceeds because of the gradual alteration of charge on the protein with staining. The ease of diffusion may also be affected by dye ions which are already bound according to conditions described in a preceding section on the affinity of dye. For example, dye ions with residual unsatisfied charges would repel oncoming ions. Such a repulsion would be particularly effective in influencing the rate of staining if the first dye reaction occurs at or near the surface of the tissue section. The rate of dyeing may follow closely the rate of diffusion of the dye ion into the protein but also may depend upon the affinity of the dye for the protein and upon the various conditions of staining such as pH, temperature, ionic strength, and dye concentration of the dye bath. The physical state of the protein is important not only as it may interfere with movement of dye but also in other ways. Degree of orientation of the fiber has been shown to affect greatly the degree of dye binding by cotton (Preston and Pal, 1947). This effect has also been described for nylon since dyeing may be enhanced by “relaxing” the fiber with heat treatment (Fidell, Royer, and Millson, 1948). Thus, highly oriented fibers show less affinity for dye than less oriented ones. XII. THE REVERSIBILITY OF STAINING REACTIONS ; EQUILIBRIUM OF STAINING A N D OTHER FACTORS WHICH INFLUENCE STAINING It is important to stress that staining is a reversible reaction and that when the solution environment of tissue sections is changed, there is a corresponding alteration in the equilibrium concentration of dye within the tissue. Dye may then be lost to the solution or removed from it. It is possible to wash out the stain in a solution free of dye particularly if the p H is adjusted upward in the case of acid dyes or downward in the case of basic ones-pH regions which would favor dissociation of the dye-protein combination. Although the reaction may be reversed and dye washed from the stained protein structure, the extent and rate of “desorption” varies with the histological structure, with the dye and with the washing conditions. Washing from tissue in which the protein is densely packed is conceivably more difficult than where the protein is more dispersed particularly when the dye has aggregated upon binding. And, dye of great affinity for a particular protein will show greater fastness than another dye and is removed only


249

with difficulty (Gerstner, 1949). Conditions of p H which do not favor binding of acid or basic dye are most favorable for stripping the dye from the protein. As already mentioned, elevated p H is most conducive for decolorizing acid dyes and low pH basic ones. The effect of acidity or alkalinity of the washing medium on destaining is considered in the early review of Pappenheim (1901) and the more recent work of Stearn and Stearn (1928a, b). Reversibility of the staining reaction is important for another reason, namely the redistribution of dye so that final staining is relatively even in a given protein structure despite rapid staining or destaining procedures which might be expected to favor uneven localization of the dye. In the redistribution, dye ions are “desorbed” from one site and transferred to another more deeply placed one. The ease of desorption and transference determines the final uniformity of the distribution. I t is known in textile staining that the migrating or leveling power of dyes differs greatly. The leveling capacity of dyes influences the course of the reaction. Dye which satisfies surface sites without tending to shift to deeper regions delays or prevents the expression of full staining capacity of the protein. Such staining stands in contrast to that with dye which rapidly migrates and distributes itself uniformly as staining proceeds. I n the former case dyeing is uneven and slow in reaching equilibrium. Various procedures of gradual alteration in dyeing conditions are used in the textile industry to improve and hasten the leveling of dye. Of interest among these is that leveling is favored by p H regions at which the charge of the protein is not extreme. There are still other factors which operate in the staining reaction. Most of these are poorly understood and, therefore, are only briefly considered here. If the dyeing time is lengthy and the temperature is elevated, decomposition of the protein may set in. In some instances the dye has been said to have a catalytic effect on degradation of the protein (Lemin and Vickerstaff, 1947). Ionic exchange has been described for the staining of ligno-cellulose with methylene blue (Sarkar and Chatterjee, 1948) and may operate more widely. The problems of staining with dye aggregates or suspensions which are widely used in histology (for example, Congo red, azocarmine, and trypan blue) have barely been touched upon in this review. The profound influence of mordants, media other than water, the effect of specific ions and the competition between dye ions of mixtures for similar sites deserve special study. Finally, the influence of brief staining times on dyeing must be touched upon. In progressive staining low dye concentrations are employed and

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staining is allowed to proceed to equilibrium. Staining is more easily controlled under such circumstances where the time of staining is not a variable. If staining is not carried to equilibrium, then the time of immersion in the dye solution is an important factor in dye uptake. Short staining times at elevated dye concentrations are quite popular in many histological techniques. Since the rate of staining is greatest during the first few minutes of reaction, and levels off slowly thereafter, tissues may be effectively stained during brief immersion. The tissue is removed when the desired intensity of staining is reached or the tissue is overstained and then secondarily destained. The amount of dye taken up in a given time will depend on a number of factors, including the mobility of the dye ion in the tissue substrate, the speed of interaction, the leveling capacity, the affinity of the dye, and the conditions of staining. Consequently, the time of dyeing must vary with each dye. In order to compensate for individual differences in dyes the concentration of the dye may be changed or other conditions of staining varied. XIII. REFERENCES Abbot, E. B., Crook, H., and Townend, F. (1947) J . SOC.D y . Col., Bradford, 63. 462. Achard, J. (1935) 2. Zellforsch., 23, 573. Arner. Dyestuff Reporter. Proc. of Airtrr. Asso. Textile Chem. Col. (1918) drncr. Dyestuff Rep., 37, 149. Anson, M. L. (1946) Protein denaturation and the properties of protein groups. Advances in Protein Chem., 2. Atkin, R. W., and Douglas, F. W. (1924) J. Airzer. Leather Chem. Asso., 19, 528. Baker, J. R. (1945) Cytological Technique. Methuen Monog., London. Bancroft, W. D. (1914a) J. phys. Chem., l8, 1. Bancroft, W.D. (1914b) J . phys. Chem., l8, 118. Bancroft, W.D. (1914~) J. phys. Chem., 18, 385. Bancroft, W.D. (1915a) 1. phys. Chem., 19, 50. Bancroft, W.D. (1915b) J . phys. Chem., 19, 145. Bejdl, W. (1950) Mikroskopie, 6, 83. Bethe, A. (1905) Beitr. Chem. Physiol. Pathol., 6, 399. Bonin, W.,Frappier, J., and LararnCe, A. (1944) Rev. Canad. BioJ., C. R., 3. 481. Boulton, J., Delph, A. E., Fothergill, F., and Morton, T. H. (1933) J. Textile Inst., 24, 113. Bourne, G. (1951) Cytology and Cell Physiology, 2nd ed. Clarendon Press, Oxford. Briggs, T. R., and Bull, A. W. (1922) J. phys. Chem., 26, 844. Brown, R. B. (1901a) J. SOC.Dy. Col., Bradford, 17, 92. Brown, R. B. (1901b) J . SOC.Dy. Col., Bradford, 17, 125. Carlene, P. W., Fern, A. S., and Vickerstaff, T. (1947) J . SOC.D y . Col., Bradford, 0S, 388. Chapman, L. M., Greenberg, D. M., and Schmidt, C. L. A. (1927) J . biol. Chem., 72, 707.


25 1

Cohn, E. J. (1945) Blood and Blood Derivatives. Smithsonian Rep., Smithsonian Inst., Wash., p. 413. Cohn, E. J., and Edsall, J. T. (1943) Proteins, Amino Acids and Peptides. Reinhold Co., New York. Conn, H. J. (1940) Biological Stains. Humphrey Press Inc., Geneva, New York. Conn, H. J., and Holmes, W. C. (1928) Stain Tech., S, 94. Craig, R., and Wilson, C. (1937) Stain Tech., Za, 99. Dempsey, E. W., Bunting, H., Singer, M., and Wislocki, G. B. (1947) Anat. Rec., 98, 417. Dempsey, E. W., and Singer, M. (1946) Endocrin., 38, 270. Dempsey, E. W., Singer, M., and Wislocki, G. B. (1950) Stain Tech., 26, 73. Dempsey, E. W., and Wislocki, G. B. (1946) Physiol. Rev., 26, 1. Dempsey, E. W., Wislocki, G. B., and Singer, M. (1946) Anat. Rec. 96, 221. Dreaper, W. P. (1906) The Chemistry and Physics of Dyeing. London. Dubos, R. J. (1945) The Bacterial Cell. Harvard University Press, Cambridge, Massachusetts. Ehrlich, P. (1879a) Arch. Physiot., 166. Ehrlich, P. (1879b) Arch. Physiol., 571. Elod, E. (1933) Trans. Faraday SOC.,29, 327. Ender, W., and Miller, A. (1937) Melliand TextilDer., 18, 633. Fautrez, J. (1936) Bull. Histol. Appl., l8, 202. Ferry, J. D., and Morrison, P. R. (1946) Znd. Eng. Chem., 38, 1217. Ferry, J. D., and Morrison, P. R. (1947) J. Amer. Chem. SOC.,69, 400. Ferry, J. D., Singer, M., Morrison, P. R., Porsche, J. D., and Kutz, R. L. (1947) 1. Amer. Chem. Soc., 69, 409 Fidell, L. I., Royer, G. L., and Millson, H. E. (1948) Amer. Dyestuf Rep., 37, 166. Fierz-David, H. E., and Blangey, L. (1949) Fundamental Processes of Dye Chemistry. Interscience, New York. Fischer, A. (1899) Fixierung, Farbung, Bau des Protoplasmas. Jena. Fraenkel-Conrat, H. (1944) J. biol. Chem., 164, 227. Fraenkel-Conrat, H., and Cooper, M. (1944) J. biol. Chenz., 164, 237. French, R. W. (1930) Stain Tech., 6, 87. Gatenby, J. B., and Beams, H. W. (1950) The Microtomist's Vade-Mecum (Bolles Lee), Blakiston, Philadelphia. Gee, W. W. H., and Harrison, W. (1910) Trans. Faraday SOC.,6, 42. Gelmo, P., and Suida, W. (1905) Sitz. Akad. Wiss. Wien. Jan. Math.-nat. Kt. 114, Quoted from Pelet-Jolivet, 1910. Gerstner, H. (1949) Melliand Textilber., 30, 253; 302. Gierke, H. (1885) 2. m'ss-Mikr., 2, 13, 164. Gilbert, G. A., and Rideal, E. K. (1944) Proc. roy. SOC.,8182, 335. Gillet, C. (1889) Rev. gen. mat. cot., pp. 15, 189. Gillet, C. (1890) Rev. gen. mat. col., p. 339. Goldstein, A. (1949) 1. Pherm. exp. Therap., Pt. 11, 96, 102. Goodall, F. L. (1938) J. SOC.Dy. Col., Bradford, 64, 45 Goodall, F. L. (1947) Am. Dyestuf Rep., 36, 380. Grollman, A. (1925) J. biol. Chem., 64, 141. Halphen, G., and Riche, A. (1904) C. R. SOC.Biol., 140, 1408. Halphen, G., and Riche, A. (1905) Rev. gen. mat. cot., p. 200.

252

MARCUS SINGER

Harrison, W. (1Fi1) J. SOC.Dy. Col., Bradford, 27, 279. Harrison, W. (1948) J. SOC.Dy. Col., Bradford, 64, 248. Haynes, R. (1928) S t a h Tech., 8, 131. Heidenhain, M. (1902) Arch. ges. Physiol., SO, 115. Heidenhain, M. (1903) In : Enzyklopiidie mikr. Technik, 1, 335. Herrmann, H., Nicholas, J. S., and Boricious, J. K. (1950) J . biol. Chew., 184, 321. Hewitt, L. F. (1927) Biochem. J., 21, 1305. Highman, B. (1945) Stain Tech., 20, 85. Hodgson, H. H. (1933) 1. SOC.Dy. Col., Bradford, 49, 213. Hofmeister, F. (1891) Arch. exp. Path. Pharm., 28, 210. Holmes, W. C. (1929) Stain Tech., 4, 75. Ikeda, S. (1935) Folia Anqt. lap., 13, 141. Ikeda, S. (1936a) Folia Anat. Jap., 14, 107. Ikeda, S. (193613) Folio Anat. lap., 14, 175. Jones, R. McClung (1950) McClung’s Handbook of Microscopical Technique. Hoeber, New York. Kelley, E. G. (1939a) J. biol. Chem., l27, 55. Kelley, E.G. (1939b) J. biol. Chem., ia7, 73. Kelley, E. G., and Miller, E. C., Jr. (1935) J. biol. Chew., 110, 113. Kienle, R. H.,Royer, G. L., and McCleary, H. R. (1945) Aster. Dyestuf Rep., S4, 42.

Kindred, J. E. (1932) Anat. Rec., 63, 43. Kindred, J. E. (1935) Sta& Tech., 10, 7. Kitchener, J. A,, and Alexander, P. (1949) J. SOC.Dy. Col., Bradford, 6S, 284. Klotz, I. M. (1946) J. Amer. Chem. SOC.,68, 2299. Klotz, I. M., Triwush, H., and Walker, F. M. (1948) J. Amer. Chefit. Soc., 70,

2935.

Klotz, I. M., Walker, F. M., and Pivan, R. B. (1946) J. Amer. Chefit. SOC.,68,

1486.

Klotz, I. M., and Walker, F. M. (1947) J. Amer. Chem. SOC.,69, 1609. Knecht, F. (1889) Ber. dtsch. c h m . Ges., a,1120. Knecht, F. (1904) Ber. dtsch. chem. Ges., 87, 3479. Knoevenagel, H. E. A. (1911) 2. angew. Chem., 106. Langmuir, I. (1916) J. Amer. Chem. SOC.,36, 2221. Langmuir, I. (1917) 1. Amer. Chem. SOC.,89, 1848. Lemin, D.R., Vickers, E. J., and Vickerstaff, T. (1946) J. SOC.Dy. Col., Bradford, 62, 132.

Lemin, D. R., and Vickerstaff, T. (1947) J. SOC.Dy. Col., Bradford, 68, 405. Levine, N. D. (1939) Stah Tech., 14, 29. Levine, N. D. (1940) Stain Tech., 16, 91. Lewis, G. N.,and Randall, M. (1923) Thermodynamics and the Free Energy of Chemical Substances. McGraw-Hill, New York. Lilienfeld, L. (1893) Arch. Anat. PhySiol., PhySiol. Abf., 391. Lloyd, D. J., and Bidder, P. B. (1934) Tram. Faraday Soc., 81, 864. Loeb, J. (1922) Proteins and the Theory of Colloidal Behavior. McGraw-Hill, New York. Loeb, J. (1924) Proteins and the Theory of Colloidal Behavior, 2nd ed. McGrawHill, New York.


