International Review of Cytology: A Survey of Cell Biology, Volume 11

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME11

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INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

Department of Anatomy Emory University Atlanta, Georgia

Department of Zoology King’s College London, England

VOLUME 11

Prepared Under the Auspices

of

The International Society for Cell Biology

ACADEMIC PRESS, New York and London 1961

COPYRIGHT @ 1961,

BY

ACADEMIC PRESS INC.

ALL RIGHTS RESERVED

N O PART OF THIS

BOOK MAY BE REPRODUCED I N A N Y FORM,

BY PHOTOSTAT, MICROFILM, OR A N Y OTHER M E A N S , W I T H O U T WRITTEN PERMISSION FROM T H E PUBLISHERS.

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Unitgd Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 17 OLDQUEEN STREET,L O N D ~S.W. N 1

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PRINTED I N T H E UiYITED STATES OF AMERICA

Contributors to Volume 11 R ~ M U LL. O CABRINI,Department of Pathology, Hospital Ramos Mejia, Buenos Aires, Argentina ALFREDJ . COULOMBRE, Department of Anatomy, Yale University School of Medicine, N e w Haven, Connecticut

K . KUROSUMI, Department of Anatomy, Gunma University School of Medicine, Maebashi, Japan* CHARLESB. METZ, Oceanographic Institute, Florida State University, Tallahassee, Florida C. M . POMERAT, Pasadena Foundation for Medical Research, Pasadena, California D. M . PRESCOTT, Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee

ELEANOR H . SLIFER,Department of Zoology, State University of Iowa, Iowa City, Iowa

J . J . WOLKEN, Biophysical Research Laboratory, Eye and Ear Hospital, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

* Present iddress : Department of Morphology, Institute of Endocrinology, Guiiiua University School of Medicine, Maebashi, Japan.

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CONTENTS CONTRIBUTORS TO VOLUME 11 ..............................................

V

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

xi

CONTRIBUTING AUTHORS IN

PREVIOCS \‘OLCMES

Electron Microscopic Analysis of the Secretion Mechanism K . KUROSUMI

I. Introduction ..................................................... I1. The Ultrastructure of Normal Secretory Cells ..................... I11. Special Cytology and Experimentally Induced Changes in Ultrastructure of Certain Secreting Cells ................................. I V. Discussion of the Secretory Mechanism ........................... References .......................................................

1 3

58 103 117

The Fine Structure of Insect Sense Organs

ELEANOR H . SLIFER

. Introduction ..................................................... . Tactile Organs ..................................................

125 126

Auditory Organs ................................................ Plate Organs .................................................... Gustatory Organs ................................................ Olfactory Organs ................................................ Ocelli ........................................................... Compound Eyes .................................................. Summary ........................................................ References .......................................................

134 143 143 151 151 156 158

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

. .

129

Cytology of the Developing Eye ALFRED J . COULOMBRE

I. I1. I11. I V. V. VI . VII . VIII.

Introduction ..................................................... Cornea .......................................................... Sclera ........................................................... Iris ............................................................. Choroid Coat .................................................... Ciliary Body ..................................................... Lens ............................................................ Retina ........................................................... References .......................................................

161 163 170 172 173 173 174 179 190

The Photoreceptor Structures J . J . WOLKEN I. I1. I11. I V.

Introduction ...................................................... The Plant Photoreceptors ........................................ The Animal Photoreceptors ....................................... Summary ........................................................ References .......................................................

195 196 205 215 216

Use of Inhibiting Agents in Studies on Fertilization Mechanisms CHARLESB . METZ

I. I1. I11. I V. V.

Introduction ..................................................... Fertilization-Inhibiting Action of Sperm and E g g Extracts .......... Fertilization Inhibitors of Fortuitous Origin ....................... Fertilization-Inhibiting Action of Antibodies ...................... Conclusions ...................................................... References .......................................................

219 220 229 240 248 251

The Growth-Duplication Cycle of the Cell D . M . PRESCOTT

I. I1. I11. IV.

Introduction ..................................................... Induction of Division Synchrony .................................. The Growth-Duplication Cycle ................................... Concluding Remarks ............................................. References .......................................................

255 256 262 279 280

Histochemistry of Ossification

R ~ M U LLO. CABRINI I. I1. I11. 1V.

Introduction ..................................................... Material and Methods ........................................... Types of Ossification ............................................ Histochemical Reactions .......................................... V. Histochemistry of Bone Formation ................................ V I . Histochemistry of Bone Resorption ............................... VII . Histochemistry of Ossification in Endocrine Disturbances and Other Experimental Conditions ....................................... References .......................................................

283 284 286 287 297 301 303 304

Cinematography. Indispensable Tool for Cytology

C. M. POMERAT I . Introduction ..................................................... I1. Organotypic Cultures ............................................ I11. Activities of the Nuclear Membrane and of the Nucleoli ............ References .......................................................

307 308 322 333

AUTHORINDEX...........................................................

335

SUBJECTINDEX...........................................................

346

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Alphabetical List of Contributors in Previous Volumes Numbers following authors’ names are Volume Numbers Alfert, Max, 3 Andrew, Warren, 5 Asboe-Hansen, G., 3 Baradi, A. F., 2 Beale, G. H., 6 Beatty, R. A., 3 Bell, L. G. E., 1 Berthet, J., 3 Best, Jay Boyd, 9 Bisset, K. A,, 1 Borghese, Elio, 6 Bourne, G. H., 2 Bowden, J., 7 Bowyer, Freda, 6 Brattgird, Sven-Olof, 3 Bretschneider, L. H., 1 Bridges, J. B., 8 Brown, R., 1 Bucher, Otto, 3 Caldwell, Peter C., 5 Chayen, J., 2 Chevremont, M., 2 Conway, E. J., 2, 4 Coons, Albert H., 5 Cornman, Ivor, 3 Couteaux, R., 4 Cowden, Ronald R., 9 Cummins, C. S., 5 Dalton, A. J., 2 Dan, Jean C., 5 Dan, Katsuma, 9 De Duve, Chr., 3 DeLamater, Edward D., 2 Dernpsey, Edward W., 3 de Robertis, Eduardo, 8 Dick, D. A. T., 8 Doljanski, F., 10 Dounce, Alexander L., 3 Doyle, William L., 2 Duguid, J. P., 9 Dziewiatkowski, D. D., 7 Ehret, C. F., 8 Engstrom, Hans, 7 Fankhauser, G., 1 Fawcett, Don W., 7 Fingerman, Milton, 8

Firket, H., 2 Gaillard, P. J., 2 Gay, Helen, 9 Glick, David, 2 Glynn, I. M., 8 Goldacre, R. J., 1 Gomori, G., 1 Gross, J., 6 Gustafson, Tryggve, 3 Hackett, David P., 4 Hammerling, J., 2 Haguenau, Francoise, 7 Hale, Arthur J., 6 Hall, David A., 8 Harman, John W., 5 Hershey, A. D., 1 Hirsch, G. C., 5 Hoch, Frederic L., 8 Hogeboom, George H., 6 Holter, H., 8 Hughes, Arthur, 1 Huskins, C. Leonard, 1 Hydkn, Holger, 3 Junqueira, L. C. U., 5 Kasten, Frederick H., 10 Kaufmann, Berwind P., 9 Kidder, George W., 1 Kopac, M. J., 4 Kuff, Edward L., 6 Kurnick, N. B., 4 Lansing, Albert I., 3 Lasnitzki, Ilse, 7 Lessler, M. A., 2 Lima-de-Faria, A., 7 Lowenstein, Leah Miriam, 8 McDonald, Margaret R., 9 Mahler, Henry R., 2 Makino, Sajiro, 6 Mandelstam, J., 5 Marshak, Alfred, 4 Marsland, Douglas, 5 Matoltsy, A. Gedeon, 10 Moe, Harald, 4 Monroy, A., 6 Montagna, William, 1 Mudd, Stuart, 2

xi

xii

CONTRIBUTING AUTHORS IN PREVIOUS VOLUMES

Miihlethaler, K., 4 Nagatani, Yoshimi, 10 Nath, Vishwa, 5, 9 Oberling, Charles, 8 O’Connor, R. J., 6 Pearse, A. G. Everson, 3 Pollister, Arthur W., 6 Pollister, Priscilla F., 6 Powers, E. L., 8 Prankerd, T. A. J., 5 Preston, R. D., 8 Rhodin, Johannes, 7 Rinaldini, L. M. J., 7 Rodyn, D. B., 8 Rosenberg, Th., 1 Rothschi,ld, Lord, 1 Rouiller, Ch., 9 Schechtman, A. M., 5 Schneider, Walter C., 6 Sharma, Archana, 10 Sharma, Arun Kumar, 10 Siebert, G., 6 Singer, Marcus, 1 Sjostrand, Fritiof S., 5

Sloper, J. C., 7 Smellie, R. M. S., 6 Spear, F. G., 7 Sutcliffe, J. F., 2 Swann, M. M., 1 Swift, Hewson, 2 Trowell, 0. A., 7 Vallee, Bert L., 8 Vendrely, C., 5 Vendrely, R., 4, 5 Vincent, W. S., 4 Wagge, L. E., 4 Waymouth, Charity, 3 Weiss, Leonard, 9 Weiss, Paul, 7 Wersall, Jan, 7 Wilbrandt, W., 1 Wilkinson, J. F., 9 Williams, Robley C., 6 Williams, Roy G., 3 Wilson, G. B., 9 Wischnitzer, Saul, 10 Wolman, M., 4 Wolpert, Lewis, 10

Electron Microscopic Analysis of the Secretion Mechanism K. KUROSUMI Department of Anatomy, Gztnma University School of Medicine, Maebashi, Japan* Page I. Introduction .................................................... 1 11. The Ultrastructure of Normal Secretory Cells .................... 3 A. The Nucleus ................................................ 3 B. The Cytoplasm ............................................. 12 C. The Cell Surface ........................................... 42 111. Special Cytology and Experimentally Induced Changes in Ultrastructure of Certain Secreting Cells .............................. 58 A. The Exocrine Pancreas ...................................... 58 B. The Gastrointestinal Mucosa ................................. 63 C. The Skin Glands ........................................... 78 D. The Thyroid Gland ......................................... 89 E. The Endocrine Pancreas and the Adenohypophysis ............ 95 IV. Discussion of the Secretory Mechanism .......................... 103 A. Ingestion of the Secretory Material .......................... 103 B. Synthesis of the Secretory Substance ........................ 106 C. Extrusion of the Secretory Product .......................... 111 Acknowledgments ............................................... 116 References ...................................................... 117

I. Introduction The term “secretion” usually means the action of cells by which a new substance is produced in the cell and then eliminated from it. The product may also be called “secretion.” To do this raw material must be taken into the cell from the surrounding media, especially from the blood stream. This is the first step of the whole process of secretion, that is, the ingestion of material. The second is the synthesis of the secretory substance, and the third is the extrusion of the product. The third process may be omitted in some cells such as ova, in which yolk is produced, stored, and finally consumed but not extruded. Thus in the usual sense, yolk is not called secretion ; but the formative process of yolk is very akin to the production of secretory substances in ordinary glands. Secretion is a very widespread phenomenon among various kinds of animal cells, not only in tissues specifically differentiated for this activity, namely, the glands-both exocrine and endocrine, but also in some of the mesenchymal and nervous tissues. For instance, the production of collagen by the fibroblast, antibodies by the plasma cells, heparin by the mast cells, and certain neurosecretory activities occurring in both hypothalamic and

* Present address : Department of Morphology, Institute of Endocrinology, Gurima University School of Medicine, Maebashi, Japan. 1

2

K. KUROSUMI

caudal neurosecretory neurons. Similarly, other phenomena of biological synthesis such as the daboration of yolk, fat, and pigment may also be considered as equivalent processes. Therefore, the elucidation of the secretory mechanism has long been an attractive subject of immense interest for most cytologists, because they expected that research on this subject might bring them to the final resolution of the vital cellular processes. Their vision, however, was severely limited while the only instrument was the ordinary light nlicroscope, which made the discussion of this subject rather imaginative and imperfect. A modern powerful instrument increasing our vision for minute objects, the electron microscope, appeared and soon became one of the most reliable weapons for biological research. Thus a considerable number of investigations with the electron microscope have revealed many important facts on a number of aspects of the mechanism of secretion. For example, the uptake of material for secretory activity, the knowledge of which was meager as obtained by means of light microscopy, has been visualized and explained from studies with the electron nlicroscope (Kurosumi and Kitamura, 1958; Kurosumi et al., 1959a). In spite of its superior resolving power, the electron microscope has some unavoidable disadvantages for its biological application. These are the relative difficulty in preparing the specimen, the extreme narrowness of the observable field, the lack of technique of selective staining, and the impossibility of observation on living material. Except the last, most of these defects may be overcome to some extent by the comparison of both images by electron and light microscopy with accompanying cytochemical tests. Moreover, biochemical assays using ultracentrifugation may aid the chemical identification of ultrastructural entities revealed by electron microscopy. The last of the defects above enumerated, however, is really unavoidable, and thus it affects most critically the interpretation of the functional significance of cell ultrastructure. For, in view of the cell secretion, this shortcoming in electron microscopy means that there is no control whatever indicating the time relationship of the continuous changes which have occurred in the cell. Such a weakness of electron microscopy has brought some confusion into the interpretation of the secretion mechanism. Above all, the formative origin of secretory granules is most intensely disputed but is unsettled: for example, in exocrine pancreatic cells Sjostrand and Hanzon ( 1954b), Haguenau and Berhhard ( 1955), Farquhar and Wellings (1957), Palay (1958), and Y. Watanabe et d. (1959) postulated that the zymogen granules were produced in or by the Golgi apparatus ; while Weiss ( 1953), Palade (1956a), Siekevitz and Palade ( 1958a), and I. Suzuki

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

3

( 1958) claimed that these originated from the ergastoplasm (endoplasmic reticulum). Moreover, Challice and Lacy ( 1954) stated that the zymogen granules were formed from mitochondria. Such a confused situation proves that electron microscope cytology is still in the cradle and numerous unknown facts as well as erroneous interpretations of electronic images are still left. Consequently, the final resolution of this problem should be left till later, after a more extensive accumulation of various findings, which might be our present task. Rather recently a review on the niorphology of secretion was presented by Palay (1958), but the results of electron microscopy on which the discussion was based were restricted to a few varieties of gland cells, and the discussion was therefore somewhat affected. This review is an attempt to survey the ultrastructure of secreting cells as widely as possible and also their probable significance in function, but it is impossible to give a full review of the vast literature on this subject. Hence, it will be restricted to some of the main results of this field, especially those published by Japanese investigators. 11. T h e Ultrastructure of Normal Secsetory Cells

A. THENUCLEUS 1. General Morphology of the Nucleiis Each secretory cell contains one or more nuclei. In some glandular cells, the multinucleation is rather common (Fig. 1A) ; for example, the parietal cell of the gastric gland and the basal cell of the human eccrine sweat gland are occasionally binucleated, and the hepatic cells are often multinucleated : we observed four nuclei in a single hepatic cell by electron microscopy. The follicular epithelium of ovarian follicles which may secrete the follicular fluid is known as a syncytium, having many nuclei (Kurosumi, 1957c), some of which show a deep indentation or constriction suggesting a mechanism of amitotic nuclear division. The entire form of the nucleus is also variable. In most serous gland cells such as the body chief cells of the stomach and the pancreatic acinar cells, the nucleus is usually seen to be regularly spherical or in the shape of a slightly flattened ellipsoid. The sweat gland cells of human beings belong to this type. Mucous or mucoid secreting cells, however, possess rather irregular nuclei. Examples of this type may be given in the mucous neck cell and the surface epithelial cell of the stomach (Kurosumi et al., 1958b), as well as in the goblet cell of tracheal (Rhodin and Dalhamn, 1956) and intestinal mucosae (Hartman et al., 1959). The nuclei of cells in the pig’s carpal organ ( a special type of the eccrine sweat glands) are

FIG.1. Electron micrographs of nucl'ei of the hepatic cells of a snake (Natriz tigrina tigrilza). Arrows indicate pores of the nuclear envelope. A. Survey picture of nuclei of a binucleated liver cell. ( X 7500.) B. Enlarged view of the portion outlined in the above picture. N, nucleoplasm; NE, nuclear envelope composed of double membranes studded with particles ; M t , a mitochondrion ; Er, rough-surfaced endoplasmic reticulum (ergastoplasm). ( X 33,000.) C. A nucleus containing a dense inclusion body ( I ) . N1, nucleolus. ( X 8500.) (N. Watari and K. Kurosumi.)

ELECTRON MICROSCOPIC A N A L Y S I S

OF SECRETION

5

also characterized by their irregular form (Kitamura, 1958). Such an irregularity of the nuclear outline has been interpreted by some light microscopists as a result of compression by secretory granules. In some cells such as the gastric surface epithelium, however, irregularly lobated nuclei are prominent, although no secretory granule is found in the perinuclear zone. Accordingly, the irregularity in the outline of the nucleus cannot be explained by the compression of any formed elements in the cytoplasm. Nor is the irregularity presumed to be caused by the artificial shrinkage effect during the entire course from the fixation to the embedding, because light microscopic observations of the same cell type with different preparatory procedures indicate the same tendency for the nucleus to be irregular. Thus in some secretory cells the irregular outline of the nucleus may be normal and an essential characteristic of such cells; further it may be a means of cell identification in electron micrographs. As a tentative speculation, it may be considered that the irregular folding of the nuclear surface may make possible an active nucleocytoplasmic interaction by providing much greater surface areas of the nuclei. The position of the nucleus depends on the shape and functional condition of the cell. In exocrine gland cells, the nucleus is usually situated in the basal cell region, because the apical part of the cell is occupied by secretory products. When the secretory substance has been depleted, the nucleus is localized at the center of the hypotrophied cell. In the case of some endocrine cells, for instance the pancreatic islets, the nucleus is situated at about the center of the cell.

2. The Nuclear Envelope or Karyotheca The nuclear membrane seen by light microscopy is a simple thin membrane, but in electron micrographs it is observed to consist of many membranous components and associated structures such as holes, attached particles, and so on (Fig. 1B). Hence the term “nuclear envelope” or “karyotheca” may be more suitable than the term “nuclear membrane” for such a complicated membrane system. High resolution electron microscopy has been able to demonstrate the double structure of the nuclear envelope in a variety of animal cells (Callan and Tomlin, 1950 ; Bairati and Lehmann, 1952 ; Hartmann, 1953 ; Afzelius, 1955, etc.) . In the exocrine pancreas cells, however, Sjostrand and Hanzon (1954a) observed that the nuclear membrane is a smooth single membrane but a single a-cytomembrane is associated parallel to it. The a-cytomembrane adjacent to the nuclear membrane directs its smooth side to the latter. The description of the nuclear envelope as being a double structure means that the single nuclear membrane and the associated a-cytomem-

6

li. KUROSUMI

brane are combined as a single unit of the karyotheca. In this connection, Watson (1955) and Palade (1955b) ’ claimed that the double nuclear membrane is an integral part of the endoplasmic reticulum ; thus the interspace between the outer and inner nuclear membranes is homologous with the cavity of the endoplasmic reticulum. They referred to this cavity as the “perinuclear cisternae.” I t is often observed that the outer nuclear membrane bulges up into the cytoplasm. The outer surface of the outer nuclear membrane possesses the small particles known as ribonucleoprotein particles, and thus is identical in morphology with the rough-surfaced lamellae of the endoplasmic reticulum (Fig. 1B). The reason why Sjostrand considered that the nuclear membrane of the pancreatic cell is not double might be the fact that in this cell the endoplasmic reticulum (a-cytomembranes) is much too crowded to make it possible to recognize the double structure. But in the case of some cells containing very little or no endoplasmic reticulum, the karyotheca is clearly observable as a double membrane. The existence of pores in the nuclear envelope was first described by Callan and Tomlin (1950) in teased material from certain amphibian oocytes, and later observed in more detail by Afzelius (1955), Watson ( 1955), and Wischnitzer ( 1958), using ultrathin sectioning. Afzelius demonstrated in sea urchin oocytes that the pore is closed by a single delicate diaphragm and ringed by a cylindrical extension of dense material (annulus). Recently, Wischnitzer ( 1958) showed that this cylindrical tube is made up of about 8 microcylinders which are seen in cross section as subannuli. Our observations on some gland cells revealed the pores in some but not all (Figs. l B , lC, and 28). Even in cases where the pore is evident, the diaphragm is not always seen ; free communication between the karyo- and cytoplasm is very distinct at some pores. The cylinder is also inconstant. In the human apocrine sweat gland the diameter of the pore (the maximum length of discontinuities of the nuclear envelope in normally cut sections) measures about 1000 A. (Kurosumi et al., 1959a), which is approximately the outside diameter of the cylindrical hem described by Afzelius (1955) and by Wischnitzer (1958). The interval between the neighboring pores is very widely variable. At the rim of the pore, the inner nuclear membrane is joined with the outer one forming a rounded edge. In the course of vast electron microscopic studies of many kinds of glandular cells, we found that the nuclear pores did not always exist. The occurrence of pores may be related to the condition of the karyoplasm and also to the functional stage of the nucleus. In fact, when the karyoplasm is seen as homogeneous, the nuclear pores are rarely observed; but the

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

7

nucleus in which chromatin is aggregated peripherally along the nuclear envelope possesses numerous pores. The latter condition suggests that the coiling of chromonemata is not completely loosened in such chromatin clumps and also suggests that the nucleus may be in a condition either just before or after the mitotic nuclear division. It is also noted that the area immediately inside the pore is always clear corresponding to the gap between the chromatin clumps (Fig. 28). Kurosumi ( 1956, 1958) observed the successive stages of mitosis in sea urchin blastomeres and concluded that the nuclear envelope might be reproduced at telophase from the microsomes (endoplasmic reticulum). Amano and Tanaka ( 1957) and Yasuzumi (1959) reported that the endoplasmic reticulum extends and wraps the chromosomes and becomes the nuclear envelope at the end of mitosis. It may be reasonably assumed that the nuclear envelope immediately after its reconstruction may have many discontinuities, but these may be reduced in number by the fusion of each perinuclear cisterna, since a long period is spent by the nucleus in the interkinetic stage. On the other hand, it is speculated that the pores may depend on the nucleocytoplasinic interaction. Anderson and Beams ( 1956) showed the nuclear (nucleolar ?) material passing through the nuclear pores. Bennett ( 1956) published a hypothesis that the ribonucleoprotein particles produced in the nucleus might attach to the inner surface of the inner nuclear membrane, and then the membrane carrying the particles might flow and reflect out along the pores in a manner similar to a conveyor belt. The facts that the inner and outer membranes are continuous at the edge of the pore and that the outer membrane is studded with R N P particles and continuous with the endoplasmic reticulum are true. The inner nuclear membrane, however, is not the same in appearance as the outer membrane. Particles occasionally attached to the inner surface of the nuclear envelope are not always the same in size as the outside particles. It cannot be determined, therefore, whether or not the outside particles are the same in nature as the inside ones. Furthermore, the inner nuclear membrane is usually thicker than the outer membrane, and on some occasions the inner membrane is observed as double-layered (Fujiwara, 1957b). Consequently, the nuclear envelope becomes trilaminar in this case, although the inner pair is not so distinct as the usual double nuclear membranes. It may be explained that this trilaminar envelope is constituted of an apposition of secondary membranous substance to the inside surface of the doublelayered perinuclear cisternae. At any rate, it must be noted that the inner nuclear membrane is different in structure from the outer ones, and hence the hypothesis by Bennett (1956) cannot be applied to all the cases of the nuclear envelope.

8

K. KUROSUMI

3. The Karyoplasnz Most papers so far .published dealing with electron microscopic observations described the karyoplasm (nucleoplasm) as being made up of the material in which minute granules are diffusely or irregularly dispersed. In fact, low magnification electron microscopy reveals the nucleus as if it were of a homogeneous granular composition, and thus some authors referred to this appearance as occurring because osmium tetroxide was not fitted to the fixation of the karyoplasm. For example, Sjostrand (1956) said, “The poor structural pattern of the nuclei might well be the result of improper preservation and it seems too early to discuss further the structure of the nucleus on the basis of its appearance in the electron microscope.” Our observation on human sweat gland cells (Kurosumi et al., 1959a), however, revealed the highly organized structure in the resting nuclei, which were quite homogeneous in low power electron micrographs and light microscopic preparations. In slightly higher magnification views with the electron microscope, the karyoplasm seems to consist of many tortuous strands of about 30G500A. in width. More highly magnified pictures show that the tortuous strands appear to be cross-striated, and each striation a thin thread composed of small particles of about 50 A. lined up like a string of beads (Fig. 2). The small unit particles may be called “chromomeres,” a row of which makes a thin thread designated as the “primary chromonema.” A cross-striated strand made up of the side-by-side alignment of primary chromonemata is named the “secondary chromonema.” It is often observed that the secondary chromonema appears to split into two along the long axis, looking like a paired strand. This may be explained by comparing it to a hollow cylinder cut longitudinally through the axis. An occasional occurrence of a ring or semicircular pattern may be seen in the transverse section. Thus the secondary chromonema may comprise primary chromonemata which wind helically in a hollow coil (Fig. 4). The actual length and number of the primary chromonemata constituting the secondary chromonema are not determined. The secondary chromonemata are also helical in shape, but in the resting nuclei the helix of most chromonemata is loosened. During mitosis, the helix of the secondary chromonemata may be tightly wound and make up the chromosomes (Kurosumi, 1958). Thus the chromosome may be of compound helical structure, although the gross helix may be loosened at the interkinetic stage. An occasional aggregation of the chromatin may take place in some nuclei in which the clumps are apt to be arranged along the inside surface of the nuclear envelope or around the nucleolus. Such chromatin clumps

ELECTRON MICROSCOPIC A N A L Y S I S O F SECRETION

9

are not artifacts caused by coarser fixation and may be interpreted by the possibility that the helix of the chromonemata is not yet perfectly loosened in such regions, because the so-called chromatin aggregation can be observed in early prophase and in late telophase, and the internal appearance of such clumps is very akin to that of the mitotic chromosomes.

FIG.2. High magnification micrograph of a part of the karyoplasm from the nucleus of a human apocrine sweat gland cell. Many strands (secondary chromonemata, CH,) show the cross-banding (primary chromonemata, C H I ) which are beadlike chains of granules (chromomeres). A shows the axial longitudinal section of a secondary chromonema, looking as if paired strands. ( X 90,000.) (K. Kurosumi et al.. 1959a.)

4. The Nzicleolus The nucleolus has long been considered as a homogeneous and structureless mass in the nucleus by many light microscopists. Rather recently Estable and Sotelo (1951) discovered filamentous structures in some nucleoli using a silver impregnation method with the light microscope. They named such structures as “nucleolonema,” and the remainder of the nucleolus they described as the “pars amorpha.” Electron microscopy revealed these structures in many cell nucleoli in studies by Borysko and Bang (1952), Bernhard et al. (1952a, 1955), Horstmann and Knoop (1957), Kurosumi and Akiyama (195S), and Yasuzumi et al. (1958). Porter (1954) and Bernhard et al. (1955) recognized that the nucleolo-

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K. KUROSUMI

nenia consisted of small particles of the same size as those attached to the endoplasmic reticulum and composed perhaps of ribonucleoprotein. Palay and Palade (1955) described in a study on neuron fine structure that the filaments of the nucleolus (nucleoloneinata) appeared as dense aggregations of fine granules which were frequently disposed in linear and rather parallel arrays. Kurosumi and Akiyania (1958) as well as Yasuzunii et al. (1958) suggested the presence of a helical arrangement of thin threads within the nucleolonemata.

FIG.3. High magnification micrograph of the nucleolus from a human apocrine sweat gland cell. It seems like a glomerulus made up of tangled thick strands (secondary nucleolonemata, N L , ) , within which parallel rows of beaded threads (primary nucleolonemata, N L , ) are observed. ( X 90,000.) (K. Kurosumi et al., 1959a.) I n human apocrine sweat gland cells, Kurosumi and his collaborators (1959a) observed that the nucleolonema was composed of many small particles about 150A. in diameter, and these were arranged in rows like strings of beads in like manner as were the chromomeres (Fig. 3 ) . Such strings are frequently aligned in parallel rows which are transverse or oblique to the long axis of the nucleolonema. These authors designated the subunit of the nucleolonema as the “primary nucleolonenia” and the thick strand originally found by Estable and Sotelo ( 1951) they designated as the “secondary nucleolonema” (Fig. 4). In this study the authors considered that the secondary nucleolonema might be constructed of helically

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

11

arranged primary nucleolonemata. However, the problem of whether the nucleolonema is similar in composition to the chromonema, i.e., whether the nucleolonema is a hollow coil or a solid spiral, was not settled. In the course of a study of spinal ganglion cells, Kurosumi and Akiyama (1959)

\

200 A

300.-

100-150 Chronionema

Nucleolonema

FIG.4. A diagrammatic illustration showing the submicroscopic organization in the nucleus of a gland cell of the human apocrine sweat gland. T w o different features of the chromonema show a longitudinal section through the central axis of a hollow cylindrical coil (below)and a lateral longitudinal section through a rather peripheral part (above). The lower corresponds to the chromonema labeled A in Fig. 2. (K. Kurosumi et al., 1959a.)

found that the nucleolonema is not a hollow cylinder but is a spirally twisted bundle of many threads (primary nucleolonemata) , and consequently it is solid. An occasional node formation along the nucleolonema was explained by a strong twisting of such thread bundles.

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The other type of nucleolus is a homogeneous mass made up of tightly packed small granules of about the same size as those found in the nucleolonema. Such a mass is either found alone or accompanied by nucleolonemata. I n the latter case, the electron density of both parts of the nucleolus is almost equal. In niost cases of somatic cells, such homogeneous masses take an irregular outline, and are considered to correspond with the pars artiorpha of Estable and Sotelo (Bernhard et al., 1955; Kurosumi and Akiyama, 1958; Wischnitzer, 1958). In egg cells of various animals, the pars amorpha is prominently developed and sometimes takes a remarkably regular globe shape, which is particularly called pars splzneroidea by Kurosumi and Akiyama ( 1958). No investigator could observe a limiting membrane surrounding the nucleolus.

5 . T h e Nuclear Inclzrsions Peculiar inclusions in the nucleus of the hepatic parenchymatous cell of a snake (Natrix tigrina tigrina) were found by light and electron microscopy (Kurosumi and Watari, 1960) (Fig. 1C). These nuclear inclusions are spherical bodies of various sizes (usually as large as 2-3 p in diameter). They are intensely stained with iron-hematoxylin or with eosin after hematoxylin-eosin staining, but are negative in PAS, Feulgen, and lipid reactions. Electron microscopic observations revealed that these inclusions are very dense, regular, round bodies which resemble zymogen granules of the exocrine pancreas cell. They are often surrounded by a quite empty space or a halo, the appearance of which may be considered, however, to be a result of artificial shrinkage of the inclusions themselves or the karyoplasm surrounding them. The localization of the inclusion may be associated with neither the nuclear envelope nor the nucleolus. We found such inclusion bodies in the hepatic cell nuclei of Natrix captured in May and June, but in the same animal in the other seasons the liver cells contain no nuclear inclusion. Their origin and fate are yet unknown. It is probable, however, that the inclusion may be equivalent to the secretory granule of a specific nuclear secretion, although extrusion of the inclusion out of the nucleus has not been clearly observed. The essential nature of this body is still under pursuit.