253

Magnus, W. (1903) Zellchemie. In: Encyklopadb mikr. Technik., 2, 1365. (See especially p. 177, Farbunganalytische Methoden). Mallory, F. B. (1944) Pathological Technique. W. B. Saunders Co., Philadelphia. Maneval, W. E. (1941) Stain. Tech., 16, 13. Mann, G. (1902) Physiological Histology. Methods and Theory. Oxford University Press. hlathews, A. (1898) Amer. J . Physiol., 1, 445. hlayer, F. (1934) Chemie der organischen Farbstoffe. Springer, Berlin. McCalla, T. M. (1941) Stain Tech., 16, 27. McCalla, T. M., and Clark, F. E. (1941) Stain Tech., 16, 95. Meyer, K. M. (1928) Melliand Textilber., 9, 573. Michaelis, L. (1900) Arch. mikr. Anat., 66, 558. Michaelis, L. (1901) Dtsch. med. Wchsch., 27, 219. Michaelis, L. (1911) Theorie des Farbeprozesses. In : Oppenheimer : Handb. d. Biochemie, Bd. 2. Michaelis, L. (1920) Arch. mikr. Anat., 94, 580. hlichaelis, L. ( 1926) Hydrogen ion concentration. Williams and Wilkins, Baltimore. Michaelis, L. (1947) Cold Spr. Harb. Symp. quant. Biol., 12, 131. Michaelis, L., and Granick, S. (1945) J . Am. Chem. SOC.,67, 1212. Miescher, F. (1874) Verh. Naturf. Gesellschaft, Basel, 6, 138. v. Mollendorff, F., and v. Mollendorff, M. (1924) Ergebn. Anat. Entw., 26. v. Mollendorff, W. (1923) Derm. Wchsch., 1417. v. Mollendorff, W., and Krebs, H. A. (1923) Arch. Amt., 97, 554. v. Mollendorff, F., and v. Mollendorff, M. (1924) Ergebn. Anat. Entw., 26. Mommsen, H. (1927) Folb Haematol., 54, 50. Naylor, E. E. (1926) A w r . J. Bot., 13, 265. Neale, S. M. (1933) J . SOC.C h m . Znd., 62, 88. Neale, S. M. (1946) Trans. Faraday SOC.,42, 473. Neale, S. M. (1947) J . SOC.Dy. Cot., Bradford, 63, 368. Neale, S. M., and Stringfellow, W. R. (1933) J . Textile Znst., !U, 145. Neurath, H., Greenstein, J. P., Putnam, F. W., and Erickson, J. 0. (1944) Chem. Rev., 34, 157. Nietzke, R. (1901) Farbstoffe. J. Springer, Berlin. Quoted from Mann (1902). Nishimura, T. (1934) F o l k A m t . Jap., l2, 357. Noble, E. I. (1945) J . SOC.Dy. Col., Bradford, 81, 328. Ochs, G. W. (1928) Folia Haematol., 37, 241. Pappenheim, A. (1901) Grundrisz der Farbchemie zum Gebrauch bei mikroskopischen Arbeiten. A. Hirschwald, Berlin. Pappenheim, A. (1917) Folia Haemtol., 92, 18. Pelet- Jolivet, L. (1910) Theorie des Farbeprozesses. Th. Steinkopf, Dresden. Peters, L., and Speakman, J. B. (1949) J . SOC.Dy. Cot., Bradford, 65, 285. Peters, R. H. (1945) J . SOC.Dy. Col., Bradford, 61, 95. Pfeiffer, H. (1929) P r o t o p l a m , 8, 377. Pfeiffer, H. (1931) 2. miss. Mikr., 48, 88. Pischinger, A. (1926) 2. Zellforsch., 5, 169. Pischinger, A. (1927a) Pfliiger’s Arch. ges. Physiol., 217, 205. Pischinger, A. (1927b) 2. Zellforsch. mikr. A m t . , 6, 347. Pratt, L. S. (1947) The Chemistry and Physics of Organic Pigments. Wiley, New York.

254

MARCUS SINGER

Preston, J. M., and Pal, P. (1947) J. SOC.Dy. Col., Bradford, 63, 430. Pulcher, C. (1927) Boll. Sac. ital. Biol. Sperm., 1, 223. Rakusin, M. A. (1928) Biochm. Z.,la,167. Kawlins, L. M. C., and Schmidt, C. L. A. (1929) J. biol. Chem., a,709. Rawlins, L. M. C., and Schmidt, C. L. A. (1930) J. biol. Chcni., 88,271. Rideal, E. K. (1950) Foreword to the Physical Chemistry of Dyeing (Vickerstaff) Imperial Chem. Ind., London. Robbins, W. J. (1923) Anw. J . Bot., 10, 412. Robbins, W. J. (1924) J. Sen. Pkysiol., 6, 259. Robbins, W. J. (1926) Univ. of Missouri Stud., 1, 3. Columbia, Missouri. Rose, F. L. (1935) Private Communication to Vickerstaff, 1950. Rose, R. E. (1942) Amer. Dyestuff Rep., 31, 204. Rowe, F. M. (1924) Colour Index. Sac. Dy. Col., Bradford, England. Royer, G. L., Zimmerman, C. L., Walter, H. J., and Robinson, R. D. (1948) T0.rtile Res. J., 18, 598. Sarkar, P. B., and Chatterjee, H. (1948) J . SOC.Dy. Col., Bradford, 64, 218. Schirm, E. (1935) I. fir&. Chem., la, 69. Schmidt, C. L. A. (1945) The Chemistry of the Amino Acids and Proteins. Charles C. Thomas, Springfield, Illinois. Schwalbe, C. G. (1907) Die neueren Farbetheorien. Stuttgart. Schwarz-Karsten, H. (1927) Dtsch. med. Wchsch., 65, 1820. Seki, M. (1933a) Folia Anat. Iap., 11, 377. Seki, M. (1933b) 2. Zellforsch. mikr. Anat., 18, 21. Seki, M. (1933~)2. Zellforsck. mikr. Anat., 18, 1. Seki, M. (1934) 2. ges. exp. Med., 94, 655. Seymour, R. B., Agnew, W., Crumley, J. A., and Kelly, A. J. (1948) A m r . Dycstuff Rep., 37, 689. Sheppard, S. E., Houck, R. C., and Dittmar, C. (1942) J. Phys. Chem., 46, 158. Singer, M. (1949) Proc. N . Y . Path. SOC., 90. Singer, M., and Morrison, P. R. (1948) I , biol. Chem., 176, 133. Singer, M.,and Wislocki, G. B. (1948) Anat. Rec., lOa, 175. Skinner, B. G.,and Vickerstaff, T. (1945) J. SOC.Dy. Col., Bradford, 61, 193. Sokolova, N. V. (1948) I . app1. Chem. (U.S.S.R.), 21, 966. Speakman, J. B.,and Clegg, H. (1934) I . SOC.Dy. Col., Bradford, 60, 348. Speakman, J. B.,and Elliott, G. H. (1943) J. SOC.D y . Col., Bradford, 59, 185. Speakman, J. B.,and Hirst, M. C. (1933) Trans. Faraday Soc., 29, 148. Speakman, J. B., and Peters, L. (1949) J. SOC.Dy. Col., Bradford, 66, 63. Speakman, J. B.,and Smith, S. G. (1936) J. SOC.Dy. Col., Bradford, 63, 121. Speakman, J. B.,and Stott, E., (1934) Trans. Faraday SOC.,30, 539. Speakman, J. B., and Stott, E. (1935) Trans, Faraday SOC.,31, 1425. Spiro, C. (1897) Uber physikalische und physiologische Selektion. Habilitationschrift. Strassburg. Stearn, A. E. (1931) I. biol. Chem., 91, 325. Stearn, A. E. (1933) J. Bact., 26, 21. Stearn, A. E.,and Stearn, E. W. (1924) Amer. J. Pub. Health, 14, 409. Stearn, A. E.,and Stearn, E. W. (1929) Stain Tech., 4, 111. Stearn, A. E.,and Stearn, E. W. (1930) Stairz Tech., I,17. Stearn, E. W., and Stearn, A. E. (1925) I . Bact., 10, 13.


255

Stearn. E. W., and Stearn, A. E. (1928a) Amer. J. Pub. Health, 18, 174. Stearn, E. W., and Stearn, A. E. (1928b) Stain Tech., 3, 81. Stearn, E. W., and Stearn, A. E. (1928~) Stain Tech., 3, 87. Stearn, E. W., and Stearn, A. E. (1929) Stain Tech., 4, 10.5. Steinhardt, J. (1940) Ann. N . Y. dcad. Sci., 41, 287. Steinhardt, J. (1942) I. Res. nut. Bur. Stand., a8, 191. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940a) J. Res. Ifat. Birr. S t a d . , 26, 519. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940b) Textile Res., 11, 72. Steinhardt, J., Fugitt, C. H., and Harris, M. (1940~) Amer. Dyestiif Rep., 29, 607. Steinhardt, J., Fugitt, C. H., and Harris, M. (1941a) J. Res. nat. Btw. S t a i d , 26, 293. Steinhardt, J., Fugitt, C. H., and Harris, M. (1941b) Atner. Dyestuff Rep., SO, 223, 250, 288. Steinhardt, J., Fugitt, C. H., and Harris, M. (1942) Anter. Dyestuf Rep., 31, 77. Steinhardt, J., and Harris, M. (1940) J. Res. nat. Bur. Stand., 24,335. Stockinger, L. (1950) Mikuoskopie, 6, 79. Sturm. K. (1935) 2. mikr.-anat. Forsch., 57, 595. Thomas, A. W., and Kelly, M. W. (1922) J. Amer. Chon. SOC.,44, 195. Tolstoouhov, A. V. (1927) Proc. N. Y. Path. Sac., 26, 147. Tolstoouhov, A. V. (1928) Stain Tech., 3, 49. Tolstoouhov, A. V. (1929) Stain Tech., 4, 81. Townend, F., and Simpson, G. G. (1946) J. Soc. Dy. Col., Bradford, 62,2, 47. Unna, P. G. (1928) Chromolyse. Aberhalden’s Handb. der Biol. Arbeitsmeth., Abt. V, pp. 1-62. Veller, E. A. (1948) J. a j p l . Chem. (U.S.S.R.),21, 1147. Vickerstaff, T. (1948) Rate of Dyeing. Imp. Chem. Ind. Technol. Monograph, No. 1, Manchester, England. Vickerstaff, T. (1949) Amer. Dyesfuf Rep., 36, 305. Vickerstaff, T. (1950) The Physical Chemistry of Dyeing. Imp. Chem. Ind., London. Weber, C. 0. (1894) J. Soc. Chem. Znd., 13, 120. Wislocki, G. B., and Singer, M. (1950) J. camp. Neurol., 92, 71. Wislocki, G. B., Singer, M., and Waldo, C. M. (1948) Aaat. Rec., 101, 487. Wislocki, G. B., Weatherford, H. L., and Singer, M. (1947) Anat. Rec., 99, 265. Wood, J. K. (1913) The Chemistry of Dyeing. London. Yasuzumi, G. (1933a) Folia Anat. Jap., 11, 267. Yasuzumi, G. (1933b) Folia Anat. Jup., 11, 415. Yasuzumi, G. (1934) Folia Anat. Jap., 12, 1. Yasuzumi, G., and Matsumoto, S. (1936) Foliu Anat. Jap., 14, 101. Zacharias, P. D. (1908) Die Theorie der Farbevorgange, Berlin. Zeiger, K. (1930a) Z . Wiss. Mikr., 47, 273. Zeiger, K. (1930b) 2. Zellforsch., 10, 481. Zeiger, K. (1938) Physikochemische Grundlagen der Histologischen Methodik. Th. Steinkopf, Dresden und Leipzig.


The Behavior of Spermatozoa in the Neighborhood of Eggs LORD ROTHSCHILD Department of Zoology, University o f Cambridge, Cambridge, England

CONTENTS I. Introduction ......................................................... 11. The Block to Polyspermy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Chemotaxis of Spermatozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. References ...........................................................

Page 257 258 260 263

I. INTRODUCTION The fact that only one spermatozoon normally participates in the fertilization reaction is one of the most striking and characteristic features of reproduction in many animal phyla. Monospermy implies that the fertilizing spermatozoon initiates a change in the egg surface such that other spermatozoa cannot fertilize or refertilize the egg. The nature of this block to polyspermy has been the subject of speculation for many years; but before these speculations can have any value it is necessary to try and find out, as quantitatively as possible, what the egg has got to contend with to prevent di-, tri-, and polyspermy. When an egg is placed in a suspension of spermatozoa, it is reasonable to assume that sperm-egg collisions take place. Apart from the reasonableness of this assumption several workers have mentioned in their papers that they have observed such collisions. It is natural to ask the question how many sperm-egg collisions occur ? The following treatment of this question, though defective in a number of respects, some of which will be mentioned later, is the nearest to a quantitative approach that has so far been found possible. Suppose that a container has in it a number of particles moving about in random directions. The number of particles per milliliter is n and their mean speed is C. If now a sphere of radius a is introduced into the container, the number of collisions 2,sustained by the sphere per second, is

Z=?ra2n?

(1)

Translating this result into terms of sea urchin eggs in suspensions of spermatozoa, the radius of a sea urchin egg (Psammechinus miliaris) is 50 microns, the mean speed of a sperm suspension is of the order of 200 microns per second at 18”C, for dilute suspensions, while the density n of the sperm suspension is under the control of the experimenter. If sea urchin semen (formerly called “dry sperm”) is diluted with sea water so that the concentration of spermatozoa in an egg suspension is lo6, lo6, 257

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or lo7 per milliliter, the number of collisions an egg will sustain per second is 0.16, 1.6, or 16. 11. THE BLOCKTO POLYSPERMY Although suggestions have on several occasions been made that the block to polyspermy is propagated over the egg surface at a high speed, in a fraction of a second, and is similar to an action potential in stimulated muscle, nerve, or plant cells, no experimental evidence has been adduced in support of this suggestion while some evidence exists in the contrary sense (Rothschild and Swann, 1949). The fastest post-insemination change which has so far been observed takes about 20 seconds to be progagated over the surface of the egg of P. miliaris at 18°C. This change in surface structure is most easily observed and its time course measured with dark ground illumination, though it is possible to see it with normal illumination or polarized light. Under dark ground illumination, the cortex of the egg scatters more light after this change has passed over the egg surface than before; the time relationships of this change in cortical structure are not inconsistent with the hypothesis that it is caused by the diffusion of a substance, mol. wt. + 10,0-20,000, derived directly or indirectly from the surface of the adhering sperm head, through the cytoplasm, the cortex of the egg being affected by this substance from the inside (Rothschild, 1949). I t can be shown that during the passage of this change only about half the number of spermatozoa colliding with the egg will in fact hit unaffected parts of the egg surface. This means that at sperm densities of 106, lo8, or 10’ per milliliter, an egg will sustain 1.6, 16, or 160 potential polysperniy-producing collisions during the passage of this cortical change. At a sperm density of lo8 per milliliter, which corresponds to an initial semen dilution of about 1 in 25,000, the incidence of polyspermy is very low, though not perhaps quite so low as the casual observer, who does not do counts, may imagine. Assuming for the moment that this “kinetic theory” of fertilization is not too wide of the mark, it is obvious either that the time relationships of the block to polyspermy are not similar to those of the 20-second cortical change, or that the probability of a sperm-egg collision being successful, in the sense that fertilization follows, is low. There are therefore four possibilities regarding the block to polyspermy and sperm-egg collisions : (1) that there is a high-speed block to polyspermy (a fraction of a second) and a high probability of a successful collision; (2) that there is a high-speed block to polyspermy and a low probability of a successful collision; (3) that there is a lowspeed block to polyspermy (10-20 seconds) and a high probability of a successful collision ; (4) that there is a low-speed block to polyspermy

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and a low probability of a successful collision. W e can dismiss the third alternative at once as, if this were correct, eggs inseminated with sperm suspensions of density lo7 per milliliter would on the average be more than lOO-spermic, which is not the case. W e can dispose of the first and second possibilities by means of the following experiment, which was carried out in collaboration with Dr. M. M. Swann. Spermatozoa were added to a vessel containing unfertilized sea urchin eggs so that the final concentration of the sperm suspension was 3 x lo6 per milliliter. (The concentration of eggs was less than one hundredth of this figure.) After the eggs had been in contact with this sperm suspension for 25 'seconds, the spermatozoa were suddenly killed but the eggs allowed to continue developing. Counts of monospermic, unfertilized and polyspermic eggs were made at the first cleavage stage. The results were: monospermic eggs, 85 per cent; unfertilized eggs, 13 per cent; polyspermic eggs, 2 per cent. In a second vessel exactly the same procedure was carried out, except that instead of killing the spermatozoa after they had bombarded the eggs for 25 seconds, more spermatozoa were added, bringing the final sperm concentration up to 3 X lo8. Counts at the first cleavage in this experiment showed the following percentages : monospermic eggs, 54 per cent; unfertilized eggs, 2 per cent; polyspermic eggs, 44 per cent. Now there are more than three times as many polyspermic eggs in the second experiment as there were unfertilized eggs in the first experiment. rn other words nearly half of the eggs which were fertilized in 25 seconds had not finished propagating their block to polyspermy in that time and became polyspermic because of the new sperm bombardment they received after the 25-second period was terminated. It may therefore be concluded that the block to polyspermy is relatively slow, of the order of 20 seconds, and that the low incidence of polyspermy is due in part to a low probability of a successful sperm-egg collision. This type of experiment, the novel feature of which centers round subjecting eggs to a known number of sperm-egg collisions, may enable the probability of a successful collision to be estimated, possibly both for heterologous and homologous fertilizations. Calculations however depend on the validity of the assumption that a suspension of spermatozoa can be treated analytically as if it were an assemblage of gas molecules. Spermatozoa undoubtedly do not collide with each other or with eggs elastically, but these factors are unlikely to interfere seriously with the analysis.