B. THECYTOPLASM 1. The Secretory Granules

The actively functioning secretory cells usually contain within the cytoplasm a great deal of secretory product which appears as granules or droplets (Figs. 21 and 22). These are mostly observable with the light

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microscope, but the stainability in light microscopic preparations and the electron density are not always parallel. Some of the dark staining granules are seen as being less dense or almost empty in electron micrographs and vice versa. In exocrine cells, secretory granules are usually situated in the apical part of the cell but are sometimes extended to para- or infranuclear regions. The shape, size, and density of the granules are very widely variable and chiefly related to the type of the gland or the chemical nature of the secretion. Thus the secretory granules of one cell type are roughly uniform, but are quite different from those of other cell types. Secretory granules of pancreatic exocrine cells were first noted by electron microscopy (Dalton, 1951b; Bernhard et al., 195213; Weiss, 1953; Sjostrand and Hanzon, 1954a) ; they are spherical granules with extremely high density and relatively large size (0.6 p in average diameter). Paneth cells of intestinal mucosa have similar secretory granules of high density (Hally, 1958). Although the parotid and some other salivary glands (Onok et al., 1957; Nakanishi, 1959) as well as the body chief cells of the gastric glands (Kurosumi et d., 1958b) are similarly serous cells, they have less dense secretory granules ; from these various steps in the transition to clear, empty secretory vacuoles are observed (Shibasaki, 1959) (Fig. 19). Some of the secretory vacuoles are fused with each other becoming an irregularly outlined large vacuole, but some are never fused (body chief cells of human stomach). Mucous droplets of the goblet cell (Rhodin and Dalhamn, 1956) and of the mucous neck cell of the stomach (Kurosumi et QZ., 1958b) are relatively large oval bodies of medium density (Fig, 21 ) . The surface epithelium of the stomach which is known to secrete the niucoid substance possesses denser granules than those of mucous neck cells (Fig. 22). They are rather small disc-shaped granules with relatively high density. But the immature form of these granules is less opaque and is slightly larger than the mature ones. Kitamura (1958) reported that the carpal organ of the pig, which is one of the sweat glands, has two distinct cell types, the dark and clear cells. The names of the cell types were based on the light microscopic observation, by which the secretory granules of dark cells are darkly tinted after iron-hematoxylin stain of Heidenhain as well as periodic acid Schiff reaction. Electron microscopy of this organ, however, revealed that the dark cell granules are very clear or almost empty; thus the dark cell is seen rather clearer than the clear cell. According to our experiences, the secretory granule of any cell type possesses a limiting membrane in its full-grown or slightly immature stage,

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but in the overripened granules which may have entered into the step of dissolution prior to the discharge, when.some of them are transformed to the secretory vacuoles, the limiting membrane becomes indistinct or disappears (Fig. 19). In such a stage, the fusion of neighboring granules may often occur. Most of the limiting membranes are smooth and monolayered but some are rough surfaced, i.e., studded with small particles on their outer surfaces, and some others are double-layered smooth membranes. Such a morphology of the limiting membrane surrounding the secretory granule may suggest, to some extent, the probable genesis of the secretory granule of a given cell type. Certain gland cells are noted by the fact that they possess two or more different types of secretory granules. For example, eccrine and apocrine sweat gland cells of the human being are laden with at least two different types of granules, one of which is a less dense, regular, spherical or oval granule surrounded by a double smooth membrane, and another is a very dark irregular-shaped granule with a single smooth limiting membrane (Fig. 26). The former is perhaps of protein nature, whereas the latter may be of lipid nature and probably contains iron and pigment. Kurosumi et al. (1959a) and Iijima (1959) designated the former as light secretory granules and the latter as dark secretory granules, whereas Charles (1959) called them smooth and rough secretory granules, respectively. In the mouse thyroid, two distinct types of big granules which simultaneously occurred and were situated at the apical cell zone were noted by Ekholm and Sjostrand (1957), although they hesitated to call them secretory granules. In the mammary gland, two types of secretory granules are also observed (Bargmann and Knoop, 1959; Hollmann, 1959). One of these is a large body with a corrugated outline containing either dense homogeneous material or less dense granulate substance, and is referred to as lipid droplets. The other is a small round granule of high electron density and is thought to be a protein or lipoprotein granule. Scott and Pease (1959) noted two different types of zymogen secretory granules in parotid acinus cells, but every gradation exists between them ; the less opaque ones may probably correspond to the secretory vacuoles found in stomach body chief cells (Kurosumi et al., 1958b; Shibasaki, 1959). The lipid secretory granules including those of steroid hormones are usually opaque to the electron beam and are frequently irregular in outline. A representative of this sort of granule may be found in the adrenal cortex (Lever, 1955 and 1956) and corpus luteum (Kurosumi, 1957c), as well as in sweat glands (Kitamura, 1958; Kurosumi et al., 1959a, b) (Figs. 25 and 26). In the sebaceous gland, however, Rogers (1957) and Kitamura

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and Kurosumi (1959) observed that lipid droplets are empty vacuoles enveloped by one or more smooth membranes (Fig. 27). Some of them contain a small amount of debris which appears as an irregular network. As Rogers has stated, such an empty appearance of lipid granules must have resulted from the removal of most of the lipid during the course of the specimen preparation. The author often encountered such fully removed or half dissolved fat droplets in sea urchin eggs (Kurosumi, 1957b) and in liver cells of various animals. The dissolving away of lipid is not only related to the technical conditions, but is also dependent on a slight difference in chemical composition of the lipid, because in the same cell cytoplasm some droplets are totally or half dissolved away but others are quite intact, and, moreover, the fat droplets of certain cell types are never observed as becoming the vacuolar form such as those in fat-storing cells of the liver (Yamagishi, 1959). Except for the thyroid and steroid-secreting organs, most endocrine cells contain dense, relatively small granules. These are often called the “specific granules” instead of the “secretory granules,” but the secretory nature of these granules cannot be denied. The secretory granules in Langerhans islets, the adrenal medulla, and anterior pituitary cells are high in electron density. Most of them are round, but beta granules of dog’s (Lacy, 1957a, b) and cat’s islets (Bencosme and Pease, 1958) and alpha granules of carp’s Brockmann body (endocrine pancreas) (A. Watanabe, 1960) are bizarre in shape. These are rod-shaped or lamellated ring or horseshoe-like crystalloids (Fig. 30). It may be considered that such a crystallization of the secretory granule is perhaps one of the storage forms of the secretory substance. The Occurrence of the crystalloid besides the secretory granules in chick thyroid cells was noted by Yoshimura and Irie (1959b), but the essential nature of such a crystalloid is not yet clarified (Fig. 29).

2. The Cytoplasmic Membrane System or Endoplasmic Reticulum Earlier light microscopic observations revealed fine filamentous or lamellar structures in various cells. These were variously designated by many investigators such as “Basalfilamente” ( Solger, 1894), “Basallamellen” (Zimmermann, 1898), “protofibrillae” (Saguchi, 1920), “Mikrosomenfiinden” (Morita, 1931), and “cytoplasmic fibrils” (MonnC, 1945). In addition, these filaments and associated amorphous substances intensely stained with basic dyes were called either “ergastoplasm” (Garhier, 1897), “Nebenkern” (Gaule, 1881, and Nussbaum, 1882), or “chromidia” (Hertwig, 1902 ; Goldschmidt, 1905 ; Monnk, 1948). The first description of these structures seen with the electron microscope was done by Porter, Claude, and Fullam (1945) on tissue cultured

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cells, in which they were described as a lacelike reticulum. This finding was extended by Porter and Kallman (1952) and named “endoplasmic reticulum.” On the other hand, Hillier ( 1950), Dalton et al. ( 1950), and Bernhard et al. (1951, 1952b) observed similar structures using the thinsectioning technique and referred to them as “filaments.” Dalton ( 1951b) described them later as “lamellae” in pancreas and gastric body chief cells. Palade and Porter (1952) observed this system in sections and identified it with the endoplasmic reticulum previously described in cultured cells. They described the system as showing morphological variations ranging from isolated vesicles to complicated networks of canaliculi. Sjostrand (1953) studied tissue sections from the pancreas and kidney fixed by both osmium tetroxide and freeze-drying, and noted a highly organized system of double membranes in both cell types. This system could not be considered to represent fixation artifacts, because specimens preserved through two quite different fixations displayed the same morphological pattern, and living cells revealed an anisotropy under the polarization optical analysis. H e speculated that the two constituent membranes of a pair represented layers of mainly protein nature and the space between might be a multilayer of lipid molecules. Then Weiss (1953) found a saclike dilatation of the space between double membranes of the exocrine pancreatic cells. H e thought that the double membranes are actually sacs which were often pressed on one another and flattened like sheets, and thus he called them the “ergastoplasmic sac.” Furthermore he noted that the membrane of the ergastoplasmic sac was granular. A study on the same material by Sjostrand and Hanzon (1954a) clearly revealed that the membrane of this system was studded with opaque particles of about 150 A. on one side. Sjostrand (1953) and Sjostrand and Rhodin (1953) observed, on the other hand, that the membrane system occurring in the kidney tubule cells was smooth on both surfaces and was not identical to the membrane system found in the pancreas. These smooth membranes were identified later by Rhodin (1954) and Pease (1955) as an invagination of the surface plasma membrane. The pleomorphism of membrane-bounded sacs has been known successively in various animal cell types : double membranes (Sjostrand, 1953 ; Sjostrand and Rhodin, 1953 ; Sjostrand and Hanzon, 1954a), tubules (Bradfield, 1953 ; Kurosumi, 1954), and round or irregular sacs (Weiss, 1953 ; Y. Watanabe, 1955) were noted. Dalton and Felix (1953, 1954) found a specialized area of the cytoplasm composed of smooth membranes which might correspond to the Golgi apparatus of classic cytology. They described the Golgi apparatus as con-

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sisting of lamellae, vacuoles, and granules, all of which were bounded by smooth dense membranes. As briefly reviewed above, various types of membranes or membranebounded spaces were found within the cytoplasm by electron microscopy, but the nomenclature of these membrane systems is not yet standardized. Palade ( 1955b, 1956b), Porter (1955), and their associates proposed the term “endoplasmic reticulum” to include all the membrane systems of the cell. Palade divided the system into two different categories in view of a purely morphological standpoint, “rough surfaced variety” and “smooth surfaced variety.” The former corresponds to the “ergastoplasm” adopted by Weiss (1953) and French authors (Bernhard and Rouiller, 1956; Haguenau, 1958) and is characterized by the fact that the membranes of this variety are studded with granules on one side of the membrane. As the granules associated with the endoplasmic reticulum are known to contain a high amount of R N A (Palade and Siekevitz, 1956a,b), the rough-surfaced variety corresponds well to the cytoplasmic basophilia described by light microscopists. Another type of the endoplasmic reticulum, namely, the smooth-surfaced variety, includes many different types of membrane systems such as small vesicles, Golgi apparatus (centrosphere region by Palade’s expression), and plasma membrane infoldings, but the common characteristc of this variety is the absence of attached granules, namely, the smoothness of the membrane surface. Sjostrand (1956) prefers to classify the several types of membrane system with noncommittal terms, a-,(3-, and y-cytoniembranes, which may correspond respectively to the ergastoplasm or rough-surfaced endoplasmic reticulum, the Golgi apparatus, and the invaginated plasma membrane. Recently Schulz and de Paola (1958) introduced a new system of membranes found in gill epithelium of salamanders, looking like a myelin sheath of the nerve fiber and proposed the term “6-cytoniembrane.” This nomenclature using the Greek alphabet, however, does not cover all the variants of membrane system which may be found within the cytoplasm. For instance, vesicle-like or reticular bodies in the oxyntic cells of the stomach, sebaceous gland cells, spermatocytes, and striated muscle fibers are all smooth-surfaced, but cannot belong to any type of a-, p-, y-, or even 8-cytomembrane. They might be described as the smooth-surfaced endoplasmic reticulum in a proper or narrow sense, although the last of them has been often called the “sarcoplasmic reticulum” (Bennett and Porter, 1953 ; Porter, 1956 ; Porter and Palade, 1957). The endoplasmic reticulum according to the Rockefeller group is a reticulum widespread throughout the entire cell, which is consonant with the old concept of “cytoskeleton.” It must be noted, however, that the

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endoplasmic reticulum may vary in amount, shape, and distribution, depending upon the cell function as well as the cell age. Additional findings by Kurosumi (195713) and Kurosumi et al. (1958a) proved that the cell organelles and inclusions may easily be moved about by centrifugation without destruction of cell components and of the entire cell. The centrifuged cell survives and soon recovers from the stratification of cytoplasm caused by centrifugation. If the endoplasmic reticulum were a firm skeleton within the cell, the stratification of cytoplasm would not be carried out unless the destruction or death of the cell occurred. Thus the endoplasmic reticulum may be a labile structure and reversibly dispersed into vesicles or reorganized. In a strict meaning, the term “endoplasmic reticulum” is unsuitable because the system is neither actually a reticulum nor observed only in the endoplasm of the cell. But this term is most widely used throughout the world, not only in America but also among many authors in Europe and in Asia. Sjostrand’s nomenclature is also incomplete as stated above, and used only by Swedish investigators. W e usually adopt the term “endoplasmic reticulum,” but we do not agree completely with the opinion expressed by Palade (1956b) and other authors of the Rockefeller school.

3. Rough-Surfaced Variety of Endopla-smic Reticulum, Ergastoplasm, or a-Cytomembrane Serous gland cells such as exocrine pancreas, salivary glands, Paneth cells of intestine, and body chief cells of the gastric gland are loaded with the most abundant basophilic substance. Early electron microscope studies reported such a basophilic substance or ergastoplasm as fibrillar or lamellar structures (Hillier, 1950; Dalton et al., 1950; Dalton, 1951b; Bernhard et al., 1951, 1952b). a. Morphological Identification of the Ribonucleoprotein in the Cytoplasm. Some cytochemists, for instance, Caspersson ( 1950) and Brachet ( 1950), considered that the cytoplasmic basophilic substance might contain ribonucleic acid and be intimately concerned with protein synthesis within the cell. In the early days of electron microscopy Dalton (1951b) and Porter (1953) also indicated that these lamellar or reticular structures might contain RNA. Claude (1943), by means of differential centrifugation of rat liver homogenate, revealed submicroscopic particulate components (50-200 mp) called “microsomes,” which were rich in R N A and phospholipid. This finding was based upon observations using dark-field microscopy, but not with the electron microscope, and was obtained from broken cells. H e and his collaborators, however, subsequently endeavored to find the same com-

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ponents within intact cells using the electron microscope. Following the work of Porter, Claude, and Fullam (1945) who found a reticular structure in a cultured fibroblast, Claude and Fullam (1946), using high speed microtomy, concluded that the subniicroscopic particles or microsomes of the guinea pig liver were embedded within a fibrous texture of the cytoplasm which appeared to be the endoplasmic reticulum. These studies became the pioneer work for the foundation of the possible synthetic role of the endoplasmic reticulum. Submicroscopic granules found in the ground substance of the cytoplasm were often described as microsomes (Kurosumi, 1954, 1957b; Morita, 1958). Kurosumi ( 1957b) classified the microsomes into two types, solid and vesicular microsomes, and Kurosumi et al. (1958a) discussed the relationships between the microsomes and the endoplasmic reticulum. The granularity of the bounding membrane of saclike bodies in the ergastoplasm was first noted by Weiss (1953), and the granules which might be free or attached to the membrane of the endoplasmic reticulum were described in detail by Palade (1955a). Kuff et al. ( 1956) and Palade and Siekevitz (1956a,b) found that the microsomes described by Claude were nothing but the fragments of endoplasmic reticulum, and the ribonucleic acid of the microsome fraction was located in the small granules (ultramicrosomes or postmicrosowl fraction). However, the possibility that R N A might be contained in constituents other than the granules of Palade (1955a) was shown by Chauveau et al. ( 1957). They isolated RNA-rich fractions which appeared in electron micrographs as membranes devoid of granules. This result may be in good agreement with the fact that in some cells, especially in egg cells, smoothsurfaced membranes correspond to the yolk nuclei which show a strong affinity to basic dyes (Rebhun, 1956; Kurosumi et al., 1958a). b. The Ultrastructure of the Ergastoplasm. In cells of various serous glands, the ergastoplasm ( rough-surfaced endoplasmic reticulum) displays a roughly uniform appearance, but the minute structural patterns may vary to a considerable extent from cell to cell. They usually occupy the basal portion of the cell and often extend to the parts lateral to the nucleus. It is frequently observed that the ergastoplasm consists of many double membranes studded with granules on the outer surfaces of the membrane pair. Double membranes are often packed parallel to each other in the direction vertical to the basal cell surface (Fig. 5 ) . This orientation is reminiscent of the old description by Ziniiiiermann (1898), who called it “Basallamellen.” Sometimes the lamellae of double membranes are oriented parallel to the nuclear surface and in other cases may appear as whorl-like or concentric lamellae, which show a resemblance to finger prints.

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FIG.5. A part of a body chief cell of the rat gastric gland (a starved rat). SV, secretory vawole ; G, Golgi apparatus ; Er, lamellated ergastoplasm (rough-surfaced endoplasmic reticulum) ; N , nucleus. ( x 25,000.) (S. Shibasaki, 1959.)

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According to Weiss (1953), the double membranes of the ergastoplasm are. flattened and pressed sacs. H e explained a concentric lamellae by postulating that many closed sacs were invaginated as though they were pushed with a finger and then cut in a plane perpendicular to the finger. To test the assumption that these structures were actually sacs, he tried the following experiments: he took pieces of fresh pancreas and put them in hypotonic solution, and found in electron micrographs that the flattened sacs became increasingly swollen with a rounded cross section. W e observe in fact that the ergastoplasm (endoplasmic reticulum) is transformed partially or totally into many round vesicles, whose limiting membranes are also studded with granules on their outer surfaces (Fig. 6A). This transformation may be caused in some by the changes in tonicity during fixation or other preparatory treatments. But in some other cases, the change may occur within life according to the functional condition of the cell, because in the same block of tissue neighboring tells frequently show quite different patterns of the endoplasmic reticulum. Sjostrand and Hanzon (1954a) described in detail this structure from the mouse pancreas as “the intracellular cytoplasmic membranes,” basing their observations on the most excellent electron micrographs of high resolution among those hitherto published. According to these authors, the membrane consists of a thin basic membrane with small opaque particles attached to one side of it. These particles, apparently identical to the RNAcontaining granules studied later by Palade (1955a) and Palade and Siekevitz (1956a,b), were noted by Sjostrand and Hanzon as being 140 A. in diameter and in various forms from irregularly rounded to rectangular or triangular, the one side facing the basic membrane. The distribution of the particles is fairly regular and the distance between the centers of any adjacent granules measures 150-450 A. The mean thickness of the basic membranes is 40 A. In this study, Sjostrand and Hanzon recognized that the two membranes of a pair joined, closing the space in between the smooth sides of the membranes, but they did not consider the structure as a sac at that time, being unaware of the work by Weiss (1953) ; and they stated, “The width of this space varies with the quality of the fixation. When less well fixed the membranes are split apart and empty spaces of varying width are formed.” Dilatation of the space in between a-cytomembranes, however, was later described by Sjostrand (1956) as a result of observation on thyroid cells. Ekholm and Sjostrand (1957), as well as Ekholm and Edlund (1959), asserted that the distension and rounding of the ergastoplasmic sacs (the space in between the a-cytomembrane) might be attributed to functional factors.

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FIG.6. Electron micrographs of the cells from the rat gastric gland, showing rough and smooth types of the endoplasmic reticulum. A. A body chief cell. EY, vesiculated sacs of the ergastoplasm ( rough-surfaced endoplasmic reticulum) ; G, Golgi apparatus with many vesicles and vacuoles (smooth-surfaced) ; A[, a mitochondrion. ( X 25,000.) B. A parietal cell. N, nucleus; SC, intracellular secretory capillary ; V , vesicles without attached particles (smooth-surfaced endoplasmic reticulum) ; M , mitochondria with extremely close packing of cristae mitochondriales. ( X 27,000.) (K. Kurosumi et al., 1958b.)

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Palade (1956a) found, in guinea pig pancreas, dense granules similar to the zymogen granules contained within a moderately dilated space of the endoplasmic reticulum. H e named these “intracisternal granules” and assumed the participation of endoplasmic reticulum in the production of secretory granules (Fig. 10). This finding apparently indicates that the endoplasmic reticulum is a cavitary system in contrast to the concept of Sjostrand and Hanzon ( 1954a). Weiss (1953) had early suggested the possibility of the involvement of the ergastoplasm in the elaboration of zymogen granules as well as of its saclike nature. More detailed discussions concerning the relationships between the ergastoplasm and the secretory function will be given in Section IV, B. The development of the rough-surfaced variety of endoplasmic reticulum may be related to the type of secretory cells ; as already mentioned, in the serous gland cells the development is most prominent, in mucous cells, intermediate, while in some other gland cells such as sweat glands, lipid secreting cells, and parietal cells of the stomach, the structure is very scanty or almost absent. In mesenchymal tissue, the plasma cell is known to contain very well-developed ergastoplasm ; in fibroblasts the amount is moderate, but macrophages contain very little of this structure (Y. Watanabe et al., 1956 ; Kajikawa and Hirono, 1959). c. The Genesis of the Ergastoplasm. The genesis of the ergastoplasm has been repeatedly disputed by many authors and the origin of the ergastoplasm has been ascribed to various cellular components. A review by Haguenau (1958) is available which treats this subject as well as the morphological and biochemical data of the system. Weiss (1953) studied the formation of ergastoplasm in mice which had been fasted and then refed. The main sites of the formation are, according to Weiss, in cytoplasmic centers removed from both the nuclear and plasma membranes. I t is, however, possible that new sacs are formed, in small numbers, in apposition to the nuclear membrane and also to the plasma membrane. The possibility of the formation of ergastoplasmic sacs (endoplasmic reticulum) from the nuclear envelope was first suggested by Weiss (1953) as above, but soon afterward many authors followed: Watson (1955) described the nuclear envelope as being continuous with the endoplasmic reticulum ; Palade (1956b) and Bennett ( 1956) suggested the probable formation of endoplasmic reticulum from the nuclear envelope as well as the surface plasma membrane; and Y. Watanabe (1957a) also suggested that the “intracytoplasmic sacs” (= endoplasmic reticulum) might originate from the outer nuclear membrane by the “protrusion” phenomenon.

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Bernhard and Rouiller (1956) observed the regenerating process of the ergastoplasm in rat -liver after a prolonged starvation, partial hepatectomy, and intoxication with carbon tetrachloride. The reappearance of ergastoplasm occurs under a close topographical relation to the niitochondria, and the membranes generally precede the appearance of the granules. They assumed that the membranes might be derived from invaginations of the cell surface as Palade (1956b) had suggested, and the granules from the nucleolus. Perhaps they did not regard the difference in thickness between the ergastoplasmic membranes (about 40 A.) and the plasma membrane (60-80 A . ) . The author and his collaborators (Kurosumi, 1957d; Kurosumi et al., 1958a), studying sea urchin eggs, put forward a hypothesis that the RNA-containing granule (solid microsome) might grow larger, synthesizing new substance within it and become a vesicle (vesicular microsome), and that the further expansion or coalescence might yield a saclike endoplasmic reticulum. It must be noticed that very young embryonic cells or rapidly growing tumor cells have no organized endoplasmic reticulum, but the dispersed granules probably containing R N A are frequently observed only in small clusters. Such granules or clusters of granules were called “growth granules” by Porter and his collaborators (Porter and Thompson, 1947, 1948 ; Porter and Kallman, 1952 ; Porter, 1955). Thus endoplasmic reticula of differentiated cells must be newly formed in some stage of development. I t is not certain whether the regenerating process faithfully repeats the process of normal ontogenic differentiation. Hay (1957, 1958) described the mode of formation of endoplasmic reticulum in differentiating cartilage cells as being such that numerous small vesicles might coalesce to form new cisternae. In the preliminary report, she stated, “At this stage, many of the cytoplasmic granules appear to be arranged in circles, and transitions from such small circles to small vesicles can be distinguished.” But lately she has inclined to the opinion that the vesicles may originate from pre-existing elements in the Golgi region. Munger ( 1958a) studied normal morphogenesis in pancreatic acinar cells and stated that the ergastoplasmic cisternae seemed to merge into areas of cytoplasm containing closely packed tiny dense granules, and the membranous sacs might be formed by a condensation of particulate material. In spite of his careful observations, the problem of how the granules are converted into membranes is not completely depicted and is left for future research.

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4. The Golgi Apparatus a: Historical. The internal reticular apparatus ( Apparato reticolare interno) of the cell, first observed by Golgi (1898) in nerve cells with a technique of silver impregnation, was subsequently found also in epithelial and gland cells (Kolster, 1913 ; Nassonow, 1923 ; Bowen, 1924; Kopsch, 1926). The apparatus can also be demonstrated by osmium impregnation (Kopsch, 1902), and may, in shape, be either reticular or dispersed. Hirschler (1927) called the former “komplexe Fortn” and the latter “diffuse Form.” In the secretory cell the Golgi apparatus is usually localized between the nucleus and the luminal cell surface as the complex form, but a few exceptions such as the oxyntic cell of the stomach and the basal cell of human eccrine sweat glands are known to have the apparatus of the diffuse form. The polarity of the Golgi apparatus usually seen in secretory cells and the close topography between the Golgi apparatus and the secretory granules attracted cytologists to consider that the secretory substance might be elaborated within the Golgi apparatus (Bowen, 1924 ; Hirsch, 1939). Since the Golgi apparatus could be demonstrated only with silver or osmium impregnation and no detectable method in living cells had been established, some cytologists doubted the real existence of the apparatus in life : as, for example, the vacuome theory of Parat and Painlevk ( 1924), the lipochondria theory of Ries (1935) and of Baker (1944, 1951), and the myelin figure theory of Palade and Claude (1949). The classic reticular appearance of Golgi apparatus was ascribed to artifacts by these authors. Electron microscopy was first applied to the observation of Golgi apparatus by Dalton (1951a) in hepatic and intestinal cells. Dalton and Felix ( 1953, 1954) demonstrated Golgi apparatus of classic reticular form in living unstained cells from mouse epididymis and duodenum by phase microscopy, and simultaneously its ultrastructure with the electron microscope as well. In addition, they suggested that isolated Golgi bodies might contain polysaccharide and ribonucleic acid. No more opinion, skeptical of the real existence of the Golgi apparatus, has appeared after the work of Dalton and Felix. b. The Ultrastructure of the Golgi Apparatus. According to Dalton and Felix (1954), the Golgi apparatus consists of three distinct components : large vacuoles, lamellae, and small granules. The lamellae are composed of paired membranes each of 70 A. thickness, and the interspace is also 70 A. The membranes are not adorned with attached particles as is the ergastoplasm (Figs. 5 and 6 A ) . Sjostrand and Hanzon (1954b) observed the Golgi apparatus in pan-

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creatic exocrine cells and to this ascribed the probable origin of zymogen granules. They described it as follows : “Three elementary components are distinguished. (1) Golgi membranes, arranged in pairs and with 2-5 membrane pairs closely packed except for some intercalated vacuolar spaces. ( 2 ) Golgi ground substance, a homogeneous finely granulated and reticulated material. ( 3 ) Golgi grmules of varying form, size, and opacity. The Golgi membranes and granules are embedded in the Golgi ground substance.” Golgi membranes which were designated later as y-cytomembranes by Sjostrand (1956) are 60 A. thick, and the space between two membranes of a pair measures 60 A., too. The Golgi granules represented dimensions from 40 A. in diameter to the size of zymogen granules and were assumed to transform into the latter. Haguenau and Bernhard (1955) agreed with Sjostrand and Hanzon in both notions about ultrastructures and secretory participation. They enumerated, however, as three main components, vacuoles, membranous structures, and granules or microvesicles. It was the same opinion as that of Dalton and Felix (1954) , and has been widely accepted thereafter. The ground substance of the Golgi apparatus, which was specially emphasized by Sjostrand and his co-workers (Sjostrand and Hanzon, 1954b ; Rhodin and Dalhamn, 1956), contrasts with the general ground substance in pancreatic cells, because the latter is filled with a bulk of ergastoplasm (a-cytomembranes) and none of it enters the Golgi area. But in other cell types with little ergastoplasm, the Golgi ground substance is frequently indistinct from the general cytoplasmic matrix. In the course of a study on the neuron fine structure, Palay and Palade (1955) noted smooth membrane structures and called them “agranular reticulum,” which was identified with the Golgi apparatus by Honjin ( 1956). Palade ( 1956b) classified the Golgi apparatus with other smooth membranous structures to belong to the smooth-surfaced variety of the endoplasmic reticulum, although he preferred the term “centrosphere region” instead of the Golgi apparatus or Golgi bodies. H e described that all possible intermediates were encountered between the supposedly typical profiles of the Golgi complex and the usual profiles of the endoplasmic reticulum in both types of smooth- and rough-surfaced varieties. However, the membrane of the Golgi apparatus differs in thickness from those of the endoplasmic reticulum. The Golgi membrane (y-cytomembrane) is 60-70 A. thick, but the membranes of endoplasmic reticulum (a-cytomembranes) are 40 A. in thickness. Even in low power electron micrographs, the Golgi meqbrane is seen thicker or denser than the membranes in the ergastoplasm, and thus the distinction between the two membrane types is rather easy. Palade did not refer to this fact. Even though

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the Golgi apparatus might reasonably belong to the endoplasmic reticulum, the difference between the smooth and rough membranes is not limited only to the presence or absence of attached opaque particles. c. Mutual Relationships between Each of Three Main Components. Weiss (1955) stated in a study of absorptive epithelium of mouse duodenum that the Golgi complex is a system of flattened sacs and vacuoles enclosed by smooth membranes, and the sacs give rise to the Golgi vacuoles and vesicles by dilatation of larger or smaller terminal segments. H e suggested that this might not be the only mode of formation of Golgi vacuoles, and that smooth membranes of the cell might be formed in connection with ergastoplasm, although it could not be clearly determined. Dalton and Felix ( 1956) showed micrographs from earthworm spermatids suggestive of budding of Golgi vesicles from the paired membranes. Grass6 and Carasso (1957) published a similar concept that the osmiophile vesicles arise from the saccules (identical with the so-called lamellae), becoming loose from the edges of the saccule, or that the saccule breaks wholly or partly into fragments which constitute as many vesicles. They stated also that the large vacuoles were far from being constant. In our experience, however, the small vesicles or granules are found within the Golgi apparatus of all types of glandular and epithelial cells examined, but either vacuoles or lamellae are not always observed. For example, in gastric surface epithelium the lamellae are well developed but no vacuole is found (Fig. 22) , while in human axillary apocrine glands the vacuoles predominate over the lamellae. Moreover, the bounding membrane of the large vacuole and a single membrane of the lamellae are quite the same morphologically as those limiting the small vesicles. Except in cases where the secretory substance has accumulated in Golgi vacuoles, the content of large vacuoles and small vesicles, as well as that in the space between the paired membranes, is equally transparent. Golgi microvesicles are mostly uniform in size, ranging from 50 to 100 mp, but the smallest one is seen to be solid and approximates in size the R N P particles which are either free or attached to the membrane of the ergastoplasm. Thus we consider that the primary fundamental structure of the Golgi apparatus may be the small granule or vesicle, at least irrespective of whether they contain RNA, and they may grow into vacuoles of various sizes, which may be flattened, being pressed on one another (Kurosumi et al., 1958a,b, 1959a). Afzelius (1956a) stated an opinion that the Golgi granules or vesicles might become larger or elongated, manifesting Golgi vacuoles and lamellae. Sjostrand (1958, unpublished data) stated in his address in Tokyo that the R N P granules probably migrated from the ergastoplasm, possibly

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turned into more or less transparent granules (Golgi vesicles), and then coalesced and grew into-large Golgi vacuoles. Recently Nagano ( 1959) has published the same opinion. Hay (1958) described the fact that the Golgi apparatus of young undifferentiated cells of regenerating salamander limbs consisted of aggregates of small vesicles ; on the other hand, flattened vesicles (lamellae) , which were a rather constant feature of differentiated cells, had not been observed in undifferentiated cells. Her study may serve as a strong support for the opinion that the small vesicIes are primary in nature among all components of the Golgi apparatus. d. Participation of Golgi Apparatus in the Secretion Activity. This probability was repeatedly speculated on by light microscopists ; for instance, Hirsch (1939) presented the theory that the Golgi apparatus is the site of congregation of cytoplasmic products from which zymogen granules may arise (Junqueira and Hirsch, 1956). Electron microscope studies of many glandular tissues, both exocrine and endocrine, have revealed a vast evidence that the secretory granules may be produced in or by the Golgi apparatus (Figs. 18 and 31A). The most conspicuous evidence to suggest this possibility is that a substance similar in density to the secretory granules appears within the Golgi vacuoles (Sjostrand and Hanzon, 1954b; Farquhar and Wellings, 1957; Palay, 1958; Hally, 1958; Kurosumi et al., 1958b, 1959a; Y. Watanabe et d.,1959; I. Suzuki, 1959; Ichikawa, 1959; Fujita and Kano, 1959; Sano and Knoop, 1959). Detailed criticism concerning the secretory mechanism will be given later. e. Lipochondria. W e found, during studies on eccrine sweat glands, a new type of granule with a bizarre morphology closely related to the Golgi apparatus (Kurosumi et al., 1958c; Iijima, 1959). These are spherical bodies of 1-4 p in diameter which contain numerous vesicles, looking like bubbles (Fig. 7). Though they somewhat resemble artifacts of electron microscopy such as holes through the supporting film, it is clear that they are pre-existing cytoplasmic structures, since the same or similar bodies have been reported by many light microscopists : Melczer (1931), Nagamitsu (1941), and Toshio Ito (1943) observed round bodies either uni- or polyvesicular, which were blackened with osmium impregnation. Ito ( 1943) called these polyvesicular fat droplets (polyvesikuliire Fettropfen). Ito and Watari (1958) recently observed the same bodies in the cells of human pancreatic islets. They identified this structure with the “lipochondria” of Baker (1944, 1951), who had postulated that the precipitation of silver or osqium around and between lipochondria manifested the classic Golgi apparatus in reticular appearance. According to It0 (1943), Iwashige (1952), and Ito and Watari (1958), the lipo-

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FIG.7. A series of electron micrographs showing various steps (A-E) from the arising to the breakdown of lipochondria (polyvesicular fat droplets) ( L P ) found in the human eccrine sweat gland. In A and B, transformation of Golgi vesicles into small dense g r a d e s is observed (arrows). I n B, enlargement of these granules (fat droplets) is obvious, and in C and D, small clear vesicles appear within them. L P in D is a mature lipochondrion, and in E, breakdown of lipochondria and outflow of vesicles and their content are clearly observed. (Magnification, A, B, x 21,000; C, D, X 16,000; E, x 7000.) (T. Iijima, 1959.)

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chondria (polyvesicular fat droplets) are demonstrable simultaneously with the Golgi apparatus of the so-called classic form after the Kolatschev method, and the lipochondria are products derived from the Golgi apparatus ( Verfettung des Golgi-Appumtes) . Iijima ( 1959) has carefully observed the lipochondria of human eccrine sweat glands with the electron microscope and found the successive steps from their arising until their breakdown (Figs. 7 and 8). Initially small dense granules may appear within the Golgi area, probably being trans-

n

FIG.8. A diagrammatic illustration showing the serial transformation of lipochondria (polyvesicular fat droplets) from the time when they arise from Golgi vesicles until they disintegrate. (T. Iijima, 1959.) formed from Golgi granules or microvesicles. These are slightly larger than Golgi vesicles but may soon increase their own size and become very similar to ordinary fat droplets. In the next step, a few small vesicles appear within the granule, and the size and number of vesicles may rapidly increase. The typical lipochondria are thus formed. They are bounded by a delicate dense membrane, and many vesicles within them are also bounded by similar dense membranes and contain completely transparent contents. Interstices among the vesicles are as osmiophilic as those of €he interior of the initial solid granules. But such dark interstices are markedly reduced as the granule grows larger. Finally the external limiting membrane of the lipochondria ruptures, and the inside

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vesicles are released into the general cytoplasm, and simultaneously most vesicles liberated may also be broken, and thus the transparent content probably of liquid nature is diffused away. Yokoh et d. (1959) found this structure in the human pancreatic islet cells and ascribed its origin to the mitochondria, but no clear-cut evidence was given. D. Lacy (1953, 1954) observed similar structures in pancreatic exocrine and endocrine cells by light microscopy and suggested that they (lipoidal bodies) might be correlated with the production of secretory substance. It is now still in doubt whether or not the lipochondria may participate in the secretion activity.

5. Smooth-Surfaced Variety of Endoplasmic Reticulum or Cytoplasmic Vacuoles

Palade’s notion of smooth-surfaced endoplasmic reticulum includes the Golgi apparatus and the plasma membrane infoldings ( Palade, 1956b). As these special types of agranular endoplasmic reticulum are independently dealt with in this review, only smooth membrane sacs noncorrelated with these are mentioned under this item. Electron microscopy of the gastric parietal cell revealed no typical Golgi apparatus (Kurosumi et al., 19581, ; Hally, 1959a,b). Instead, many vesicular or vacuolar bodies limited by single smooth membranes containing electron-lucent substance are observed in this cell (Sedar, 1955 ; Challice et al., 1957; Kurosumi et d.,1958b; Hally, 1959a,b) (Figs. 6B and 20). Palade (1956b) and Kurosumi et d. (1958b) considered that the vesicular bodies of this cell may belong to the smooth-surfaced variety of endoplasmic reticulum. But Hally negated this classification, because the vacuoles in parietal cells are not interconnected to form the reticulum. These vesicles are frequently spherical but sometimes polyhedral or somewhat elongated. The size ranges from 30 to 600 mp in diameter. They are either randomly distributed, grouped in clusters, or chained in rows, and never display a parallel orientation as do some of the roughsurfaced profiles. The vesicles are often gathered around the intracellular secretory capillaries, and features suspicious of a communication are observed between the cavity of the vesicle and the lumen of secretory capillary. It is suggested, therefore, that the secretory product of the parietal cell may primarily be produced by or accumulated within these vesicles. Similar vesicles of various sizes are observed in the cells of the human sebaceous gland (Fig. 27), and are considered to be closely associated with the secretory function of the sebaceous cells (Kitamura and Kuro-

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sumi, 1959). These are absent or very few in the youngest cells situated in the outermost layer of the acini. As the cell grows, the vesicles multiply very rapidly, filling up all the space left by lipid droplets. Frequently expanded secretory droplets may press the vesicles against the cell periphery or to the surface of the droplets themselves, resulting in an occurrence of lamella-like patterns of superposition of collapsed vesicles. Palay ( 1958) suggested, from the result of an observation on rat’s Meibomian glands, that these might correspond to the Golgi apparatus. However, these are quite different from the Golgi apparatus, which is very inconspicuous in human sebaceous glands. Palay showed a highly organized crystal-like pattern on the smooth membranes surrounding the lipid droplets. It consists of interlaced tubuleformed cisternae of “agranular reticulum.” Such a crystal-like lamellated body which might be part of the smooth-surfaced endoplasmic reticulum was observed also in the pigment epithelium of the retina of the bat (Yamada, 1958).

6. The 8-Cytomembranes and Lamellar Bodies Schulz and de Paola (1958) discovered a new system of membranes and named it “8-cytomembrane.” It may be part of the smooth-surfaced endoplasmic reticulum, but it has a peculiar shape being composed of very tightly packed lamellae either straight or rolled up as a spiral, and hence it is considered to be specifically differentiated. In intermediate cells of the gill epithelium of the salamander (Amblystomu mexicanum) , they appear as fingerprint-like spirals which consist of membranes of 30-45 A. in thickness and intermembranous spaces of about 60 A. (Fig. 9). The 8-cytomembranes may be differentiated in the perinuclear region of the cytoplasm from a homogeneous dense substance, and may develop into the lamellar bodies (lmelliire Cytosomen), which contain rolled up 8-cytomembranes and a system of compartments filled with either moderately dense homogeneous material or much clearer substance. I n superficial cells, the lamellar body no longer has stratified membranes but contains a number of clear secretory vacuoles bounded by single or double limiting membranes. Comparing with results of histochemical tests, Schulz and de Paola considered that the 8-cytoniembranes and the lamellar bodies might exert an important function in the synthesis of mucopolysaccharide and might play a role in mucus secretion. Stoeckenius (1956) noted a similar structure of lamellae in basophilic granules of tissue mast cells, As the mast cell is known to produce heparin (Holmgren and Wilander, 1937), the lamellar granule is likely to be concerned with the synthesis of this substance. Pease (1956b) and Toru Ito

FIG.9. Electron micrograph of a section through the intermediate cell of the gill epithelium of a salamander (Amblystoma mexicanurn). Almost all the area of the figure is occupied by a large lamellar body (lamelliires Cytosom), which contains lamellary rolled 6-cytomembranes (arrows), homogeneous substance ( X ) , and clear substance ( Y ) . Cym, outer limiting membrane of the lamellar body; L, fat droplets ; P, melanin pigment; M, a mitochondrion. ( x 40,000.) (Courtesy of H. Schulz and D. de Paola, 1958.)

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( 1958) observed lamellar structures in specific granules of basophile myelocytes and leucocytes. Ito’s finding .is very akin to the lamellar body of Schulz and de Paola, having a dense homogeneous substance accompanying concentric lamellae. Such a structure is also reminiscent of those in precursors of cortical granules of sea urchin eggs (Afzelius, 1956b ; Kurosumi, 1957a). These showed a marked lamellar pattern in early stages, but in later stages of egg maturation, the lamellae are replaced by honiogeneous round granules. In these bodies, it may be evident that a sort of biological synthesis occurs. Characteristic lamellar membranes which may be the same as or very similar to the 8-cytomembranes have been reported by some authors, and a close relationship to the lipid, and further to the Golgi apparatus or mitochondria, was postulated. NiIsson ( 1958b,c) demonstrated such a membrane system in mouse uterine surface epithelia after the injection of estrogen. These were found in close apposition to lipid granules. In his micrographs a typical Golgi apparatus was depicted near the area occupied by a complex of mingled lamellar membranes and lipid granules. Nilsson ( 1 9 5 8 ~ ) discussed the possibility that the membrane system might be concerned with fat metabolism in this cell. Bargmann and Knoop (1959) presented a case of similar lamellae closely attached to the surface of the lipid droplets of rat’s mammary gland cells. Chou and Meek (1958) observed lipid globules in neurons of Helix and divided them into three types. One of them, the “blue globule,” is an ovoid body possessing a marked lamellar structure along its periphery, looking very similar to the “lamellare Cytosomen” reported by Schulz and de Paola. Chou and Meek stated that osmium-calcium fixation might well preserve the whole structure of round lamellar globules but ordinary osmium fixation might break it and would give appearance of straight lamellae which corresponded to the features of the so-called Golgi body. Thus they argued that the “Golgi apparatus” was the artifact derived from the distortion of the “blue globules.” On the other hand, Clark (1957) considered, in a study of developing renal epithelium, that similar dense bodies with marked lamellae might arise through the concentration of dense or osmiophile substances within mitochondria. The true nature as well as the genesis and the probable functions of lamellar structures are not precisely, known, and it is still unsettled whether all the cytoplasmic bodies with a similar appearance of lamellae may belong to one and the same unit of the cell structure or represent a merely superficial resemblance of varied unrelated entities.

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7. The Mitochondria a. Historical. The mitochondria are the most prominent organelle of the cytoplasm, and are observed as either threads, rods, granules, or chains of granules by light microscopy. They were discovered in the middle piece of mouse spermatozoon by Benda (1898) and named “mitochondria,” but many other names such as “chondriosomes” and “plastosomes” ( Meves, 1908) have also been used. Nowadays, especially in the literature of electron microscopic research, the term “mitochondria” is exclusively used. The mitochondria can be found in all cells not only from animals, but also from plants (Meves, 1904), and are rather easily observable in living cells, using either supravital staining with Janus green B (Sorokin, 1938) or phase contrast microscopy. Differential centrifugation of cell homogenates enabled the finding that the mitochondria are the sites of respiratory enzymes and thus play an important role in energy release for cell metabolism (Claude, 1954). Electron microscopic observations before the advent of the ultrathin sectioning technique revealed the mitochondria merely as shadows of long filamentous bodies in thinly spread cells cultured in vitro (Porter et al., 1945; Porter and Thompson, 1947, 1948). The visualization of internal structures of mitochondria thus had to await the establishment of ultramicrotomy. Pease and Baker (1950), who introduced the first effective method to cut sections thin enough, observed mitochondria in kidney tubule cells of rats, and recognized a transverse banding as well as a limiting membrane. b. T h e Ultrastructure of the Mitochondria. Palade (1952) successfully demonstrated, for the first time, the highly organized internal structures of the mitochondria. H e found that the mitochondrion possesses (1) a limiting membrane 7 to 8 mp thick, ( 2 ) a mitochondrid matrix which appears structureless except for occasional granules, and ( 3 ) a system of internal ridges or folds protruding from the inside surface of the limiting membrane toward the interior of the organelle. H e proposed the term “cristae mitochondriales” to indicate the internal ridges, which are oriented more or less perpendicular to the long axis of the mitochondrion and lie parallel to one another (Fig. 10). According to his description, the cristae are not complete septa, but a central channel is always left free of the cristae, extending along the long axis of the organelle. I n that report, Palade noted the trilaminar structure (two denser layers each 5-7 mp thick and a central light layer of 8-10 mp in thickness) of each crista, but he did not refer to a similar structure of the limiting membrane. In a later paper, however, Palade (1953a) pointed out the fact that the

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mitochondria1 limiting membrane is also double (trilaminar) and the cristae are folds of the inner layer of the limiting membrane. In the same year, Sjostrand (1953) and Sjostrand and Rhodin (1953) published more detailed and different descriptions of the mitochondrial

FIG.10. A mitochondrion ( M t ) and intracisternal granules (ZG) situated in the cavity of rough-surfaced endoplasmic reticulum (ER) from the pancreatic acinar cell of a guinea pig after injection of secretin. ( x 43,000.) (Courtesy of I. Suzuki, 1958.)

structure. According to them, each mitochondrion is surrounded by an outer limiting double membrane and in the interior is a system of internal double membranes, being oriented parallel to one another and chiefly transversely to the long axis of the organelle. The thickness of each single membrane was calculated as 45 A. and the space between the two single

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membranes as 70 A., for both the inner and outer mitochondrial menibranes. Within the matrix among the internal double membranes appear dark areas of size varying from 200 to 700 A. Sjostrand and Hanzon (1954a) showed, in the mitochondria of pancreatic exocrine cells, that the inner membranes are slightly thicker than the outer one, and that the inner membranes are usually in contact with the outer membrane only at one end. The chief difference between the opinions of Palade and of the Karolinska school was as follows : Sjostrand and his associates argued that no direct communication exists between the inner and outer double membranes, and nothing corresponding to the central clear channel reported by Palade (1952) is present. Sjostrand (1956) discussed the fact that the interpretation by Palade (1953a), that cristae are folds of the inner layer of double limiting membrane, had not been supported by any detailed analysis on high resolution micrographs. Moreover, he ascribed the central channel of Palade to an artifact that might be manifested by the fragmentation and shrinkage of cristae caused by the post-mortem changes. On the contrary, Low (1956) and Freeman (1956) presented evidence in human leucocytes for the direct continuation between the outer limiting membrane and the internal cristae. Y. Watanabe ( 1957b), Ekholm (1957a), and later Ekholm and Sjostrand (1957) presented the same evidence. But the last authors stated that the central light layers of the outer and inner membranes are separated at the place of contact by an opaque layer, although the free communication between the spaces in cristae and in the limiting membrane had been shown by Low and others. According to Freeman (1956), the individual denser layers of the cristae as well as of the outer mitochondrial membrane were further resolved into three strata, two outer dense lines 15-17 A. thick and an electron-lucent core 20-23 A. thick. Sjostrand (1956) also negated a direct continuity between the space bounded by the two dark layers of the inner mitochondrial membranes and the surrounding cytoplasmic milieu. However, Powers and his collaborators (1955, 1956) indicated in Paramecium mitochondria that the cavity of the crista, which is a tubule in this animal, opens to the cytoplasm outside the organelle. Fujiwara (1957a) revealed clear-cut evidence of the opening of the space in a crista to the outside, from sections of frog’s striated muscle. As already mentioned, Palade and Sjostrand both maintained that the internal structures of the mitochondria are disclike sheets which are disposed in parallel array generally transverse to the long axis of the mitochondrion. But irregularity in shape of cristae, i.e., bifurcation and

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anastomosis, has been frequently observed (Low, 1956). Many other forms of the internal structure have been reported in mitochondria of varying cell types from various animal species. Above all, three groups of variation may be remarked : ( 1) A longitudinal lamellar structure was noted by Beams and Tahmisian (1953, 1954) and Powers et al. (1956) in mitochondria of male germ cells of Helix. ( 2 ) A tubular shape of the crista was seen in various cell mitochondria, such as Bradfield (1953) reported in some insect cells, Kurosumi (1954) reported in sea urchin eggs, and Powers et al. (1955) and Sedar and Rudzinska ( 1956) reported in some protozoa. In higher vertebrates, mitochondria of steroid secreting cells (Belt and Pease, 1956) as well as of parenchymal liver cells (Fawcett, 1955) possess tubule-shaped cristae projecting from the limiting membrane chiefly in a radial orientation and being reminiscent of microvilli. This is a rather common characteristic in spheroidal mitochondria. ( 3 ) Granular or vesicular internal structures were also reported (Hartmann, 1953; Kurosumi, 1954, 1957b; Bargmann et al., 1955; Kakinuina et al., 1955, Rhodin and Dalhamn, 1956; Nagano, 1959), but the artificial image distortion by which the cristae may be fragmented must be checked carefully. c. Functional Significance of Mitochondria in the Secretion Activity. As the secretion mechanism will be dealt with in a later section, probable functions of the mitochondria are briefly mentioned here. Differential centrifugation technique revealed the mitochondria to contain a series of respiratory enzymes, and to be the sites of active energy generation. Therefore, the mitochondria may play a most important role in every step of secretion activity, but their participation in the secretion mechanism is indirect in most gland cells without showing any morphological changes. In some secretory cells, however, there is postulated the possibility that the secretory granules might originate from mitochondria, for example, in pancreas (Challice and Lacy, 1954), in sweat glands (Kitamura, 1958; Kurosumi et d.,1959a,b; Iijima, 1959), in sebaceous glands (Rogers, 1957; Kitamura and Kurosumi, 1959), and in lipid (steroid) secreting cells (Lever, 1955, 1956). d . Proliferation and New Formation of Mitochondria. In some secretory cells, mitochondria appear to be consumed, and to be transformed into secretory substance. Moreover, in rapidly proliferating tissue, the relative amount of chondriome of a single cell may be gradually reduced in reverse pioportion to the increase of the cell population, unless the proliferation of mitochondria occurs. Therefore, the number of mitochondria must be increased in some way.

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Fawcett (1954, 1955) found features to suggest a splitting of mitochondria, namely, in liver cells of the rat fasted and subsequently refed, many mitochondria appeared to be divided into approximately equal halves by a transverse partition. Napolitano and Fawcett ( 1958) observed budding from the sides as well as the tips of mitochondria in rat brown adipose tissue. Within such buds, no cristae were found in most cases; and the authors stated that these might be newly formed growing tips which had not yet developed their internal structure. The cristae may arise de novo from the amorphous matrix instead of being formed by plication of the inner layer of mitochondrial membrane, according to these authors. The above findings may support the view that mitochondria originate from nzitochondria. However, some evidence was presented for the new formation of mitochondria from other cellular components. Rouiller and Bernhard (1956) suggested that the mitochondria might be formed through a transformation from the “microbodies,” which had been first described by Rhodin (1954) and were characterized by a single membrane, a finely granular matrix, and average dimensions below those of mitochondria. The microbody of regenerating hepatic cells usually bears a central core, in which a series of double membranes may sometimes occur, recalling the mitochondrial cristae. Thus they said, “The microbodies are the precursors of mitochondria.” However, neither Rouiller and Bernhard nor Rhodin referred to the origin of the “microbody.” Takagi (1959) performed a similar experiment on liver cells and generally agreed with Rouiller and Bernhard (1956). Moreover, he has suggested that the microbody may be produced from the smooth-surfaced endoplasmic reticulum. Another theory of the new formation assumes the microsomes ( R N P granules) to be the origin of mitochondria. Eichenberger (1953) suggested this possibility, and Morita (1958) expressed a similar opinion. The author observed in cells of the developing corpus luteum of the rabbit, that mitochondria were consumed, turning into lipid droplets on the one hand, while, on the other hand, a vast number of small mitochondria were newly formed (Fig. 11). The smallest one is a round vesicle of about 50 mp in diameter, containing one or two dense particles, which are very similar to the R N P granules. As it becomes larger, the dense particles increase in number and are disposed in rows reminiscent of cristae mitochondriales. A thorough series of transitions from particlecontaining vesicles to the typical mitochondria are observed. This observation may suggest that the mitochondria may develop from small vesicles or granules in this cell.

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FIG.11. A part of the cytoplasm of a cell in a corpus luteum of the rabbit in an early stage of gestation. F , fat droplet; M , mitochondria. Small granules containing dense particles are abundant. These are tentatively presumed to be precursors of mitochondria. ( x 18,OOO.) (K. Kurosumi.)

8. The Centriole

The centriole is the organelle that plays an active role during mitosis and may serve in the development of cilia and flagellae, through which the active movement of the whole or part of the cell may occur. No essential correlation of the centriole with the cell secretion is recognized. However, the centrioles sometimes may be observed in glandular cells in the interkinetic stage. They are found at the supranuclear region, being surrounded by the Golgi apparatus. The ultrastructure of the centriole as represented by the basal corpuscle of cilia was first noted by Fawcett and Porter (1954), who described the basal corpuscle as a cylindrical body whose wall is composed of nine parallel tubules. The centrioles not concerned with the cilia, either in the interkinetic or kinetic cells, were described by Yamada (19561, De Harven and Bernhard (1956), and Tanaka et al. (1956, 1957), and were known to be not fundamentally different from the basal corpuscle. The centrioles of the glandular cells were observed by Irie (1960) in chick thyroid cells (Fig. 12). According to De Harven and Bernhard (1956), the centriole is a hollow cylinder with a diameter of about 150 mp and a length of 300-500 nip.

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FIG.12. Parts of the cytoplasm of follicular epithelial cells of a chick thyroid. Centrioles cut either longitudinally (C) or transversely (C’ in the inset) are observed. G, Golgi apparatus; M, mitochondria; S G , secretory granule; ER, endoplasmic reticulum of rough-surfaced type. ( x 25,000 in both figures.) (Courtesy of F. Yoshimura and M. Irie.)

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Its osmiophilic wall consists of approximately 9 filaments, which are oriented parallel to the long axis of the cylinder and to one another. Each of 9 units of longitudinal filaments is often composed of 2-3 subunits which are seen as tubules. Tanaka et al. ( 1957) postulated that the Golgi canaliculi (Golgi lamellae in the ordinary sense) are nine in number and may be produced by the extension of nine tubules of the centriole. Although the centriole is usually situated in the Golgi region in the resting cell stage, the direct continuation between the centriolar elements and the Golgi elements is not clearly observed. Therefore, the hypothesis of Tanaka et al. is nowadays not accepted by all the investigators in this field.

C. THECELLSURFACE 1. The Plarvvaa Membrane In electron micrographs of cell sections, a dense demarcating line is always observed along the cell surface. The thickness of the line is less than 100 A. and hence is below the limit of the resolving power of the light microscope. This is the reason why, in light microscopic cytology, it is often stated that most animal cells have no definite cell membrane. But the existence of a membrane has been presumed in view of the fact that the cell surface acts physiologically as a membrane having a selective permeability. Such a presumed membrane has been frequently called “plasma membrane” instead of “cell membrane,” because the latter often means the light-microscopically visible membrane of plant cells chiefly made of cellulose. Microdissection experiments revealed the presence of a plasma membrane which is considerably resistant and highly elastic (Kite, 1913; Carlson, 1952). A delicate dense line encircling the cell body as observed by electron microscopy apparently corresponds to the “plasma membrane,” the term proposed by the physiologists and now commonly adopted among many electron microscopists. But a few of them prefer the term “cell membrane.” Recent electron microscopic knowledge teaches the fact that the protoplasm is completely surrounded by a plasma membrane, at the present level of resolution, and no discontinuity exists under the normal condition of cell physiology and under satisfactory preservation by suitable fixation. In most cases, the plasma membrane is seen as a single dense line in micrographs of sections cut perpendicularly to the membrane surface. The thickness was measured as 60 A. (Sjostrand and Hanzon, 1954a) or 80 A. (Sjostrand and Rhodin, 1953). However, a local difference in the thickness has been noted, the membrane being thicker where it covered the microvilli and at the terminal bars. In such parts the double membrane

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or trilaniinar structure was described ; for example, Zetterqvist ( 1956), Ekholm and Sjostrand (1957), Nilsson (1958b), and other authors indicated the double structure of microvillous membranes. According to Robertson (1957b), in almost all cell types, the cell is covered with a plasma membrane about 75 A. thick, which consists of two parallel dense lines less than 25 A. wide, separated by a light zone less than 25 A. wide. H e (1957a, 1958) demonstrated excellent electron micrographs showing the double membrane structure in a single unit of the plasma membrane of the Schwann cell from frog sciatic nerve, and reported that fixation with permanganate (Luft, 1956) and embedding in araldite (Glauert et al., 1956) may definitely visualize this structure, but the osmium fixation and methacrylate embedding may produce more obscure pictures. Recently Yasuzumi (1959) also pointed out the doubleness of the surface plasma membrane. The surface of the exocrine gland cells may be divided into three parts, the basal cell surface abutting the basement membrane and the connective tissue thereon, the lateral cell surface contiguous to the neighboring cell of the same nature, and the apical or luminal free surface facing the gland lumen. In some cases the gland lumen may extend into or between the gland cells as an intra- or intercellular secretory capillary, respectively. The latter appears as a dilatation of the space between the two apposed lateral cell membranes, but is continuous with the main lumen. In the glands composed of stratified (sebaceous gland) or pseudostratified epithelium (eccrine sweat gland), some of the glandular cells are lacking in one or two of the above enumerated parts (apical and basal). Many gland cells possess complicated extensions and depressions in all or part of the cell surface, i.e., microvilli in apical, interdigitation in lateral, and infoldings in basal surfaces.

2. The Lateral Cell Surface The contact surface of two neighboring cells is shown as two parallel dense lines separated by a less opaque space of uniform thickness (Fig. 13A). Each of the double membranes is an integral part of the plasma membrane of one of the neighboring cells, and the light intermembranous space is essentially extracellular. According to Sjostrand ( 1956), the thickness of this space is strikingly uniform and measures 110-130 A. It is likely that some kind of substance fills this space, and such a substance has been tentatively called “cement substance.” Coman ( 1954) suggested that the substance may contain calcium which is responsible for the cell adhesiveness. Sjostrand (1956) assumed that the light space between the dense plasma membranes corresponds to an organized layer of

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lipid, and that this represents a part of the plasma membrane. But this assumption was disproven by Robertson ( 1957a). In some epithelia, such as of respiratory passages (Rhodin and Dalhamn, 1956) and of the epidermis (Selby, 1955), relatively wide intercellular spaces are brought about between epithelial cells. This is a very rare case, however, among glandular epithelia except for the occurrence of intercellular secretory capillaries. a. The Intercellular Interdigitation. In human sweat glands, for example, the laeral cell surface is not straight, but an irregular folding is clearly observed. This tendency is more remarkable in the eccrine sweat gland (Fig. 13B, C) than in the apocrine gland (Fig. 13A), and is more elaborated at the basal part of each lateral cell boundary (Kurosumi et ul., 195&, 1959a; Hibbs, 1958). A small amount of cytoplasm of one cell invades the neighboring cell as a fingerlike projection, which is either vertical or oblique to the contact surface. In an extreme case, the projection lies parallel to the cell boundary. Such a structure was noted by Dalton ( 1 9 5 1 ~ ) in kidney tubule cells and by Weiss (1953) in the duodenal epithelium, and was referred to as “intercellular interdigitation.” Fawcett ( 1955) observed a specified region probably significant for cell adhesion in hepatic parenchymatous cells. This is a knoblike projection of cytoplasm that exactly fits a concavity on the surface of the neighboring cell. This structure apparently belongs to the intercellular interdigitation, but is somewhat specific, because it is only rarely observed and occurs singly, unlike the multiple foldings observed in the sweat glands. A similar interdigitation to those reported by Fawcett was found in the mouse thyroid by Ekholm and Sjostrand (1957). The interdigitations of the type found in sweat glands were also found in many other epithelia, such as intestinal epithelium ( Weiss, 1953), gastric surface epithelium (Kurosumi et d.,1958b), uterine epithelium (Nilsson, 1958a), ependyme of choroid plexus (Maxwell and Pease, 1956), distal convoluted segment of the frog nephron (Fawcett, 195S), bile duct epithelium (Kurosumi and Yamagishi, unpublished), and duct of salivary glands (Seki, 1959). The gastric gland bears this structure, but it is ill-developed. The intercellular bridges of the stratified epithelium also consist of interdigitated processes of the neighboring cells, although the intercellular space is markedly large (Fawcett, 1958; and our own unpublished observation). Hence the intercellular bridges may belong to the intercellular interdigitation in a broader sense. When the intercellular space is wide, in such a manner does the difference between microvilli and interdigitations become obscure (Scott and Pease, 1959). The interdigitations are not thought to represent a cell shrinkage caused

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FIG.13. Lateral configuration of the human sweat gland cells. A. From an apocrine sweat gland. The lateral intercellular boundary is provided with many adhesion plates (arrows) and relatively simple interdigitations (ID). ( x 15,000.) (T.Kitamura.) B and C. From eccrine sweat glands. The upper picture shows elaborately folded interdigitations, while the lower depicts rather simple microvilli-like interdigitations. (Magnification, B, x 13,000; C, x 12,000.) (T. Iijima, 1959.)

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by inappropriate fixation, and may be concerned with the firmness of cell attachment. The fact that the surface-covering epithelia of the gastrointestinal tract and of the skin bear the interdigitations and bridges may indicate the significance of this structure as mechanical reinforcement rather than the secretory function. In the sweat gland, the contraction of well-developed myoepithelium may exert a considerable force upon the glandular epithelium. This may be the reason for the occurrence of complicated interdigitations. b. The Terminal Bar and Adhesion Plates. At the uppermost part of the lateral cell margin occurs a thickening of the plasma membrane 0.5-1.0 p long. Iron-hematoxylin staining reveals this structure as a black line outlining the free surface of an epithelial cell. This is called the “terminal bar.” Under electron microscopy, the terminal bar is shown as a local thickening and increase in density of the plasma membrane (Fig. 21). The interstice between two thickened membranes is left clear, as are those in other portions of the intercellular space, but the interstice at the terminal bar is slightly narrower than those in others and was measured as 50 A. in tracheal mucosa by Rhodin and Dalhamn (1956). Ekholm and Sjostrand (1957) in the thyroid, and Ekholm and Edlund (1959) in the exocrine pancreas, observed, however, the fact that the clear interstice at the terminal bar is broadened as compared with those in the other parts of lateral intercellular boundaries. The thickened membrane of the terminal bar may sometimes be resolved to a double membrane (Ekholm and Sjostrand, 1957). The cytoplasm immediately adjacent to the thickened plasma membranes is denser than the general cytoplasm, fading off into the lighter area of cytoplasm without any sharp demarcation (Yamada, 1955 ; Kitamura, 1958) (Fig. 17A). But in some cell types (human sweat glands), little or no associating dense material is observed. As well as at the corner between the apical free surface and the lateral intercellular boundary, at the edges of the intercellular secretory capillary the terminal bar appears as observed in the gastric gland (Kurosumi et al., 1958b), eccrine sweat gland (Iijima, 1959), and at the bile capillary (Yamagishi, 1959). The terminal bar is structurally the same as the desmosome (node of Bizzozero or knot of Ranvier) of the intercellular bridge found in stratified epithelium (Selby, 1955; Odland, 1958), the intercalated disc of the heart muscle (Fawcett and Selby, 1958; Sjostrand et al., 1958), and synaptic plates in various nerve terminals (De Robertis and Bennett, 1955). These structures are considered as a special differentiation of the

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plasma membrane which is significant for a firm intercellular adhesion. “Adhesion plates” is used as a term including all of these structures. In glandular and covering epithelia, numerous adhesion plates may appear beside the terminal bar (Fig. 13A). It was recognized in rat gastric gland that mitochondria may attach to this thickening of the plasma membrane (Kurosumi et al., 195813) (Fig. 21A). When mitochondria of two adjacent epithelial cells adhere to the same portion of intercellular plasma membrane, they look like twin mitochondria. Owing to the direction of the cutting plane, the attached mitochondrion on one side is sometimes smaller than that on the other side, or it appears unilaterally. This phenomenon occurs in a high percentage of cases (about 45% in 149 observed cell boundaries). Considering the thinness of the section, it may be supposed that this phenomenon might occur in another part of the same cell, even though it is absent in a given plane of section. This attachment of mitochondria occurs at a point shortly apart from the terminal bar, but has never been observed at the very point of the terminal bar or any parts of far more basal territories of the lateral cell border. Nor is it seen at the apical or basal surfaces. This curious phenomenon was also found in epithelium of interlobular bile ducts of rabbit liver (Kurosumi and Yamagishi, unpublished) (Fig. 17A). It is quite interesting that the cell surface to which mitochondria attach is similar in morphology to the terminal bar and the desmosome. At the desmosome of stratified squamous epithelia, attachment of tonofilaments to the thickened plate of the plasma membrane has been noticed (Selby, 1955). Furthermore, it was reported in some of the ciliated cells that rootlet fibrils of cilia attach to the lateral cell boundaries (Kanda and Tanaka, 1959). Thus the thickened part of the plasma membrane, which is thought to be a special device for cohesion of epithelial cells, may possess an unknown power to attract some bodies in the cell. In this case, the mitochondria might be one of the attracted bodies.