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.m...m... FIG.1. a. Movements of bracken spermatozoa in tap water. The circles indicate

where each track begins. Numbers at the beginning and end of each track refer respectively to time of start and duration of track in seconds. b. Movements of

111. CHEMOTAXIS OF SPERMATOZOA

The most serious objection concerns the possibility that a substance called Fertilizin or Gynogamone I, which attracts spermatozoa chemotactically, diffuses out of eggs of the same species. If this were so, the “kinetic” analysis, which assumes random sperm movement, might be seriously in error. The claim that chemotaxis occurs has been made for many years, though it is noteworthy that in her article on Fertilization in the 1946 Edition of the Encyclopaedia Britannica, Dr. Ethel Browne Harvey states (p. 189) that “The meeting of the egg and sperm is generally believed in animals to be by chance, not by chemical attraction.” Most workers, with the exception of Loeb (1914), have assumed that a substance which makes spermatozoa swim more quickly will act as an attractive agent. The argument runs rather like this: if a spermatozoon happens to be swimming in the direction of increasing concentration of

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J

bracken spermatozoa after insertion of pipette (diameter 30 microns), containing 1 per cent sodium L-malate in agar-tap water gel, into the same sperm suspension as in Fig. la. Numbers at the beginning of tracks indicate time in seconds after insertion of pipette.

the activating substance, it will swim more quickly and therefore get nearer the source of the substance. If a spermatozoon happens to be swininiing in the direction of descending concentration of Fertilizin, it will swim more slowly and therefore get less far from the Fertilizin source. This argument is fallacious as can be seen from the following rather childish example. Suppose that in a drop of water containing spermatozoa we suddenly create an area which is lethal to spermatozoa. For the purposes of the argument it is assumed that the poisonous substance does not diffuse out of the lethal area. At the beginning of the experiment the spermatozoa, swimming in random directions, are uniformly distributed in the drop of water. Whenever a spermatozoon enters the lethal region by chance, it is killed, that is to say its movement is slowed up a great deal, and it stays there. In due course, therefore, nearly all the spermatozoa accumulate in a region where they swim more slowly:

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conversely, they will in these circumstances be repelled from a region where they are stimulated to swim more quickly. One implication of this argument is that it might be better from the egg’s point of view to make spermatozoa swim more slowly, rather than more quickly. Although many workers have found that egg secretions increase the O2 uptake of sea urchin spermatozoa, Hayashi (1946) found exactly the reverse effect, that the addition of egg secretions decreased oxygen consumption. Dr. E. S. Guzman Barr6n (1949, p. 45) says that Hayashi’s “techniques of measurement of 0 2 uptake and of dilution were faulty,” but I ought to mention that I have confirmed Hayashi’s results this year, using the standard Warburg technique. A slowing-up action of Fertilizin, if it occurred, would hardly be a sensitive enough mechanism to account for the alleged cases of chemotaxis. An alternative mechanism, which contains the necessary ingredients to account for the variability of the phenomenon, i.e., some people have observed chemotaxis toward egg water while others have not, was suggested to me by some ,experiments of Vasseur (1950) on the effect of egg jelly and calcium ions on the 0 2 uptake of sea urchin spermatozoa. When, for example, calcium is suddenly added to a suspension, there is a sudden increase in 0 2 uptake followed by an exponential decline to about the original rate. If the 0 2 uptake of sea urchin spermatozoa is a reflection of their speeds of movement, this type of response,. with an exponential adaptation to a basal speed-Orthokinesis with Adaptation-may cause the spermatozoa to move up a gradient of the stimulating substance, while the intensity of the effect will depend, inter alia, on the rate of decline in response. If, for example, adaptation to the stimulus is very slow, the mechanism will be very feeble. Before investigating the alleged chemotaxis of sea urchin spermatozoa toward Fertilizin, I thought it advisable to examine the phenomenon in a case where it is known to occur, in fern spermatozoa. Pfeffer (1884) showed qualitatively that fern spermatozoa are positively chemotactic towards L-malic acid. This phenomenon has now been examined quantitatively (Fig. 1). I have noted that they are also attracted by D-malic acid and nialeic acid, but not by fumarate, or several of the other participants or near participants in the tricarboxylic acid cycle, such as succinate, acetate, oxalacetate, pyruvate, or lactate. None of these substances, including D- and L-malate and maleate, appear to have any effect on the velocities of fern spermatozoa with the possible exception of lactate, which may have a slight inhibitory action. Dihydroxymaleic acid, which is directly oxidized by peroxidase is, on the other hand, toxic ; but it is not a chemotactic agent. No such behavior is observed when sea urchin spermatozoa are subjected to a gradient of egg secretions.

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IV. CONCLUSION From what has been said, it seems doubtful whether chemotaxis, at any rate in the case of sea urchin gametes, is likely to make the “kinetic” approach to fertilization seriously in error. There remains the jelly round the egg in those cases where it has not been removed. This is a subject which we have been examining in recent months. The results have not yet been analyzed. V. REFERENCES Barrbn, E. S. G., Gasvoda, B., and Flood, V. (1949) Biol. Bull. Woods Hole, 97, 44. Harvey, E. B. (1946) Encyclobaedia Britannica, 9, 188. Hayashi, T. (1946) Biol. Bull. Woods Hole, 90, 177. Loeb, J. (1914) J. ex#. Zool., 17, 123. Pfeffer, W. (1884) Unter Bot. Inst. Tiibingen, 1, 363. Rothschild, Lord (1949) J. exp. Biol., 20, 177. Rothschild, Lord, and Swann, M. M. (1949) J . exp. Biol., 20, 164. Vasseur, E. (1950) Ark. Kcmi. Min. Geol., 1, 393.


The Cytology of Mammalian Epidermis and Sebaceous Glands WILLIAM MONTAGNA Departmerit of Biology, Brown University, Providence, Rhode Islarid CONTENTS

I. Introduction . . . . .. . .. . . . .. . . . . ... . . . .. . . . . . . . . . . ...... ....... . ... .. .. 11. The Epidermis .... . . ... .. . .. . . . . . .. ... .... ... .......... . ... . . .... . . . . 1. General Description . . . . . . . . . . . . . . . . . . . . . . . . ............. 2. Intercellular Bridges and Tonofibrils . . . . . . . . . . . . . . . . . . . . . 3. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Golgi Element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . 5. Lipids . . . . . . . . . . . . . . . . . . . . . ....................................... 6. Keratinization . 7. Sulfhydryl Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Basophilia . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Glycogen ...................................................... .. . . . .. 11. Phosphatase and Lipase . . . . . . . . . ................................ 12. Mineral Substances . . . . . . ................................ 13. Pigment . .. . . . . . . . 14. Mitotic Activity . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Sebaceous Glands ... . . .... . . . . . . . . . .. . . . . . . ...

4. Lipids

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.. . . . .. . . . . . . . . . . . . . . . . . . . 297 8. Peroxidase ................. 9. Phosphatases and Lipases . . . ...................... 10. Growth and Proliferation . ...... . .... . . ... . . . . . .. . .. ... . ... . . .. 298 ........ .. . ... ,....................... . . . ... 299 IV. References . . . . . . . . . .

I. INTRODUCTION During recent years, research in our laboratory has been directed toward mammalian skin. Attention was at first focused on the sebaceous glands, but it soon became evident that neither sebaceous glands nor other cutaneous appendages could be studied alone. It was found, for instance, that when the sebaceous glands of the mouse are eradicated with one percutaneous application of methylcholanthrene in benzene, they regenerate from the cells of the external root-sheath of hair follicles only if these

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follicles contain actively growing hair bulbs (Montagna and Chase, 1950). In addition, the epidermis of mice is much more resistant to injury by x-ray and/or methylcholanthrene, when the skin contains growing hairs, than when the hairs are in the club, or resting stage (Chase and Montagna, 1951). These findings and others draw attention to the skin as an integrated functional system. Although the literature which deals with the cytology, chemistry, and physiology of skin and cutaneous appendages is voluminous, there is a lack of harmony among the different findings. The study of the cytology of skin has been burdened by excessive nomenclature, disharmony, and hasty conclusions. This review considers only the cytology of the epidermis and of the sebaceous glands. The cytology of sudoriparous glands, of hair follicles (which deserve a thorough review in their own right) and of the dermis will be omitted. 11. THEEPIDERMIS

1. General Description Mammalian epidermis is composed of two principal layers : an inner stratum germinativum, which rests upon the dermis, and a horny superficial layer, the stratum corneum. The stratum germinativum has been called the mucous layer, stratum Malpighii, rete or mucus Malpigliii, rete mucosum, etc., by various authors. I n agreement with Cowdry (1932) and Hoepke (1927), stratum germinativum is to be preferred, since it is the germinal layer of the epidermis. It is composed of a lowermost layer of cells above the dermis, the stratum basale (stratum cylindricum of Kolliker), or basal layer, and a layer of variable thickness above the latter, the stratum spinosum, or spinous layer. The upper cells of the spinous layer show a progressive accumulation of granules, readily stainable with ordinary histological methods ; these cells form a layer called the stratum granulosum or granular layer. In the epidermis of the palms and soles, there is a hyalin layer above the stratum granulosum ; this layer is seldom colored by histological stains and was named stratum lucidum by Oehl (1857, cited from Martinotti, 1924). In the thinner epidermis of the general body surface, the stratum lucidum is seldom present. The outer cornified layer of the epidermis is composed of dead, flattened cells which when dissociated, resemble squamae (Kolliker, 1853). In the “pressure areas” of the skin, palms, and soles, the stratum corneum is very thick. The thickness of the epidermis is variable in different mammals and in different parts of the body of the same animals. In man it is relatively

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thick. Among the laboratory mammals, the normal epidermis of the mouse and the hamster is very thin and consists of a stratum germinativum two or three cells in thickness ; the spinous and granular layers are not distinct, and the stratum mrneum varies in thickness from 5 to 10 layers of cells. In the rat and guinea pig the epidermis is thicker and one can distinguish well-defined basal, spinous, and granular layers in the stratum germinativum. In the epidermis of the paws and digital pads of the cat, rat, guinea pig, and others, all the layers described, including the stratum lucidum, are present. In human epidermis, the cells in the basal layer are usually cuboidal or fusiform, although they may be columnar. Those of the stratum spinosum are polyhedral and become increasingly flattened as they ascend to the stratum granulosum (Schafer, 1912), where the cells are elongated horizontally. In the stratum lucidum and corneum the cells are flattened squamae. In histological sections the dermoepidermal junction of human skin appears as an undulating line. Epidermal cones and ridges (rete pegs) project into the dermis, enclosing between them highly vascular “dermal papillae.’’ When the epidermis is separated from the dermis, it becomes apparent that the epidermal cones and ridges seen in sections are in reality a series of branching ridges. The architecture of these epidermal ridges is more complex in skin areas which constitute “pressure areas,” palms and soles, than in the skin elsewhere in the body (Hoepke, 1927 ; Odland, 1950). In the normal skin of most laboratory mammals, such as the mouse, rat, and rabbit, epidermal ridges are usually lacking, and the dermoepidermal junction is fairly straight. The cells of the basal layer in human epidermis send into the dermis a number of delicate protoplasmic processes which provide a close union between the dermis and the epidermis (Schafer, 1912). Favre (1950) demonstrated in human skin that the spiral filaments of Herxheimer (vide infro) in the basal cells form radicles which project into the surface of the corium. The union between the basal cells and the corium is very intimate, and Martinotti ( 1914a,b), believed that delicate collagenic fibers from the corium are insinuated between the cells of the basal layer, and that the condensation of these fibrils forms a basement membrane. This concept, as we shall see below, is not correct. The cells of the basal layer are separated from the corium by a poorly defined basement membrane (Cowdry, 1932). Studies on the nature of the basement membrane by Herxheimer ( 1916), Laguesse ( 1919a,b), Born (1921), Frieboes (1920, 1921, 1922), Busacca (1922), Hoepke

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(1924), Martinotti (1924), and many others are not in agreement with one another. Hefxheimer (1916) believed that a translucent basement membrane is intimately associated with the protoplasmic processes of the basal cells which project into the dermis. Frieboes (1920, 1921, 1922) demonstrated that the basement membrane is actually a complex argyrophilic reticulum whose meshes contain the cytoplasmic processes of the basal epidermal cells. Studying this problem, Dick (1947) concluded that it is difficult to determine whether reticular fibers form an open meshwork with interstices into which the cytoplasmic processes of the basal cells fit, or whether the meshwork forms a series of fibrils which run up to the cells and fix them either by entering them, or by filling the spaces between their basal processes. Odland (1950), in an excellent reinvestigation of this problem, has demonstrated that the argyrophilic reticulum “forms a continuous meshwork of delicate fibrils. Where the meshwork is penetrated by basal epithelial processes, the constituent reticular fibrils are compacted to form a network of coarse strands compressed between adjacent cell processes.” Odland also observed that the dermo-epidermal junction is morphologically adapted to “variable shearing forces to which the skin is exposed.” In palms and soles, this adaptation is “reflected by the relatively long basal epidermal processes as well as by the extensive development of the reticular net between the cell processes.” Moreover, in such areas, “epidermal ridges and cones attain a greater depth.” In contrast, in the skin of thighs or abdomen, the epidermal ridges are shallower and the union of the epidermis with the dermal reticulum is less distinct. 2. Intercellular Bridges and Tonofibrils Schultze ( 1864) first described the characteristic cytoplasmic processes which appear to connect the cells in the stratum germinativum. These intercellular bridges give dissociated epidermal cells the appearance of small burrs, and for this reason they have been called “prickle cells” or %pinous cells.” Chambers and de RCnyi (1925) believe they have deinonstrated that the intercellular bridges are real protoplasmic exteiisions which connect adjacent cells, since in human epidermis the effect of injury to a single cell is quickly transmitted to other cells connected to it by protoplasmic bridges. In human skin intercellular bridges are best defined in the stratum spinosum, and they gradually become obliterated in the stratum granulosum (Shapiro, 1924). Roughly midway between two cells, each protoplasmic bridge possesses a spindle-shaped swelling, the node of Bizzozero (Bizzozero, 1871). Studies in other animals have revealed well-defined intercellular bridges and nodes of Bizzozero, particu-

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larly in the lip of the horse and ox, and in the epidermal lamina in the hooves of ungulates. Although the normal epidermis of the mouse does not contain visible intercellular bridges and nodes, these become visible in the skin of mice with avitaminotic dermatosis (Montagna, 195Oa), or in skin treated with such irritants as podophyllin and methylcholanthrene (Paletta et al , 1941 ; King, 1949). Epidermal cells contain in their cytoplasm delicate fibrils (first described by Ranvier, 1879) which appear to sweep from cell to cell by way of the intercellular bridges (see Shapiro’s, 1924, review of the literature). These fibrils, which in fresh or formalin-fixed frozen sections are anisotropic (Schmidt, 1937; Litvac, 1939), and which in paraffin sections are stainable with Heidenhain’s hematoxylin or with Mallory’s acid phosphotungstic hematoxylin, are the so-called tonofibrils of the epidermis. In human epidermis, tonofibrils are most distinct in the cells of the stratum germinativum ; they are lesI: clear in the stratum granulosum, and appear to be absent from the stratum corneum. The older literature which deals with these elements is reviewed by Weidenreich (1900), Rosenstadt (1910), Shapiro (1924), and Patzelt (1926). There is confusion concerning the nature and origin of tonofibrils, and much of it has stemmed from the discovery by Herxheimer (1889) of thick, undulating or spiral filaments in the cells of the stratum basale. Branca (1899) and Argaud (1914) considered the spiral filaments of Herxheimer to be intracellular fibrils. Favre and Regaud (1910a,b) and Regaud and Favre (1912), on the other hand, have stated that these structures are actually mitochondria of the basal cells, and suggested that the tonofibrils might develop from them ; Favre (1950) now denies that tonofibrils are related to the spiral filaments. Firket (1911 ) objected to such an interpretation and assumed that since the basal cells contain Herxheimer filaments as well as mitochondria, the spiral filaments must be young tonofibrils just formed from mitochondria. A different concept of the origin of tonofibrils was presented by Martinotti (1914a,b) who believed that they arise from a coalescence of cytoplasmic granules, and that they multiply by splitting longitudinally. Neither Martinotti nor other authors (Branca, 1899 ; Firket, 191l ) , who also considered that a multiplication of tonofibrils occurs by longitudinal splitting, actually observed this process. Studying the effect of podophyllin and methylcholanthrene on the skin of mice, King (1949) found that tonofibrils, which are scanty in the normal animal, undergo rapid hypertrophy and proliferation in skin rendered hypertrophied by these agents. Furthermore, tonofibrils are produced in spite of severe morphologic disturbances in the cytoplasm and nucleus of

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the epidermal cells. King suggested that tonofibrils are intimately associated with the process of keratinization, a concept shared by many authors (vide infra) . In summary, tonofibrils are structures which seem to extend from cell to cell by way of the intercellular bridges. Their origin, exact nature, morphology, and significance are unclear, although they seem to be associated with keratinization. Although often confused with tonofibrils, the spiral filaments of Herxheimer, as will be shown later, appear to be mitochondria. 3. Mitochowidria Before discussing the mitochondria in epidermal cells one must agree with Cowdry (1918) that “the terminology of mitochondria is unnecessarily complicated and confusing. The confusion has resulted from incoordination and from hasty individual action in elaborating new names, often only to discard them in a new paper in favor of some other.” In a discussion of mitochondria and their shapes it is profitable to recall the observations of Lewis and Lewis (1915) in living tissue culture cells “that every type of mitochondria is continually changing shape and may assume as many as fifteen or twenty shapes in ten minutes.” In this review the term mitochondria will be used to designate those intracytoplasmic elements which are revealed in fresh tissues by supravital staining with Janus green, and in paraffin sections of postchromed tissues stained with Regaud’s or Heidenhain’s hematoxylin. As implied above, confusion has arisen from the existence of recognizable mitochondria, spiral filaments of Herxheimer, and tonofibrils in the same epidermal cell. In the cells of the stratum basale of human epidermis, Herxheimer (1889) described thick, spiral filaments which he first considered to be intracellular lymphatic spaces. Later, however, he interpreted them as shrunken parts of the cell membrane resulting from fixation artifact (Herxheimer and Miiller, 18%) ; this concept was shared by Schultze ( 1896). Most subsequent authors, among them Kromayer ( 1892), Rabl (1897), Firket ( 1911), and Kollmann and Papin ( 1914), concluded that the filaments of Herxheimer are the forerunners of epidermal fibrils. Favre and Regaud (1910a,b and Regaud and Favre, 191’2) demonstrated that the filaments in the basal layer of human epidermis are revealed by Regaud‘s method for mitochondria and that Herxheimer filaments are coexistent with typical mitochondria. By lengthening the period of postchromation, Favre (1920a,b; 1924) demonstrated spiral filaments in the cells of the spinous layer as well as in the basal layer. In mitochondria preparations, Favre found that the nodes of

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Bizzozero are stained selectively together with mitochondria and spiral filaments, and concluded that the nodes of Bizzozero are a part of the mitochondria1 complex. This point he reemphasized in two recent papers (Favre, 1946, 1950). From a lifelong study of mitochondria in human epidermis, Favre reveals several significant facts. H e has convincingly demonstrated that Herxheimer spiral filaments are actually mitochondria, although not all mitochondria are in this form. While postchromation of skin for one month is sufficient to demonstrate the spiral filaments in the stratum basale, longer postchroming is necessary to reveal them in the upper strata. In palmar and plantar epidermis, the spiral filaments are larger and more numerous and show less polymorphism than in the general body skin. In both types of epidermis they are aligned parallel to the long axis of the cells. In cells of the basal layer, the spiral filaments form a bed of radicles which extends into the dermis by way of the basal processes. Spiral filaments are found throughout the stratum germinativum, but in the upper strata they become thicker and less numerous. In the stratum granulosum they show a progressive fragmentation, but even in the superficial cells of this layer, recognizable fragments are scattered among the keratohyalin granules. From these observations the author concluded that mitochondria are directly concerned with the process of keratinization, and that since this is so, a process of insensible keratinization begins in the basal layer. Mitochondria are very sensitive indices of cellular change and damage. In psoriasis, and in other inflammatory conditions, for example, the nuclei of the epidermal cells in the stratum germinativum are displaced distally and the fragmented and polymorphic mitochondria become subnuclear. In epidermal neoplasms, mitochondria become strikingly polymorphic, and the nodes of Bizzozero disappear. Favre ( 1950) illustrates basal-cell carcinoma in which the mitochondria resemble those of normal spinous cells while in squamous-cell carcinoma they resemble those in normal basal cells. Parat ( 1928) cautions that mitochondria1 preparations often give deceiving and unreliable results. In the skin of newborn rats, stained supravitally with Janus green, Parat found mitochondria whose appearance and distribution correspond to the spiral filaments described by Favre. Even in the stratum granulosum he found numerous flexuous mitochondria and batonettes. Fixed and stained preparations gave results which were comparable to those stained supravitally. Parat’s studies with supravitally stained material give greater credence to Favre’s belief that the spiral filaments are mitochondria.

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In summary, mitochondria are large and numerous in the basal layers of the stratum germinativuni ; in the stratum granulosum they become fragmented and dispersed. They are polymorphic, appearing either in the form of spiral filaments or batonettes. The work of Favre and Regaud gives abundant evidence that Herxhejmer filaments are mitochondria and that the nodes of Bizzozero contain mitochondria1 elements.

4 . Golgi Element Concerning the study of the Golgi apparatus in epidermis, the papers of Deineka (1912) , Cajal (1915), DaFano (1921), Tello (1923a,b), Ludford (1924, 1925), Cowdry and Scott (1928), and Parat (1928) are instructive. This puzzling organelle has been demonstrated almost exclusively with silver or osmium tetroxide impregnation methods. Cowdry and Scott (1928) and Parat (1928) appear to be the only ones who have stained the epidermis supravitally with neutral red for the study of the Golgi element. Ludford (1925) demonstrated the Golgi element in the cells of the epidermis of mice under normal and pathologic conditions with osmium tetroxide. H e described the Golgi apparatus in cells of the basal layer as a juxta-nuclear network or group of rodlets in the distal end of the cell (the mitochondria being heaped up at the proximal end). This precise polarity is lost in the upper cells of the spinous layer where the Golgi element is dispersed irregularly as are also the mitochondria. Golgi elements become fragmented and scattered at the onset of keratinizalion, and in cells laden with keratohyalin granules, they are no longer demonstrable with osmium tetroxide. Parat ( 1928), studying fresh epidermis of the newborn rat, stained supravitally with neutral red, found in the cells of the basal layer a supranuclear, compact mass of neutral red vacuoles which often descends along the sides of the nucleus (this is the “vacuome” of Parat). In the cells immediately above the basal layer, the system of neutral red stained vacuoles is as dense as in the basal cells; in cells nearer the stratum granulosum, the mass of vacuoles tends to become subnuclear and somewhat dispersed, but it remains always in the proximity of the nucleus. The mitochondria do not have such intimacy with the nucleus. Although the smaller vacuoles readily become colored with neutral red, the larger ones do so less easily. In the stratum granulosum, the masses of vacuoles become dispersed among the keratohyalin granules, and oftentimes keratohyalin granules are encircled by neutral red stained material. In the stratum granulosum, the larger vacuoles are located basally and along the sides of the nucleus, while the smaller ones are found apically. With continued keratinization the “vacuome”

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becomes more “rarefied” and disappears. This is in contrast with supravitally stained flexuous mitochondria1 filaments which are still present among the keratohyalin granules. Parat’s silver impregnation preparations show in the lower cells of the stratum germinativum a classical Golgi element which he described as a mass of compact rodlets and tubules. These structures, which comprise the “dictyosome,” have the same orientation as the “vacuome” ; ascending toward the surface cells they become dispersed (as also described ’by Cajal, 1915, Tello, 1923a,b, and Ludford, 1925). I n the stratum granulosum, scattered Golgi elements appear as “appendages” of the keratohyalin granules. These observations strengthen Parat’s belief that the Golgi element (vacuome or dictyosome) is involved in the formation of keratohyalin granules. Although it is not possible to determine from his work whether or not keratohyalin granules arise from the Golgi element, Parat has made an important contribution to the understanding of the nature of this organelle. He was the first cytologist to look for a Golgi net in living cells, and found instead a series of vacuoles. The subsequent work of Hirsch (1939), Worley (1944 ; see also Worley’s excellent review, 1946), Baker (1944, 1949), Cain (1947, 1949), Thomas (1948), and Palade and Claude (1949a,b) on the Golgi element in other cells has substantiated Parat’s observations and added to them. The classical Golgi net is an artifact which develops at the site of a system of spherules. One fact brought out especially by the school of Baker, and by Palade and Claude (1949a,b), is that Golgi vesicles are lipoidal in nature. Parat (1928) using Sudan I11 and Scharlach R found lipid droplets in the basal layer of the epidermis of the newborn rat, but riot in the cells of the more superficial layers. These granules, however, did not correspond to the “vacuome”; on the contrary, they seemed to coincide with granules which were supravitally stained with the “nadi” reagent (Parat interprets granules stained by the “nadi” reagent as lipids; this is an odd interpretation since according to Keilin, 1925, they should represent sites of cytochrome oxidase) . Nicolau ( 1911) demonstrated lipid granules in the basal layers of human epidermis, and Kollmann and Papin (1914) demonstrated osmiophobic but sudanophilic granules in the basal cells in the lining of the esophagus of the guinea pig. None of these authors, however, observed whether these granules correspond to the Golgi element. Montagna (1950b) studied the distribution of lipids in the epidermis of several laboratory mammals by coloring frozen sections with Sudan black. H e observed in the cells of the stratum germinativum a series of perinuclear lipid spherules which lie close to the nuclear membrane and usually cluster at the distal pole of

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the nucleus. The cells of the stratum granulosuni contain only barely visible lipid granules. In the epidermal cells of the monkey, phospholipid granules, demonstrated with Baker’s (1946) acid hematein test, are identical with the perinuclear sudanophil bodies. Cain ( 1949) demonstrated comparable phospholipid bodies in the skin of the guinea pig, an animal not studied by Montagna. In preparations impregnated with silver or with osmium tetroxide, rodlets or granules appear in the same general areas in the cells occupied by the lipid granules. The perinuclear lipid granules in the epidermal cells, then, are comparable to the “vacuome” of Parat ( 19289, and must, but not without caution, be considered either as the Golgi complex or a part of it. Since the name “Golgi apparatus” has come to denote a variety of things to cytologists, Baker (1950) proposed the name “lipochondria” for these lipoidal spherules which are stainable supravitally with neutral red and which apparently correspond to Parat’s “vacuome.” 5. Lipids In addition to the perinuclear lipids described above, the epidermis often shows lipid granules in the intercellular spaces. One is cautioned against too literal an interpretation of these intercellular lipids. In poorly fixed tissues, or in tissues kept in the fixative longer than one month, the number of intercellular lipid droplets increases pari passu with the decrease of intracellular lipids. The nodes of Bizzozero in frozen sections treated with Sudan black also show some lipoidal content. In some unpublished observations on the skin of mice simultaneously treated with methylcholanthrene and x-radiated with lo00 r, we find that four days after treatment, the perinuclear lipid bodies discussed above (Montagna, 1950a,b) become fragmented and diffuse. Furthermore, numerous lipid particles are found in the intercellular spaces, a situation which is not encountered in the normal skin of the mouse. On the other hand, from seven to twelve days after treatment most of the epidermal cells, which have become tremendously hypertrophied, contain so much lipid that they resemble sebaceous cells. These cells resemble those in the epidermis of mice treated with tar, described by Ludford (1925). Lipids in the stratum corneum were first described by Ranvier (1898). These lipids must come from three sources: (1) intrinsically from lipophanerosis in epidermal cells ; (2) from the secretion of sebaceous glands ; and (3) from the secretion of sweat (Levin e t d.,1940, and Mickelsen and Keys, 1943, have demonstrated lipids in sweat ; Hoepke, 1927, Bunting et al. 1948, and Bunting, 1948, have demonstrated lipids in the secretory cells of sweat glands). Unna and Golodetz (1909) have shown in human


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skin that the cells of the stratum spinosum contain a relative abundance of free cholesterol but a paucity of cholesterol esters. The stratum corneum, on the other hand, contains approximately equal amounts of free and ester cholesterol. These authors state that while the cholesterol secreted by sebaceous and sweat glands is easily oxidized, the intracellular cholesterol of the epidermis is not. Kvorning (1949), in an analysis of lipids secreted upon the face of normal subjects, finds only small amounts of cholesterol. The stratum corneum in skin areas which contain no sebaceous glands is also sudanophilic, and the lipids must be derived from lipophanerosis and from sweat, if sweat glands are present. I n the skin of biotin-deficient mice where the sebaceous glands are plugged (Montagna, 1950a), the stratum corneum is sudanophilic, as is also the case in the skin of mice where sebaceous glands have been completely eliminated by local applications of methylcholanthrene (Montagna and Chase, 1950). Since mice have no other skin glands, these lipids must come from the unmasking of bound lipids in the epidermal cells. However, the sudanophilia of the stratum corneum in skin deprived of sebaceous secretion is not homogeneous as in the skin of normal animals, but appears granular. When frozen sections of skin of man (Montagna et al., 1948), or hamster (Montagna and Hamilton, 1949), or of all the other mammals studied are treated with Nile blue sulfate, the stratum corneum is colored pink, indicating perhaps the presence of neutral lipids. The stratum corneum is always Schultz-positive (indicating the presence of cholesterol or its esters), it is colored with Baker’s acid hematein test for phospholipids, and it is brilliantly birefringent (after extraction with organic solvents the intense birefringence is partially lost, the residual birefringence being a property of keratin). I n summary, the demonstrable lipids in the stratum germinativum are principally the perinuclear sudanophil bodies. The nodes of Bizzozero are mildly sudanophilic. The stratum corneum contains histologically demonstrable cholesterol esters, and possibly also neutral fats and phospholipids.