3. The Basal Cell Surface a. Infoldings of the Basal Plasma Membrane. I n some glandular cells yielding a secretion with a high amount of water such as in the sweat gland, salivary gland, and choroid plexus, the plasma membrane at the basal cell surface may be invaginated into the cytoplasm (Fig. 14). This structure is rather well developed in some absorptive epithelia as well, for irlstance, kidney tubule cells in which the structure was first noted. Pease and Baker (1950) found “tubular sheaths” surrounding mitochondria at the basal cytoplasm of the proximal convoluted tubule. Dalton ( 195lc) referred to them as “filament-like structures” or “intracellular

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lamellae.” Then Sjostrand (1953) and Sjostrand and Rhodin (1953) disclosed that this structureis actually a double membrane, which was later designated as “P-cytomenibrane” by Sjostrand (1956) ; Rhodin (1954) and Pease (1955) subsequently determined that such a membrane system is a

FIG.14. Electron micrograph of the basal part of a glandular cell of the human apocrine sweat gland. Infoldings ( I F ) of the basal plasma membrane and small vesicles ( S V ) continuous to them may be noted. MT, mitochondria; LSG, light secretory granules ; DSG, dark secretory granules ; EDG, encapsulated dark granules considered as a younger form of the dark secretory granule. ( x 13,000.) ( K . Kurosumi et al., 1959a.)

heap of infolded basal plasma membrane. Pease (1955) suggested from this study that the infolding may be associated with the transport of water in order to reabsorb urine. H e and his collaborator (Pease, 1956a; Maxwell and Pease, 1956) extended the observations on this structure to many other tissues and confirmed to a considerable extent the above-nien-

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tioned concept. Not only in kidney tubules of some mammalia, but also in the excretory organs of many lower animals was the structure found, such as amphibian kidney tubules (Bargmann et al., 1955; Arai, 1956), hlalpighian tubule of insects (Beams et al., 19SS), crayfish nephron tubule (Beams et al., 1956), and coxal gland of the scorpion (Rasmont et al., 1958). In exocrine glands, Weiss (1953) was the first who noted this structure. H e described the presence of basal infoldings in pancreatic acinar cells, but he ascribed this to the origin of the ergastoplasm and did not refer to a possible relation to water transport. The structure was noted in serous cells and duct epithelia of salivary glands (Pease, 1956a; Ichikawa and Irie, 1957a; Seki, 1959), ependymal cells of the choroid plexus (Millen and Rogers, 1956; Maxwell and Pease, 1956; Honjin and Yaniato, 1958), epithelium of the ciliary body (apical parts of the cell only in this case) (Holmberg, 1956; Pease, 1956a), epithelia of the utricle and stria vascularis of the inner ear (Smith, 1956, 1957), and follicular epithelium of the thyroid (Ekholm and Sjostrand, 1957). Epithelia of these various organs are all concerned with water transport, either absorption or secretion. Kurosumi and Kitamura (1958) first observed this structure in sweat glands (pig’s carpal organ). Then it was confirmed that the structure is highly developed in human sweat glands, both eccrine and apocrine (Kurosumi et d.,1958c, 1959a; Hibbs, 1958; Charles, 1959). In gastric glands, this structure occurs in the parietal cell although to a lesser extent (Kurosumi et al., 1958b; Hally, 1959b). W e believe that the infolded basal plasma membrane plays a role in absorption of water and watersoluble substances from the blood stream or extracellular fluid in the connective tissue surrounding the gland. Ruska et al. (1957) pointed out the possibility that the basal infolding of renal tubular cells may act in the reabsorption of urine, depending upon the hydrostatic pressure occurring in the space between the infolded membranes. They suggested that the mitochondria situated among the infoldings may participate in this mechanism as an energy generator for pushing the water into the blood vessel against the pressure gradient. In secretory cells no mitochondria are inserted among the infolded membranes. Such a difference may be reasonably interpreted as indicating that the water flow in gland cells is the reverse of that in renal tubular cells and therefore no power is required to push the water, namely, water may easily flow from blood capillaries into the gland cells with a negligible amount of energy consumption. In the sweat glands, it was often observed that small vesicles are aligned in a row which follows the infolded plasma membrane (Kurosumi

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et al., 1959a; Iijima, 1959) (Fig. 14). Bargmann et d. (1955) observed such rows of vesicles in the frog’s renal tubule, but they did not refer to the functional significance of this structure. Palade (1953b) found in capillary endothelial cells that many vesicles are concentrated immediately under the cell membrane and some of them appear to open at the surface. H e concluded that these vesicles may act for transporting fluids across the capillary wall. Later he also found rows of small vesicles continuous with the infolded plasma membrane in the splenic macrophage and assumed that these may show pinocytosis (Palade, 1956b). Smith (1957) has revealed beaded vesicles as well as basal infoldings in the marginal cells of guinea pig stria vascularis that is widely known as the site of endolymph formation. Our observation on sweat gland cells is quite similar to those by Palade and Smith. It may be assumed in the case of sweat glands that small vesicles may arise by pinching-off at the bottom of the infoldings or by multiple constrictions of them, and then migrate upward through the cytoplasm, transporting water from the basal to apical cell territories. This concept is in good agreement with the hypothesis of “membrane flow and membrane vesiculation” presented by Bennett (1956). Rhodin (1958) considered that the so-called basal infoldings of convoluted renal tubules are actually the ridgelike extensions protruding from the basal half of the cell, which are interlocked with those of the neighboring cell. H e demonstrated a schematic illustration in which the basal infoldings were represented as cogs of interlocking gears. In this regard the structure may not be distinguished from the interdigitation. But, in the same paper, Rhodin showed the presence of pure noninterlocking infoldings in the collecting tubule. Rhodin’s concept is discordant with Y . Suzuki’s finding (1958), on the morphogenesis of the infolding in developing proximal convoluted tubules, that vesicles in the cytoplasm may fuse into a broad flattened sac which in turn opens to the basal surface. Our observations revealed that most of the infoldings occurring in sweat gland cells are not interlocked, but are pure invagination of the basal plasma membrane into its own cell body. But the fact that the invaginated membrane is further invaginated, and the fenestration and anastomosis of infolded membranes manifest the strong complexity of basal infoldings. The three-dimensional shape of the structure was illustrated diagrammatically by Iijima (1959) (Fig. 15). b. The Basement Membrane. T h e glandular epithelium is invested with a common basement membrane which separates the epithelium from the connective tissue. If myoepithelial cells exist, the basement membrane commonly covers both the glandular epithelium and the myoepithelium.

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The electron microscope may disclose the basement membrane as a single dense membrane of varying thickness, for example, 150 A. in the exocrine part of the pancreas (Sjostrand and Hanzon, 1954a), 300 A. in the apocrine sweat gland (Kurosumi et al., 1959), and 490 A. in the thyroid gland (Ekholm and Sjostrand, 1957), the outlines of which do not appear sharply defined. Between this membrane and the basal plasma

FIG.15. A diagrammatic illustration showing the three-dimensional structure of the basal infolding of sweat gland cel!s. (T. Iijima, 1959.)

membrane of the glandular or myoepithelial cells, a clear space of constant thickness is always observed. I t is as wide as 250 A. in human apocrine glands (Kurosumi et al., 1959a) or 200-300 A. in the case of the epidermis (Ottoson et d.,1953). This space is not empty and clear but slightly dark, and thus is considered probably to be made up of a certain cementing substance. This space is continuous with the space between two apposed plasma membranes at the lateral adjoining surface of two neighboring gland cells and with the space between infolded 'membranes, if present. The basement membrane follows the invagination of the myoepithelial cell

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membrane but does not follow the infolded plasma membranes of the glandular cells, nor does it dive into the lateral intercellular space. It is doubted that the invagination of the surface of the myoepithelium may be caused by its contraction, and therefore it is not the essential feature. The fibrillar structure of the basement membrane is argued by some authors (Karrer, 1956), but is indistinct in basement membranes of glands. Although the controversy in this regard may be set aside in the present review, it must be noted that the diffusion through the basement membrane is far easier than through the plasma membrane, since no morphological change corresponding to the infolding and vesiculation of the plasma membrane is recognized at the surface or within the basement membrane. This fact may suggest a sieve-like structure for the basement membrane, although clear-cut evidence is not yet established. Findings on blood capillaries in some tissues, that pores exist through the endothelial lining but that the basement membrane underneath the endothelium is always continuous (Ekholm and Sjostrand, 1957 ; Stoeckenius and Kracht, 1958 ; Ekholm and Edlund, 1959), may support this assumption to some extent.

4. The Apical Free Surface a. The Microvilli. The free surface of epithelial cells is often modified to form a specific surface layer, such as the “brush border” of kidney tubule cells and of cells in sweat glands (It0 et al., 1956) and the “striated border” of intestinal epithelial cells, which were all demonstrated with the light microscope. Kolliker (1855) and Welcker (1857) postulated that the border of the intestinal epithelium might be perforated with numerous canaliculi, while Brettauer and Steinach (1857) and Heidenhain (1858) argued that the border consisted of threadlike processes protruding from the cytoplasm. The latter view was ascertained with the electron microscope by Granger and Baker ( 1950) and by Dalton et al. (1951). In thyroid glands, Monroe (1953), Braunsteiner et ul. (1953), and Dempsey and Peterson (1955) observed such filiform projections which may serve for the absorption of hormone. The best example of such a surface modification of protoplasm may be represented by the brush border of proximal convoluted tubules of the kidney, to which Pease and Baker (1950) extended an electron microscopic observation and found as cytoplasmic thin projections. Sjostrand and Rhodin (1953) gave an erroneous interpretation of this structure, that it might consist of honeycomb-like tubules. However, Rhodin (1954) and Pease (1955) corrected this assumption and confirmed the previous

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view by Pease and Baker. The projections were later called “microvilli” and .were also observed in other absorptive epithelia such as gall bladder epithelium (Yamada, 1955) and the surface of liver cells facing the sinusoid (Fawcett, 1955 ; Wasserman, 1958 ; Yamagishi, 1959). They were tentatively connected with the absorptive function through a vast increase of surface area available for the absorption. However, the microvilli are not restricted to the absorptive epithelium, but are observed on secretory cell surfaces, for example, the surface of liver cells facing the bile capillary (Fawcett, 1954, 1955; Coman, 1954; Yamagishi, 1959), the luminal surface of the pancreatic acinar cell (Sjostrand and Hanzon, 1954a ; Ichikawa, 1958), surfaces bounding the main lumen and intra- and intercellular secretory capillaries of various glandular cells of the stomach (Dalton, 1951b; Sedar, 1955; Challice et d.,1957; Ishimaru and Kada, 1956; Kurosumi et al., 1958b; Hally, 1959b) (Fig. 6B), the luminal surface as well as the surface facing the intercellular secretory capillary of sweat gland cells (Kurosumi and Kitamura, 1958 ; Kurosumi et a/., 1958c, 1959a) (Fig. 13A), and the surface of the goblet cell (Rhodin and Dalhamn, 1956; Palay, 1958). Parotid acinus cells of the rat are the exceptional case, extending microvilli not only from the luminal surface but also from the lateral and basal cell surfaces into the spaces between two adjacent cells or between the cell body and the basement membrane (Scott and Pease, 1959). The microvilli are cylindrical extensions of the cytoplasm with blunt tips. The cross-section diameter of microvilli is roughly uniform and measures usually about 80 mp, but the length is very widely variable, ranging from a slight elevation of surface protoplasm to what is as long as 1.5 p. The concentration is far more variable: the microvilli of the kidney tubule cell, the ependymal cell in the choroid plexus, and the intestinal epithelium are so closely packed to form the brush or striated border, that almost no space is left between the neighboring microvilli, while in some other epithelia only two or three villi can be observed on the whole area of the luminal cell surface in a given longitudinal section. In secretory epithelia, the form as well as the profusion of microvilli may be apparently concerned with the functional state of the cell. An active discharge of the secretion from the cell is usually accompanied by a disappearance or a decrease in number and in length of microvilli. Branching of microvilli is sometimes observed. .Yamada (1955) described in a study of the gall bladder epithelium that the heads of microvilli, “capitulum microvilli” as he designated them, were appreciably greater in density than those of the main shafts of microvilli, and from the tips as well as the distal parts of the shafts many

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delicate lacelike filaments radiated, “antenulae microvillares.” Nilsson (1958a) demonstrated a similar feature; in uterine surface epithelium, of a fine reticular luminal substance being gathered around microvilli. The possibility cannot be totally excluded that a coagulation of some material in the lumen near the free cell surface may bring about such a feature, the so-called antenulae. Zetterqvist ( 1956) showed the plasma membrane surrounding microvilli of the striated border of jejunal epithelium resolved into a triple-layered or double membrane structure, viz., two dense layers and an interposed clear layer. The total thickness of the plasma membrane is 105 A., the thickness of the two opaque layers is 40 A. in each, and the width of the clear space is 25 A. Ekholm and Sjostrand (1957) in the thyroid, Nilsson (1958b) in uterine surface epithelium, Hally (1958) in the Paneth cell of the intestine, and Hally (195913) and Kada (1959) in gastric parietal cells, revealed a similar double structure of the microvillous membrane. Some of these authors indicated that the plasma membrane lining the inter-microvillous crypts is similarly a double membrane (Ekholm and Sjostrand, 1957). In the microvilli on the wall of intracellular canaliculi of the mouse gastric parietal cell, Hally (1959b) found an additional membrane underlying the double plasma membrane. In the microvilli of the same portion but from different animal species, Kada (1959) reported that a dense granulate substance, probably the secretory product, may be contained within the space either between outer and inner microvillous membranes, in the case of dogs, or inside the inner membrane, in the rabbit microvilli (Fig. 16). H e postulated that the pinching off of microvillous tips containing the secretion might represent a chief mechanism in the secretion release by the parietal cell. Such a probable mechanism of the secretion release has been reasonably discussed about a considerable number of variants of the microvilli that are frequently shaped like polyps. Maxwell and Pease (1956) observed specifically-differentiated microvilli with extremely expanded tips in the ependymal cells of the choroid plexus and designated such a border composed of polyplike microvilli as the “polypoid border” (Fig. 17C). Van Breemen and Clemente (1955) suggested that the pinching off of the rounded tips of polypoid microvilli may represent a sort of apocrine secretion at the submicroscopic level. Similar features of microvilli were detected in pig’s carpal organ (Kitamura, 1958) and in the intrahepatic bile ducts (Kurosumi and Yamagishi, unpublished) (Fig. 17A). I t is noted that one or more round vesicles are contained in an expanded tip of the polypoid microvillus as well as in round or ellipsoidal bodies floating in

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the lumen in both cases of the choroid plexus and of the carpal organ (Millen and Rogers, 1956 ; Kitamura, 1958). b. The Crust. In human apocrine sweat glands, a dark glassy layer is observed underlying the brush border with the light microscope (Minamitani, 1941). Montagna ( 1956) called this layer the “apical hyaline layer” or “hyaline terminal border.” The term “crust” (Crusta) was adopted for this structure by Toshio Ito (1949) and now is widely accepted. Rabbit

.

Dog

FIG.16. A diagrammatic illustration of microvilli occurring on the wall of the intracellular secretory capillaries of the gastric parietal cells from the rabbit and dog. (Courtesy of K. Kada, 1959.)

The crust is characterized in electron micrographs by its relatively high density, the presence of abundant tiny granules and vesicles, and the absence of mitochondria and secretory granules (Kurosumi et al., 1959a). The essential nature of the granules scattered in this area is unknown, but possibly they are some material belonging to the secretory substance (iron and pigment ?). Various-sized vesicles bounded by smooth single membranes are also scattered. The contents of the vesicles are quite clear. Smaller vesicles similar in appearance to those are present in the middle and basal cytoplasm, either freely distributed or aligned in rows successive to the basal infolding. The vesicles may arise by a pinching off from the infolded plasma membranes and migrate upward through

FIG.17. Electron micrographs of the polypoid border and the apocrine secretory projection. A and B. Epithelium lining the intrahepatic bile duct of the rabbit. L, lumen of the duct ; P, expanded tips of polypoid microvilli, some of which are pinched off and floating in the lumen; N, nucleus; G, Golgi apparatus. Arrows indicate attachment of mitochondria to the adhesion plates on the lateral intercellular membranes. AP, a stout projectioii extendipg from the cell surface of the bile duct epithelium. (Magnification A, X 7000; B, X 12,000.) (K. Kurosumi and M. Yamagishi.) C. The polypoid border of the choroid plexus in the third ventricle of the rat brain. ( X 15,000.) (Courtesy of the late H. Mitomo.)

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the cytoplasm, and may be finally accumulated at this apical layer, increasing their sizes. They are tentatively considered to be water-containing vesicles, and are also found in the eccrine sweat glands although the socalled “crust” is not formed in this gland (Hibbs, 1958; Iijima, 1959). According to Ito and Iwashige (1951), the thickness of the crust is proportional to the secretory activity of the cell. This fact strongly suggests that this specialized layer of the surface cytoplasm may be the accumulation of secretory substance, unlike the cuticular border of the other kinds of epithelium such as the sensory epithelia in the inner ear (Wersall, 1954; Smith and Dempsey, 1957 ; Engstrom and Wersall, 1958), where the cuticle may play a role in the mechanical reinforcement. c. The Apocrine Projection. The apical cell surface of the gland cell is more or less convex, and sometimes shows a strong bulging or projection which may be called the “apocrine projection” or “secretory extension’’ (Fig. 17B). It has long been believed that the extension may become decapitated or ruptured, and through the opening the secretion together with a small amount of cytoplasm may be discharged. These projections were observed in the thyroid gland (Braunsteiner et al., 1953; Ichikawa and Irie, 1957b), eccrine and apocrine sweat glands (Kitamura, 1958; Kurosumi et al., 1959a; Iijima, 1959), gastric parietal cells (Kurosumi et al., 1958b; Shibasaki, 1959), epithelia of bile ducts (Kurosumi and Yamagishi, unpublished), and those of excretory ducts of the submaxillary gland (Nakanishi, 1959). The content of such a huge projection is either watery clear or finely granulated, and sometimes contains small globular vesicles. The general outline and the size of the projection is variable. I n bile duct epithelium, various steps of size ranging from a stout microvillus with a rounded tip to a wide projection covering the whole area of the free cell surface were observed (Fig. 17A, B). Irregularly shaped projection in the apocrine sweat gland are very similar to the pseudopod of the leucocyte in amoeboid movement, not only in outline but also in texture of the contents (Low and Freeman, 1958). It is quite reasonable to assume that the formation of the apocrine projection may be associated with a local solation in colloidal state of the apical cytoplasm. The low density of the interior of the projection implies the high water content in this region. The absence of microvilli on the surface of the secretory extension (Braunsteiner et al., 1953; Kurosumi et al., 1958b, 1959a; Yoshimura and Irie, 1959a) may be interpreted by a possibility that a strong tension may be exerted on the covering plasma membrane. ’

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111. Special Cytology and Experimentally Induced Changes in Ultrastiucture of Certain Secreting Cells A. THEEXOCRINE PANCREAS

1. The Norwtal Structure of Pancreatic Acinar Cells One of the most typical serous glands is the exocrine portion of the pancreas, the cells of which are characterized by an abundant load of ergastoplasm and secretory (zymogen) granules. The ergastoplasm (rough-surfaced endoplasmic reticulum) tends to localize in the basal part of the cone-shaped cell, but may extend also to the para- or supranuclear regions. The ultrastructure of the ergastoplasm varies from cell to cell owing probably to the functional state of the cell, i.e., in some cells they are arranged in parallel lamellae, while in others they take a form of isolated sacs of various sizes either round or irregular. Zymogen granules about 0.5-0.7 p in diameter are spherical dense bodies and are accumulated in the apical cytoplasm immediately beneath the luminal free surface. Long filamentous mitochondria with transversely oriented cristae are seen in a random disposition, but in almost all the cases they are oriented in the direction parallel to the long axis (basal to apical) of the cell. Round or oval mitochondria were noted in human materials (Ekholm and Edlund, 1959). A small region of the cytoplasm just above the round nucleus is occupied by the Golgi apparatus. Three components of the apparatus, viz., the Golgi lamellae, the Golgi vacuoles, and the Golgi granules or microvesicles, are observed. Small numbers of zymogen granules, which are small in size and hence considered as immature granules, are situated in the Golgi region (Fig. 18A). On the other hand, small granules with the same density as that of zymogen granules are often contained within sacs (cisternae) of the rough-surfaced endoplasniic reticulum. These have been noted in guinea pig pancreas and called “intracisternal granules” (Palade, 1956a) (Fig. 10). Such a feature was observed in pancreatic cells of fishes (Kurosumi et d.,1959b) and of frogs (Ogiso, 1959) as well. Ogiso (1959) asserted, however, that this feature might not represent the immature form of zymogen granules but represent a step of disintegration of these. The lumen of the acinus is relatively narrow, and the luminal surfaces of gland cells are adorned with microvilli. The acinar lumen is filled frequently with a dense substance of essentially the same appearance as the content of zyniogen granules (Siekevitz and Palade, 1958a ; Ekholm and Edlund, 1959), and a direct continuation between the content of a zymogen granule and the luminal substance was noted by Y. Watanabe st d.(1959). The lateral and basal cell boundaries are almost smooth,

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but a few interdigitations and basal infoldings can be observed (Sjostrand and Hanzon, 1954a ; Weiss, 1953 ; Ekholm and Edlund, 1959).

2. Changes Caused by Starvation and Refeeding In order to determine the formation of the ergastoplasm and its probable functions in the secretory activity, Weiss (1953) carried out the following experiment. Mice were placed on an insufficient diet, “a fasting diet.” At the end of 7 days’ fasting, some animals were killed and the survivors were fed their regular feed. At frequent intervals up to 6 hours following this first feeding, they were killed and the pancreas was examined. According to Weiss, there is a marked decrease in fasting animals in the number of zymogen granules, mitochondria, and ergastoplasmic sacs, the cavities of which become so reduced that the sacs appear as fibers. After the refeeding, ergastoplasmic sacs reappear in cytoplasmic centers, or in apposition to the nuclear and also plasma membranes (cf. p. 23). The base of an acinar cell of a mouse killed 90 minutes after postfasting feeding contains lamellarly arranged ergastoplasmic sacs, at both ends of which small buds are pinched off. Near the base of the cell, the spheres free from the parent sacs are small and contain electron-lucent material. As the apex of the cell is approached, the spheres become larger and some of them contain electron-dense material. All gradations may appear between empty spheres at the base and black secretion granules at the apex. The membranes surrounding the small spheres are granulated. However, as the sphere becomes larger, and its content becomes increasingly electron dense, the surrounding membrane becomes smoother, like those surrounding the mature zymogen granules. From this result Weiss concluded that the zymogen granules are essentially products of the ergastoplasmic sacs. Sjostrand and Hanzon ( 1954a) performed an experiment of starvation for 24 hours on mice, but they detected no change in pancreatic exocrine cells. Perhaps the time of starvation carried out by them might be too short to manifest any changes within the cell. Integrated morphological and biochemical studies with electron niicroscopy and ultracentrifugation were carried out on the guinea pig pancreas by Siekevitz and Palade (1958a). Two materials were used: as the first the gland was removed from animals starved for 48 hours, and as the second the pancreas was excised 1 hour after the beginning of a meal that ended a fast of 48 hours. The result in morphology were described as follows : “The pancreatic exocrine cells of starving guinea pigs were distinguished by an endoplasmic reticulum, the cisternae of which showed minimal lumina and extensive preferential orientation. Intracisternal

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granules were absent or only exceptionally present. By contrast, many of the exocrine cells of the guinea pigs killed 1 hour after feeding showed large accumulation of intracisternal granules in an endoplasmic reticulum characterized by distended cavities and little or no preferred orientation.” Biochemically, it was known that the microsomes from starved animals were found to account for 5 2 0 % of the trypsin-activatable proteolytic activity and ribonuclease activity of the whole cell, whereas in fed animals they contained 30% of these activities. It may be worth while to note that the specific enzymic activities of a heavy microsomal subfraction rich in intracisternal granules are almost equal to those of isolated purified zymogen granules. As the conclusion, Siekevitz and Palade (1958a) considered that the postprandial increase in microsomal enzymic activity might be due to the synthesis of new enzymes by the rough-surfaced part of the endoplasmic reticulum, and that the intracisternal granules represented precursors of the zymogen granules. From similar experiments on guinea pigs, however, I. Suzuki (1959) arrived at a diverse conclusion, that the zymogen granules of animals refed after starvation might be produced within the Golgi vacuoles (Fig. 18A). Ogiso (1959) studied the pancreas of frogs (Rana nigromaculata) and compared the pancreatic ultrastructure of the normally-fed frog with that of the animal under hibernation, which might be considered a sort of naturally occurring starvation. In the latter case, she found the decrease and fragmentation of ergastoplasmic sacs as well as an extreme reduction of zymogen granules. These results are very closely akin to those in starved frogs, except for one difference which is the occurrence of large fat droplets (she called them “colloid”) in pancreas cells of the starved animal. No remarkable change has been noted on mitochondria under various conditions such as hibernation, fasting, refeeding, and pilocarpine stimulation. She suggested the origin of zymogen granules from microsomes, but neither from mitochondria nor from Golgi apparatus.

3. Changes Induced by the Administration of Chemicals To this field of experimental cytology, electron microscopy was first applied by Sjostrand and Hanzon (1954a), who administered pilocarpine to mice, but they obtained only a negative result. Ichikawa (1958) experimented in a similar way on male rats, which were starved- for 24 hours followed by a stimulation with pilocarpine. Zymogen granules in pancreatic acinar cells of the injected animals are all extruded by 1% hours after the pilocarpine injection, and thereafter new formation of granules is markedly observed. At the first hour the frag-

FIG.18. Electron micrographs of the Golgi apparatus ( G ) of exocrine pancreas cells. Features suggestive of the new formation of zymogen granules at the Golgi region are depicted. A. From the pancreas of a guinea pig twice refed after fasting for 72 hours. The arrow indicates a dense granule, probably of a younger form of the zymogen granule appearing in a Golgi vacuole (intravacuolar granules). ( X 40,000.) (Courtesy of I. Suzuki, 1959.) B. From the pancreas of a rat injected with ethionine. Golgi vacuoles are enlarged and converted into the so-called “empty secretion granules” ( e S G ) . ( x 35,000.) (Courtesy of Y. Watanabe, K. Arakawa, and I. Yamanioto, 1959.)

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mentation and vesiculation of endoplasmic reticulum was noted. At the second hour new zymogen granules appeared in the Golgi region. Besides these, granules with the same density as that of the zymogen granule appeared within vacuole-shaped endoplasmic reticula (rough-surfaced) . Mitochondria in the apical cell zone were swollen and began to show an irregularity in the form of cristae suggesting a breakdown of the organelles. Thus three main organelles of the cell, the Golgi apparatus, the ergastoplasm, and the mitochondria, are all changed as a result of the administration of pilocarpine which may cause also a marked increase of secretory activity. H e concluded that all the three organelles are concerned with the formation of zymogen granules, though an occurrence of the so-called intracisternal granules was especially remarked upon. In the mouse killed 4 hours after an injection of pilocarpine, Palay ( 1958) observed an occurrence of peculiar polyvesicular bodies consisting of ergastoplasmic vesicles and cytoplasmic matrix together with numerous RNP granules. These bodies are not bounded by a distinct membrane, but are separated from the surrounding ergastoplasm by a clear space. The significance of these bodies is not known, but Palay suggested that these were exaggerated physiological changes rather than manifestations of toxic drug action. I. Suzuki (1958) studied the pancreas of guinea pigs injected with 1 mg. secretin, which is known to accelerate the pancreatic secretion. The intracisternal granules were very rarely observable in the pancreas of adult normal guinea pigs, but in the pancreas of secretin-injected animals numerous intracisternal granules could be observed which were apt to be grouped (Fig. 10). At one hour or more after the injection, intracisternal granules become reduced in number but mature secretory granules increase. Some granules suggestive of an intergrade between the intracisternal granules and mature zymogen granules are observed: this is the granule which completely fills the interior of a cisterna of the endoplasmic reticulum, whose membrane is apparently rough-surfaced. The result of this experiment suggests that the zymogen granules may originate from the rough-surfaced endoplasmic reticulum. On the same animal, however, he obtained a different result from a series of experiments of post-starvation feeding (I. Suzuki, 1959). H e observed granules with a similar density to that of the zymogen granule within the Golgi vacuoles of pancreatic cells of guinea pigs starved for 72 hours and subsequently refed twice in an interval of 6 hours, and he called them “intravacuolar granules” (Fig. 18A). H e concluded, “The intracisternal and intravacuolar granules are both considered to be immature forms of the secretory granule. -The results may suggest the formation of secretory

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granules from the Golgi apparatus as well as from the rough-surfaced variety of the endoplasmic reticulum, and may support the view by Palade (1956b) that the Golgi apparatus is only a local differentiation of the endoplasmic reticulum.” From the dog’s pancreas he also obtained a similar result through various experimental procedures. The acinar cells of animals whose pancreatic duct had been ligated contained many intracisternal granules, but the membrane of the cisternae was smooth-surfaced. In these cells the endoplasmic reticulum as well as the R N P granules were markedly reduced although the zymogen granules became more numerous. In the pancreas of dogs that have been injected with vagostigmine, which stimulates the vagus nerve and increases the secretion from the pancreas, the endoplasmic reticulum decreases while the Golgi apparatus becomes prominent. The Golgi vacuole in such an animal frequently contains a dark granule, the “intravacuolar granule.” In these respects, the origin of secretory granules may be ascribed to the Golgi apparatus. On the other hand, Y . Watanabe et al. (1959) arrived at the same conclusion from the experiment in which DL-ethionine was administered to male rats after feeding with diet insufficient in protein for 2 weeks. This chemical is known to inhibit protein synthesis as an antagonist in metabolism to methionine. Degenerated pancreas cells due to ethionine administration show remarkable changes in ergastoplasm, mitochondria, and zymogen granules. The ergastoplasm decreases in amount and is fragmented, the mitochondria suffer from deformation, and normal zymogen granules decrease in number or disappear entirely. Among these degenerated cells are observed some regenerating cells, in which many large vacuoles containing a substance of low electron density were present. The authors referred to these vacuoles as “empty secretion granules,” between which and the Golgi vacuoles various intergrades in form and dimension (Fig. 18B) were observed. Thus Y . Watanabe concluded that the secretion granules of pancreatic acinar cells may develop from the Golgi apparatus at least in the case of such experimental animals.

B. THEGASTROINTESTINAL MUCOSA The mucous membrane of the fundus and body of the stomach is provided with tubular glands called “gastric or fundic glands,” which are composed of four different types of glandular cells: (1) the body chief, (2) parietal or oxyntic, (3) mucous neck, and (4) argyrophile cells. The surface epithelium of the stomach is an independent cell type possessing a secretory function, too. The intestinal mucosa also consists of various cell types: (1) the absorptive cell, (2) the goblet cell, (3) the Paneth cell,

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and ( 4 ) the chromaffine cejl. Except for the first, all the others arc secreting cells. Comparing the gastric and intestinal mucosae, similarities of secreting cells are found between both of the mucous membranes.

1 . Zywogenic Cells The gastric body chief cell and Paneth cell of the intestine are very similar to the pancreatic exocrine cells and to the serous cells of salivary glands. All of these are interpreted as producing digestive enzymes, and hence they are often called zyrnogenic cells. a. The Gastric Body Chief Cell. The secretion granules of this cell are less opaque than the zymogen granules of the pancreatic acinar cell, and various grades of variation in size and electron density of the secretory granules and vacuoles are observed (Fig. 19). In the normal condition of the cell, the apical cytoplasm is usually occupied by many secretory vacuoles filled with an electron-lucent substance (Kurosumi et al., 1958b). One of the previous authors, Shibasaki ( 1959), subsequently studied the stomach of rats starved for several days and then refed, and extended our knowledge on the secretion mechanism in this cell. The body chief cell of the starved animal is somewhat hypotrophied, but the secretory vacuoles are still left in the apical cytoplasm, and the ergastoplasm shows marked parallel orientation of lamellae in the basal part of the cell (Fig. 5 ) . The Golgi apparatus can be seen in the supranuclear region. The cell from the animal killed at 30 minutes after the first feeding shows an almost complete expulsion of the remaining secretory vacuoles, and the new formation of secretory granules begins. At 1-3 hours after the feeding, the production of secretory granules and their transformation into vacuoles are remarkable. Newly formed secretory granules appear at the Golgi region. They are round, slightly dense bodies, covered with a smooth limiting membrane. Smaller ones are comparable in size with the Golgi vesicles, whereas larger ones have the same size as the secretory vacuoles. The electron density of the interior of smaller granules is relatively high, but is always less than that of the pancreatic zymogen granules. As the size of the granule increases, the density of the interior decreases markedly until becoming the same as that of the secretory vacuoles, whose limiting membranes have partly or totally disappeared (Fig. 19). The fusion of neighboring vacuoles is often observed. Simultaneously with the occurrence of dark secretory granules in the G l g i region, the ergastoplasmic lamellae convert into many round sacs bounded by the particle-studded membrane. Such a vesiculation of the ergastoplasm is most remarkable at 3 hours after the refeeding. The size of rounded ergastoplasmic sacs increases as they ascend to the supra-

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nuclear region approaching the Golgi apparatus. The internal space of each sac is denser than the general cytoplasmic matrix and the original space between the double membrane of the ergastoplasm. Therefore, an accumulation of some material within the sac is evident. Juxtaposition of rough-surfaced vacuoles derived from the ergastoplasm and smooth-surfaced ones from the Golgi apparatus was frequently observed, but the confluence between these two types of vacuoles could not be determined. However, the diffusion or exchange of some of the substance can-

FIG.19. Supranuclear region of a body chief cell of the gastric gland from a rat at 3 hours of refeeding after starvation for 3 days. N, nucleus; L, lumen of the gland; M, mitochondria; G, Golgi apparatus. 1 indicates slightly enlarged Golgi vesicles, which may be the first step of the formation of secretory granules, 2-4 indicate the increasing steps in size of secretory granules, and 5-7 show dissolution of secretory substance becoming the secretory vacuoles. Complete transition from 1 to 7 is observed. Many ergastoplasmic sacs and mitochondria surround the Golgi region. ( x 22,000.) (S. Shibasaki, 1959.)

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not be negated. As clusters of free R N P granules are abundant in the apical cell zone, it is possible that the disappearance of membrane leaving the granules might occur on the ergastoplasmic sacs migrated hither. In a previous paper on the normal rat stomach (Kurosumi et al., 1958b), we postulated that the secretory granules and vacuoles might be produced through an expansion of vesicular rough-surfaced endoplasmic reticulum. But more recent study by Shibasaki (1959) revealed the apparent participation of the Golgi apparatus in the formation of secretory granules as described above. Additionally, the ergastoplasm (roughsurfaced endoplasmic reticulum) is also involved in the elaboration of secretory material in refed rats after a long period of starvation. It seems likely that the first step of the synthesis of the enzyme may be carried out in the ergastoplasm, and the product may be transported to the Golgi apparatus by the vesiculation and movement of the endoplasmic reticulum. In the Golgi region, the secretory granules are formed and grow, and then are liquefied very rapidly. Mitochondria do not participate directly in the secretion mechanism. Usually they are situated in the periphery of the cell lying parallel to the lateral cell surface. But in the most active stage of the formation of secretory granules at about 3 hours after refeeding, some mitochondria appear in the vicinity of the Golgi apparatus, suggesting their indirect participation in the granule formation. The mode of extrusion of the secretory substance into the lumen is quite unknown. The disappearance of limiting membranes of the secretory vacuoles is very conspicuous, and therefore diffusion of liquid secretion through the intact cell membrane is most probable. b. The Paneth Cell. It has been known that most of the digestive enzymes, especially peptidase of the intestinal juice, are secreted from the Paneth cell (Van Weel, 1937) which is the cell situated at the bottom of the intestinal crypt. The general morphology of the Paneth cell is very closely akin to that of pancreatic exocrine cells, i.e., the apical cell zone is filled with very dense secretory granules, and the base of the cell is packed with abundant lamellar ergastoplasm (Honjin et al., 1957; Hally, 1958). For this description we are chiefly indebted to Hally. The nucleus shows, however, a conspicuous irregular form, which differs remarkably from the relatively regular round nuclei of pancreatic and gastric zymogenic cells. The secretory or Paneth granules are dense spherical bodies of 0.75-1.5 p in diameter, each lying in a vacuole of 1-2 p in diameter. The vacuole is not a constant feature, however, as there are occasional cells where the space surrounding the granule is filled with a moderately dense substance.