6. Keratinization The process of keratinization in epidermis has interested cytologists since the early descriptions by Langerhans (1873) and by Ranvier ( 1879). In human epidermis Ranvier recognized a “keratogenic” layer which Unna later called the stratum granulosum. Ranvier called the granules of the stratum corneum “dlkidine en graine” and the content of the cells of the stratum lucidum “ilkidine. en flaques.” Waldeyer (1882) called the granules in the stratum granulosum “keratohyalin” granules. In the

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stratum granulosum, keratohyalin granules are usually aggregated at the poles of the nucleus. The granules may be stained with most basic dyes as well as with acid dyes such as Congo red, acid fuchsin, and “wasserblau.” Several morphologically detectable changes in the cells of the stratum granulosum accompany the formation of keratohyalin granules. There is a decrease in the number of mitochondria and Golgi elements; an apparent decrease and fragmentation of tonofibrils ; an apparent extrusion of nucleolar material ; and an increase in the cell volume. It is not known with certainty which cell component or components are responsible for the elaboration of keratohyalin granules. It is remarkable that a study which has engaged the talents of so many cytologists should have borne so few tangible results. That the keratohyalin granules develop at the expense of mitochondria is a thesis which has been pursued by Regaud and Favre since 1910. In his latest paper, Favre (1950) presented a detailed account of the metamorphosis of mitochondria in the different layers of the epidermis and concluded that since mitochondria are responsible for the elaboration of keratohyalin granules, the process of keratinization must actually begin, although imperceptibly, in the basal layer of the stratum germinativum. Parat (1928), on the other hand, implicated the “vacuome” or Golgi apparatus. Kollmann and Papin (1914) believed that keratohyalin granules represent transformed nucleolar extrusions. Ludford (1924) agreed in part with Parat and in part with Kollmann and Papin but stated that although the Golgi apparatus and the nucleolus are partially involved, the process of keratinization is essentially a function of the ground cytoplasm of the cell. Martinotti (1914a,b, 1915, 1921), in a series of studies based upon a battery of different stains applied to human skin, concluded that keratohyalin is formed from several sources : (1) epidermal fibrils by a process of “fibrillorhexis”; (2) from the ground cytoplasm, probably the basophilic granules in the cells of the stratum germinativum ; (3) from the nucleus by “karyolysis” ; and ( 4 ) from the cell membrane. Branca (1907) and Firket (1911) considered that keratinization begins upon the tonofibrils and then proceeds to the interfibrillar cellular substance and to the cell membrane. More recent literature (King, 1949) tends to support this view. Keratin can be demonstrated histochemically by the application of the Millon reagent or by the xanthoproteic test. Presumably, these tests are specific for proteins which contain tyrosine in their molecule, but actually a color reaction is given by nearly all proteins and phenolic compounds (Serra, 1946). Unna and Golodetz (1909) identified two types of keratin in epidermis :


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keratin A, which is insoluble in nitric acid and in a mixture of sulfuric acid and hydrogen peroxide, and keratin B, which is soluble in these reagents. According to Hawk et al. (1%7), the keratin molecule consists of closely packed polypeptide chains which are held together by the disulfide bond of cystine, the resistance to solvents and enzymes being associated with the close packing of the chains. The major portion of hair, horn, hoof, feather, nails, and the stratum corneum of the skin is made up of albuminoid proteins. The keratin of hair, nails, and other appendages contains from 3 to 5 per cent sulfur, while that of skin contains from 1 to 3 per cent, nearly all of which is cystine (Hawk ,et al., 1947). Keratin, according to Block and Vickery (1931), “is insoluble in dilute alkalies, in water and in organic solvents, and . . . on acid hydrolysis, yields such quantities of histidine, lysine, and arginine that the molecular ratios of these amino acids are respectively approximately as 1 :4 :12.” Wilkerson (1934) found that these amino acids are in a molecular ratio of 1 :5 :15. Wilkerson (1935) has shown further that the isoelectric points for keratin, hair, and nails are 3.70; 3.67; 3.78. He suggested that since the isoelectric points are practically the same and the basic amino acids are present in approximately the same molecular ratios, possibly the amino acids responsible for the acid groups are also present in a definite molecular ratio in these three chemically, physically, and embryologically related structures. These figures show the remarkable unity which exists among the keratins from different cutaneous appendages. If they are correct, they throw some doubt upon the conclusions of Martinotti (1914a,b, 1915, 1921) and others, who, on the basis of painstaking cytological studies concluded that there are several “different” types of keratins. It is pertinent here to mention the findings of Litvac (1939) from observations on skin cultured in vitro ; she found that in very young cultures all the epidermis is digested by pepsin and trypsin. In 10-day cultures, the young keratin is digested only by pepsin, and the mature keratin in 3-week old cultures is resistant to both enzymes. Astbury (1933) demonstrated that most mammalian keratins, in the normal state, give approximately the same x-ray diffraction diagrams, indicating a periodicity of 5.15 A, characteristic of the a form. When keratin is stretched, changing from a- to p-keratin, there is an extension of the polypeptide chains of about 100 per cent (Astbury and Woods, 1930). Alpha- and @-keratins seem to be stereoisomers corresponding to two different structures having similar molecular configurations. Derksen and Heringa (1936), studying the lip of the ox, Derksen e t d. (1938) the human nail, and Giroud and Champetier (1936) the chestnut of the

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horse and the hoof of the calf, all report the same x-ray diffraction pattern of a-keratin. Derksen and Heringa, in agreement with the findings of Astbury, report that these keratins when placed in warm water give the diffraction pattern of @keratin. These authors conclude that the polypeptide skeleton possessing the periodicity of 5.15 A is preexistent in the stratum germinativum even before keratinization begins, and that the tonofilks are probably the elements involved. Champetier and Litvac (1939) studying thick sections of hoofs from embryonic calves note that the birefringence of the thick stratum corneum (produced by a-keratin, or keratin B of Unna) is destroyed in sections placed in chilled 2 per cent potassium hydroxide. The keratinized cell membranes (keratin A of Unna), on the other hand, remain intact and are isotropic. After digestion with pepsin and trypsin the tonofibrils in the stratum germinativum are destroyed, but these enzymes do not digest a-keratin in the keratinized region. This is explained by the fact that the keratinized cell membranes (keratin A of Unna), which are resistant to proteolytic enzymes, protect the enclosed a-keratin. Studies of the x-ray diffraction patterns of untreated epidermis confirm the findings of the authors named above in that diagrams corresponding to a-keratin are obtained where the tonofibrils are present, either in the non-keratinized stratum germinativum or in the keratinized upper layers. They conclude that the tonofibrils are responsible for the characteristic x-ray diffraction pattern of a-keratin. Alpha keratin under pressure, or after exposure in the autoclave, is transformed to /3 keratin. X-ray diffraction diagrams of re!atively pure (extracted with KOH) membrane keratin (keratin A of Unna) are unlike those of either a or 3, keratin, and must represent something quite different. These papers present interesting data, but whether or not the tonofibrils represent stages in keratinization or a stage in the polymerization of keratin molecules, as stated by King (1949), has not been conclusively shown.

7. Sulfhydryl Groups The presence of sulhydryl groups in the epidermis is closely related to the process of keratinization. After treatment of tissues with sodium nitroprusside, there is a moderate coloration in the stratum germinativum, a strong reaction in stratum granulosum and lucidum, and no reaction in the stratum corneum. Kaye (1924) believes that this reaction in the skin is due to glutathione. Walker (1925) denies the presence of glutathione and suggests that the nitroprusside reaction is due to a substance similar to, or identical with the thermostable sulfhydryl constituent of muscle, since pretreatment of sections of skin with alkyl isothiocyanate abolishes


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the reaction. Giroud and Bulliard (1930, 1934, 1935), on the basis of their results with nitroprusside, conclude that there are two sulfhydryl substances involved in two types of keratinization : one soluble, which is probably glutathione, involved in soft keratinization in the skin ; the other, in much larger quantities, associated with hard keratinization. Chhremont and Frederic (1943), utilizing a new technique (modification of the prussian blue reaction, whereby the -SH groups reduce ferric ferricyanide to ferric ferrocyanide to give a blue color), repeated the observations on the distribution of -SH groups in the skin of guinea pigs and of man. In the stratum germinativum there is a delicate color reaction. I n the stratum granulosum the keratohyalin granules are always intensely colored as are also the “flaques d’C1Cidine.” The stratum corneum is completely negative. These authors deny the presence of two types of keratinization proposed by Giroud and Bulliard. Frederic (1949) using the prussian blue reaction, studied the distribution of -SH groups in x-radiated skin of guinea pigs. If the injury is not too grave and the stratum granulosuin is still present, it shows no diminution of histochemically reactive -SH substances, although the reaction in the stratum germinativum is much weaker. Control skin of non-irradiated areas in the same animal also shows a diminution of the reaction in the stratum germinativum. During regeneration and repair of the injury, the epidermis shows an augmentation in the intensity of the reaction. Bennett and Yphantis (1948) have recently synthesized a reagent, 1-4 (chloromercuriphenylazo) -naphthol -2, which is apparently specific for the demonstration of sulfhydryl groups. Mescon and Flesch ( MS ) have applied this reagent to thin sections of fresh and fixed human skin and obtained results essentially similar to those revealed by the nitroprusside method. They report good staining in the stratum basale, a less intense staining in the stratum spinosum and minimal or no staining in the stratum corneum. Staining in the latter is intensified by longer exposure of sections to the reagent. The nuclei of cells of the stratum germinativum stain quite well. The staining of the stratum corneum, however slight, is interesting since these authors have shown by direct chemical methods, in agreement with Rudall (1946) and Gustavson (1949), that human horny scales contain some free sulfhydryl groups. Two possible roles might be attributed to the sulfhydryl groups in epidermis: one, that they may be concerned with cell division and proliferation ; the other, that they may undergo a transformation to disulfide substances in the stratum corneum. Hammett (1931), by the application of benzyl mercaptan, a sulfhydryl containing compound, to the skin of mice,

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induced hypertrophy of the epidermis, accompanied by abundant mitotic activity and differentiation of a distinct stratum spinosum (normally not apparent in the epidermis of the mouse). H e inferred that the percutaneously applied sulfhydryl compound was responsible both for the accelerated mitotic activity and for epidermal differentiation. Admitting these facts, one must remember that both of these conditions can be induced by the application of almost any irritant. Concerning the second point, that sulfhydryl compounds may be concerned with keratinization, Litvac (1939) found that addition of cysteine to the culture media caused no acceleration of keratinization in epidermis cultured in vitvo.

8. Ascorbic Acid In conjunction with the studies on keratinization, Giroud, Leblond, and Ratsimamanga ( 1935a,b) have also studied the presence of ascorbic acid in epidermis. By the use of chemical and cytochemical methods these authors have demonstrated in the chestnut of the horse and in the hoof of the pig, as well as in the epidermis of the guinea pig, a considerable amount of ascorbic acid in the stratum germinativum, but only traces in the cornified layer. It is not without interest to find this correspondence between the distribution of this substance and that of the -SH groups in epidermis. 9. Basophilia The cells in the stratum germinativum are intensely basophilic when sections of skin are stained with basic dyes. The ground cytoplasm of the cells in the stratum granulosum is much less basophilic but the keratohyalin granules usually stain intensely. The stratum lucidum and the stratum corneum stain very weakly or not at all. Treatment of sections of skin before staining with a solution of ribonuclease buffered to pH 6.7 abolishes all the cytoplasmic basophilia (the basophilia of the nucleolus is also abolished) but the intact nuclei and the keratohyalin granules still stain clearly. This is presumptive evidence that the basophilia which is eliminated by the enzyme is due to ribonucleic acid. These observations can be extended also to the cells of the outer root-sheath of hair follicles. Dempsey et d. (1950) have shown that oxidation of sections of skin (frontal skin of the deer) with periodic acid before staining with toluidin blue, greatly enhances the basophilia normally present in epidermis. This induced basophilia does not correspond to the basophilia presumably due to ribonucleic acid. When nucleoproteins and/or acid mucopolysaccharides were destroyed by treating sections with hydrochloric acid and the sections then oxidized with periodic acid, and stained with toluidin blue, the epi-

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28 1

dermis showed no diminution of dye uptake when compared with sections oxidized with periodic acid without acid hydrolysis. Sections treated only with hydrochloric acid and stained without oxidation showed complete loss of cytoplasmic basophilia, but the keratinized areas were still basophilic. Since these facts indicate that acid groups other than nucleic acids or mucopolysaccharides exist in the epidermis, the authors concluded tentatively that the newly formed basophilia is related to the sulfur content of proteins. Oxidation of disulfide in keratin and of sulfhydryl groups in the stratum germinativum might conceivably lead to the formation of sulfonic acids, responsible for the induced basophilia. There are, then, in the stratum germinativum three biologically active substances : sulfhydryl groups, ascorbic acid, and ribonucleic acid. Ascorbic acid might mediate the transformation of -SH groups in the stratum germinativum to -S-S- groups in the keratinized area. Perhaps nucleic acid plays a role in protein synthesis (Caspersson, 1947), a role which might also be played by the -SH groupings.

10. Glycogen Whereas chemical analyses of the skin (Folin et al., 1927, and Calvery et a,!., 1946) show that it contains glycogen, this substance is not generally demonstrable by cytochemical methods in the normal epidermis of man and laboratory mammals. In human epidermis, occasional cells in the upper stratum spinosum and granulosum do have some cytochemically demonstrable glycogen (Montagna et al., 1948), but most of those epidermal granules which recolor the Schiff reagent are visible even after previous treatment of sections with saliva or diastase. These substances, then, may represent polysaccharides other than glycogen (this is in striking contrast with the epithelial lining of the oral and buccal cavities, and of the vagina, whose epithelial cells are usually laden with glycogen granules). It is possible that the high glycogen values obtained in chemical analyses of skin correspond not to the glycogen of the epidermis, but rather to the glycogen stored in the cells of the outer root-sheaths of hair follicles and the glycogen in the sudoriparous glands. In the skin of all mammals studied, the cells of the outer root-sheath, up to the level of the sebaceous glands, contain variable amounts of demonstrable glycogen (in man, Lombardo, 1907; Sasakawa, 1921 ; and Montagna et al., 1948; in the rat, Johnson and Bevelander, 1946; in the rabbit and other mammals, Bolliger and McDonald, 1949). Glycogen is particularly abundant in the heavy sheaths of thick hairs (in human skin), and it is absent from the sheaths of follicles which contain resting “club” hairs.

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In the skin of embryonic rats, Parat (1928) found glycogen in all the layers of the epidermis, except in the most superficial cells of the stratum corneum. In the basal cells it is present as large plaques, either infra- or supranuclear. Glycogen granules are numerous and large in the lower cells of the spinous layer; in the upper cells of this layer the granules decrease in size but they increase in number. I n the cells of the stratum granulosum, the whole cytoplasm shows diffuse glycogen content. Glycogen diminishes in the stratum lucidum and disappears in the stratum corneum. I n human embryos (Lombardo, 1907 ; Sasakawa, 1921) , during the first six months of fetal life, there is abundant glycogen in the epidermis and in all of the cutaneous derivatives. After-the sixth fetal month, glycogen diminishes in the epidermis and becomes restricted to those structures where it is found in postnatal life, i.e., in external root-sheaths of hair follicles and in the secretory epithelium of sudoriparous glands. I n an analysis of glycogen in skin under different pathologic conditions, these authors find that a stimulus, of whatever nature, inducing excessive epidermal proliferation causes a reappearance of glycogen in variable quantities. Under such conditions, there is thus an apparent reversion to the state observed during the first part of fetal life.

11. Phosphatases a d Lipme Gomori (1941) in his first report on the distribution of alkaline phosphatase in tissues, stated that with the exception of its capillaries, skin contains no demonstrable alkaline phosphatase. Bourne ( 1943) found strong phosphatase activity only in the sebaceous glands and hair follicles. Fisher and Glick (1947) described a slight amount of alkaline phosphatase activity only in the stratum granulosum of human epidermis. I n the epidermis of the mouse, Biesele and Biesele (1944) found an increase in epidermal alkaline phosphatase after treating the skin with methylcholanthrene. At best, then, there is a scant amount of alkaline phosphatase in the epidermis of all mammals studied. Unpublished observations on the distribution of alkaline phosphatase in the epidermis of the cat and rabbit show that the enzyme is present in irregularly scattered foci, usually around the pilosebaceous orifices. Thompson and Whittaker (1944), studying the skin of man and rat manometrically, have demonstrated two esterases. One is a specific active cholinesterase, the other, a non-specific aliphatic esterase. Cytochemically, neither cholinesterase nor non-specific esterases (lipase) have been demonstrated in the epidermis. Although lipase activity has been demonstrated by the method of Gomori (1946) in the sebaceous glands of man (Mon-


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tagna et d.,1948), mouse (Kung, 1949), and the hamster (Montagna and Hamilton, 1949) only small amounts were reported in the epidermis of man and hamster where it was confined to the stratum corneum. I t should be pointed out that considerable lipase (esterase) activity can be demonstrated cytochemically in the subcutaneous fat. One wonders from these scant data if the esterases described in slices of skin by Thompson and Whittaker may not correspond to the esterases revealed in the sebaceous glands and in the subcutaneous fat. 12. Mineral Substances

Epidermis normally contains minerals. These minerals have been studied both by chemical methods and by examination with dark field illumination of incinerated tissue sections. Using chemical methods, Suntzeff and Carruthers (1945) found that the average amounts of K, Na, Mg, and Ca per 100 mg. of human epidermis are 0.322, 0.122, 0.018, and 0.015 mg. respectively. MacCardle et d. (1941) studied the mineral content of human skin under normal and pathologic conditions by means of spectrophotometric analysis, expressing their values for Ca, Cu, Mg, I, P, and Zn in relative intensities of spectral lines. Relatively large amounts of Mg were found in normal skin. Study of skin by microincineration has made possible a partial understanding of the localization of these minerals. Unfortunately, specific identification of the individual elements is .not practical since nearly all of them leave a whitish ash, with the exception of iron whose ash is yellow to red. Furthermore, it is likely that after such treatment the original topographic relationship of these elements is disturbed. Since Na and K are the principal metals found in epidermis, it is reasonable to assume that the white ash residue represents both of them. Scott (1933) found in normal epidermis of man and other mammals a concentration of mineral ash in the distal-most layers of the epidermis. Cowdry and Andrew (1950) found large amounts of mineral matter in human skin, especially in the basal layer of the epidermis. The spinous cells contain visibly less ash than the basal layer, and ash is again abundant in the superficial layer of the stratum germinativum, and in the stratum corneum. The mineral ash content of the epidermis of the mouse, under normal and pathologic conditions, has been discussed in a review by Cowdry (1943). MacCardle et al, (1943) in an excellent account of mineral ash in human skin summarize their findings in the following way: “In the skin of infants under 1 year old, the stratum corneum, stratum granulosum and superficial spinous layer leave a bluish ash indicative of the presence of sodium and

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potassium, while the deeper spinous, basal, and dermal layers contain much white ash of calcium and magnesium. In persons from 10 to 50 years of age all layers of the skin are heavily mineralized with white ash except the basal layer, which leaves mostly bluish ash. The basal cells, as well as the spinous cells, in the skin of persons between the ages of 10 and 50 years all possess thick perinuclear mantles of calcium and magnesium, except the ‘clear cells’, whose ash is blue and whose nuclei contain large amounts of iron. In children between infancy and 10 years of age it is only the stratum corneum and stratum granulosum that are constant in their heavy deposits of white ash. The other layers may or may not be heavily mineralized. At all ages there are single clear cells more or less ashless scattered about in the spinous and basal layers. The nuclei of these clear cells contain an unusual amount of iron.”

13. Pigwent The five pigments responsible for the color of normal skin are melanin, in the deeper layers of the epidermis; melanoid, a substance allied, $0 melanin, allegedly found throughout the epidermis ; carotene, in the stratum corneum, as well as in the subcutaneous fat; and reduced hemoglobin and oxyhemoglobin in the blood vessels of the dermis. The relative abundance of these pigments in human skin has been studied spectrophotometrically by Edwards and Duntley (1939 ; see also brief review by Jeghers, 1950). Of the five, melanin is the only one which lends itself completely to cytological studies. Melanin is a yellow to black pigment which is related in some way to the metabolism of tyrosine. It is resistant to nearly all chemical agents and is not modified even by concentrated acids. It is, however, somewhat soluble in concentrated KOH, or NaOH, and is bleached by hydrogen peroxide, potassium permanganate, and other reducing agents. Melanin is an argentaffin substance, reducing ammoniacal silver nitrate without the intervention of a reducing agent, and it is easily impregnated by all histological silver methods (Fontana, Bielchowski, del Rio-Hortega, Achucarro, etc.) (see Lison, 1936, for a diagnostic characterization of melanin and other pigments). The problems of pigment and melanogenesis in skin have been reviewed by Becker (1927, 1948), Meirowsky (1940), Masson (1948), Rawles ( 1948), and Lerner and Fitzpatrick (1950). In normal human epidermis, melanin is present in varying amounts in the basal layers of the stratum germinativum, but it may be present also throughout the stratum spinosum, and rarely, in the stratum granulosum and corneum. When abundant, melanin granules are evenly and diffusely distributed in the cytoplasm


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of epidermal cells, especially in those of the basal layer; when less abundant, the granules form supranuclear caps. Pigmented dendritic cells occur in the epidermis couched between the basal cells, and resting against the basement membrane. These cells have been studied critically by Masson ( 1948), Billingham ( 1949) , and Zimmermann and Cornbleet (1948) in human skin, by Billingham and Medawar (1950) in the skin of guinea pigs, and by Chase et al. (1951) in the skin of the mouse. Billingham and Medawar consider that these cells form a definite and specific system which they have called the “epidermal glial system.” Since both dendritic cells and epidermal cells contain melanin granules, the presence of dendritic cells is masked in histological preparations. In scantily pigmented epidermis, dendritic cells are readily seen in silvered paraffin sections or under the phase contrast microscope (Chase et al., 1951). Dendritic cells can be demonstrated selectively by the application of the “dopaJJ (3-4 dihydroxyphenylalanine) reaction of Bloch ( 1917). This reaction was perfected by Laidlow and Blackberg (1932) and later modified by Becker (1935). After the use of dopa, the cytoplasm of the epidermal dendritic cells is stained selectively gray, grayish brown, or black, whereas the epidermal cells remain uncolored. The specificity of this technique is based on the assumption that an enzyme (dopa oxidase) present in the dendritic cells, but lacking in the epidermal cells, converts the substrate, dopa, into a dark pigment, dopamelanin (for critical comments on the specificity of this reaction see Rawles, 1948, and Lerner and Fitzpatrick, 1950). In naturally occurring melanogenesis, this same dopa oxidase presumably transforms natural chromogens in the cytoplasm of dendritic cells into melanin, Thus, any cell which is dopa-positive is considered as a cell which is capable of elaborating melanin and should, therefore, be called a melanoblast, with the exception of the granular leucocytes which are also dopa-positive. Those cells which contain melanin but which are dopa-negative are called melanophores, the assumption being that they have received their melanin from nearby melanoblasts. Masson believes that there is a sequence of centripetal maturation of melanin in the dendritic melanoblasts of human skin, the most distally located dendritic granules being the most mature. In some melanoblasts, whose cell bodies are clear and presumably contain only “premelanin,” there is a progressive accumulation of demonstrable melanin granules toward the ends of the dendrites. If the epidermal melanoblasts are the only source of epidermal melanin, this substance must be transferred from the melanoblasts to the epidermal

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cells (melanophores) . Masson’s view is that the melanoblasts are comparable to glandular cells, which secrete their product by virtually injecting it by way of their dendrites into epidermal cells, a process for which he has coined the term “cytocrine.” Billingham ( 1949), Billingham and Medawar ( 1950), and Zimmermann and Cornbleet ( 1948) also believe in the “cytocrine” concept of pigment distribution. Despite the plausibility of this, no one has actually observed the “cytocrine” process in mammalian epidermis. In our laboratory, when melanogenesis is induced by x-radiation or by carcinogenic agents in naturally non-pigmented epidermis of the mouse, fine melanin granules are found around the nucleus and coarse ones at the periphery of the epidermal cells. In addition, pigment granules are often found in the nuclei of epidermal cells. These observations suggest that the nucleus is implicated in melanogenesis. The deceptiveness of histological preparations and the fact that these cells have been injured, would not justify such a conclusion. Meirowsky et al. (1950) state that in epidermal cells “melanin first appears in the nucleolus and spreads to the linin frame work of the nucleus. It appears in intracellular vacuoles and finally involves the entire nucleus and the membrane. It leaves the nucleus by way of the chromidial derivatives.” These authors give a good summary of the main investigations which deal with melanin formation in the nucleus of epidermal cells. Danneel and Lubnow (1936) and Lubnow (1939) have an entirely different view of the formation of pigment. They deny altogether the existence of dendritic cells. They assert that pigment is formed within the epidermal cells in association with, or at the expense of, the Golgi element or “lipochondria,” since these elements disappear at the same pace as pigment granules appear and increase in the cells. It is difficult to evaluate the relative merits of these three concepts. The fact that most authors believe in the “cytocrine” method of melanin distribution does not make it valid. It is conceivable that all these proposed processes may play a role in melanization. Singularly significant contributions to the problem of melanogenesis, have been made by Rothman et d. (1946) and Flesch ( 1949). They have obtained from human epidermis and from homogenates of rabbit skin a water-extractable, heat-stable, dialyzable, non-protein-like sulfhydryl compound which inhibits melanin formation in the tyrosine-tyrosinase system. These authors suggested the possibility that this substance occurs normally in melanoblasts and inhibits the action of the enzyme on the substrate. Melanogenic stimuli such as sunshine, x-rays, heat, and inflammatory


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diseases would oxidize or destroy these sulfhydryl compounds of the epidermis, leaving the enzyme free to act upon melanin precursors. 14. Mitotic Activity

Epidermal cells possess one spherical nucleus (occasionally two). In flattened or fusiform basal cells the nuclei become ovoid or elongated. The basophilic nuclear membrane encloses a granular, or finely reticulated chromatin, one or more nucleoli, and a variable number of chromatin nucleoli. The nucleolus has a dense basophilic membrane, a homogeneous, delicately basophilic content, and one or more peripheral heterochromatic granules. In the basal layers of the stratum germinativum, nuclei are more basophilic than they are in the upper layers. The apparent scantiness of mitotic activity in epidermis has always puzzled histologists. It did not seem plausible that the few mitotic figures encountered in the stratum germinativum could provide an adequate mechanism for the replacement of cells lost from exfoliation of the stratum corneum. Andrew and Andrew (1949) believe that a transformation of lymphocytes into cells indistinguishable from those of the epidermis make up for the relatively low numbers of mitotic figures seen in normal epidermis. Although it is possible that such transformations may occur, it would seem unlikely that they are to be considered seriously as a major factor in epidermal growth and repair (vide infra) . Flemming (1884) observed mitotic division in the deepest three layers of the epidermis of the snout of the pig. In human skin, Patzelt (1926) found them principally in the basal layer, but occasionally also in the second layer, and Pincus (1927) asserts that division occurs only in the basal layer. Thuringer (1924)) studying the epidermis of the scalp, found mitotic activity in the basal layer and throughout the stratum germinativum. The number of mitotic figures is greater in the middle and outer one-third than in the deeper layers. In the epidermis of the human prepuce, Thuringer (1928) encountered the majority of mitotic figures in the stratum spinosum; only 10.75 per cent of all mitotic figures were found in the basal layer. Since the axes of mitotic figures in the basal layer appeared to be parallel to the basement membrane, Thuringer believed that these cells do not move into the stratum spinosum. In this study he found focal groupings of mitotic figures which suggested to him the presence of “growth waves.’’ Cowdry and Thompson ( 1944), studying the foot pads of mice, found maximal mitotic frequency in the proximal and middle third of the stratum spinosum. In the epidermis of the rabbit, stimulated topically with testosterone propionate, Montagna et al. ( 1949)

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also found mitotic figures more frequently in the lower layers of the stratum spinosum than in the basal layer. In agreement with Cowdry and Thompson, they concluded that in the basal layer, daughter cells may remain upon the basement membrane, or move up into the stratum spinosum. Thus, not only the basal cells but also those of the lower twothirds of the spinous layer must be considered as the germinal elements of the epidermis. The riddle of the scantiness of mitotic activity in the epidermis began to be solved by the work of Ortiz-Picon (1933) who found more abundant mitotic activity in mice killed at noon than in those killed a t night. Carleton (1934), also in the epidermis of the mouse, discovered a rhythmic mitotic activity with a maximum from 8 o’clock in the evening to midnight, and a minimum about noon. On the contrary, Cooper and Franklin (1940) found that the period of greatest mitotic frequency in mouse epidermis occurs at 10 o’clock in the morning, and that of least frequency at 10 o’clock at night, the number of mitotic figures in the morning being more than twice that at night. (These data agree essentially with those of Blumenfeld, 1939, on the epidermis of the rat.) Mice and rats are nocturnal, and their diurnal period of rest and sleep coincides with tissue repair. In human epidermis there is also a rhythmic mitotic cycle which, unlike that of nocturnal animals, is higher in the night hours than in the morning hours (Cooper and Schiff, 1938; Cooper, 1939; Broders and Dublin, 1939). The most thoroughgoing studies on mitotic activity in the epidermis of the mouse are those of W. S. Bullough (1946, 1948a,b, 1949a,b,c,d,e, 1950a,b; Bullough and Green, 1949; and Bullough and Van Oordt, 1950). In this series of papers, Bullough has studied not only the rhythmicity of epidermal mitotic activity, but also the physiologic factors which are responsible for the rhythmicity. In the diurnal cycle he found that in males, as well as in females, mitotic frequency extends from 10 A.M. to 4 P.M. with a peak at approximately 1 P.M. ; thus confirming that during bodily activity, the mitotic rate is low, and during sleep and rest it is high. Excessive muscular exercise is followed by an abnormal depression of the mitotic rate ; the same effect can be induced by extreme cold. In mice placed on starvation rations, mitotic activity drops considerably below that of normally fed controls. Since in these situations there is a drain of the sugar reserves in the body, it was suspected that an abundance of sugar might be a critical factor in the development of a high mitotic rate. It was demonstrated that injections of starch solution subcutaneously causes a marked rise in mitotic rate. Injection of a substance which inter-


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feres with phosphorylation, such as phloridzin or insulin, depresses mitotic activity, whereas injections of disodium hydrogen phosphate together with starch induce greater mitotic activity than starch alone. There is a remarkable drop in mitotic rate in animals which are subjected to ischemic shock, and it has been pointed out that although shock raises the blood-sugar level, there is a coincident fall in the total oxygen consumption, indicating that less sugar is being oxidized. These studies indicate that the number of resting cells entering division at any moment is in direct proportion to the amount of sugar being oxidized. In the studies of mitotic rate in normal males and females and in animals treated with androgenic and estrogenic hormones, it was shown that these hormones also have a profound effect upon cell division. I n a study of the diurnal mitotic rate of male mice during each of the first twenty months of life, Bullough found that the life of the animals can be divided into four periods. During infancy the animals are still growing and their epidermal mitotic rate is generally high. During mature age, the mitotic rate is lowered; during the middle age which follows, mitotic rate increases, and in senility, it is again reduced. Coincident with these changes, there are changes in spontaneous bodily activity. In female mice, H. F. Bullough (1943, 1947) and W . S. Bullough (1946, 1948a,b, 1949a,b,c,d,e, 1950a,b) have shown that the epidermis undergoes a cyclic growth similar to that of the reproductive organs, and that the epidermal mitotic rate can be stimulated by injections of oestrone. During the estrous cycle, peaks of mitotic activity are obtained in the third day of diestrous and again in early estrus. These peaks coincide with the normal diurnal peaks at approximately 1 P.M. W. S. Bullough, in comparing the effect of glycogen and oestrone, reported that while both substances are mitogenic, the maximum stimulation obtained by an increased glycogen concentration is small compared with the stimulation obtained with estrone. I n males and females, the duration of each mitotic division was established at 2% hours. While glycogen and androgen increase the mitotic rate, neither has an effect on the duration of each division, which remains constant at 2% hours. Estrogen, on the other hand, not only increases the number of divisions, but also reduces the duration of each to less than one hour. These points and others demonstrate the complexities encountered in a study of mitosis. Reports of mitotic indices which do not take into account diurnal periodicity, state of nutrition and activity, the sex, age, and general physiological status of the animal, as well as physical environment, are difficult to assess.