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Regarding the mechanism of the formation of secretory granules, Hally described, “The secretory granules develop from a vacuole arising within the Golgi complex. First the vacuole incorporates Golgi vesicles through a deficiency in its enclosing membrane. Secondly, it comes to contain finely granular substance in addition to the cluster of vesicles. Thirdly, by further accumulation of granular substance it becomes a small secretory granule in which vesicles can still be seen embedded in the mass of granular substance.” The most conspicuous characteristic of secretory granules of the Paneth cell may be the clear “halo” or “vacuole” around the granule. The true nature of such a halo was not determined, but Hally demonstrated that this halo was unaltered by changes in the tonicity of the fixative or washing fluid, or by a 10 minutes delay in fixation. A similar halo was observed around granules in the fish pancreas and noted as one of the “intracisternal granules” (Kurosumi et al., 1959b). In both cases, the halo is sometimes replaced by a substance of intermediate density. This finding may suggest that the halo does not appear totally as an artifact. However, a halo around the nuclear inclusion of snake’s liver cells (cf. p. 12) is considered as an artifact caused by shrinkage of the karyoplasni. Therefore, the halo around the Paneth granule s e e m to be produced by an extraction of peripheral substance during the specimen preparation, and enhanced to some extent by a small degree of shrinkage of the surrounding cytoplasm or the granule itself. It is still unknown whether such a feature means the dissolution process of the mature granule, or a step of maturing in which the peripheral space is destined to be filled with the secretion material.

2. Oxyntic Cells The parietal cell of the gastric gland is the only example of the oxyntic or acid-secreting cell of the human and higher animal bodies. Electron microscopic observations on this cell have been reported by relatively many authors such as Dalton (1951b), Sedar (1955), Ishiniaru and Kada (1956), Challice et al. (1957), Umetani ( 1957), Kurosunii et a.1. ( 1958b), Hally ( 1959a,bj , Kada (1959), and Shibasaki (1959). Oxyphile granules which characterize the parietal cell were regarded as the secretion granules by some earlier researchers (Muller, 1898; Zimmermann, 1898), but some other light microscopists postulated them as mitochondria (Lim and Ma, 1926; Beams and King, 1932). The latter view is completely endorsed by electron microscopy as recorded by all the investigators above mentioned. Mitochondria of this cell, however, are specifically differentiated, namely, they are twice as large as the mito-

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chondria of the other cells, are very abundant in number, and contain an extremely close packing of cristae mitochondriales (Fig. 6 B ) . They are usually round or elliptical in form, but are sometimes rodlike and branched. One of the conspicuous characteristics of this cell is the presence of many vesicles bounded by a smooth single membrane (Figs. 6B, 2OA). These were first observed by Sedar ( 1955), and classified into the smoothsurfaced variety of the endoplasmic reticulum by Palade ( 1956b). These are 30-600 mp in diameter and are either randomly distributed or grouped in clusters or chained in rows. The content is very transparent. Preferential disposition of vesicles gathering adjacent to the intracellular canaliculi has been noted (Kurosumi et al., 1958b; Hally, 1959b). In the stomach of the starved rat the vesicles are abundant, but they are markedly reduced in number at 30 minutes after feeding (Fig. 20A and B ) . Instead, small particulate components freely scattered in the cytoplasm are numerous. During 1-3 hours the number of vesicles increases gradually, but free particles diminish in reverse proportion to the multiplication of vesicles ( Shibasaki, 1959). Therefore, the vesicles of smooth-surfaced endoplasmic reticulum are closely related to the secretory function of this cell. W e postulate that the secretion of this cell, a precursor of hydrochloric acid, may be produced by or accumulated in these vesicles, and then released into the lumina of intracellular canaliculi, or to the main lumen through openings of the vesicles occurring at the bottom of the intermicrovillous crypts. The mitochondria are also related to some extent indirectly to the secretory function, because the variation of the mitochondria population is proportional to the number of the smooth-surfaced vesicles. Hally (1959a) pointed out that the “vacuole-containing bodies,” which consist of a vacuole-0.2-0.4 p in diameter-bounded by a single membrane containing small 500 A. vacuoles, were found one or two in each section in the fasting mouse, but became more numerous and much larger in the parietal cell of pilocarpine-injected mice. From these observations, he suggested that this peculiar body might be associated in some way with the secretory state of the cell. In this cell type, no typical Golgi apparatus was recognized (Kurosumi et al., 1958b; Hally 1959b). Sedar (1955) and Challice et al. (1957), however, described the existence of the Golgi apparatus. It seems likely that a localized close-packing of vesicles or intracellular canaliculi whose lumina were obliterated might be erroneously referred to as a typical Golgi apparatus. Rough-surfaced cisternae of the endoplasmic reticulum are absent or only exceptionally found, but freely scattered small particles ( R N P

FIG.20. Two different states of secretory function of the gastric parietal cells of the rat. A. From a starved rat. The cytoplasm is filled with numerous vesicles belonging to the smooth-surfaced endoplasmic reticulum. Intracellular secretory canaliculi (C) have almost collapsed. I t may he a state of strong retention of secretion within the cell. ( x 7000.) B. From a rat at 30 minutes after the onset of refeeding, when the secretion discharge is markedly active. Vesicles of smoothsurfaced endoplasmic reticulum are very few and small, but small dense particles are relatively abundant. Lumina of the intracellular canaliculi (C) are dilated. ( x 10,000.) (S. Shibasaki, 1959.)

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granules?) can be observed, These are tentatively assumed to be the origin of the smooth-surfaced vesicles (Kurosumi et al., 1958b ; Shibasaki, 1959j . The intracellular canaliculi or secretory capillaries are the tubular invagination of the surface plasma membrane. Dalton (1951b j first found the microvilli on the lining membrane of the canaliculi and described them as “striated border” or “filamentous border.” In the rat parietal cell these are of regular cylindrical form with an average diameter of 80 nip and the maximum length of about 800 mp. Hally (1959b) described the microvilli of the mouse parietal cell as having an additional membrane underlying the double plasma membrane. Kada (1959) found a dense granular substance contained either within the space between the double microvillous membranes (dogs j or inside the inner membrane (rabbits) (Fig. 16). In the case of .the rabbit, atropine administration makes an increase in number of the microvilli, while pilocarpine administration causes a decrease in number as well as disappearance of the dense substance of the microvilli. In the case of the dogs, pilocarpine injection induces a change so that the microvilli are expanded and lose the dense substance between the double surface membranes. Kada postulated that the dark substance found in the microvilli is the secretory substance, which may be derived from the mitochondria and released into the lumen by a pinching off of the microvilli. In rat’s parietal cells, however, we could observe neither such a dense substance nor a pinching-off phenomenon. IVe found a stout projection of cytoplasm extending from the free cell surface into the lumen like a tongue. No microvilli could be observed on the surface of this projection. Its contents are generally less dense, small particulate substance fills it and it contains no mitochondria. A few small vesicles probably identical with the smooth-surfaced endoplasmic reticulum are scattered among the particles at the base of the projection. I t is considered that this projection may be constricted off at the base and may form a part of the secretion of the gastric gland, namely, the secretion mechanism of apocrine type may exist in the parietal cell besides the eccrine type secretion which may be transported through the intracellular canaliculi. As Shibasaki found that apocrine projections are numerous in starved rats, such a mode of secretion may probably be one of some abnormal or stressed cell activities.

3. Mucus-Secreting Cells a. The Goblet Cell. The goblet cell of the intestinal mucosa was studied by Palay (1958). The apical cytoplasm of the full-grown goblet cell is entirely occupied by secretory, mucous droplets forming the socalled goblet. Mature mucous droplets are mostly spherical or oval bodies

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of 0.5-1.5 p in diameter with no limiting membrane. The electron density of the mucous substance is rather low, and sometimes a floccular or bubbled appearance is perceived. The ergastoplasm and mitochondria are usually pushed away by the growing goblet to the cell periphery. The nucleus is also pushed downwards. Relatively well-developed Golgi apparatus is observed at the supranuclear region. In growing goblet cells, both the Golgi apparatus and the ergastoplasm proliferate. In such Golgi apparatus, large vesicles or vacuoles possessing a content somewhat denser than the cytoplasmic matrix are often observed. Thus, Palay (1958) emphasized the origin of mucous droplets as the Golgi apparatus. The droplets accumulating in the apex of the cell gradually distend it so that the microvilli at the surface become flattened out. Finally the apex of the cell bursts open, and the contents of the goblet, consisting of coalesced mucous droplets, flows out into the lumen of the intestine. An electron microscopic study of the goblet cell of the tracheal mucosa by Rhodin and Dalhamn (1956) arrived at a similar conclusion. They discussed that the Golgi membranes might be widened to become vacuoles, resembling very much the mucous granules, and moreover, the Golgi membranes were seen in very intimate contact with the mucous granules, so that there remained a possibility that the Golgi apparatus could be involved in the formation of mucus. They offered another possible source concerning the mucus production, the “large opaque granules,” which were reported only in the tracheal goblet cells. These granules vary in size from 0.4 to 1.0 p, and were classified into three types. The first type is characterized by one large intensely opaque inner granule and several smaller ones with an occasional limiting membrane. The second type is bordered by a single membrane separated from the inner opaque mass by a narrow clear space. The third type consists of rather small granules with concentrically arranged membranes. The last somewhat resembles the lamellar body composed of S-cytomembranes which was described by Schulz and de Paola (1958), and postulated as the place of origin of mucus in the gill epithelium of the salamander. According to Rhodin and Dalhamn, the number of the “large opaque granules” is inversely proportional to that of the mucous droplets, suggesting a probable participation of these granules in the formation of mucus. Additionally, the electron microscopic criteria of the goblet cell, among various cells of the tracheal mucosa, were indicated by these authors as the very dense cytoplasm and the irregular lobated nucleus. b. The Gastric Mzicous Neck Cell. The neck chief or mucous neck

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cell is found mostly at the neck of the gastric gland, and is known to secrete mucus, in spite of f i e similarities in gross morphology to the body chief cell. Under light microscopy an irregularity of the nuclear outline, which is also detected in other mucus-secreting cells such as the goblet cell, characterizes the mucous neck cell. This fact is also proved by electron microscopy. Electron microscopic morphology of this cell generally agrees with that of the goblet cell (Kurosumi et al., 1958b). But in this cell, a circumscribed oval area filled with mucus, the so-called goblet, is not formed, i.e., mucous droplets are widely spread in the apical cytoplasm (Fig. 21A). The Golgi apparatus is frequently situated in the midst of a heap of the inucous droplets. Such a feature cannot be seen in the goblet cell. The appearance of mucous droplets (secretory granules) is very closely akin to that in the goblet cell, namely, these are oval or spherical bodies with the roughly uniform size of about 1.0-1.5 p in diameter. The content is moderately dense like the goblet cell droplets, slightly darker than that of the secretory vacuoles of the body chief cell. Unlike the secretory granules of the latter, no gradual variation of the density was observed among mucous droplets within a single cell. However, the density of droplets may vary from cell to cell, due probably to the fixation influence. The texture of the content of the droplets is not homogenous, but a faint reticular or foamy appearance is observed. The limiting membrane is not provided in most granules, but some small immature granules are bounded with a single smooth membrane. The latter are found near or in the Golgi apparatus (Fig. 21C). Each of the mature droplets is frequently encircled by a clear space which is often made up of chains of clear foams. This was considered as an artifact probably caused by shrinkage of the droplets or bubbling around them occurring during fixation or embedding (polymerization of plastic). It is often observed that several pairs of the Golgi membrane are closely applied to the surface of the mucous droplet (Fig. 21B). Kurosuini and his collaborators (1958b) concluded, “The real source of production of secretory granules is difficult to determine, but the close topographical relationship between the Golgi double membranes and the secretory granules and the fact that small droplets with distinct membranes are frequently observed near or in the Golgi area are sufficient to suspect an intimate association of the Golgi apparatus with the formation of secretory granules.” This conclusion coincides with those of‘ Palay (1958) and of Rhodin and Dalhamn (1956) on the secr,etory mechanism in the goblet cell. The gastric mucous neck cell has a considerable amount of ergastoplasm, which is composed of parallel lamellae lying along the cell periphery or

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FIG.21. Electron micrographs of the mucous neck cells of the normal rat gastric gland. A. Apical part of two contiguous mucous neck cells. Arrows indicate the attaching of mitochondria (MT) to the adhesion plates on the lateral cell boundary. TB, terminal bar; L, lumen of the gland; N, nucleus; SG, secretory granules; M V , microvilli. ( X 8400.) B. Golgi lamellae (G) closely applying the secretory granules. ( x 20,000.) C. Golgi apparatus ( G ) with lamellae and vacuoles. The arrow shows an immature secretory granule contained within the Golgi area. N, nucleus ; M , mitochondrion ; SG, secretory granule. ( x 15,000.) (K. Kurosumi et al., 1958b.)

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along the nuclear envelope. Both the mitochondria and the ergastoplasin are pressed against the celf boundary by a ‘bulk of accumulation of mucous droplets. Very long filamentous mitochondria were often observed. c. The Gastric Surface Epithelium. Epithelial cells lining the internal free surface of the gastric mucosa as well as the gastric pits have long been believed to be mucus-secreting cells. The nature of the mucus produced by this cell, however, is known to be slightly different from that secreted by the ordinary mucous glands or the goblet cells. Not only the staining behavior, but the electron density of the secretory granules, differ from that of mucous droplets of the mucous neck and the goblet cells. Thus this cell is reasonably called frequently the “mucoid cell.” In the normal condition of the cell, apical cytoplasm is usually occupied by a multitude of closely packed secretory granules. Electron density is highest among the secretory granules of various secretory cells of the gastric mucosa (Kurosumi et al., 1958b) (Fig. 22A). The size and shape of the granules are variable ; the more basally situated, the larger and more regularly spherical are the granules. At the superficial layer of the cytoplasm, many granules of rodlike or rectangular shape with rounded corners are observed, among which round ones are mixed. Rodlike profiles measure about 1.0 p in length and 0.3-0.5 p in width, and the round profiles are 1.0-1.5 p in diameter. Sometimes several rodlike profiles of secretory granules are aligned facing one another with their broad sides, looking like a rouleau of erythrocytes. Therefore, the three-dimensional form of this granule may be assumed to be a disc like a red blood cell. More basally situated granules, especially within the Golgi area, however, show rounded forms, never being observed as such rod-shaped ones. These round granules are relatively low in density, large in size, and less crowded (Fig. 22B, C). The Golgi apparatus is well developed in this cell, and is composed of straight or hairpin-like curved lamellae and numerous tiny vesicles. Large vacuoles are not observed at all. There are small round granules of various sizes ranging from those equal to the Golgi vesicles up to those as large as the secretory granules, situated within the Golgi area surrounded by lamellae (Fig. 22B, C). These granules show an enhanced density along the surface suggesting a limiting membrane, which is quite obscure around the mature granule. It is most probable that the secretory granules may arise from the Golgi vesicles, and that the immature ones may migrate into the more apical region of the cytoplasm and may change their form and density owing presumably to the condensation of the contents and to the close packing. The lumen of the foveola gastrica is seen dark in a striking contrast to

FIG.22. Electron micrographs of the surface epithelium of the normal rat gastric mucosa. A. A survey picture. Each epithelial cell contains many dark secretory granules of somewhat irregular shape at the apical portion of the cytoplasm. The nucleus is irregularly contoured, and the intercellular interdigitation is conspicuous. ( X 6000.) B and C. The Golgi regions of surface epithelial cells. Lamellae are markedly observed, but no vacuole is seen. Inside the .Golgi area, dense spherical granules of various sizes are contained. These may be the younger form of the secretory granule. Between these and small granular or vesicular components of the Golgi apparatus, there exist many transitional intergrades. ( x 16,000.) (K. Kurosumi et al., 195813.)

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the clear apical cytoplasm. It is often observed that some secretory granules, approaching the surface, become larger and irregular in form, but are somewhat lesser in density, resembling the luminal substance (Fig. 22A). Sometimes a direct continuity was observed between the surface-located granule and the luminal substance. The endoplasmic reticulum of rough-surfaced type is less developed in this cell, but freely scattered R N P granules are found in abundance. Mitochondria are either filamentous or round. The lateral intercellular boundary, especially its basal half, is strongly folded forming an elaborate interdigitation, which is thought to be useful in reinforcement against a mechanical injury.

4 . Chromafine and Argyrophile Cells A specific basal granulated cell has been noticed among various cells of the intestinal crypt. Granules of this cell are well preserved and yellowish tinted with chromium-containing fixatives, and hence the term “gelbe Zellen” or “chromaffine cell” is adopted for this cell. The chromaffine cell is also positive in the argentafine reaction of Masson (1928). The gastric glands of some mammals but not rats also possess chromaffine or argentaffine cells. Uchida (1958), however, found the basal clear cells in all the mammalian stomachs examined. These are clear owing to the absence of any stainable granules after ordinary staining procedures and are negative in argentaffine reaction. The third type is the acidophile basal granulated cell of Kull ( 1912) and Tehver (1930). The last is also negative in argentaffine reaction. However, the argyrophile reaction of BodianHamperl ( Hamperl, 1952) revealed small specific granules (argyrophile granules) in almost all of the three cell types including the chromaffine cell. Uchida proposed an inclusive term, “gelbe Zellen-System” for all of the three cell types. Kurosumi et al. (1958b), in a paper on electron microscopy of the normal rat stomach, used the term “argyrophile cell” as the representative for the system, because the argyrophile reaction is the commonest characteristic of this system and the so-called chromaffine cell (gelbe Zellen) is absent in the rat stomach. The enterochromaffine (intestinal argentaffine) cells have been observed by Christie (1955) and Honjin et d. (1957). Christie showed that the granules are spherical dense bodies of approximately 0.3 p, and that the cytoplasm is extremely dense as compared with those of columnar absorptive cells. As the technique for preparing electron microscopic specimens was not improved at that time, the detailed structure of this cell was left only incompletely clarified. Recently, the cells were observed by Nagano (personal communication)

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

77

FIG.23. Electron micrographs of the argyrophile cells (AR) of gastrointestinal tract. In both the intestinal and gastric cells, tiny specific granules with variable density and slender mitochondria are contained. Golgi apparatus is indistinct and the ergastoplasm is poorly developed. A. Intestinal chromaffine cell from the rat duodenum. ( x 7000.) (Courtesy of T. Nagano.) B. Gastric argyrophile cell (basal clear cell) of the rat. ( X 11,000.) (S. Shibasaki, 1959.)

78

K. KUROSUMI

and Taylor and Hayes (1959), and in general the architecture coincides with the gastric argyrophile cell reported by us (Kurosumi et al., 1958b). The specific granules of both gastric and intestinal argyrophile (chromaffine) cells are small round granules ranging in diameter from 70 to 200 nip (120 to 210 mp according to Honjin et al. (1957) and 100 to 250 mp according to Taylor and Hayes (1959) (Fig. 23). These are either far below or just at the limit of the resolving power of the light microscope, so that the usual preparations have often failed to demonstrate these granules, but the silver impregnation, which may somewhat enlarge each granule and enhance the contrast, has successfully demonstrated them under the ordinary light microscope. The electron density of the granules is variable, some are extremely opaque, but others are much clearer. Spherical or filamentous mitochondria of this cell system are always small and slender, measuring about 150 mp in diameter or width, which is half or one-third as much as that of mitochondria of zymogenic or mucous cells. Round or irregular contoured dense bodies probably identical to the fat droplets are found frequently. Rough-surfaced endoplasmic reticulum and Golgi apparatus are poorly developed, but small granular and vesicular components are richly contained and evenly distributed throughout the cell. The argyrophile cell system is considered to be one of the endocrine cells, for the gastric argyrophile cells are always situated basally, apart from the gland lumen, closely abutting the basement membrane ; and the enterochromaffine cells are characterized by the peculiar accumulation of granules in the basal cell zone which is never observed in any of the exocrine secretory granules. Furthermore, the electron microscopic morphology of this cell system, for instance, small specific granules and slender mitochondria, is very closely akin to that of some cell types of Langerhans’ islet or of the adenohypophysis (cf. p. 95). Some authors argued that the chromaffine cell might produce serotonin (enteramine) (Erspamer, 1953 ; Barter and Pearse, 1953). Shibasaki (1959) found no remarkable change in gastric argyrophile cells during a long period of starvation and refeeding thereafter, in which exocrine cells of the gastric gland showed marked alterations.

C. THESKINGLANDS 1. The Apocrine Sweat Gland Two reports of electron microscopy on the human axillary apocrine sweat gland have been published (Kurosumi et al., 1959a; Charles, 1959). The secretory portion of the gland consists of a complicated coiled tubule, which is lined with a simple columnar or cuboidal epithelium. Myoepithe-

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

79

lial cells are interposed between the glandular epithelium and the basement membrane (Fig. 24). The mitochondria are mostly spherical or oval, and the cristae mitochondriales are less developed and situated at the periphery of the organelle in a form of arch (Fig. 13A). The rough-surfaced variety of the endoplasmic reticulum is generally less developed, and a small number of rough-surfaced cisternae are closely applied to some mitochondria. Smooth-surfaced vesicles, however, are extremely abundant in these gland cells and accumulate in the crust, a layer just beneath the luminal free surface. Similar vesicles are often arranged in rows successive to the tips of infolded basal plasma membranes (Fig. 14). It was assumed that the vesicles might arise by pinching off from the tips of the basal infoldings or multiple constrictions of them, and then migrate upwards through the cytoplasm, transporting water from the basal to the apical cell zone (Kurosumi et al., 1959a). The Golgi apparatus is well developed and usually situated in the supranuclear region. I n this gland the Golgi lamellae are only rarely observed, but grouped small vesicles and large vacuoles are recognized. The human apocrine sweat gland has two distinct types of secretory granule (Fig. 2 5 ) , one of which is a less dense, regularly spherical or oval granule designated as “light secretory granule” by Kurosumi et al. ( 1959a), and as “smooth secretory granule” by Charles ( 1959). Thq former authors argued that this granule might be derived from the mitochondria. The density of the content is either equal to or slightly lower than that of the mitochondrial matrix. The limiting membrane is frequently resolved into double smooth membranes. Furthermore, straight or curved double membranes are contained within the granule, being assumed as the residue of cristae mitochondriales. Therefore, it may be reasonably concluded that the light granules are nothing but the modified mitochondria with increased internal matrix. But the difference in morphology from the typical mitochondria is apparently correlated with the polarity of the gland cell, i.e., the so-called light secretory granules are accumulated in the apical cytoplasm, whereas the typical mitochondria are numerous in the basal part (Fig. 14). Thus such a transformation from the mitochondria can be considered to be concerned with the active secretory function of the cell. The second type of secretory granule is characterized by its extremely high density. This type is called “dark” (Kurosumi et al., 1959a) or ‘--\/

Al koline phosphatase

Acid phosphotase

Beta-glucuronidase

s - -

/--

ye/

Esterase

Succinic-dehydrogenase

Cytochrome oxidase

FIG.1. Diagrammatic representation showing the distribution of several substances in direct ossification. a.o., adult osteocyte ; Y.o., young osteocyte ; o.b., osteoblast ; o.c., osteoclast ; P.o., preosteoblast.

The calcification of the cartilaginous matrix is associated with important histochemical modifications (Fig. 1). Ground substance loses its affinity for metachromatic staining, modifying its nietachroniasia ( Sylven, 1917a,b ; Rubin and Howard, 1950). It has been considered that these changes might be useful for the precipitation of calcium salts (calcifying ability). With these extracellular modifications, large quantities of alkaline phosphatase, phosphorylase, and esterases are observed, the latter being exclusively extracellular. W e must suppose that these enzymes, and also the modifications in the ground substance, are somehow related to the precipitation of calcium salts, and any study of a biochemical nature must be connected in some way to the presence of these substances, whose true significance

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in the decalcifying process of cartilage must be clarified. There are some theories on the mechanism of calcification which take into account these histochemical data. Another histochemical fact, which has been given great importance in the process of cartilaginous calcification, is the loss of glycogen when the hypertrophic cells become calcified ; these facts, together with others, such as the presence of phosphorylase and the biochemical determination of whole series of enzymes and intermediary substances found in anaerobic glucogenolysis ( Gutman and YU, 1950 ; Gutman, 1951 ; Albaum et al., 1952), induced the Gutman group to develop a theory on calcification based on a mechanism related to the glycogenolytic process. Without going into the purely biochemical details, we wish to point out that in many cases the calcification of cartilage (slow growth) is not associated with a significant diminution of glycogen in cartilaginous cells. On the other hand, the disappearance of glycogen could be considered as an involutive phenomenon, as there are facts sufficiently clear to suppose that when the cartilaginous calcification zone is large, necrobiosis is inevitable (Ham, 1953). This viewpoint is strengthened by some significant histochemical data : loss of enzymes (acid and alkaline phosphatase, P-glucuronidase, succinic dehydrogenase) , and other substances such as DNA, RNA, and glycogen (Fig. 1) . B. BONETISSUE The formation of bone tissue is related to the presence of various histochemically detectable substances. These substances, including enzymes, are found in osteoblasts and also in neighboring cells, but very little can be detected in bone matrix. Osteoblasts in full activity are rich in various enzymes (alkaline phosphatase, P-glucuronidase, phosphorylase, succinic dehydrogenase, and cytochronie oxidase) (Fig. 2). These enzymes probably act in the elaboration or metabolism of ground substance; alkaline phosphatase has been linked with the calcification mechanism (Robison, 1933; Robison and Rosenhein, 1934; Waldman, 1950), and lately it has been supposed that its function may be connected with the elaboration of proteins, as occurs in other parts of the organism, especially in the formation of collagen. Furthermore, R N A appears to be related to the formation of proteins (Cappellin, 1948). In other tissues cytoplasmic (3-glucuronidase is found in zones of active proliferation and also in neoplastic growths (Fishman et al., 1947), but it is difficult to suppose that it plays a similar role in osteoblasts ; perhaps it is also linked with the elaboration of mucoproteins (Cabrini and

300

ROMULO L. CABRINI

Schajowicz, 195s). Some authors, on the other hand, consider that its role is that of a link with the precipitation.of calcium salts (Monesi, 1958). Acid phosphatase, succinic dehydrogenase, and cytochrome oxidase exist in small proportions in the osteoblast. Since they are to be found in a large number of cells of the organism, it can be considered that in the osteoblasts they are connected with the general metabolism, but do not play a part directly in any specific function.

Alkaline phosphatase

Acid phosphatase

0

8 Beta-glucuronidase

Succinic dehydrogenase

Phosphorylase

0

8 Cytochrome oxidase

FIG.2. Diagrammatic representation of some enzymes in enchondral ossification. r.c.c., resting cartilage cell ; P.c.c., proliferative cartilage cell ; h.c.c., hypertrophic cartilage cell ; h.c.c.c., hypertrophic calcified cartilage cell ; c.t.. chondroclast ; o h . , osteoblast ; o.t., osteocyte.

Phosphorylase may play a part in the precipitation of calcium salts ; however, a secondary function in the metabolism of carbohydrates cannot be rejected. The distribution of glycogen in osteoblasts is interesting and worth while studying. While in direct calcification glycogen is found in the osteoblasts or in neighboring cells in great quantities, in rapid endochondral ossification it does h o t appear in appreciable quantities ; nevertheless, this only appears to be a contradiction because the sectors of rapid endochondral ossification are in those zones where there is enzymic loss of glycogen

HISTOCHEMISTRY OF OSSIFICATION

301

which settles in the hypertrophic cartilage. It is very probable that osteoblasts need, in order to function, some substance vinculated with the degradation of glycogen, but not precisely glycogen itself. This view is upheld by the absence of glycogen in the cytoplasm of hypertrophic osteoblasts in zones of direct ossification, accompanied by large quantities of glycogen in the neighboring mesenchynial cells. Glycogen, in osteocytes, appears to be a reserve substance similar to that found in the cells of adult cartilage and in dense fibrous tissue of the tendons. The study of numerous cases of pathological processes (tumoral, dystrophic, and inflammatory) in human bone tissue is an efficient method for the confirmation and widening of our knowledge of normal ossification. In many cases, the specific activity of a tissue can be observed better under pathological rather than under normal conditions ; in general and without going into details, we can affirm that the data given for normal material can be extended and confirmed in pathological processes (Schajowicz and Cabrini, 1954a, 1958a).

VI. Histochemistry of Bone Resorption Resorption of bone tissue is linked with two anatomical facts, the presence of giant cells and the existence of erosive lines. Resorption of calcified cartilaginous tissue is especially linked with the erosion vessels and with cells similar to osteoclasts : chondroclasts. Up to the present, histochemical data refers, in particular, to giant cells showing the presence of a large quantity of chemical substances and enzymes (mucoproteins, RNA, acid phosphatase, $-glucuronidase, succinic dehydrogenase, cytochrome oxidase, etc.). On the other hand, some of these substances have a variable disposition and appear in different quantities, creating in this way osteoclasts and chondroblasts of different types which would appear to be the consequence of different metabolic stages, that is to say, different functional phases. It is surmised that RNA takes some part in proteic metabolism (Morse and Greep, 1960). In general, the presence of mucoproteins has been looked upon as phagocytic material which would correspond to the eliminated ground substance. The possible role of enzymes found in osteoclasts has been studied very little as these facts are still very recent; they constitute an important field for future investigations. Despite the presence of large quantities of some enzymes (acid phosphatase, succinic dehydrogenase, cytochrome oxidase, and $-glucuronidase), this is not an important proof as regards the explanation of the destruction or elimination of calcium. Investigations which are at present being carried out in our laboratory (in collaboration with F.

TABLE I HISTOCHEMICAL DISTRIBUTION OF S O M E SUBSTANCES PRESENT I N BONEAND CARTILAGE CELLS Bone tissue Osteoblast Glycogen RNA Mucoprotein Alkaline phosphatase Acid phosphatase Phosphorylase fi-Glucuronidase Succinic dehydrogenase Cytochrome oxidase

++++ ++++ + ++++

f or

+f

+++ +++

+ +

Osteoclast

DURING

Osteocyte

Chondrocyte in proliferation

Hypertrophic chondrocyte

+++ ++

++++ +++

f

f

-

-

-

++++ ++++ ++++ ++++

-

+ +

++++ +

-

+

r

-

-

f

-

++++ +++

OSSIFICATION

PROCESS

Cartilage tissue

-or

+

-

+

Chondrocyte in calcified area -or++

0

f

&

s

++++

r

n

f

-

H

+

56

E 0

F

-

-

z 56

1:

+I

HISTOCHEMISTRY OF OSSIFICATION

303

Schajowicz, F. A. Carranza, Jr., and E. C. Merea) show clearly that the resorption of cellulosic foreign bodies is accompanied by a reactional granuloma rich in giant cells containing large quantities of the same enzymes ; this has been noticed in wound healing where histiocytes conimencing their macrophagic activities appear to acquire a high enzymic activity. (See Table I.)

VII. Histochemistry of Ossification in Endocrine Disturbances and Other Experimental Conditions Histochemical data are still very recent and therefore not many studies have been made on the histochemical behavior of bone in experimental conditions which are in some way linked with the formation of bone tissue ; it is understood that this type of work has to be performed under welldefined and correctly controlled conditions. In general, it cannot be expected for the moment that the histochemical variations should show other than little or no alterations in those cases where there is normal histology. Histochemical techniques, on the other hand, show a certain inconsistency which has to be evaluated before conclusions on experimental series can be made. Experimental hyperparathyroidism has been used for the study of certain variations in mucoprotein and glycogen content ( Heller-Steinberg, 1951) which we have already analyzed in Section IV,A, but further investigations are necessary in order to amplify and confirm these studies with other substances and enzymes. Coinciding with the involution of osteoblasts in scurvy, it has been possible to prove a loss of alkaline phosphatase; the appearance of some zones rich in fibroblastic cells with a glycogen content has also been observed (Bourne, 1942 ; Follis, 1950b ; Zorzolli and Nadel, 1953). Vitamin D deficiency does not cause a loss of alkaline phosphatase, and this fact would seem to confirm the participation of this enzyme in the formation of proteic material rather than in the precipitation of calcium salts (Morse and Greep, 1947, 1949). An increase in the PAS reaction and nietachromasia in the cartilaginous matrix has been observed (Hirschman, 1954) in rats maintained on a rachitogenic diet. In experimental fluorosclerosis, variations in alkaline phosphatase have been observed in accordance with the close; with high doses, a reduction of the enzyme was reported (Ichikawa, 1954). ACKNOWLEDGMENTS The author wishes to acknowledge his appreciation to Drs. Fermin A. Carranza, Jr. and Fritz Schajowicz for reading the manuscript and for helpful suggestions. He also wishes to express his gratitude to the Department of Biology and Medicine of the Atomic Energy Commission, Buenos Aires, where part of this review was done.