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111. THE SEBACEOUS GLANDS 1. General Description The distribution of sebaceous glands in man has been studied by Benfenati and Brillanti (1939). They are most numerous in the scalp, forehead, face, and chin, where there may be 400 to 900glands to each square centimeter of skin. Over the rest of the body there are fewer than 100 glands per square centimeter. They tend to be larger and more numerous in the midline of the body, especially of the back. They are lacking in the palms and soles and in the dorsum of the foot. The glands are largest where they are most numerous. In other mammals, sebaceous glands are widespread over the body skin, and they are largest and most numerous in the external auditory meatus and in the perianal region. Most sebaceous glands are appendages of the external root-sheath of hair follicles and open to the surface of the skin together with hairs. The size of the glands often varies inversely with the diameter of the hairs with which they are associated, the largest glands being found where hairs are small or wanting. This, however, does not always apply, since large glands may be associated with coarse hairs and vice versa, In addition to hair-associated glands, there are free sebaceous glands with excretory ducts opening directly onto the surface of the skin. In man, free sebaceous glands are found in the palpebrae (Meibomian glands), occasionally in the buccal mucosa, in the nipples (Perkins and Miller, 1926), in the preputium (Tyson’s glands), occasionally in the glans penis (De Sousa, 1931) and in the labia minora. In other mammals, aggregates of free sebaceous glands form preputial glands (in the rat and mouse), inguinal glands (rabbit), scent glands (shrew), and anal and circumanal glands (rodents, carnivores). The morphology, physiology, and growth of sebaceous glands have been reviewed by Schaffer (1927), Hoepke (1927), Pincus (1927), and Clara (1929). Sebaceous glands are holocrine, multiple acinar glands ; their general configuration is determined by their relative abundance (crowding), and by the nature of the dermis in which they lie (Clara, 1929). Whether or not sebaceous glands are associated with hair follicles, their fine morphology is similar. The details which follow are based primarily upon hair-associated glands, but they apply also to free sebaceous glands. The acini of the glands converge toward a common excretory duct which opens into the upper part of hair follicles. The stratified squamous epithelium of the excretory duct is continuous with the external rootsheath, which in turn, is continuous with the surface epidermis. The sebaceous acini are composed of closely packed, large, misshaped and

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moribund central cells, and small peripheral cells which are non-sebaceous, usually flattened, and resemble those of the epidermis. Most acini contain a lumen which is filled with sebum, a substance composed of lipids and cell debris. Not all the acini in the same glandular unit are in the same state of sebaceous maturity; in some, only the central cells are laden with lipid droplets, and there may be no lumen; in others there is a lumen, and lipid accumulation may extend to the periphery of the acinus. Just as keratinization Characterizes the end point in epidermal cells, lipid accumulation and fragmentation characterizes that of sebaceous cells.

2. Mitochondria Although mitochondria are discernible in sebaceous cells, there is very little information concerning them. They were described briefly by Nicolas et al. (1914), who believed that the lipid droplets first appear within the mitochondria1 filaments. As the lipid droplets become larger and coalesce, the accompanying mitochondria decrease in number. Such an interpretation is probable since Murray (1916) observed the formation of lipid globules within the mitochondria in the cells of a transplantable sarcoma of the guinea pig. Ludford (1925), in normal and pathologic skin of the mouse, found that mitochondria increase in numbers at the beginning of sebaceous lipid storage. With continued sebaceous accumulation they become fewer, and in mature cells there are only scattered fragments between the lipid droplets. Ludford did not find transition stages between mitochondria and lipid globules and is inclined to doubt such a relationship. Observations made in our laboratory on the mitochondria of sebaceous glands are not very informative on this point, but they are in agreement with those of Ludford. 3. Golyi Element Observations on the Golgi element were made by Ludford (1925) in the glands of the mouse, Bowen (1926, 1929) in the white inguinal glands of the rabbit, and Melczer and Deme (1943) in human sebaceous glands. The non-sebaceous cells at the periphery of the acini, and in the excretory ducts, contain perinuclear osmiophilic Golgi bodies. As lipid accumulation begins, the osmiophilic bodies increase in numbers, and the stored lipid droplets appear to develop within them. Melczer and Deme (1943) believe that the early lipid globules correspond to the Golgi internuni. As the lipid globules increase in size, the osmiophilic bodies become reduced to curved or crescentic rods or shells around them (Ludford, 1925). When the sebaceous cells attain maturity, there is practically no Golgi material left. The early lipid droplets in the center of the Golgi bodies are osmio-

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phobic, but with continued growth they show a progressive blackening with osmium tetroxide (Ludford, 1925). Short treatment of osmicated sections with turpentine or potassium permanganate bleaches the lipid droplets but not the accompanying Golgi elements (Ludford, 1925 ; Melczer and Deme, 1943). In the sebaceous glands of the cat (Montagna, 1949a), the non-sebaceous peripheral acinar cells contain perinuclear osmiophilic bodies which at the onset of lipid storage appear as osmiophilic ringlets with an osmiophobic center. In mature cells, the large, osmiophobic lipid globules are surrounded by a delicate osmiophilic ring. In the degenerating sebaceous cells, there are minute osmiophilic fragments among osmiophobic lipid masses. This sequence of events was also described in the sebaceous glands of the hamster (Montagna and Hamilton, 1949). The only difference between the results of Montagna and those of the authors named above, is that the lipid droplets in the sebaceous glands in his preparation were nearly always osmiophobic. This discrepancy might be explained by the fact that Montagna’s preparations were post-osmicated a shorter period of time. Although osmium is not a specific cytochemical reagent, these results are constant, and they are similar to those obtained with Baker’s acid hematein test for phospholipids. In the sebaceous glands of man (Montagna et al., 1949; Suskind, 1951), the hamster (Montagna and Hamilton, 1949), and the dog (Montagna and Parks, 1948), there are acid hematein positive elements whose shape and distribution resemble those of the osmiophilic Golgi bodies. Montagna and Chase (1950) observed a transformation of sebaceous cells from the cells of the external root-sheath in the skin of mice treated with methylcholanthrene. When the skin of the mouse is painted with one application of this carcinogen, its sebaceous glands become fragmented and disappear within four days. They will regrow within one week from the cells of the external root-sheath, if the follicles contain actively growing hairs. In frozen sections colored with Sudan black, the cells of the external root-sheath possess discrete perinuclear sudanophil bodies similar to those described in epidermal cells. An increase in the size and number of perinuclear sudanophil bodies in focal groups of cells in the external root-sheath indicates the first sebaceous transformation. Young sebaceous cells are distinguished from non-sebaceous cells only by their increased lipid content. Since the perinuclear sudanophil bodies seem to be identical with the osmiophilic granules and the acid hematein-positive elements, one must assume that they are the Golgi bodies or “lipochondria.” From these observations, it is apparent, in agreement with Ludford, Bowen, and Melczer and Deme, that the Golgi element is implicated in some way in the process of lipid storage in sebaceous cells.


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4 . Lipids Quantitative chemical analyses of sebum are few and contradictory. For example, while Butcher and Parnell ( 1947), studying the surface sebum of the scalp, found that cholesterol makes up as much as 10 per cent of the lipids, Kvorning (1949) in a much larger number of subjects found only very small amounts of this substance. Since it is not possible to obtain for chemical analyses sebum which is free of corneal exfoliation, chemical values must be viewed with caution. Melczer and Deme (1942) studied the distribution of lipids in human sebaceous glands employing histochemical methods of doubtful specificity. They demonstrated four concentric layers of lipids. At the periphery of the glands, they found fatty acids, probably oleic acid. The next layer inward contained eicosyl alcohol, allegedly formed by a reduction of fatty acids. I n the third layer, comprising the cells undergoing sebaceous breakdown, they found triglycerides, and in the sebum an admixture of fatty acids, neutral fats, and esters of arachyl alcohol. Neither Montagna et d. (1948) nor Suskind (1951) found such layering in normal human sebaceous glands. The progression of lipid accumulation in young sebaceous acini begins in the center, and proceeds toward the periphery as the acini become mature (Montagna and Noback, 1946b, 1947). I n moderately mature acini the central cells are laden with large lipid globules ; these diminish in size and in numbers toward the periphery, and the outermost cells may contain only small perinuclear sudanophilic bodies. In fully mature acini even the most peripheral cells may be inflated with large lipid spherules. These observations hold good for the sebaceous glands of all the animals studied. For an overall study of lipids, Sudan black is excellent since it colors lipids indiscriminately. Sudan I11 and IV, being less powerful lipid dyes, may occasionally be more instructive. For example, in the sebaceous glands from the costovertebral pigmented spot of the hamster, Sudan I V shows a concentric stratification of lipids, but Sudan black does not (Montagna and Hamilton, 1949). When frozen sections of skin of man, mouse, hamster, rabbit, cat, and rat are treated with secondary osmication, only the sebum in the excretory ducts of the sebaceous glands becomes blackened. The newly formed sebum in the center of the acini, and the lipid droplets in sebaceous cells, are osmiophobic. I n the glands of the rabbit, the lipids in the peripheral acinar cells are weakly osmiophilic (Montagna, 1949b). Thus, the old sebum, being osmiophilic, may differ chemically from the sebum just formed and from the lipid droplets in the sebaceous cells both of which are osmiophobic.

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Nile blue sulfate colors the mature sebum pink or red; the newly formed sebum is usually colored purple, and the sebaceous lipid droplets, pink. The peripheral cells contain purplish or blue lipid droplets. Although Nile blue is not a specific cytochemical reagent, a rose color usually indicates the presence of neutral lipids. One might surmise, then, that sebum and sebaceous cells contain appreciable amounts of triglycerides. Since the Fischler method for the demonstration of fatty acids is not a specific test (Lison, 1936), the results obtained with its use are of little value. Only the fact that in sebaceous glands the new sebum shows a positive reaction (blue-black), while the old sebum does not, is of some interest (Montagna and Hamilton, 1949). I n addition, when sections treated with this method are subsequently colored with Nile blue, the previously uncolored old sebum, as well as the discrete lipid droplets in the sebaceous cells, becomes pink. I n the skin of the rat treated with the Smith-Dietrich method for phospholipids, Montagna and Noback (1947) described a positive reaction at the periphery of the sebaceous acini. The more specific acid hematein test of Baker (1946; see also Cain’s critiques, 1947, 1950), indicates that phospholipids are present in the spongy cytoplasm of the mature and degenerating sebaceous cells, and in the sebum, as well as in the granules and rodlets which are comparable to the Golgi elements. These observations were made by Montagna and his associates in the glands of man, dog, cat, and hamster, and by Suskind (1951) in a large number of human sebaceous glands. It is not surprising that phospholipids are revealed in the sebum, since Engman and Kooyman (1934) in an analysis of surface lipids found that they contain slightly more than 1 per cent phospholipids. The application of the Schultz method for the demonstration of cholesterol or cholesterol esters shows a positive blue-green reaction, principally in the sebum. I n man (Montagna et al., 1948; Suskind, 1951), in the rat (Montagna and Noback, 1947), the dog (Montagna and Parks, 1948), and the hamster (Montagna and Hamilton, 1949), only the sebum and degenerating sebaceous cells become colored ; in the cat and rabbit (Montagna, 1949a,b) and in the monkey (Morisuye, 1950) a positive reaction is found throughout the mature glands. In the preputial glands of the rat, Montagna and Noback (1946b) found that only the sebum in the excretory ducts reveals digitonide crystals after treatment with digitonin. Free cholesterol combines with digitonin to form acetone-insoluble birefringent crystals. Digitonide crystals were not found in the gland of the general skin in the rat or other animals studied. Normal human sebaceous glands contain no free cholesterol but


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the stagnant sebum in comedones and in early acne cysts contains free cholesterol in abundance (Suskind, 1951) . When viewed under polarized light, sebaceous glands reveal variable amounts of anisotropic lipids. With minor exceptions, the distribution of these lipids corresponds to the color reaction obtained with the Schultz test. In human sebaceous glands, only the sebum shows consistent birefringence (Montagna et al., 1948). Suskind (1951) observed birefringent lipids in the glands of 30 out of 45 samples of human skin. In most of the 45 specimens the sebum in the pilosebaceous exits and in the excretory ducts was anisotropic. Suskind observed that those specimens in which sebaceous acini exhibit marked anisotropy also contain glands which give the most intense Schultz reaction. In the preputial glands of the rat (Montagna and Noback, 1946b) and in the sebaceous glands of the hamster (Montagna and Hamilton, 1949), only the sebum is consistently birefringent. In the glands of the rat, dog, and cat (Montagna and Noback, 1947 ; Montagna and Parks, 1948 ; Montagna, 1949a), birefringence is more extensive, being present in the degenerating and mature sebaceous cells as well as in the sebum. In these glands the sebum appears as a birefringent homogeneous mass ; in sebaceous cells birefringent lipids are in the form of spherules and acicular crystals. In the glands of the rabbit (Montagna, 1949b), the sebum is weakly birefringent, but the peripheral sebaceous cells contain abundant anisotropic spherules and acicular crystals. These birefringent lipids are not colored with Sudan IV, Nile blue sulfate, or Baker's acid hematein test. After short treatment with Sudan black, the spherocrystals are colored pink, but the acicular crystals either remain colorless or they are a light blue. With longer treatments in Sudan black the birefringent lipids become deeply colored and isotropic. After secondary osmication there is no birefringence in the peripheral lipids, but the blackened sebum shows increased anisotropy. Molisch fluid does not alter the peripheral birefringence, but enhances that of the sebum. The spherocrystals, but not the acicular crystals, are dissolved by 90 per cent alcohol ; 95 per cent alcohol or acetone removes all birefringent lipids. Heating to 60" C. destroys birefringence, but it reappears virtually unchanged as the sections are cooled to room temperature. Cooling to -2" C. increases the anisotropy of all sebaceous lipids, but the induced anisotropy is lost when the sections are rewarmed to room temperature. Anisotropy is not a physical property which allows a specific identification of lipids, but the manipulations mentioned above give it some characterization. The parallelism between birefringence and the Schultz

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test strongly suggests that the birefringent lipids in sebaceous glands represent esters of cholesterol. Sebaceous glands, under near-ultraviolet light (3600 A ) , emit a yellow to orange light which is usually comparable in distribution to the birefringence. Montagna and his associates (1946b, 1947, 1948, 1949a) have studied the autofluorescence in the sebaceous glands of several species of animals. The sebum in the exits of the sebaceous ducts emits a brilliant yellow light, the new sebum and the degenerating sebaceous cells emit yellow or white light of low intensity which fades toward the periphery of the glands. I t is possible that the anisotropic Schultz-positive cholesterol esters are responsible for the emission of the autofluorescent light. There is in sebaceous glands an orderly progression of events which leads to the formation of sebum. Sebum contains histologically demonstrable cholesterol esters, phospholipids, and possibly triglycerides. The blocked sebum in comedones and in sebaceous cysts contains, in addition, free cholesterol. Cytochemically demonstrable differences appear between sebum in the excretory ducts and sebum just formed in the center of the glands. 5 . Glycogen Adult sebaceous glands contain practically no glycogen. Lombard0 (1907) and Sasakawa (1921) found glycogen in the sebaceous glands of human fetuses up to six months; after this age it disappears. In the adult, under several pathologic conditions, sebaceous glands as well as the epidermis may reacquire glycogen. Montagna et al. (1949) found some glycogen in the center of young sebaceous acini, and only traces in the sebaceous cells of the human external auditory meatus. In the adult glands of all animals studied there was no glycogen.

6. Basophilia The cytoplasm of the non-sebaceous cells in sebaceous glands is strongly basophilic. This has been described by Montagna and associates in the glands of several animals. The cytoplasmic basophilia is abolished by ribonuclease and is apparently due to ribonucleoproteins. Young sebaceous acinar buds are intensely basophilic. In more mature acini the peripheral cells stain clearly with basic dyes ; the more central cells do not. There is a progressive centripetal loss of basic staining, and in cells laden with lipid droplets the spongy cytoplasm appears acidophilic.