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Morse, A., and Greep, R. 0. (1960) Arch. Oral Biol. 2, 38. Nachlas, M. M., Tsou, K., Soqza, E., Cheng, C., and Seligman, A. M. (1957) J. Histochem. and Cytochem. 6, 420. Parvisi, V. R. (1938) Arch. ist. biochem. ital. 10, 281. Pearse, A. G. E., and Macpherson, C. R. (1958) J. Pathol. Bacteriol. 76, 69. Pearson, B., and Defendi, V. (1954) J . Histochem. and Cytochem. 2, 248. Pritchard, J. J. (1952) J . Anat. 88, 259. Pritchard, J. J. (1956) I n “The Biochemistry and Physiology of Bone” (G. H. Bourne, ed.), p. 179. Academic Press, New York. Riley, J. F. (1959) “The Mast Cells.” Livingston, Edinburgh. Robison, R. (1933) Biochm. J . 17, 286. Robison, R., and Rosenheim, A. H. (1934) Biochem. J. 28, 694. Rosa, C., and J. T.Velardo (1954) 1. Histochem. and Cytochem. 2, 110. Rosin, A. (1952) Acta Anat. 16, 29. Rossi, F., Pescotto, G., and Reale, E. (1951) C. Atti. SOC.Ztal. Anat. 13th Congr. Sociate p. 1. Rubin, P. J., and Howard, J. E. (1950) Trans. 2nd Josiah M a y , Jr. Conf. Metabolic Interrelations 2, 155. Schajowicz, F., and Cabrini, R. L. (1954a) I. Bone and Joint Surg. W A , 474. Schajowicz, F., and Cabrini, R. L. (1954b) Bol. SOC.Arg. Ort. y Traum. 19, 148. Schajowicz, F., and Cabrini, R. L. (1955) J . Histochem. and Cytochem. 3, 122. Schajowicz, F., and Cabrini, R. L. (1956a) Stain Technol. 31, 129. Schajowicz, F., and Cabrini, R. L. (1956b) Rev. Ort. y Traum. Latino am. 1, 1. Schajowicz, F., and Cabrini, R. L. (1957) Rev. SOC. Arg. BioZ. 33, 257. Schajowicz, F., and Cabrini, R. L. (1958a) J. Bone and Joint Surg. 40A, 1081. Schajowicz, F., and Cabrini, R. L. (1958b) Rev. Ort. y Traum. Latino am. 3, 213. Schajowicz, F., and Cabrini, R. L. (1958~) Science 127, 1447. Schajowicz, F., and Cabrini, R. L. (1959) Stain Technol. 34, 59. Schajowicz, F., and Cabrini, R. L. (1%0) Science 191, 1013. Screebny, L. M., and Nikiforuk, G. (1951) Science 113, 560. Seligman, A. M., Tson, K. C., Rutemberg, S. H., and Cohen, R. B. (1954) J . Histochem. and Cytochrm. 2, 209. Sibert, R. S. (1951) J. Exptl. died. 93, 415. Sognnaes, R. F. (1955) Ann. N . Y . Acad. Sci. SO, 545. Steedman, H. F. (1950) Quart. J . Microscop. Sci. 91, 477. Sylven, B. (1941) Acta. Chir. Scartd. 86, 1. Sylven, B. (1947a) J . Bone and Joint Surg. 29A, 973. Sylven, B. (1947b) 1. Bone and Joint Surg. 29A, 745. Sylven, B. (1948) J . Bone and Joint Surg. 3OA, 158. Sylven, B. (1954) Quart. J. Microscop. Sci. 96, 327. Takamatsu, H. (1939) Trans. SOC.Pathol. Japoit. 29, 437. Takeuchi, T. (1956) J. Histochem. and Cytochem. 4, 48. Tonna, E. A. (1958a) J . Gerontol. 13, 14. Tonna, E. A. (1958b) Nature 181, 486. Urist, M. R., and McLean, F. C. (1957) A.M.A. Arch. Pathol. 63, 239. Utsunomiya, S., and Matsuda, S. (1953) Zgaku to Seibutsugaku 26, 213. Waldman, J. (1950) Trow. 2nd Josiah Macy, Jr. Conf. MetaboZic Interrelations 2, 203. Zorzolli, A. (1948) Anut. Record 102, 445. Zorzolli, A., and Nadel, E. M. (1953) J . Histochem. and Cytochem. 1, 362.

Cinematography, Indispensable Tool for Cytology’e2 C. M. POMERAT Pasadena Foundation for Medical Research, Pasadena, California

I. Introduction ..................................................... 11. Organotypic Cultures ............................................ A. Kidney ...................................................... B. Dorsal Root Ganglia ........................................ C. Sensory Organs ............................................. 111. Activities of the Nuclear Membrane and of the Nucleoli ............ A. Nuclear Rotation ........................................... B. Activities of the Nuclear Membrane .......................... C. Experimentally Produced Changes in the Shape and Density of Nucleoli .......................................... Acknowledgment ................................................ References ......................................................

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I. Introduction The investigator who presents evidences of cellular activity as revealed by time-lapse cinematography often finds that the transcription of dynamic projection material to the static printed page is made difficult because abstracts of selected film sequences in many instances fail to demonstrate complex serial events unless they are of adequate size and excursion distance, at the magnification employed. Beyond calling attention to a few significant contributions in the general area of cell biology, the value of cinematographic analyses will be concentrated at two morphological levels, ( A ) the organotypic and (B) the organoid, with special reference to the nuclear membrane and to nucleoli. By way of limiting the scope of these themes, the mitotic process will not be discussed. Dr. George Rose, who originated the method employed for the first group of studies, has prepared a paper entitled “Variations of the Cellophane Strip Technique for Tissue Culture and the Effects of Cytodifferentiation” which is intimately related to the point of view presented here. 1 This paper was illustrated by 1800 ft. of 16 mm. film when it was presented in Section 9 as “The Application of Cinematography to the Study of Cells,” at the 10th International Congress for Cell Biology in Paris, in September, 1960. 2 This investigation was supported in part by the U. S. Army Medical Research and Development Command, Department of the Army, under Research Contract NO. DA-MEDDH-61-12.

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11. Organotypic Cultures

The evolution of techriical procedures for the management of cell systems in vitro has provided a wide variety of methods which include cell cultures, in which undifferentiated metazoan cells behave like and are managed like microorganisms ; tissue cultures, where a proportion of the elements retain their original histiotypic properties ; and organ cultures, in which organotypic arrangements may be maintained, or further differentiation may be observed. It has long been known that frequent transfer of cell aggregates with its attending trauma favors rapid multiplication and the loss of characteristic specialized morphology. Conversely, the preservation of organotypic features is encouraged in systems which remain well encapsulated, without being subjected to undue mechanical disturbance. The emigration of cells from explants may also produce graded transitions of cultures from the organotypic to the undifferentiated cell type. Grobstein (1959) has reviewed the evidence which demonstrates the fallacy of considering that the decline in overt differentiation is related to simplification of the cellular system. H e concludes that . . The conditions under which organ cultures-are precisely those differentiation occurs in vitro-in which favor maintenance of mass and integrity.” The problem of the apparent loss of differentiation has led to renewed interest among students working with clone cell lines, as witnessed by the discussions at the Decennial Review Conference on Tissue Culture in 1956. While the concept of true dedifferentiation as such remains unpalatable, investigations in this area, and particularly in relation to tumorigenesis, continue to attract investigators. Thus, evidence for entirely different hereditable characteristics has been found in cells from two clonal cell lines by Sanford and her associates (1959), who employed the method of isolating single cells with capillary pipettes. These authors have placed considerable emphasis on the character of the culture medium employed to obtain their results. Attention is drawn to changes in nutritional habits or antigenicity of microorganisms accompanying changes in media. Among the hypotheses offered, emphasis is given to the importance of removing cells from the physiological controls of the host to a situation which, perforce, must bring about a drastic change in cell metabolism. The most favorable of culture media “. . . cannot possibly contain all factors present in the tissue fluids and must induce metabolic changes in the cell. Specific substrates can influence enzyme formation and activity and mediate synthetic changes, thus acting as differentiating agents. Such a permanent change in metabolic pattern may occur, just as in embryonic differentiation cells acquire relatively permanent and irreversible differences in nlorphology and function” (Sanford et d., 1959). ‘I.

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That proteins in the immediate environment of the cell may be important to the establishment of clonal lines is underlined by the condition of restricted diffusion which obtains with the use of isolations employing the capillary method. To this, Harris’ (1957) emphasis on the possible role of proteins in stabilizing cell properties bears upon the immediate cell environment which may be associated with the maintenance of the organotypic state in vitro. The presence of extra-cytoplasmic substances involved in the process of chick metanephric tubular formation has received brilliant experimental proof with the use of Millipore filter barriers (Grobstein, 1955, 1956) and as a result of studies of such systems with electron microscopy (Grobstein and Dalton, 1957). A review of various methods of organ culture, including Fell’s watch glass technique and its modifications by Gaillard (1951) and by Martinovitch (1951), and the more recent practice of employing cellulose acetate rafts (cf. Paul, 1959), indicates that there are certain essential conditions required to maintain tissue architecture. (1) The object to be studied must be sufficiently small to prevent irreparable central necrosis. The movement of nutrients and of waste products must be assured across limited distances. (2) Disorganizing migration of cells must be discouraged by employing well-encapsulated embryonic organs or by favoring the development of a cellular envelope around the excised fragment (Gaillard, 1950). ( 3 ) In attempting to cultivate explants of adult tissues, Trowel1 ( 1959) has emphasized that embryonic tissues are remarkably resistant to anoxia; nonetheless, gas exchange probably is one of the controlling factors in differentiating cell systems. ( 4 ) Limitation of diffusion of intercellular fluid in the cell complex would seem to be favored by the situation in which an air-fluid phase is always present. The liquefaction of plasma clots, with the consequential reduction of proximity to the air phase, appears unfavorable to explanted embryonic organs except for relatively brief periods. In the course of exploiting the possibilities of a sandwich-type culture chamber (Rose, 1954), difficulties were encountered with clot liquefaction when various tissues were employed. Furthermore, in attempting to cultivate thyroid explants in the absence of plasma, strips of cellophane cut from Visking dialysis tubing were employed for the fixation of the tissue to the glass surface. I n the course of developing this method, it became obvious that the character of the culture differed in areas covered by the dialysis membrane from those in which the cells extended freely on the glass surface.

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The purpose of the following section is to add observations with the aid of phase contrast cine time-lapse records to those already reported for muscle by Rose and associates (1958) and by Capers (1960) ; for the thyroid by Rose and Trunnell (1959) ; and for bone by Rose and Shindler (1960). Moreover, it is suggested that the cellophane strip Rose chamber method may be ideal for producing conditions in which large molecules may be retained close to the cellular complex, while free diffusion of smaller chemical entities is permissible due to the pore size of the dialysis membrane. This is approximately 24 A. for the Visking tubing. Significant for the thesis at hand is the fact that the optical properties of this method are highly favorable for phase microcinematog raphy in contrast to hanging drop, Maximow slides, or culture dishes such as those used in experimental embryology, where air phases and glass curvatures interfere with the condition essential for phase microscopy. The setup for organotypic cultures in Rose chambers employing dialysis iiiembrane technique is shown in Fig. 1. Of special value for cinematographic recording is a timer illuminated by a stroboscopic lamp in line with a prism on the side of the camera (Fig. 2). A single film frame showing the time record on its margin is shown in Fig. 3. Temporal relations were found to be more easily tabulated with this system than the more usual photographing of a clock. A.

KIDNEY

At low magnifications, the value of the method is suggested by a single film frame from a record of metanephric development from a 10-day chick embryo which was photographed at the rate of one frame every 5 minutes. After 3 days the culture (Fig. 4) appeared healthy with a more definitive outline suggestive of continued differentiation in Vitro. Cine records were made at one-minute intervals of kidney tissue cultures from a newborn rabbit on the first day (Fig. S ) , the fifth day (Fig. 6), and the fifteenth to the eighteenth day in vitro (Figs. 7-22). This series illustrated the optical properties of the method as well as the opportunity of following not only the course of differentiation, but also of the physioFIG.1. Central portion of Rose chamber setup for organotypic cultures. E, explant; D, dialysis membrane; A , air bubble; M , metal outerplate. The culture area is 25 mm. in diameter. FIG.2. Apparatus for recording time relations on the film record. T, timer; S, strobe lamp ; P, prism; C, camera ; V , viewing periscope. FIG.3. A single 16 mm. film frame of a microglial cell in a culture of a human pituitary tumor showing the time marker, the middle figure indicating the time in minutes.

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FIG.4. Metanephric complex of a 10-day chick embryo. From a 3-day record photographed at 1 frame per minute. FIGS.5-9. Newborn rabbit kidney. FIG.5. Explanted tissue photographed during the first day in vitro. FIG.6. The same area as shown in Fig. 5 but on the fifth day in vitro. FIG.7. Typical organotypic culture showing tubular epithelium, a clear basement membrane, and connective tissue with characteristically active histiocytes. Fifteenth day of culture. FIG.8. The same preparation as shown in Fig. 7 but at higher magnification. FIG. 9. Tubular structures showing numerous loops. A record of this field made at 1 frame per minute was particularly useful for demonstrating “peristaltic” activity.

FIGS.10-12. Newborn rabbit kidney cultivated under a dialysis membrane in a Rose chamber. Sixteen days in vitro and photographed at 1 frame per minute. The arrows call attention to a tubule whose lumen opened (Figs. 10, 12) and closed (Fig. 11). FIG.10, zero time; FIG.11, 5 hours and 28 minutes later; FIG.12, 9 hours and 16 minutes later. (Scale indicated on Fig. 10 applies to entire plate.)

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logical state of the tubular systems. Comparison of Figs. 5 and 6 shows the increase in interstitial tissue and the beginning of separation of the tubular mass. These phenomena which occurred for the same culture on the fifteenth day are shown in Figs. 10-12. Higher magnifications (Figs. 7, 8 ) illustrate the relations of epithelium, basement membrane, and connective tissue. In the film sequence of the culture from which Fig. 7 was abstracted, the activities of histiocytes were very conspicuous. Changes in the shape of tubules suggestive of a peristaltic wave were clearly evident in the area, including tubule loops as shown from a single film frame (Fig. 9). The most advantageous record for demonstrating possible changes in the functional state of a tubule has been abstracted. Repetitive evidence of the appearance and disappearance of a lumen in the tubule is indicated by the arrows (Figs. 10-12). With careful attention to focusing, it could be established with certainty that the phenomenon was real and not due to drifting of the focal plane. Figures 13-22 were prepared to illustrate changes in the outline of a tubular structure in an explant of the cortex of the newborn rabbit kidney as a function of time. The cine projection provided dramatic evidence of alterations in the cross sectional diameter of the segment. While these observations are, as yet, poorly documented, they may serve to suggest test objects for studies on the effect of electrolytes and hormones in a situation where direct recordings can be made of tissue elements under highly controllable conditions. The problem of renal interstitial fluid (cf. Swann et al., 1956), and current controversies concerning the functional state of various segmental regions of a particular nephron, might be profitably explored with the use of these preparations.

B. DORSALROOTGANGLIA A culture which typifies the appearance of groups of neurons under a dialysis membrane in Rose chambers is illustrated in Fig. 23 which was made after 41 days’ incubation of a ganglion obtained from a newborn FIGS.13-22. Newborn rabbit kidney. Same source as that shown in Figs. 10-12 but of a critical area photographed on the 17th and 18th days of culture. Changes in the cross sectional diameter of the lumen of a tubule is suggested by changes in its outline. In the cine projection, the phenomenon is best described as that of a peristaltic wave. (Scale indicated on Fig. 13 applies to entire plate.) FIG.13, zero time; FIG.14, 15 minutes later; FIG. 15, 2 hours 55 minutes later; FIG.16, 3 hours 30 minutes later; FIG.17, 5 hours 20 minutes later ; FIG. 18, 8 hours 15 minutes later; FIG. 19, 8 hours 55 minutes later; FIG.20, 13 hours 25 minutes later; FIG.21, 15 hours 25 minutes later; FIG.22, 25 hours 50 minutes later.

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rat. While niyelin is not shown in this image, the formation of this substance is frequently observed after approximately one month in vitro. The phenomenon of nuclear rotation, which was reported by Nakai (1956) for dissociated neurons, is very commonly observed in the type of

FIG.23. Doha1 root ganglion from a newborn rat cultivated under a dialysis membrane in a Rose chamber. Forty-one days in vifro-unstained preparation showing disposition of neurons. Photomontage of photomicrographs made with a dark phase objective.

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FIGS. 24-27. Neuron in a 13-day culture. The prominent nucleolus serves to describe the rotation of the nucleus. In Fig. 24 it is located at the 270" position, one hour later (Fig. 25), at 360"; at 3 hours (Fig. 26), at 90";and at 12% hours, at 180" (Fig. 27). (Scale indicated on Fig. 24 applies to entire plate.)

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culture preparations described here. Figures 24-27 illustrate how such measurements are made possible. The 'circular migration of the eccentric nucleolus can be plotted as a function of time. In this instance, a revolution of 360" required some 20 hours. Generally, complete revolution of the nucleus is accomplished in one to two hours. The value of the Rose chamber for obtaining continuous records with phase contrast optics of a given culture preparation, without the necessity of dismounting the cover glass, or filling the air space in depression slides with oil to overcome the effect of plano-concave glass, and preserving the index of refraction for the light beam, is illustrated with two series of experiments. In Figs. 28-30 abstracts of cine records are shown of the same culture, newborn rat dorsal root ganglion, which was photographed at 3 points in time during a 3-month period. Peterson and Murray (1955) have reported, in a brilliant analysis of manifestations of injury and the repair of neurons for explanted chick dorsal root ganglia, that after an initial displacement to the margin of the perikaryon the nucleus is restored to its central position. In the course of a series of experiments dealing with the relative radioresistance of neurons to irradiation, cine records have been made of the responses of cells from the dorsal root ganglia of 10-day chick embryos and of newborn rats. Figures 31-33 are concerned with the persistence of newborn rat neurons following irradiation with 10,000 r from a cobalt60 source. While non-neuronal elements, with the exception of occasional macrophages, appeared to be rapidly destroyed, little evidence of structural or activity manifestations could be observed in the neurons recorded under time-lapse cinematography on the first (Fig. 31), tenth (Fig. 32), and twenty-first (Fig. 33) day post irradiation. However, Bodian preparaFIGS.28-33. Selected film frames from cultures of newborn rat dorsal root ganglia which serve to illustrate the opportunity offered for recording cellular responses with phase contrast optics as a function of time without disturbing the preparation. (Scale indicated on Fig. 28 applies to entire plate.) FIGS.28-30. Abstracts from cine photomicrographs of the same culture made during the first week (Fig. 28), the first month (Fig. 29), and during the third month iir nitro (Fig. 30). FIGS.31-33. Abstracts of records made to establish the viability of neurons following irradiation with 10,000 r from a cobalt-60 source. FIG.31, 24 hours after irradiation; FIG.32, 10 days after irradiation; FIG.33, 21 days after irradiation. Nuclear rotation persisted even on the 21st day post irradiation. However, preparations treated with Bodian's method following this record showed evidences of injury to the neurofibrillar apparatus of the perikaryon.

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tions made of cultures fixed on the twenty-second day showed damage to the neurofibrillar apparatus in the area of the perikaryon. It is of interest that this method of culture appears to offer an important challenge in the study of neuronal radio-resistance in a situation where the nutrition is not dependent upon a relatively vulnerable capillary bed.

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The method proved to be of particular value for the analysis of the rhythmic pulsatile activity-of Schwann cells. The changes in form of an individual cell are illustrated in Figs. 34-39. This phenomenon was described on the basis of an analysis of a series of film records by Poinerat (1959). The cycle of contraction which was shown to require from 4 to 18 minutes was of the order of magnitude described for cells in oligodendrogliomas by Canti et al. (1937) and by Pomerat (1955), as well as in non-neoplastic tissue by Lumsden and Pomerat (1951) and by Pomerat ( 1958a). While the role of rhythmically active neuroglia remains obscure, their response to chemical influences such as serotonin and certain of its antagonists (Benitez et d.,1955), and for chlorpromazine and serotonin (Nakazawa, 1960), makes in vitro methods especially useful for neurophysiological studies. Although results obtained with the dialysis membrane technique in Rose chambers have not excelled those reported with the use of the cover slip roller tube method or with the Maximow assembly technique (cf. Bornstein and Murray, 1958), its great advantage has been in providing the opportunity of making repetitive observation with high power phase optics without disassembly of the culture.

C. SENSORY ORGANS Preliminary studies of the development on the isolated lens of 4-day chick embryos have been made by John Y. Harper, Jr. (unpublished). Activity along stellate rays of the explants has yielded film records which may prove valuable for the understanding of the genesis of fiber systems, and their injury as a result of the application of ionizing radiation. Indeed, the study of various structures of the eye (cf. Pomerat and Littlejohn, 1956), using culture methods favoring organotypic growth, may prove very rewarding. Illustrations are provided of 2-day cultures of the 4-day chick embryo lens at low (Fig. 40) and higher magnification (Fig. 41). In a systematic approach to the behavior of the otic complex of the chick embryo, John P. Reinecke, of Northwestern University Medical School, has demonstrated continued development of various portions of the sensory apparatus together with excellent maintenance of neurons. Figures 42 and 43 are representative of results obtained after 2 days’ incubation of material obtained from a 2-day embryo (unpublished data). The examples which have been given for organotypic cultures of kidney, dorsal root ganglia, the lens, and otic complex serve to illustrate the use of cinemafographic methods for analyses of the course of events in descriptive morphology. Cultures in perfusion chambers, especially when employed with the use of split film frame technique, as illustrated by

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FIGS. 34-39. A Schwann cell showing rhythmic pulsatile activity from a phase cine record made at high magnification. (Scale indicated on Fig. 34 applies to entire plate.) FIG.34, zero time; FIG.35, 1 minute; FIG. 36, 2 minutes; FIG.37, 3 minutes; FIG.38, 5 minutes; FIG.39, 9 minutes.

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recent work on Staphylococcus toxin and immune bodies (Felton and Pomerat, in press), and m the work which will be described in the next section for studies on the effect of A T P on the nucleolus (Yoshida and Pomerat, 1960), add importance to photographic procedures for analysis of cell behavior.

111. Activities of the Nuclear Membrane and of the Nucleoli The fact that the nuclear membrane is readily stainable, easily seen in living preparations, particularly in neurons, and that micrurgical technique demonstrated it to be tough, undoubtedly influenced workers of a recent era to look upon it as a relatively impassable barrier during interkinesis. The study of this structure with the use of electron microscopy has been highly rewarding. A review of current opinions regarding pore systems in the nuclear membrane is beyond the scope of the present report. Citation of a single paper may suffice for the purposes to which this section is directed (cf. Watson, 1959). Even as the existence of devices for permitting exchanges between the nuclear and cytoplasmic areas had had to be postulated, mobility manifestations of the nucleus, especially in blood cells, had been long appreciated. However, the presence of deep folds and inpocketings commonly observed in ultrathin sections of the nuclear membrane, and the recognition of the plasticity of the nucleus even in cells which are not characterized by active locomotion, invite speculation concerning intracellular traffic. Current views on the possible role of nucleolar RNA in regulating synthetic activity in the cytoplasm are that it cannot be looked upon as exclusively dependent upon a one-way traffic. The goal of this portion of the present paper is to call attention to phenomena which may be implicated in the entrance or exit of nuclear substances. ROTATION A. NUCLEAR The first phase in the description of this activity, which could be shown to be subject to quantitative analysis, in cultures of human nasal mucosa (Pomerat, 1953) consisted of demonstrating that it was common to many FIGS.40 and 41. Lens from a 4-day chick embryo at low (Fig. 40) and higher magnification (Fig. 41) after 2 days in culture. (Kindly provided by J. Y . Harper, Jr.) FIGS.42 and 43. Organotypic cultures from the otic complex of 2-day chick embryo after 2-days in vitro. A portion of what is believed to be a sensory structure (Fig. 42) and a group of adjacent neurons (Fig. 43). (These illustrations were obtained through the courtesy of Mr. John P. Reinecke.) (Scale indicated on Fig. 42 applies also to Fig. 43.)

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species of elements. Leone et al. (1955) studied the phenomenon in cells of the HeLa strain, and-Hintzsche (1956) in cultures of the mouse kidney papilla. It was described as taking place in spindle elements from the rat kidney which were presumed to be fibroblasts (Pomerat, 1958b), for neurons of embryonic chick dorsal root ganglia (Nakai, 1956), and in muscle fibers (Cooper and Konigsberg, 1959; Capers, l%O) . Studies on the mechanism responsible for nuclear rotation, and its consequence in the economy of the cell, are urgently needed.

B. ACTIVITIESOF

THE

NUCLEAR MEMBRANE

Cells with considerable mobility, and classed by Willmer (1954) as amebocytes, are particularly useful for demonstrating the plasticity of the nucleus. In the course of studying the effect of irradiation on cells of strain lines (Pomerat et al., 1957), deep folds were observed in nuclear membranes. Cinematographic records revealed twitching contractions in such areas. Selected film frames from a record of nuclear activity in a cell of the HeLa S3 clonal line, after 10 days’ incubation, are shown in Figs. 44-49. The culture had received 2000 r of gamma irradiation from a cobalt-60 source 8 days before the record was prepared. Changes in marginal outline are shown for a period covering 2 hours and 20 minutes. The progressive inward movement of a narrow segment of the nuclear membrane, as shown in Figs. 50-55, appeared to produce a spherical body which may have passed into the interior of the nucleus (see Fig. 54). This was also a cell of the S3 clonal HeLa line, but in this instance a sonic preparation of H . pertussis had been introduced 3 days after the culture had been growing for 11 days. The observation was made in the course of studies on cytopathogenic effects of pertussis antigens on living cells in vitro (cf. Felton et d.,1954). In a study of Staphylococcus toxin and antiserum, such phenomena have led to the suggestion that pinocytosis may be involved in the process of antigen-antibody reactions (Felton and Pomerat, in press). It is possible that antigens may even reach into the nucleoplasm. Cell destruction by an anti-HeLa serum was shown with the aid of cine technique to be accompanied by a dramatic shrinkage of the nucleus (Miller and Hsu, 1956). The use of fluorescein labeling to follow the migratory path of antibodies in cell culture might prove valuable in determining whether the nuclear membrane is involved in the actual penetration of substances from the cytoplasm (cf. Goldstein et al., 1959). In examining the activities of the nuclear membrane, careful focusing often reveals the pattern of profiles of indentations which are seen to be long folds “on the flat.” These features are demonstrated in Figs. 56-58,

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FIGS.44-49. HeLa clone S3. 10-day culture photographed 8 days after receiving 2000 r of gamma irradiation from a cobalt-60 source. (Scale indicated on Fig. 44 applies to entire plate.) FIG.44, initial selected film frame; FIG.45, after 7 minutes. Note the deep indentations which have developed in the margin of the nucleus at the upper edge of the photograph. FIG.46, after 13 minutes; FIG.47, after 17 minutes; FIG. 48, after 2 hours; FIG.49, after 2 hours and 20 minutes.

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FIGS.50-55. HeLa clonal S3. Cultivated for 11 days following which it received a sonic extract of H. pertussis. The film record was made 3 days later. Indentation of the nuclear margin is suggestive of the formation of an inwardly moving bud which can best be seen in Fig. 54 at the point marked by an arrow. (Scale indicated on Fig. 50 applies to entire plate.) FIG.50, zero time; FIG.51, 15 minutes later; FIG.52, 18 minutes later; FIG.53, 31 minutes later; FIG.54, 57 minutes later ; FIG.55, 1 hour later.

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FIGS.56-61. Cells of the HeLa S3 line showing indentations and folds of the nuclear membrane suggestive of changes in the content of the nucleus. Fifteen-day culture. Cine projections of such sequences demonstrated continuous alterations in the configuration of the nuclear surface. FIGS.56-58 were designed to show changes in the pattern as a result of varying the plane of focus. FIGS.59-61 represent typical examples of radiating folds which frequently showed angular bends. Twelve-day culture. (Scale indicated on Fig. 56 applies also to Figs. 57-59; scale on Fig. 60 applies also to Fig. 61.)

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in which the focal plane was allowed to drift at various levels through the nucleus of a HeLa cell. -Figure 59 shows radiating pleats in the nuclear membrane of a HeLa cell which often showed angular bends, as if the volume of the nuclear content were considerably reduced. Such patterns

FIG.62. The nuclear membrane of a HeLa cell 8 days following irradiation with 1000 r as seen with electron microscopy. Deep folds probably reflect the pattern which is revealed in part in living preparations with the use of phase microscopy of si&'ilarly irradiated material as shown in Figs. 4 4 4 9 and in related studies discussed in the text. (This micrograph was kindly contributed for this report by James K. Koehler of the Donner Laboratory, University of California, Berkeley, California.)

in the same cell are shown from film abstracts in Figs. 60 and 61 which, in the course of projection, were suggestive of the partial deflating of a bag with a relatively rigid wall. However, such cells survived for at least 2 weeks beyond this observation.

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W e are indebted to James K. Koehler for providing an illustration (Fig. 62) of an ultrathin section of the nuclear membrane of a HeLa cell which had received loo0 r of irradiation 8 days before fixation. The pattern of folds seen with the electron microscope is believed to reflect evidence of the complex systems which may be related to the movement of materials between nuclear and cytoplasmic areas. I n a record of the activity of cells of the human skin clonal line NCTC 2414, 12 days after irradiation with 2000 r from a cobalt-60 source, not only was vigorous nuclear folding observed, but nuclei were frequently seen to rotate (Figs. 63-66). The combination of these complex events was further complicated by evidences of intranuclear cytopathology. This post irradiation effect has been described briefly for this cell line (Pomerat, 1959).

PRODUCED CHANGES IN C. EXPERIMENTALLY DENSITYOF NUCLEOLI

THE

SHAPE AND

In the course of investigations on cancer chemotherapy, changes in the density of the nucleoli were noted, following contact with Actinomycin C, in perfusion chamber cine records. Similar observations have been published by Cobb and Walker (1958). Figures 67-69 show progressive changes in the shape of the nucleoli in primary cultures of human carcinoma of the bladder treated with 0.001 gamma/ml. of Actinomycin C. More typical changes in the density of the nucleoli are shown in Figs. 70-72, in which cells of the Chang conjunctival line were produced following contact with O.OOO.5 gamma/ml. of Actinomycin C. Interest in this problem naturally is focused on the possibility that the mechanism of action of this antibiotic involves a decrease in the R N A content of both the nucleoli and of the cytoplasm (Rounds et al., 1960). Changes in the architecture of the nucleolus, as revealed with electron microscopy following Actinomycin D, have been reported by Journey ( 1959). Alterations in the optical properties and size of nucleoli were also encountered in the course of another study directed at other goals. The application of 1.8 mg./ml. of A T P regularly was found to alter these nuclear organoids after 2 4 3 0 hours. A preliminary report of this work has been presented by Yoshida and Pomerat (1960). Figures 73-78 represent unpublished data on the effect of ATP on HeLa and the conjunctival cell lines recorded simultaneously, on a split frame 16 mm. film in a setup employing a comparison eyepiece. The appearance of the two test cell preparations immediately before introducing 1.8 mg./ml. of A T P for a 2-hour period is shown in Fig. 73.

FIGS.63-66. Nuclear membrane activity in a cell of the human skin cell line NCTC 2414 12 days after irradiation with 2000 r of gamma irradiation from a cobalt-60 source. The membrane showed deep infoldings, while the nucleus rotated in a counterclockwise direction. (Scale indicated on Fig. 63 applies to entire plate.) FIG.63, zero time; FIG.64, 8 minutes later; FIG.65, 16 minutes later; FIG. 66, 1 hour and 22 minutes later.

FIGS.67-72. Nucleolar changes in living cells following treatment with Actinomycin C as seen with phase microscopy. FIGS.67-69. Carcinoma of the human bladder. Seven-day culture treated with 0.001 gamma/ml. Actinomycin C. FIG.67, zero time; FIG.68, 1 hour and 21 minutes later; FIG.69, 2 hours and 33 minutes later. Note the change of nucleoli from an irregular to a rounded form. (Scale indicated on Fig. 67 applies also to Figs. 68 and 69.) FIGS.70-72. Chang’s conjunctival strain showing ringlike forms of nucleoli following con,tact with 0.0005 gamma/ml. Actinomycin C. FIG.70, zero time; FIG.71, 57 minutes later; FIG.72, 5 hours and 24 minutes later. (Scale indicated for Fig. 70 applies also to Figs. 71 and 72.)

FIGS.73-78. Comparison of the effect of A T P on two cell lines. The record was made of cells in two Rose chamber cultures on a single 16 mm. film with the aid of a B & L comparison eyepiece. Attention is drawn to the fact that in each numbered figure a pair of narrow lines serve to separate the two experimental records. Cells of the clonal S3 HeLa line are presented on the left side, while those of the Chang conjunctiva are on the right half of each figure. Both were 5-day cultures which were photographed a t 4 frames per minute for 3 days. (Scale indicated on Fig. 73 applies to entire plate.) FIG.73. Appearance of cells immediately before the addition of 1.8 mg./ml. of ATP. FIG.74. Eighteen and one-half hours after a 2-hour contact with the drug. FIG.75. Appearance of the cells 44 hours after treatment. Note mitoses in both species of cells.- FIG.76. Record at 46% hours. Note that the nucleoli of the conjunctival elements (right) are considerably larger than those of the HeLa cells (left). FIG.77. Fifty-one hours after treatment with ATP. FIG.78. Sixty hours post treatment. Note that while the nucleoli of the conjunctival cells appear to have recovered, those of the HeLa series remain unduly small.

CINEMATOGRAPHY, I N D I S P E N S A B L E TOOL FOR CYTOLOGY

333

After 18 hours and 30 minutes the nucleoli in all cells had become markedly reduced in size (Fig. 74). In contrast to their characteristic irregular outline and varying optical density, they were spherical with sharply delimited edges. At this and at later times in the course of making the record, the dimension of the nucleolar size was greater and recovery was more rapid in the conjunctival cells. As has been observed in previous records, A T P did not appear to interfere with completion of mitoses. In Fig. 75, made 44 hours after contact with the test chemical, mitotic figures were recorded simultaneously in both species of cells. Nucleolar recovery was clearly evident 46% hours post treatment in the conjunctival series, but lagged in the corresponding strain (Fig. 76). These conditions persisted at 51 (Fig. 77) and 60 hours (Fig. 78). Robineaux et al. (1958) have reviewed the evidence for nucleolar lesions produced by adenosine, adenylic acid, and A T P (Hughes, 1952), and by agents such as Actinomycin C. Further studies are needed to understand these mechanisms. It would appear that direct observation of nucleoli would continue to serve importantly in the course of the biochemical quest.

ACKNOWLEDGMENT Grateful appreciation is due to Dr. John Y. Harper, Jr. and Messrs. J. P. Reinecke and J. K. Koehler, who permitted the use of valuable, previously unpublished illustrations and data. Dr. Donald Rounds made important suggestions and Mrs. Josephine Fowler was responsible for the typescript. C. George Lefeber and David Pearson prepared the photographs.

REFERENCES Benitez, H., Murray, M., and Wooley, D. W. (1955) Proc. 2nd Intern. Congr. Neuropathol., London, 1955, P t . I I , p. 423. Bornstein, M. B., and Murray, M. R. (1958) J . Biophys. Biochem. Cytol. 4, 499. Canti, R. G., Bland, J. 0. W., and Russell, D. S. (1937) Research Publs. Assoc. Research Nervous Mental Disease 16, 1. Capers, C. R. (1960) J. Biophys. Biochern. Cytol. 7 , 559. Cobb, J. P., and Walker, D. G. (1958) J . Natl. Cancer Inst. 21, 263. Cooper, W. G., and Konigsberg, I. R. (1959) Anat. Record 133, 368. Felton, H. M., and Pomerat, C. M. J. Exptl. Med. in press. Felton, H. M., Gaggero, A., and Pomerat, C. M. (1954) Texas Repts. Biol. and Med. 12, 960. Gaillard, P. J. (1950) Proc. Acad. Sci. Ainsterdam SS, 1300. Gaillard, P. J. (1951) Methods in Med. Research 4, 241. Goldstein, M. H., Hiramoto, R., and Pressman, D. (1959) J . Natl. Cancer Inst. 22, 697. Grobstein, C. (1955) J . Exptl. 2001.1s0, 319. Grobstein, C. (1956) Exptl. Cell Research 10, 424. Grobstein, C. (1959) In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Chapt. 11, p. 462. Academic Press, New York.

334

C. M . POMERAT

Grobstein, C., and Dalton, A. J. (1957) J . Exptl. 2002.135, 57. Harris, M. (1957) J. Natl. Cancer Inst. 19, 507. Hintzsche, E. (1956) 2. yellforsch. i f . mikroskop. Anat. 4!l, 526. Hughes, A. (1952) Exptl. Cell Research 3, 108. Journey, L. (1959) In Annual Report, Roswell Park Memorial Institute, p. 24. Leone, V., Hsu, T. C., and Pomerat, C. M. (1955) 2. Zellforsch. 21. mikroskop. Anat. 41, 481. Lumsden, C. E., and Pomerat, C. M. (1951) Exptl. Cell Research 2, 103. Martinovitch, P. N. (1951) Methods in Mcd. Rescarch 4,240. Miller, D. G., and Hsu, T. C. (1956) Cancer Research 16, 306. Nakai, J. (1956) A m . J. Anat. 99, 81. Nakazawa, T. (1960) Texas Repts. Biol. ond Med. 18, 52. Paul, J. (1959) “Cell and Tissue Culture.” Livingstone, Edinburgh and London. Peterson, E., and Murray, M. R. (1955) Ain. J . Anat. 96, 319. Pomerat, C. M. (1953) Exptl. Cell Research 6, 191. Pomerat, C. M. (1955) J. Neuropathof. Exptf. Neiwol. 14, 28. Pomerat, C. M. (1958a) In “Biology of Neuroglia” (W. F. Windle, ed.), Chapt. 10. C . C Thomas, Springfield, Illinois. Pomerat, C. M. (1958b) Federation Proc. 17, 975. Pomerat, C. M. (1959) Science 190, 1759. Pomerat, C. M., and Littlejohn, L. (1956) Southern Med. 1. 49, 230. Pomerat, C. M., Kent, S. P., and Logie, L. C. (1957) 2. Zellforsch. u. mikroskop. Anat. 47, 175. Robineaux, R., Buffe, D., and Rimbaut, C. (1958) Compt. rend. colloq. intern. sur chinriothbrap. Cancers et leuckrnies p. 225. Rose, G. G. (1954) Texas Repts. Biol. and hfed. 12, 1074. Rose, G. G., and Shindler, T. 0. (1960) J . Bone and Joint Surg. 42A, 485. Rose, G. G., and Trunnell, J. B. (1959) Endocrinology 64, 344. Rose, G. G., Pomerat, C. M., Shindler, T. O., and Trunnell, J. B. (1958) J . Biophys. Biochem. Cytal. 4, 761. Rounds, D. E., Nakanishi, Y. H., and Pomerat, C. M. (1960) Antibiotics 6.Chemotherapy 10, 597. Sanford, K. K., Merwin, R. M., Hobbs, G. L., Young, J. M., and Earle, W. R. (1959) J. Natl. Cancer Inst. 23, 1035. Swann, H. G., Valdivia, L., Ormsby, A. A,, and Witt, W. T. (19.56) 1. E%ptl. Med. 104, 25. Trowell, 0. A. (1959) Exptl. Cell Rescarch 16, 118. Watson, M. L. (1959) J . Biophys. Biocheiii. Cytol. 6, 147. Willmer, E. N. (1954) “Tissue Culture: The Growth and Differentiation of Normal Tissues in Artificial Media.” Wiley, New York. Yoshida, M., and Pomerat, C. M. (1960) Abstr. 1st Intern. Congr. Histochem. and Cytochem., Paris, p. 125. Pergamon Press, New York.

Author Index Numbers in italics represent pages on which references are listed.

A Abe, H., 38, 120 Adolph, E. F., 263, 265, 270, 280 Afzelius, B. 4., 5, 6, 27, 34, 117 Agrell, I., 272, 279, 280 Akamine, R. N., 289, 304 Akiyama, Y., 120, 121 Alagna, G., 169, 190 Albaum, H. G., 299, 304 Alfert, M., 268, 280 Alfrey, V., 106, 117 Allen, R. D., 220, 251 Amano, S., 7, 40, 42, 107, 117, 124, 278, 280 Amprino, R., 171, 190, 194 Anderson, E., 7, 49, 117, 146, 158 Anderson, K. J. T., 204, 217 Anderson, N. G., 256, 268, 280 Anderson, P. A., 261, 280 Anderson-Cedergren, E., 46, 123 Anlyan, A. J., 299, 304 Arai, M., 49, 117 Arakawa, K., 2, 28, 58, 61, 63, 107, 115, 124 Arnold, W., 205, 216 Atkinson, W. B., 289, 304 Aurell, G., 169, 190 Autrum, H., 208, 216 Aziz, S. A., 150, 158

B Bairati, A . , 5, 117 Baker, J. R., 25, 28, 117,295, 304 Baker, R. F., 35, 47, 52, 119,122 Bang, F. B., 9, 117 Baratieri, A., 294, 304 Barch, S. H., 245, 247, 253 Barden, R. B., 172, 190 Bargmann, W., 14, 34, 38, 49, 50, 84, 85, 104, 114, 115, 116, 117 Barka, T., 267, 268, 281 Barner, H. D., 257, 260, 266, 268, 280 Barter, R., 78, 117 Bauer, A., 9, 12, 117 Baylor, E. R., 156, 158, 159 Bayne-Jones, S., 263, 265, 270, 280

Beams, H. W., 7, 38, 49, 67, 105, 115, 117, 129, 136, 143, 146, 148, 150, 158, 159 Bec, P., 165, 166, 190 Beloff, R. H., 176, 190 Belt, W. D., 38, 110, 117 Bembridge, B. A., 183, 190 Bencosme, S. A., 15, 95, 96, 117 Benda, C., 35, 117 Bendall, F., 197, 217 Benitez, H., 320, 333 Bennett, H. S., 7, 17, 23, 46, 50, 117, 118 Bentzon, M. W., 257, 268, 281 Bergamini, G., 168, 191 Berggren, L., 172, 190 Bernhard, W., 2, 9, 12, 13, 16, 17, 18, 24, 26, 39, 40, 107, 117, 118, 119, 123 Bettini, G., 295, 305 Bevelander, G., 287, 288, 289, 293, 304 Bhaskar, S. N., 291, 304 Bibring, T., 278, 281 Birge, E. A., 285, 304 Bland, J. 0. W., 320, 333 Bloch, D. P., 268, 280 Bloom, G., 150, 158 Bloom, W., 111, 121 Boell, E. J., 184, 193 Bohus, Jensen, A., 235, 231 Bollum, F. J., 270, 280 Borghese, E., 293, 304 Bornstein, M. B., 320, 333 Borysko, E., 9, 117 Bottino, D., 291, 305 Bourne, G. H., 283, 293, 303, 304 Bowen, R. H., 25, 107, 11i Bowness, J. M., 208, 216, 218 Brachet, J., 18, 106,117, 290, 304 Bradfield, R. G., 16, 38, 117 Brahma, S. K., 175, 193 Br.andt, P. W., 173, 192 Branham, J. M., 237, 238, 249, 251 Brattglrd, S., 182, 190 Braunsteiner, H., 52, 57, 89, 92, 93, 117 Brettmer, J., 52, 118 Brin, C. P., 204, 217 Brini, A., 173, 190

335

336

AUTHOR INDEX

Brockhoff, V., 185, 190 Brookbank, J. W., 219, 224, 23,245, 246, 247, 251, 253 Brookhaven, Symposia in Biology, 195, 216 Buffe, D., 333, 334 Bullivant. S.. 31, 53. 67, 68, 1 I8 Bullough, W. S., 272, 280 Burke, V., 176, 190 Burkhardt, D., 156, 1.58 Burns, V. W., 260, 280 Burstone, M. S., 294, 295, 296, 297, 301 Burtner, J., 285, 305 Burton, J. F., 295, 304 Butterfasz, T., 198, 216 Butterworth, C. E., Jr., 3, 119 C Cabrini, R. L., 285, 287, 289, 290, 291, 293, 294, 295, 296, 300, 301, 304, 306 Callan, H. G., 5, 6, 118 Calmettes, L., 165, 166, 190 Calvin, M., 196, 201, 202, 205, 216 Campbell, A., 256, 280 Canti, R. G., 320, 333 Capenos, J., 152, 156,159, 208, 218 Capers, C. R., 310, 324, 333 Cappellin, M., 291, 293, 299, 304 Carasso, N., 27, 119, 186, 187, 188, 190 Carlson, J. G., 42, 118 Carlson, L., 263, 264, 265, 266, 281 Carranza, F. A., Jr., 294, 304 Casas, M., 183, 184, 192 Caspersson, T. O., 18, 106, 118, 263, 264, 265, 266, 281, 290. 304 Castellani, A., 295, 304 Castiaux, P., 49, 123 Cessi, C., 293, 304 Challice, C. E., 3, 31, 38, 53, 67, 68, 109, 118 Chambers, L. A., 242, 250, 251 Chardard, R., 198, 216 Charles, A., 14, 49, 78, 79, 118 Chase, H. B., 114, 121 Chauveau, J., 19, 118 Chayen, J., 267, 280 Cheng, C., 296,-306 Chou, J. T. Y., 34, 118 Christie, A. C., 76, 118 Clark, S. L., Jr., 34, 118

Clark, W. Le G., 150, 158 Clarke, B., 285, 304 Clarke, W. M., 177, 178, 190,191 Claude, A., 15, 18, 19, 25, 34, 35, 106, 110, 118, 122 Clayton, R. C., 205, 216 Clayton, R. M., 177, 190 Clemente, C. D., 54, 124 Clendenning, K. A., 196, 216 Cleveland, L. R., 278, 280 Cobb, J. D., 297, 304, 329, 333 Cohen, A. I., 178, 190 Cohen, R. B., 295, 306 Cohen, S. S., 257, 260, 266, 268, 280 Colmano, G., 202, 218 Colwin, A. L., 226, 249, 251 Colwin, L. H., 226, 249, 251 Coman, R. D., 43, 53, 118 Coombs, R. R. A., 241, 253 Cooper, W. G., 324, 333 Coulombre, A. J., 168, 169, 173, 181, 184, 186, 189, 190 Coulombre, J. L., 168, 169, 173,190 Crescitelli, F., 195, 205, 216

D Da Costa, A. C., 181, 182, 190 Dalhamn, T., 3, 13, 26, 38, 43, 46, 53, 71, 72, 123 Dalton, A. J., 13, 16, 18, 19, 25, 26, 27, 44, 47, 52, 53, 67, 70, 106, 107, 118, 119, 120, 309, 334 Daly, M. M., 106, 117 Dan, J. C., 222, 223, 226, 229, 249, 251 Dan, K., 277, 281 Daumer, K., 159 Davidson, E. A., 168, 192 Davies, H. G., 267, 280 Davies, R. G., 126, 159 Davson, H., 164, 190 De Bernard, G. L., 295, 304 Deeley, E. M., 267, 280 Defendi, V., 296, 306 De Harven, E., 40,118 Delamater, E. E., 259, 275, 280 de Lorenzo, A. J., 150, 159 Dempsey, E. W., 52, 57, 89, 91, 92, 93, 95, 107, 118, 123 Deodati, F., 165, 166, 190 de Paola, D. D., 17, 32, 33, 71, 108, 123

AUTHOR INDEX

De Robertis, E., 46, 110, 118, 187, 190, ,205, 207, 216 Dethier, V. G., 126, 143, 158 Detwiler, S. R., 163, 185, 186, 189, 191 de Vincentiis, M., 174, 175, 191 Devine, R. L., 49, 105, 115, 117, 146, 158 Dewey, M. M., 46, 123 Dohi, S., 40, 124 Donovan, J. E., 223, 252 Dostal, B., 138, 158 Dotti, I. B., 285, 304 Doudney, C. O., 275, 280 Drew, R. M., 267, 281 Drews, G., 198, 217 Dunnington, J. H., 164, 191

E Earle, W. R., 308, 334 Eastharn, A. B., 179, 194 Ebert, J. D., 245, 251 Edlund, Y., 21, 46, 52, 58, 59, 118 Edwards, G. A., 151, 154,158 Ehret, C. F., 37, 38, 122, 123 Eichenberger, M., 39, 118 Ekholrn, R., 14, 21, 37, 43, 44,46, 49, 51, 52, 54, 58, 59, 89, 91, 95, 104, 118 Eklof, H., 109, 118 Elbers, R. F., 200, 216 Engel, E. K., 195, 216 Engel, M. B., 289, 304 Engstrom, H., 57,118, 132,158 Erickson, J. O., 82, 121 Erickson, R. O., 272, 280 Ernster, L., 279, 280 Erspamer, V., 78, 118 Esping, U., 237, 251 Estable, C., 9, 10, 118 Eyring, A., 196, 217

F Falcone, G., 259, 280 Farquhar, M. G., 2, 28, 98, 99, 103, 107, 108, 118, 123 Faurk-Frerniet, E., 207, 216 F a d , M., 109, 122 Fawcett, D. W., 38, 39, 40, 44, 46, 53, 105, 110, 118, 122 Felix, M. D., 16, 25, 26, 27, 118 Fellinger, K.,52, 57, 89, 92, 93, 117 Felton, H. M., 322, 324, 333

337

Fernkndez-Morkn, H., 152, 153, 154, 158, 196, 205, 208, 216 Ferreira, D., 95, 96, 108, 119 Finean, J. B., 198, 216 Finkler, A. E., 241, 253 Finlayson, L. H., 157, 159 Firket, H., 267, 280 Fischer, G. J., 285, 292, 293, 305 Fiset, M. L., 241, 253 Fishrnan, W. H., 295, 299, 304 Foley, M. T., 250, 252 Follis, R. H., Jr., 287, 291, 293, 296, 303, 304, 305 Fornes-Peris, E., 166, 191 Forro, F., 263, 280 Foster, T. S., 271, 280 Fowler, I., 177, 178, 190, 191 Franqois, J.. 185, 191 Frank, J. A., 221, 228, 251 Freeman, J. A., 37, 57, 119,121 Freiman, D. G., 285, 305 French, C. S., 197, 217 Frey-Wyssling, A., 195, 196, 198, 200, 203, 204, 216, 217 Fuchs, H. M., 223, 251 Fujirnori, E., 197, 216 Fujita, H., 28, 89, 91, 95, 107, 108, 119 Fujiwara, T., 7, 37, 119 Fukarni, I., 210, 216 Fullam, E. F., 15, 19, 35, 118, 122 Furth, J., 293, 305 G Gaffron, H., 195, 216 Gaggero, A,, 324, 333 Gagnon, A., 240, 251 Gaillard, P. J., 309, 333 Gall, J., 268, 269, 270, 280 Gander, H., 110, 119 Garcia-Austt, E., 183, 189, 191 Gamier, Ch., 15, 106, 119 Gaule, J., 15, 119 Gaulden, M. E., 268, 273,278, 280 Gautier, A., 13, 16, 18, 117 Gelfant, S., 273, 280 Gemolotto, G., 169, 191 Gendre, H., 287, 305 Genis-Gilvez, J. M., 182, 191 George, L. A., Jr., 104,122 Gerzelli, G., 291, 305

335

AUTHOR INDEX

Ghiani, P., 168, 191 Giardini, G., 224, 226, 252 Giese, A. C., 258, 280 Glauert, A. M., 43, 119 Glauert, R. H., 43, 119 Glenner, G. G., 107, 119 Glock, G. E., 287, 305 Gliicksmann, A,, 182, 191 Godman, G. C., 268, 280 Goedheer, J. C., 195, 200, 216 Goldschmidt, R., 15, 119 Goldsmith, T. H., 151, 156,159,208, 216 Goldstein, L., 278, 280 Goldstein, M. H., 324, 333 Golgi, C., 25, 119 Gomori, G., 292, 293,294, 305 Goodwin, T. W., 197, 216 Gordon, E., 299, 304 Granger, B., 52, 119 Granick, S., 196, 216 Grassk, P. P., 27, 119 Gray, E. G., 130, 132, 133, 134, 136, 139, 144, 154, 158 Greco, J., 285, 287, 305 Greenblatt, C. L., 195, 216 Greenfield, P. C., 184, 193 Greep, R. O., 285, 289, 291, 292, 293, 301, 303, 305, 306 Grenacher, H., 157, 159 Grobstein, C., 308, 309, 333, 334 Gropp, A., 9, 12, 117 Gross, J. A., 197, 216 Giittes, E., 173, 179, 180, 191, 268, 271, 272, 274, 281 Gutman, A. B., 288, 299, 305

H Haddad, S. A., 263, 281 Hagstrom, B., 223, 251 Hagstrorn, B. E., 220, 223, 224, 237, 238, 240, 247, 251, 252, 253 Haguenau, F., 2, 9, 12, 13, 16, 17, 18, 23, 26, 107, 117, 119 Hahn, F. L., 285, 305 Hale, C. W., 290, 305 Hally, A. D., 13, 28, 31, 49, 53, 54, 66, 67, 68, 70, 108, 119 Ham, A. W., 111, 119, 299, 305 Hamburger, K., 258, 266, 280 Hamperl, H., 76, 119 .

Hanaoka, M., 40, 42, 124 Hannibal, M. J., 297, 305 Hanzon, V., 2, 5, 13, 16, 21, 23, 25, 26, 28, 37, 42, 51, 53, 59, 60, 107, 123 Harding, C. V., 237, 240, 250, 251 Harding, D., 239, 240, 251 Harris, H., 267, 270, 280 Harris, J. E., 164, 191 Harris, M., 309, 334 Harris, P. J., 278, 281 Harris, S. H. A., 287, 305 Harrison, J. R., 179, 191 Hartman, R. E., 3, 119 Hartman, R. S., 3, 119 Hartmann, J. F., 5, 38, 119 Harvey, E. B., 230, 251 Hase, E., 260, 282 Hashirnoto, M., 13, 122 Hathaway, R. R., 226, 251 Hay, E. D., 24, 28, 119 Haye, C., 166, 192 Hayes, E. R., 78, 124 Heidenhain, R., 52, 119 Heilbrunn, L. V., 239, 251 Heitz, E., 202, 203, 216 Heller-Steinberg, M., 287, 289, 293, 303, 305 Hellstrom, B. E., 184, 186, 191 Hendler, R. W., 107, 119 Henle, G., 242, 250, 251 Henle, W., 242, 250, 251 Henrichsen, E., 293, 305 Herrmann, H., 164, 167, 191 Hertl, M., 176, 191 Hertwig, R., 15, 119, 265, 280 Hesse, R., 157, 159 Hibbs, R. G., 44,49, 57, 82, 83, 119 Hill, R., 197, 217 Hilleman, H. H., 285, 305 Hillier, J., 16, 18, 119 Hintzsche, E., 324, 334 Hiramoto, R., 324, 333 Hirono, R., 23, 107, 120 Hirsch, G. C., 25,28, 107, 110,119,120 Hirschfeld, A., 299, 304 Hirschler, J., 25, 119 Hirschman, A., 303, 305 Hobbs, G. L., 308, 334 Hodge, A. J., 196, 202, 217 Hodgson, E. S., 126, 159

339

AUTHOR INDEX

Hogeboom, G. H., 19, 106, 120 Hollmann, K.-H., 14, 84, 85, 119 Holmberg. A.; 49, 119, 173, 191 Holmgren, H., 32, 119, 169, 190 Holt, A. S., 202, 217 Honjin, R., 26, 49, 66, 76, 78, 119 Horowitz, N. H., 293, 305 Horstmann, E., 9, 119 Hotchkiss, R. D., 287, 305 Howard, A., 267, 280 Howard, J. E., 290, 298, 306 Hsu, T. C., 324, 334 Hubbard, R., 204, 211, 213, 215, 217 Hughes, A. F. W., 273, 280,333, 334 Hultin, T., 221, 251 Hunter-Szybalska, M. E., 259, 275, 280

I Ichikawa, M., 28, 49, 53, 57, 60, 89, 98, 99, 101, 103, 108, 110, 115, 120, 303, 305 Igo, Y., 173, 191 Iijima, T., 2, 6, 8, 9, 10, 11, 14, 27, 28, 29, 30, 38, 44, 45, 46, 48, 49, 50, 51, 53, 55, 57, 78, 79, 80, 81, 82, 83, 86, 104, 109, 116, 120, 121 Iizuka, M., 31, 95, 124 Imhoff, C. E., 285, 304 Immers, J., 238, 253 Imms, A. D., 126, 159 Inami, E., 185, 191 Irie, M., 15, 40, 49, 57, 89, 91, 92, 93, 94, 95, 116, 120, 124 Ishimaru, S., 53, 67, 120 Ito, Toru, 32, 120 Ito, Toshio, 28, 52, 55, 57, 82, 84, 92, 114, 120 Ivan Sorvall, Inc., 196, 217 Iverson, R. M., 258, 280 Iwamura, T., 260, 282 Iwashige, K., 57, 84, 120 Iwashige, T., 28, 120 Izumi, S., 66, 76, 78, 119

J Jackson, B., 173, 191 Jackson, S. F., 166, 191,289,305 Jacob, E. E., 202, 217 Jahn, T. L., 258, 282 Jakus, M. A., 163, 167, 170,191

James, T. W., 259, 281 Jander, R., 156, 159 Janosky, I. D., 179, 183,191 Jorschke, H., 156, 159 Johnson, M., 272, 280 Johnson, P. L., 287,288, 289, 293,304 Jones, P., 285, 305 Journey, L., 329, 334 Junqueira, L. C. U., 28, 110,120 Just, E. E., 229, 251

K Kabat, E. A., 293, 305 Iiada, K., 53, 54, 55, 67, 70, 115, 120 Kahler, H., 16, 18, 52, 118 Kajikawa, K., 23, 107, 120 Kakinuma, T., 38, 120 Kallman, F. L., 16, 24, 122 Kamen, M. D., 197, 217 Kanaya, T., 92, 123 Kanda, S., 47, 120 Kano, K., 92, 120 Kano, M., 28, 31, 89, 91, 95, 107, 108, 119, 124 Karlin, L. J., 105, 122 Karrer, H. E., 52, 120 Kataoka, S., 183, 191 Kaudewitz, F., 264, 280 Kelly, C. D., 263, 280 Kent, S. P., 324, 334 Khalaf, K. T., 208, 217 Kido, T., 89, 119 Kikkawa, Y., 167, 191 Kimball, R. F., 263, 264, 265, 366, 267, 268, 281 King, R. L., 67, 117 Kinsey, V. E., 173, 191 Kiriyama, M., 9, 10, 124 Kitamura, T., 2, 5, 6, 8, 9, 10, 11, 13, 14, 15, 27, 28, 31, 38, 44, 46, 48, 49, 51. 53, 54, 55, 57, 78, 79, 80, 81, 82, 83, 84, 86, 87, 104, 109, 110, 114, 120, 121 Kite, G. L., 42, 120 Knaysi, G., 264, 281 Kirk, P. L., 272, 274, 282 Kitagawa, T., 52, 120 Knoop, A., 9, 14, 28, 34, 38, 49, 50, 84, 85, 104, 108, 114, 115, 116, 117, 119, 123 Koecke, H. U., 180, 191

330

AUTHOR INDEX

Kohler, K., 221, 228, 229, 241, 242, 243, 244, 245, 250, 251, 252 Kolliker, A., 52, 120 Koishikawa, S., 183, 191 Kojima, K., 184, 192 Kokomoor, K. L., 273, 280 Kolster, R., 25, 120 Konigsberg, I. R., 324, 333 Kopsch, Fr., 24, 120 Kracht, C., 52, 95, 96, 108, 123 Kramisheva, V. N., 190, 192 Krasnovsky, A. A., 204, 217 Krauss, M., 226, 252 Krauss, R. W., 260, 282 Kriszat, G., 237, 252 Kropf, A., 211, 213, 217 Kuff, E. L., 19, 106, 120 Kull, H., 76, 120 Kuroki, S., 183, 191 Kurosumi, K., 2, 3, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 22, 24, 27, 28, 31, 34, 38, 44, 46, 47, 48, 49, 51, 53, 55, 57, 58, 64, 66, 67, 68, 70, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83, 86, 87, 104, 107, 108, 109, 110, 114, 115, 116, 120, 121 Kuwabara, M., 138, 159

L Lacy, D., 3, 31, 38, 109, 118, 121 Lacy, P. E., 15, 95, 96, 121 Laden, E. L., 82, 121 Landsteiner, K., 241, 252 Langman, J., 175, 176, 177, 192 Lark, K. G., 259, 268, 275, 276, 281 Laskey, A., 285, 305 Lefebre, C. G., 104, 122 Lehmann, F. E., 5, 117 Leone, V., 324, 334 Lever, J. D., 14, 38, 109, 110, 121 Lewis, M. R., 185, 186, 192 Leyon, H., 198, 200, 203, 217 Lez, C. H . , 285, 305 Lillie, F. R., 220, 229, 249, 252 Lillie, R. D., 285, 287, 305 Lim, R. K. S.,-67, 121 Lindauer, M., 159 Lindberg, O., 279, 280 Lindegren, C. C., 263, 281 Lindeman, V., 189, 192 .

Linden, I., 82, 121 Linker, A., 168, 192 Lipman, L. N., 250, 2-52 Lison, L., 289, 290, 305 Littlejohn, L., 320, 334 Livingston, R., 197, 216 Lloyd, B., 16, 18, 52, 118 Lobitz, W. C., Jr., 114, 121 Logie, L. C., 324. 334 Lorber, M., 289, 305 Lorch, I. J., 285, 292, 293, 305 Lorenzi, C. L., 295, 304 Louderback, A. L., 258, 282 Low, F. N., 37, 38, 57, 121 Lowenstein, O., 157, 159 Luft, J. H . , 43, 121 Lumry, R., 196, 217 Lumsden, C. E., 320, 334 Lynch, V. H., 197, 217

M Ma, W. C., 67, 121 MaalZe, O., 257, 259, 268, 275, 281 Macpherson, C. R., 295, 306 Maclay, H. K., 205, 216 Magasanik, B., 274, 281 Maggio, M., 224, 226, 252 Majima, Y., 184, 192 Majno, G., 293, 294, 305 Malinsky, J., 288, 305 Malmgren, H., 275, 282 Maly, R., 202, 216 Mancini, R. E., 287, 305 Mann, I., 162, 192 Marsland, D., 273, 281 Martinovitch, P. N., 309, 334 Maruyama, Y., 261, 275, 276, 281 Masson, P., 76, 121 Mast, S. O., 205, 217 Matsuda, S., 293, 306 Maurice, D. M., 167, 168, 192 Maximow, A. A., 111, 121 Maxwell, D. S., 44,48, 49, 54, 121 Mazia, D., 245, 253,274, 277, 278, 281 McDonald, B. B., 263, 267, 281 McIndoo, N. E., 138, 159 McKeehan, M. S . , 174, 175, 192 McLean, F. C., 290, 306 McLean, J. D., 202, 217 McManus, J. F. A., 287, 305

341

AUTHOR INDEX

McMaster, R., 267, 271, 274, 282 McMaster-Kaye, R., 278, 281 McQuillen, K., 274, 281 Meek, G. A., 34, 118 Melanotte, P. L., 2%, 305 Melczer, N., 28, 121 Menke, W., 196, 217 Mercer, F. V., 202, 217 Merwin, R. M., 308, 331 Metz, C. B., 219, 220, 221, 222, 223, 226, 228, 229, 230, 231, 232, 233, 237, 238, 239, 241, 242, 243, 244, 249, 250, 251, 252, 253 Metzner, H., 197, 217 Meves, F., 35, 121 Meyer, D. B., 162, 163, 164, 166, 170, 174, 178, 192 Meyer, K., 168, 192 Michaelson, I. C., 185, 192 Micou, J., 278, 280 Millen, J. W., 49, 55, 121 Miller, D. G., 324, 334 Miller, G. L., 204, 217 Miller, W. H., 151, 159, 208, 217 Milne, L. J., 205, 217 Milne, M. J., 205, 217 Minamitani, K., 55, 81, 109, 121 Minganti, A., 220, 252 Minick, 0. T., 37, 38, 123 Minnaert, K., 200, 216 Mirsky, A. E., 106, 117 Mitchison, J. M., 263, 264, 270, 275, 281 Miyake, S., 82, 121 Mizutani, Y., 114, 121 Mohammed, C. I., 291, 304 Molesworth, J. M., 3, 119 Monesi, B., 283, 295, 300, 305 Monni, L., 15, 121 Monroe, B. G., 52, 89, 121 Monroy, A., 224, 226, 235, 252,253 Montagna, W., 55, 114, 121 Moog, F., 183, 192 Moore, D. H., 49, 123 Morita, Sh., 15, 19, 39, 121 Morse, A., 285, 289, 291, 292, 293, 303, 305, 306 Moscona, A., 170, 194 Moses, M. J., 271, 274, 281 Mouli, Y., 19, 118 Moyer, F. H., 173, 180, 192

Miihlethaler, K., 196, 203, 217 Miiller, E., 67, 121 Miiller, N. R., 196, 217 Munger, B. L., 24, 95, 96, 108,121 Murray, M. R., 318, 320, 333,334 Myers, J., 261, 282

N 224, 235, 245,

168,

276,

301,

Nachlas, M. M., 296,297,305,306 Nadel, E. M., 303, 306 Nagakawa, T., 18, 19, 24, 27, 121 Nagamitsu, G., 28, 121 Nagano, T., 28, 38, 122 Nagao, T., 109, 124 Nakai, J., 324, 334 Nakanishi, T., 13, 57, 122 Nakanishi, Y. H., 329, 334 Nakayama, K., 182, 183, 192 Nakazawa, T., 320, 334 Napolitano, L., 39, 110, 122 Nasatir, M., 274, 275, 277, 281 Nassonow, D., 25, 122 Naylor, E. J., 167, 192,193 Neetens, A., 185, 191 Neufeld, E. F., 277, 281 Neumann, H., 168, 193 Newton, A. A., 260, 281 Nicolas, J., 109, 122 Nihei, T., 260, 281, 282 Nikiforuk, G., 285, 306 Niklowitz, W., 198, 217 Nilausen, K., 185, 192 Nilsson, O., 34, 43, 44,54, 122 Nisonoff, A., 247, 250, 252 Nomura, Y., 38, 120 Nordmann, J., 173, 192 Nussbaum, M., 15, 122 Nygaard, 0. F., 268, 271,272, 274, 281 0 Oberling, Ch., 9, 12, 13, 16, 18, 117 O’Brien, R. T., 104, 122 Odland, G. F., 46, 122 Offret, G., 166, 192 Ogiso, K., 58, 60,122 Okada, S., 184, 192 Okamoto, S., 89, 91, 95, 107, 119 Okumura, T., 66, 76, 78,119 Olson, R. A., 195, 216 O’Melveny, K., 221, 241, 253 Onodera, Y., 109, 124 Onoi, T., 13, 122