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7 . Nadi Reaction Fresh or formalin-fixed sections of the preputial glands of the rat treated with the M-nadi reagent show numerous indophenol-blue granules, especially in the peripheral acinar cells (Montagna and Noback, 1946b). The cytoplasm of non-sebaceous acinar cells is virtually full of indophenolblue granules. In cells which contain lipid globules, indophenol-blue granules form a perinuclear zone. In mature cells engorged with lipids, only a few scattered granules can be seen in the cytoplasm. I n the sebaceous glands of the general-skin of the rat, there is only a trace of indophenol-blue reaction (Montagna and Noback, 1947). In the sebaceous glands of man and of laboratory animals the reaction is scant or absent. 8. Peroxidase Benzidine peroxidase, like the indophenol-blue reaction, was demonstrated in the preputial glands of the rat (Montagna and Noback, 1946b). It is particularly marked in the sebum-forming central acinar cells and in the sebum, the mature sebum being the most reactive. In all other sebaceous glands little or no peroxidase has been demonstrated.

9. Phosphatases and Lipass Alkaline phosphatase activity in sebaceous glands was first demonstrated in human skin by Bourne (1943). It was described in the glands of the rat by Johnson and Bevelander (1946), and by Montagna and associates in the glands of several animals. Although there are species differences, all the sebaceous glands observed contain some alkaline phosphatase. The enzyme is most abundant in the glands of the cat and rat and least abundant in those of the hamster. In the cat and rat enzyme activity is copious in the peripheral acinar cells and diminishes gradually toward the center, where the mature sebaceous cells show practically none. I n the hamster there are only traces of alkaline phosphatase activity in the outer peripheral cells. Some acid phosphatase activity has been demonstrated in the sebaceous glands of man and various animals. This enzyme is more labile than alkaline phosphatase, and much of it is probably inactivated during histological manipulations. The glands of the hamster show much acid phosphatase (Montagna and Hamilton, 1949). The reaction is strong throughout the acinus, but it is weak or absent at the periphery of the acini and in the center where the cells are undergoing degeneration. The newly formed sebum in the center of the acini indicates intense activity ; the old sebum shows less.

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In most sebaceous glands, including those of man, lipase activity, demonstrated by the method of Gomori, is present, but not abundant. It has been shown by Kung (1949) in the glands of the mouse, and by Montagna and Hamilton (1949) in the glands of the hamster. In the hamster, lipase activity, indicated by the presence of dark brown lead sulfide granules, is scattered sparsely throughout the acihar cells. I n those cells which are undergoing sebaceous degeneration, enzyme activity is maximal. The sebum, new and old, exhibits much lipase activity, being dark brown to black. In immature acini, which do not contain sebum, the central cells alone show copious lipase activity.

10. G r w t h and Proliferation Although holocrine glands must overcome the problem of replacing cells lost in secretion, mitotic activity appears scant in them. Some authors (Bizzozero and Vassale, 1887; Stamm, 1914; Kyrle, 1925 ; Schaffer, 1927; and others) described mitotic division in the peripheral acinar cells and believed that this provides for the replenishing of cells in the center. Others (Bab, 1904; Brinkman, 1912; Clara, 1929; and others) believed that mitotic activity occurs almost entirely in the epithelium of the sebaceous ducts at their junction with the acini, and they envision that the new cells formed there glide down into the body of the glands. Mitotic activity is, indeed, found in both places described above (Parnell, 1949), but the concept of cells gliding down into the fundus of the glands is unfounded. Sebaceous glands of mice injected with colchicine in the morning hours and sacrificed five hours later at approximately 1 P.M. show numerous mitotic figures in the peripheral cells. This not only demonstrates that in mice mitotic activity takes place in the peripheral cell, but also that it follows a cyclic rhythm similar to that of the epidermis. Bullough (1946) has shown that mitotic division in the peripheral sebaceous cells of female mice, as in the epidermis, is correlated with the ovarian cycle, being maximal in early pro-estrus and minimal in the first day of diestrus; injection of estrone induces a peak in mitotic activity. Androgens also stimulate sebaceous activity (Hamilton, 1941, 1947 ; Hooker and Pfeiffer, 1943 ; Ebling, 1948). Montagna and Kenyon (1919) induced mitotic activity in the sebaceous glands of the rabbit with topical application of testosterone propionate. Without the use of a mitotic block, they observed a sharp rise in sebaceous mitotic activity, regardless of the time of day or night the animals were killed. I n these animals, although mitotic divisions were also present in the cells of the excretory ducts, they were most numerous in the non-sebaceous peripheral cells


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of sebaceous acini. Mitotic division occurred also in sebaceous cells containing a moderate amount of stored lipids. Sebaceous glands of kittens treated similarly showed the same results. Mitotic division in the sebaceous glands of the mouse, rabbit, and cat, then, occurs principally in the periphery of the acini. Parnell (1949) confirmed this in the glands of the rat. The concept of cells gliding into old acini is also objectionable because it implies that sebaceous acini, once formed, remain unchanged. Montagna and his associates (1946a, 1947, 1949) described sebaceous acini in a constant state of change. Epithelial buds, which grow from the walls of the excretory ducts, develop sebaceous kernels in their centers and grow into new sebaceous units. As the new acini expand, they may encroach upon nearby acini, fuse with them, and become a part of larger units, In such sebaceous complexes, the periphery of the fused elements is still outlined by small epithelial cells and fibroblasts adhering to them, forming trabeculae which separate the sebaceous units into locules. Adventitial sebaceous acini also develop from tabs of non-sebaceous cells at the periphery of the acini, protruding into the dermis as appendages of the parent acini. Such lateral buds grow and engulf smaller adjacent ones. Sebaceous kernels may develop anywhere along the acini, outside or in, where there are accumulations of non-sebaceous cells. The fact that mitotic activity is abundant at the periphery of the acini, combined with the vicissitudes just described, militates against the static concepts usually described in textbooks of histology. Furthermore, the inherent growth dynamics of these glands is demonstrated by their quick regeneration from the cells of the external root-sheath of hair follicles, when the glands have been completely destroyed by chemical agents (Montagna and Chase, 1950). ACKNOWLEDGMENTS T h e work of Montagna on skin and cutaneous appendages was supported in part by the United States Public Health Service. T h e author expresses his gratitude to Miss Helen Melaragno for her invaluable help in the preparation of this review and t o his colleagues a t Brown University for their counsel and patience. IV. REFERENCES Andrew, W., and Andrew, N. V. (1949) A m t . Rcc., lO4, 217. Argaud, R. (1914) C. R. SOC.B i d , 77, 61. J - . Ceut., 110, 210. Astbury, W. T. (1933) Sci. P Y ~ J ~T7vcnl. , 126, 913. Astbury, W. T., and Wwds, H. J . (1930) N f l t ~ r cLolttf., Bab, H. (1904) Beiti-. l i l i i t . A/c,d., 1;rstschr. Seriator, 1. Baker, J. R. (1944) Quart. J . rrricr. Sci, 86, 1. Baker, J. R. (1946) Qztart. J . naicr. Sci., 87, 441. Baker, J. R. (1949) Quart. J . niicr. Sci., 90, 293.

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Baker, J. R. (1950) Proc. Liiztz. Soc. Lond., 162, 67. Becker, S. W. (1927) Arch. Drrm. Syph., 16, 259. Becker, S. W. (1935) Arch. Derm. Syph., 31, 190. Becker, S. W. (1948) Sp. Pztb. N . Y . Acad. Sci., 4, 82. Benfenati, A., and Brillanti, F. (1939) Arch. ital. D e r m Sifilo!~.b'rncrcol., 16, 33. Bennett, H. S., and Yphantis, D. A. (1948) J. A n w . Chem. Soc., 70, 3522. Biesele, J. J., and Biesele, M. M. (1944) Cancer Res., 4, 751. Billingham, R. E. (1949) J . Anet., 83, 109. Billingham, R. E., and Medawar, P. B. (1950) Heredity, 4, 141. Bizzozero, G. (1871) Centralbl. med. Wissensch., 9, 482. Bizzozero, G., and Vassale, G. (1887) I'irchozws Arch., 110, 155. Bloch, N. (1917) Arch. Derm Syph., U4, 129. Block, R. J., and Vickery, H. B. (1931) J. Biol. Chenz., 99, 113. Blumenfeld, C. M. (1939) Science, 90, 446. Bolliger, A., and McDonald, N. D. (1949) Ailst. J. e x p . Biol. J J I C ~ .Sci.,27, 223. Born, S. (1921) Derm. Z.,94, 324. Bourne, G. (1943) Qtrart. J. exp, Physiol., a,1. Bowen, R. H. (1926) Quart. J . nricr. Sci., 70, 193. Bowen, R. H. (1929) Quart. Rev. Biol.,4, 484. Branca, A. (1899) J. Anat. Physiol., 36, 764. Branca, A. (1907) J. Avat. Physiol., 43, 341. Brinkman, A. (1912) 2. ges. Ailat., Abs. 3 : Ergebn. Amt. Et~t.ro-Gcsck.,20, 1173. Broders, A. C., and Dublin, W. B. (1939) Proc. Mayo Clin., 14, 423. Bullough, H. F. (1943) J . Endocrirrol., 3, 280. Bullough, H. F. (1947) Nature, Lond., 169, 101. Bullough, W. S. (1946) Philos. Tratrs., BN1, 453. Bullough, W. S. (1948a) Proc. roy. Soc., B186, 233. Bullough, W. S. (1948b) Proc. roy. SOC.,BlM, 233. Bullough, W. S. (1949a) Nature, Lotid., 169, 680. Bullough, W. S. (1949b) J. e x p . B i d , 26, 83. Bullough, W. S. (1949~) J. exp. Riol., 26, 261. Bullough, W. S. (1949d) J . exp. Biol., 26, 287. Bullough, W. S. (1949e) Brit. J . Cancer, 3, 275. Bullough, W. S. (1950a) J. Eiidocrinol., 6, 340. Bullough, W. S. (1950b) Acta Pltysiol. Phartn. tried., 1.2, 357. Bullough, W. S., and Green, H. N. (1949) Nature, Lot~d.,164, 795. Bullough, W. S., and Van Oordt, G. J. (1950) Acta Eirdocrino?., 4, 291. Bunting, H. (1948) Anat. Rec., 101, 5. Bunting, H., Wislocki, G. B., and Dempsey, E. W. (1948) A n a t . Xec., 100, 61. Busacca, A. (1922) Arch. Dcwii. Syph., 141, 88. Butcher, E. O., and Parnell, J. P. (1947) J. Invest. Derni., 9, 67. Cain, A. J. (1947) Quart. J . P J I ~ C ~Sci., . 88,151. Cain, A. J. (1949) Oa-jord Sci., II, No. 2, 30. . 73. Cain, A. J. ( 1950) Biol. K E ~ ' .a6, . tTkIi7!. dftrdr.. 12, 127. Cajal, R. y. (1915) 7'r(16. Lab. I ~ r w s t hiol. Calvery, H. O., IJraize, J. H., and Laug, E. P. (1916) 'Phvsiol. Kelp.. 4, 195. Carleton, A. (1931) J . L'IJlfJt., 68. 251. Caspersson, T. (1947) Synrp. Soc. exp. Biol., No. 1, 127.

CYTOLOGY OF MAZI MALIAN EPIDERMIS

30 1

Chambers, R., and de Kinyi, G. S. (1925) Airier. J. Ailat., S6, 385. Champetier, G., and Litvac, A. (1939) Arch. Anat. micr., 36, 65. Chase, H. B., and Montagna, W. (1951) Proc. SOC.enp. Biol. Med., 76, 35. Chase, H. B., Rauch, H., and Smith, V. W. (1951) Physiol. Zool., 24, 1. ChCvremont, M., and Frederic, J. (1943) Arch. Biol., 64, 589. Clara, M. (1929) Ric. Morfol., 9, 121. Cooper, 2. K. (1939) J . Invest. Derm., 2, 289. Cooper, Z. K., and Franklin, C. (1940) Anat. Rec., 78, 1. Cooper, Z. K., and Schiff, A. (1938) Proc. SOC.exp. Biol. Med., 39, 323. Cowdry, E. V. (1918) Contribs. Embryol., No. 25, 8, 108. Carnegie Inst. Wash. (Pub. 271). Cowdry, E. V. (1932) Special Cytology. Vol. 1. Paul B. Hoeber, New York. Cowdry, E. V. (1943) Biol. Symp., 10, 131. Cowdry, E. V., and Andrew, W. (1950) J. Gerontol., 6, 97. Cowdry, E. V., and Scott, G. H. (1928) Arch Irist. Pasteur Tunis, 17, 233. Cowdry, E. V., and Thompson, H. C., Jr. (1944) Anat. Rec., 86, 403. DaFano, C. (1921) 7th Rep. Imp. Cancer Res. Fund, p. 67. Danneel, R., and Lubnow, E. (1936) Biol. Zbl., 66, 572. Deineka, D. (1912) Anat. Anz., 41, 289. Dempsey, E. W., Singer, M., and Wislocki, G. B. (1950) Stain Tech., a6, 73. Derksen, J. C., and Heringa, G. C. (1936) Gaz. mid. polonaise, 28 and 29 (cited from Derksen et al., 1938). Derksen, J. C., Heringa, G. C., and Weidinger, A. (1938) Acta m h l . Morph. Norm. Path., 1, 31. De Sousa, M. (1931) C. R. des sburzces, 108, 894. Dick, J. C. (1947) 1. Anat., 81, 201. Ebling, F. J. (1948) J. Endocririol., 6, 297. Edwards, E. A., and Duntley, S. Q. (1939) Amer. J . Aucat., 66, 1. Engman, M. F., and Kooyman, D. J. (1934) Arch Dlerm. Syph., 29, 12. Biol., 83, 349. Favre, M. (1920a) C. R. SOC. Favre, M. (1920b) C. R. SOC.Biol.,89, 351. Favre, M. (1924) C. R. SOC.Bwl., 91, 1220. Favre, M. (1946) Anrt. Derm. Syph., Paris, 6, 537 Favre, M. (1950) Ann. Derm. Syph., Paris, 10, 241. Favre, M., and Regaud, C. (1910a) C. R. Acad. Sci., UO,560. Favre, M., and Regaud, C. (1910b) Lyon mid. 22, 1132. Firket, J. (1911) Anat. Anz., 38, 537. Fisher, I., and Glick, D. (1947) Proc. SOC.exp. Biol. Med., 66, 14. Flemming, W. (1884) Arch. mikr. Allat., 29, 148. Flesch, P. (1949) Proc. SOC.exp. Biol. Med., 70, 136. Folin, O., Trimble, H. C., and Newman, L. H. (1927) 1. Biol. Chew., 76, 263. Frederic, J. (1949) d r c h . Biol.,60, 79. Friboes, W. (1920) Derm. Z., 91, 57. Friboes, W. (1921) Derm. z., S2, 1. Friboes, W. (1922) Arch. Derm. Syph., 140, 201. Giroud, A., and Bulliard, H. (1930) d r c h . klorpli. g i r l . exp., 29, 1. Giroud, A., and Bulliard, H. (1934) BuZZ. Histol. Tech. micr., 11, 169. Giroud, A., and Bulliard, H. (1935) Arch. Airat. wicv., 91, 271.

302

WILLIAM MONTAGNA

Giroud, A,, and Champetier, G. (1936) Bull. Sac. C h i . biol., Paris, U,656. Giroud, A.,Leblond, C. P., and Ratsimamanga, R. (1935a) C. R . SOC.Biol., lla, 321. Giroud, A., Leblond, C. P., and Ratsimamanga, R. (1935b) C. R. Asso. Allat. Montpelier, 1. Gomori, G. (1941) J. cell. comp. PhyJiol., 17, 71. Gomori, G. (1946) Arch. Path., U, 121. Gustavson, I

INTERNATIONAL REVIEW OF CYTOLOGY V1, Volume 1 (v. 1)

International Review of Cytology

International Review of Cytology