342

AUTHOR INDEX

O'Rahilly, R., 162, 163, 164, 166, 168, 170, 174, 178, 192 Ormsby, -4.A,, 314, 334 Osako, R., 23, 124 Osawa, S., 183, 192 Oshima, H., 229, 252 Otsuka, R., 13, 122 Ottoson, D., 51, 122 Ottoson, R., 275, 282 Ozanics, V., 164, 169, 193

P Padilla, G. M., 259, 281 Painlev&, J., 25, 122 Painter, R. B., 267, 281 Pakesch, F., 52, 57, 89, 92, 93,117 Palade, G. E., 2, 6, 10, 16, 17, 18, 19, 21, 23, 24, 25, 26, 31, 35, 37, 50, 58, 59, 60, 63, 68, 96, 104, 106, 122, 123, 198, 200, 203, 217, 218 Palay, S. L., 2, 3, 10, 26, 28, 32, 53, 62, 70, 71, 72, 88, 92, 105, 107, 122 Pansa, E., 171, 190 Papero, G. P., 285, 304 Pappas, G. D., 173, 192 Parat, M., 25, 122 Parry, H. B., 189, 192 Parvisi, V. R., 287, 306 Patrone, C., 169, 191 Paul, J., 309, 334 Pearse, A. G. E., 78,117, 295, 304,306 Pearson, B., 296, 306 Pease, D. C., 14, 15, 16, 32, 35, 38, 44, 47, 48, 49, 52, 53, 54, 95, 96, 104, 117,121,122,123, 196, 217 Pelc, S. R., 267, 280 Pequegnat, W., 229, 230, 232, 252 Perlmann, H., 246, 247, 248, 250, 252 Perlmann, P., 220, 228, 245, 246, 247, 248, 250, 252, 253 Perry, M. M., 158, 159 Perry, R. P., 278, 280 Pescotto, G., 306 Peterson, E., 318, 334 Peterson, H., 176, 190 Peterson, R. R., 52, 89, 91, 92, 93, 95, 98, 107, 118, 122. Pettijohn, D. E., 261, 280 Philpott, D. E., 151, 156, 159, 208, 216 Pickels, G. E., 204, 218 Pirie, A., 183, 190

Planel, H., 165, 166, 190 Plaut, W., 277, 281 Polyak, S., 162, 184, 192 Pomerat, C. M., 104, 122, 310, 320, 322, 324, 329, 333, 334 Porter, K. R., 9, 15, 16, 17, 18, 19, 24, 35, 40, 117,118,122 Porter, R. R., 250, 252 Post, L. C., 198, 218 Powers, E. L., 37, 38,122,123 Prescott, D. M., 257, 258, 261, 263, 264, 265, 266, 267, 270, 271, 274, 277, 281 Press, N., 49, 117 Pressman, D., 247, 250, 252,324, 333 Prestage, J. J., 129, 136, 143, 148, 150, 159 Prince, J., 186, 192 Pringle, J. W. S., 157, 159 Pritchard, J. J., 287, 291, 293, 306 Pumphrey, R. J., 130, 132, 159

R Rabinovitch, M. P., 277, 281 Rabinowitch, E. I., 196, 217 Rahn, O., 263, 280 Ranvier, L., 111, 123 Rapkine, L., 277, 281 Rasmont, R., 49, 123 Reale, E., 306 Rebhun, L. I., 19, 123 Rebollo, M. A., 183, 184, 186, 189,192 Redslob, E., 171, 192 Regaude, C., 109, 122 Reviews of Modern Physics, 195, 217 Reygadas, F., 285, 305 Rhodin, J., 3, 13, 16, 26, 36, 38, 39, 42, 43, 46, 48, 50, 52, 53, 71, 72, 123 Richard, G., 127, 159 Richards, A. G., 136, 138, 142, 157,159 Richards, 0. W., 126, 159 Rickenbacher, J., 182, 192 Ries, E., 25, 116, 123 Riley, J. F., 290, 306 Rimbaut, C., 333, 334 Rinehart, J. F., 98, 99, 103, 118,123 Roberts, I. Z., 274, 281 Roberts, R. B., 274, 281 Robertson, D., 43, 44,123 Robineaux, R., 333, 334 Robison, R., 292, 299, 306

AUTHOR INDEX

Rochon-Duvigneaud, A., 185, 192 Rogers, G. E., 14, 38, 49, 53, 55, 86, 88, 109, 110, 119, 123 Rosa, C., 296, 306 Rose, G. G., 309, 310, 334 Rosenheini, ,4. H., 299, 306 Rosin, .4., 293, 306 Rossi, F., 306 Roth, L. E., 37, 38, 122, 123 Rothschild, Lord, 220, 223, 252 Rouiller, C., 17, 19, 24, 39, 110, 117, 118, 119, 123, 198, 207, 216, 293, 294, 305 Rounds, D. E., 329, 334 Rubin, P. J., 290, 298, 306 Ruck, P., 151, 158 Rudzinska, M. -4., 38, 123 Runnstrom, J., 220, 237, 238, 240, 245, 252, 2.53 Rusch, H. P., 268, 271, 272, 274, 281 Ruska, H., 49, 123 Russell, D. S., 320, 333 Rutemberg, S. H., 295, 306 Ruyter, J. H. C., 168, 193

s Sabatini, D., 110, 118 Sager, R., 197, 200, 203, 205, 217 Saguchi, S., 15, 123 St. Whitelock, 0. v., 195, 217 Sakai, H., 277, 281 Sanford, K. K., 308, 334 Sano, P., 28, 108, 123 Sarnat, B. G., 289, 304 Sawada, T., 9, 10, 124 Sawano, J., 92, 123 SaxCn, L., 163, 183, 186,192 Schaechter, M., 257, 268, 281 Schajowicz, F., 285, 287, 289, 290, 291, 293, 294, 295, 296, 300, 301, 304, 306 Scheer, I. J., 208, 218 Scherbaum, 0. H., 257, 258, 266, 282 Schiebler, Th. H., 38, 49, 50, 104, 117 Schiefferdecker, P., 111, 123 Schmidt, W.J., 195, 210, 217 Schulz, H.. 17, 32, 33, 71, 108, 123 Schwarz, W., 167, 171, 193 Schwertz, F. A,, 198, 200, 201, 202, 204, 218 Scott. B. L., 14, 44, 53, 104,123 Scott: D. B., 31, 53, 67, 68, 118 Screebny, L. M., 285, 306

343

Sedar, A. W., 31, 38, 53, 67, 68,123 Sekhon, S. S., 138, 139,159 Seki, M., 44, 49, 123 Sekiguchi, H., 109, 124 Selby, C. C., 44, 46, 47, 118,123 Seligman, A. M., 295, 296, 306 Seo, S., 169, 193 Serpell, G., 185, 193 Shaver, J. R., 245, 247, 253 Sheldon, H., 165, 166, 193 Shen, S.-C., 184, 193 Sherman, J. K., 187, 193 Shibasaki, S., 3, 13, 14, 20, 2, 27, 28, 31, 44, 46, 47, 49, 53, 57, 64, 65, 66, 67, 68, 69, 70, 72, 73, 74, 75, 76, 77, 78, 104, 107, 110, 115, 116,121,123 Shibata, K., 260, 274, 282 Shin, E., 207, 218 Shindler, T. O., 310, 334 Sibert, R. S., 293, 306 Sidman, R., 181, 184, 193, 195,217 Siekevitz, P., 2, 17, 19, 21, 58, 59, 60, 106, 122, 123 Sin-ikC, T., 179, 193 Sirlin, J. L., 175, 193 Siskin, J. E., 267, 271, 274, 282 Sissakian, N. M., 197, 217 SjiSstrand, F. S., 2, 5, 8, 13, 14, 16, 17, 21, 23, 25, 26, 27, 28, 36, 37, 42, 43, 44, 46, 48, 49, 51, 52, 53, 54, 59, 60, 89, 91, 95, 104, 107, 118, 122, 123, 196, 198, 205, 216,217, 218 Slifer, E. H., 129, 136, 138, 139, 143, 144, 148, 150, 157, 159 Smelser, G., 164, 169, 173, 192, 193, 195, 218 Smith, C. A., 49, 50, 57, 104, 123 Smith, D. T., 179, 193 Smith, E. L., 204, 218 Smith, F. E., 156, 158, 159 Smith, J. H. C., 204, 218 Smith, McD., 104, 122 Smith, R. B. W., 3, 119 Smith, R. H., 289, 305 Smits, G., 167, 169, 171, 193 Snodgrass, R. E., 126, 136, 159 Sobel, A. E., 299, 304 Sognnaes, R. F., 283, 306 Solger, B., 15, 123 Solomon, A. K., 105, 123 Sonderman, R., 173, 193

344

AUTHOR INDEX

Sorokin, C., 35, 123,260, 261, 282 Sotelo, J. R., 9, 10, 118 Souza, E., 296, 306 Speakman, J., 172, 193 Spikes, J. B., 1%, 217 Stanier, N. Y., 197, 218 Stanier, R., 197, 218 Stanners, C. P., 267, 282 Stanworth, A., 167, 193 Steedman, H. F., 290, 306 Steinach, J., 52, 118 Steinman, E., 198, 200, 216 Stenstrom, S., 51, 122 Stern, H., 256, 271, 272, 274, 275, 277, 280, 281, 282 Stocking, C . R., 196, 218 Stoeckenius, W., 32, 52, 95, 96, 108,123 Stoll, R., 173, 192 Stone, L., 179, 193 Strain, H. H., 197, 218 Striebich, M. J . , 16, 18, 118 Stroeva, 0. G., 176, 193 Strother, G. K., 207, 218 Strugger, S., 202, 218 Stuhlman, H., 142, 159 Sugihara, R., 9, 10, 124 Sugioka, M., 9, 10, 124 Sullivan, N. P., 176, 190 Suzuki, I., 2, 28, 36, 60, 61, 62, 106, 107, 124 Suzuki, Y., 50, 124 Svaetichin, G., 51, 122 Svensson, G., 263, 264, 265, 266, 281 Swann, H. G., 314, 334 Swann, M. M., 223, 252, 256, 272, 273, 282 Sylven, B., 275, 282, 289, 290, 298, 306 Szybalski, W., 259, 275, 280

T Tahmisian, T. N., 38, 49, 105, 115, 117 Takagi, F., 39, 109, 124 Takahashi, T., 52, 120 Takahashi, Y., 14, 38, 58, 67, 108, 109, 121 Takamatsu, H., 292, 306 Takamatsu, M., 23, 124 Takashima, S., 204, 218 Takeuchi, T., 297, 306 Tamiya, H., 260, 282 Tanaka, A., 47, 120

Tanaka, H., 7,40, 42, 115,117,124 Tanaka, Y., 3, 13, 14, 22, 27, 28, 31, 44, 46, 47, 49, 53, 57, 64, 66, 67, 68, 70, 72, 73, 74, 75, 76, 78, 104, 107, 115, 116, 121 Takeda, K., 138, 159 Tansley, K., 189, 193 Taylor, J. H., 267, 270, 271, 274, 278, 281, 282 Taylor, J. J., 78, 124 Tehver, J., 76, 124 ten Cate, G., 176, 193 Terry, T. L., 173, 191 Thamisian, T. N., 146, 158 Thomas, J. B., 196, 198, 200, 216,218 Thompson, H. P., 24, 35, 122 Thorell, B., 275, 282 Till, J. E., 267, 282 Tobias, C. A., 275, 282 Tokuyasu, K., 185, 188, 193 Tomlin, S. G., 5, 6, 118 Tonna, E. A., 296, 306 Tosi, L., 221, 226, 252 Trowell, 0. A., 309, 334 Trunnell, J. B., 310, 334 Trurnit, H. J., 202, 218 Tson, K. C., 295, 306 Tsou, K., 296, 306 Turano, A., 152, 156,159, 208, 218 Tyler, A., 219, 220, 221, 222, 223, 224, 226, 228, 229, 235, 239, 241, 242, 245, 246, 247, 249, 250, 251,253

U Uchida, G., 3, 13, 14, 22, 27, 28, 31, 44, 46, 47, 49, 53, 57, 64, 66, 67, 68, 70, 72, 73, 74, 75, 76, 78, 104, 107, 115, 116, 121, 124 Uchino, F., 40, 124 Umetani, K., 13, 67, 122, 124 Urist, M. R., 290, 306 Utsunomiya, S., 293, 306

V Valdivia, L., 314, 334 Vallee, B. L., 210, 216 Van Breemen, V. L., 54, 124 van den Heuvel, J. E. A., 176,193 van den Hooff, A., 167, 168,193 Vandermeersche, G., 49, 123 van Doorenmaalen, W. J., 176, 178,193

345

AUTHOR INDEX

van Walbeek, K., 168, 193 Van Weel, P. B., 66, 124 Vasseur, E., 220, 252 Vatter, A. E., 202, 217 Velardo, J. T., 296, 306 Verly, W. G., 267, 280 Vertregt, N., 198, 218 Vogel, R., 134, 138, 140, 159 von Frisch, K., 134, 140, 159 Von Wettstein, D., 203, 205, 218 Vrabec, F., 172, 193

W Wacker, F., 134, 159 Waddington, C. H., 158, 159 Wald, G., 186, 193, 195, 205, 210, 211, 216, 218 Waldman, J., 299, 306 Walker, D. G., 329, 333 Walker, P. M. B., 262,275, 281, 282 Walls, G., 185, 193 Wang, L., 89, 91, 107,124 Wasserman, F., 53, 124 Watanabe, A., 14, 15, 38, 58, 67, 95, 96, 97, 108, 109, 121, 124 Watanabe, Y., 2, 16, 23, 28, 37, 58, 61, 63, 107, 115, 124 Watari, N., 12, 28, 120, 121 Waterman, T. H., 156, 159 Watson, M. L., 6, 23, 124, 322, 334 Weed, K., 176, 190 Weier, T., 196, 218 Weil, A. J., 241, 253 Weimar, V., 164, 191,193,194 Weinmann, J. P., 291, 304 Weinstock, J., 49, 123 Weiss, J. M., 2, 13, 16, 17, 19, 21, 23, 27, 44, 49, 59, 104, 106, 124 Weiss, P., 170, 171, 194 Weissman, B., 168, 192 Welcker, H., 52, 124 Wellings, S. R., 2, 28, 98, 107, 108, 118 Wendler, L., 156, 158 Wenger, B. S., 179, 183, 191 Wenner, A. M., 143, 159 Went, H. .4.,245, 253 Wersall, J., 57, 118,124, 132, 158 Whittingham, C. P., 197, 217 Wicklund, E., 237, 238, 240, 253 Wigglesworth, V. B., 126, 159 Wilander, O., 32, 119

Wildy, P., 260, 281 Willmer, E. N., 207, 218, 324, 334 Wilson, W. L., 239, 251 Winkelman, J. E., 168, 193 Wischnitzer, A., 6, 12, 124 Wislocki, G., 164, 170, 178, 184, 193,194 Wissig, S. L., 89, 91, 92, 107, 124 Wissler, F. C., 250, 252 Witt, W. T., 314, 334 Woernley, D. L., 250, 252 Wolbarsht, M. R., 143, 1958 Wolff, E., 185, 194 Wolken, J. J., 152, 156, 159, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 211, 212, 213, 215, 216, 217, 218 Woodard, J. W., 267, 271, 282 Woods, P. S., 278, 282 Wooley, D. W., 320, 333 Woolf, D., 174, 194 Wulff, V. J., 205, 218

Y Yamada, E., 32, 40, 46, 53, 124, 185, 188, 193 Yamada, H., 82, 121 Yamagishi, M., 15, 18, 19, 24, 27, 46, 53, 105, 121, 124 Yamamoto, J., 2, 28, 58, 61, 63, 107, 115, 124 Yamato, K., 49, 66, 76, 78, 119 Yanagita, T., 261, 281 Yasuzumi, G., 7, 9, 10,43, 115, 124 Yokoh, S., 31, 95, 124 Yoneyama, K., 164, 194 Yoshida, M., 183, 184, 186, 194, 322, 329, 334 Yoshimura, F., 15, 57, 89, 91, 93, 94, 95, 116, 124 Young, J. M., 308, 334 Yii, T. F., 288, 299, 305

2 Zalokar, M., 197, 217 Zetterqvist, H., 42, 54, 124 Zeuthen, E., 257, 258, 264, 266, 270, 271, 272, 274, 279, 280,282 Zimmerman, L. E., 179, 194 Zimmermann, K. W., 15, 19,67,124 Zorzolli, A., 285, 292, 303, 306 Zussman, H., 186, 193

Subject A Xcetylcholinesterase in developing retina, 184 in retinal rods and cones, 186 Actinomycin C effect on density of nucleoli, 329, 331, 333 Adenohypophy sis carp, electron micrographs of, 102 cytology of, 98-103 acidophile cells, 98 basophile cells effect of castration on, 99, 103 of thyroidectomy on, 103 gonadotroph, 99, 103 thyrotroph, 99, 103 chromophobes, 98 electron micrographs of rat cells, 100, 101 secretory granules, origin of, 108 Adenosine triphosphate effect on nucleoli, 329, 332 Adrenaline effect on thyroidal fine structure, 95 Amoeba proteus cell growth rate during growth-duplication cycle, 264 experimentally induced cytokinesis in, 263 Animals vertebrate, olfactory epithelium, electron microscopy of, 150 A4ntibodies fertilization-inhibiting action of, 240248, 250 effect on eggs, 245-248 effect on sperm, 241-245 production, site of, 1 Antifertilizin action of, 228 mechanism of, 228-229 effect on fertilizability of eggs, 228229 on jellyless eggs, 228 extraction, 221, 228 of sperm surface, 241, 242

Antiserum effect on sea urchin eggs, 245-249 A rbacia dermal secretion, 229-237 chemical nature of, 236-237 fertilization-inhibiting action of, 230, 232-236, 249 mechanism of, 232, 236 preparation, 229 fertilizing capacity of fertilizin-treated sperm, 222 sperm surface antigens, 243-245 Arthropods, see also Insects detection of polarized light by, 156 Asterias forbesi sperm, electron photomicrographs, 226, 227

B Blow fly gustatory organs, 157 electron microscopy of, 143 Body chief cell, of gastrointestinal mucosa, 64-66 electron micrograph of, 65 secretion, mode of extrusion, 112, 116 secretory granules of, 64, 66 Bone, see also Ossification and Tissues, bone histochemistry of, 283, 297-301 phase contrast cine time-lapse records for, 310 resorption, histochemistry of, 301, 303 C

Carotenoids chlorophyll and, 197 complexes with proteins and lipoproteins, 204 function, 197 retinal, 186-187 Carpal organ pig, 84 secretion, mode of extrusion, 112 Cells acid-secreting, of gastrointestinal mucosa, spe Oxyntic cells

346

SUBJECT INDEX

acinar, of exocrine pancreas experimentally induced changes in, 59-63 ultrastructure of, 58-59 argyrophile, gastrointestinal, 76, 78 electron micrograph, 78 chromaffine, gastrointestinal, 76, 78 division synchrony, induction of, 256262 chemical, 260-261 mechanical methods, 261-262 by temperature shock, 257-260 electron microscopic analysis of secretory mechanism, 1-124 of surface structure, 42-57 of exocrine glands, surface of, 43 factors determining growth and multiplication rates, 256 glandular, extrusion of secretion from, 111 invagination of basal plasma membrane in, 47-50 growth-duplication cycle, 262-282 cell growth during, 262-267 enzyme fluctuations during, 275-276 RNA synthesis during, 274-275, 276 reproduction of structures, 276-279 respiration and energy metabolism during, 272-274 hepatic, snake, electron micrographs of nuclei in, 4 mucous-secreting, of gastrointestinal mucosa, 70-76 pancreatic, lipoidal bodies in, 31 secretory granules of, 13 secretory, cytoplasm of, 12-42 multinucleated, 3 nucleus, of, 3-12 ultrastructure of, 3-57 sensory, of Planaria, 207 surface 42-57 apical free, structure of, 52-57 apocrine projection, 57 crust, 55, 57 microvilli, 52-55 basal invagination of basal plasma membrane, 47-50 lateral, structure of, 43-47 intercellular interdigitation, 44-46

347

terminal bar and adhesion plates, 46-47 zymogenic, of gastrointestinal mucosa, 63, 64-67 Centrioles, 40-42 function of, 40 reproduction in cleaving sea urchin eggs, 278 ultrastructure of, 40, 41, 42 Cltlorella synchronization of cell division in, 260261 Chlorophyll in chloroplasts, 200-202 complexes with proteins and lipoproteins, 204 isomers, 197 as photosensitive pigment, 195 Chloroplastid development of chloroplast from, 202204 Chloroplastins molecular weight, 204 nature of, 204 Chloroplasts algal, structure, 198 composition, 196-197 development of, 202-204 gene action on, 203-204 of higher plant, structure, 199 molecular structure, function and, 205 as plant photoreceptors, 195, 196-204 proteins of, 197 structure, 196 molecular, 200-202 Choroid plexus secretion, mode of extrusion, 112, 115 Chromatin, nuclear, 8-9 Chromulina eyespot of, 207 Cinematography in cytological studies, 307-334 Collagen, cellular origin, 1 in corneal stroma, 167-168 formation, alkaline phosphatase and, 299 in sclera, 171, 172 transparency and, 171 Colloid thyroidal, origin of, 107

348

SUBJECT INDEX

Compound eyes arthropod, see also Compound eyes, insect phylogenetic development, 206 types of, 209-210 visual cells of, 151ff., 207ff., see also Ommatidia, Rhabdomeres insects, 151-156 analyzer of polarized light in, 156, 210 Cornea, 163-166 Bowmans membrane of, 166 development of, 162, 163 Descemets membrane of, 170 epithelium of, 163, 164-166 basal cells of, 165 basement membrane of, 166 function of, 164 intermediate cells of, 165 squamous cells of, 164-165 cytogenesis of, 165 stroma and, 164 function of, 163 stroma of, 166-169 composition of, 166-168 metachromasia in developing, 168169 transparency and, 168, 169 Cuticle insect sense organs and, 125ff., 157-158 Cyanopsin, 210 Cytochrome oxidase in bone tissue, 296-297 Cytochromes in chloroplast proteins, 197 Cytokinesis relation between nuclear division and, 275 a-Cytomembrane, 17, 18, see also Ergastoplasm P-Cytomembrane, 48 6-Cytomembranes, 17, 32-34 electron micrograph of, 33 lamellar bodies and, 32, 34 mucus production and, 108 occurrence, 32 y-Cytomembrane; 17, 26, see also Golgi apparatus, membranes of Cytoplasm membrane system of, 15-42, see also

Ergastoplasm, Reticulum, endoplasmic intracellular, 21 terminology 15, 17-18 of secretory cells, electron microscopy of, 12-42 secretory granules in, 12-15 size, shape, and density of, 13

D DNA synthesis, during growth-duplication cycle, 267-272 in microorganisms, induction of synchrony in, 258-260 Desmoenzyme, 297 Desmosome, 46 Dragonfly ocellus of, 151 Drosop hila compound eyes of, 207-208

E Egg cells animal, pars amorpha of, 12 Egg jelly precipitants for, 228 Eggs acid-treated (jellyless), fertilizability, 224 antigenic composition, 245 effect of Arbacia dermal secretion on, 230, 232-237 dissection of egg surface and, 232, 234-236 proteolytic enzymes and, 233, 234-235 fertilizability, effect of antifertilizin on, 228-229 surface, fertilization and, 226, 229 Electron microscope analysis of cellular secretion by, 1-124 Endocrine glands, see also Pancreas, Thyroid, etc. secretory granules of, 14-15 shape of, 15 Enzymes, see also individual compounds in active osteoblasts, 299 activity, during cellular growth-duplication cycle, 275-276 bone resorption and, 301

349

SUBJECT INDEX

cartilaginous calcification and, 298, , 299-301 distribution in bone and cartilage tissue, 285, 291-297, 302 proteolytic, sensitivity of eggs to Arback dermal secretion and, 233, 234-235 respiratory, in mitochondria, 35, 38 Epithelium bile duct, secretion, mode of extrusion, 112, 114 gastric surface, secretion, mode of extrusion, 112, 115 glandular, basement membrane of, 5052 intercellular interdigitation in, 44 terminal bar and adhesion plates in, 46-47 Ergastoplasm, 18-24 genesis of, 23-24 of mammary gland, 84 occurrence, 23 in pancreatic acinar cells, 58 production of secretory granules in, 106-107 as source of thyroidal colloid droplets, 91 thickness, 24 ultrastructure of, 19-24 variations in, 19 vesiculation in gastric body chief cell, 64-65 zymogen granules and, 23 Escherichia coli induction of synchronized cell division in, 260-261 Esterases in bone tissue, 297 Euglenu chloroplasts, light and, 203 molecular structure, 200 eyespot of, 205, 207 Eye vertebrate, choroid coat, 173 embryology of, 173 ciliary body, 173-174 development of, 173-174 cribriform plate, development of, 171172

cytology of developing, 161-193, scc also Cornea, Iris, Sclera, Lens, Retina, etc. geometric relationship between OCUlar tissues, 161-162 structure, 162 trabecular meshwork, development, 172 Eyespot, 205-207 function, 205 phylogenetic development, 206 retinal rod and, 207

F Fat metabolism lamellar bodies and, 34 Fertilization mechanism, 223-224 inhibiting agents in study of, 219-253 role of egg membrane lysins in, 219 of fertilizin and antifertilizin in, 229 of trypsin-treated eggs, 235 Fertilization-inhibiting agents, 219, 220, see also Fertilizin, Antifertilizin, etc. action, possible mechanism of, 248-249 from dermal secretion of Arbacia, 229237 from egg and sperm extracts, 220-229 from Fucus, 229 unrelated to gametes, 239ff., 249, 250, see also Arbacia, Fucns, etc. “Fertilization rate” method, 223 Fertilizin combining sites, 224 inactivation by Arbacia dermal secretion, 235 fertilization-inhibiting action, possible mechanisms, 226, 228 role in fertilization, 223, 229 sea urchin, preparation of, 221 Fertilizin theory, 220 Flies, see also Insects compound eyes, electron micrographs of, 153, 154, 155 fine structure, 151ff. Fucais fertilization-inhibiting extracts of, 237239, 249 chemical nature, 237 preparation, 237

350

SUBJECT INDEX

G of human apocrine sweat gland, 79, , 81, 82 Gastric mucous neck cell, 71-34 types of, 79, 81, 82 morphology, electron microscopic, 72of human sebaceous gland, 86, 88 74 of mammary gland, 84ff. Gastric parietal cell origin of, 2 secretion, mode of extrusion, 112, 114, Grasshoppers 115, 116 auditory organs, 129 “Gelbe Zellen,” see Cells, chromaffine compound eyes, fine structure of, 152, P-Glucuronidase 156 in bone tissue, 295 olfactory organs, 143 role in bone formation, 299-300 coeloconic pegs as, 150-151 G1y cogen electron micrographs of, 144-145, in cartilage tissue, 288, 298 148, 149 cartilaginous calcification and, 298, 299, thick-walled pegs as, 143-144 300-301, 302 thin-walled pegs as, 144, 146-150 in neural retina, 183-184 tactile hairs of, 127 ossification and, 287-288 electron microscopy of, 127-129 in pigmented retinal epithelium, 179 structure of, 127-129, 156 Goblet cell Ground substance of Golgi apparatus, 26 of gastrointestinal mucosa, 70-71 Growth-duplication cycle origin of mucous droplets, 71 cellular, 255-282 of tracheal mucosa, 71 terminology, 255-256 Golgi apparatus, 16, 25-31 H of anterior pituitary cells, 98 as source of secretory granules, 98- H substance, human 99 fertilization-inhibiting action, 239, 240 components of, 26 Helix relationships between, 27 lipid globules in neurons of, 34 demonstration of, 25 Heparin formation of milk protein and, 85 fertilization-inhibiting action, 239-240 of mucous and, 71, 72 production, site of, 1 of secretory granules and, 28, 62, 63, Histochemistry 64, 65, 66, 67, 74, 98-99, 107-109 of bone formation, 297-301 lamellar bodies and, 34 of bone resorption, 301, 303 lipochoridria and, 28 of ossification, 283-306 membrane of, 26-27 histochemical reactions, 287-297 role in cellular secretion, 28 materials and methods, 284-286 as source of retinal melanin, 173, 180 in pathological conditions, 289, 303 thyroidal colloid droplets and, 91 Honey bee ultrastructure of, 25-27 compound eyes, fine structure, 152, 154, Granules 156 intracisternal, 23 plate organs, 138-143, 157 secretory, see also Zymogen granules auditory organs of locust and, 140, of anterior pituitary cells, 98-99 142, 157 species differences in, 99 electron micrographs of, 140, 141 of cytoplasm, 12-15 fine structure, derived from electron in exocrine pancreas, 59-63 microscopy, 136-140 Golgi apparatus and, 28, 62, 63, 64, as olfactory structures, 134, 136, 142 65, 66, 67, 74, 98-99, 107-109 retinene, in rhabdomeres of, 208

351

SUBJECT INDEX

Housefly retinenel in rhabdomeres of, 208 Hymenoptera sense organs, ampullaceous, 157 Hyperparathyroidism effect on bone tissue 289, 303 Hypophysectomy effect on thyroidal ultrastructure, 92

I Immune bodies, cinematic studies on, 322 Insects abdominal stretch receptors, 157 campaniform organ, 157 compound eyes of, 151-156 analyzer of polarized light in, 156, 210 fine structure, 151-156 hairs of, structure, 126-127 as sense organs 126-127, 156 photosensitive pigments in, 208 sense organs, see also Insects, compound eyes auditory, 129-134 elements of, 125 fine structure of, 125-159 gustatory, 143 ocelli, 151 olfactory, 143-151 plate organs, 134-143 tactile, 126-129 electron microscopy of, 127-129 hair as, 126-127, 156 terminology, 126 Iodopsin, 210 Iris, development of, 172

K Kidney cine records of tissue cultures, 310, 312314, 315

L Lactobacillirs acidophilus synchronization of cell division and DNA synthesis in, 260 Lamellar bodies, 32, 34 biological activity, 32, 34 electron micrograph of, 33 Golgi apparatus and, 34

heparin production and, 32 Langerhans islets, see Pancreas, endocrine Lens antigens of, 176-178 difference between early and adult, 176 localization, 177-178 chick embryo, development, cine records of, 320 development of, 174-178 of capsule, 178 cine records of, 320 early morphogenesis of, 175 induction, cytologic changes during, 174-175 Lipids cytoplasmic membranes and, 34 in secretory granules, 14-15 Lipochondria, 28-31 electron microscopy of, 29 Golgi apparatus and, 28 of human sweat glands, 30 Locust auditory organs, electron micrographs of, 136, 137, 144, 145 fine structure of, 129-134, 140, 142 plate organs of bee and, 140, 142, 157 Lysins of egg membrane, role in fertilization, 219

M Mammary gland, 84-86 apocrine sweat gland and, 84, 86 secretion, mode of extrusion, 86, 112, 114, 115, 116 products of, 84-85 secretory granules of, 14 Mast cells heparin production and, 32 Meibomian gland rat, 88-89 Melanin granules, in Golgi apparatus, 173 in pigmented retinal epithelium, 179 origin of, 180 Microorganisms cell growth rate during growth-duplication cycle, 262-267

352

SUBJECT INDEX

relation between nuclear division and cytokinesis, 275 Microsomes, 18, 19 composition, 18 endoplasmic reticulum and, 19, 24 types of, 19 Microvilli of apical free cellular surface, 52-55 occurrence, 52ff structure, 53-56 Mitochondria, 35-42 of apocrine sweat gland, 79 duplication of, 279 electron micrograph, 36 of endocrine pancreas, 98 function of, 35 number, cell volume and, 279 origin of, 39 as origin of secretory granules, 109-

110, 111 oxidative enzyme systems in, 110 pathological degeneration, 110 precursors of, 39, 40 proliferation and new formation of,

38-39 role in cellular secretion, 38 terminology, 35 ultrastructure of, 35-38 Mitosis effect of irradiation on, 278 prevention by respiration inhibitors, 273 R N A and, 277 sulfhydryl groups and, 277 Molluscs eyes of, 208 Morphology descriptive, Cinematographic methods in, 320 Moths compound eyes, electron microscopic studies on, 152, 154 Mougeotiu chloroplast, molecular structure, 200 Mucopolysaccharides acid, in bone tissues, 289-290, 302 in cartilage fissue, 302 in corneal stroma, 168 in developing retina, 183 in sclera, 171

synthesis, cytoplasmic membranes and,

32

'

Mucoproteins bone resorption and, 301 distribution in cartilage and bone tissue during ossification, 302 Mucosa gastrointestinal, argyrophile cells of,

76, 78 chromaffine cells of, 76, 78 gastric surface epithelium, electron microscopic morphology of, 74-

76 secretory cells of 63-78, see also Body chief cell, Goblet cell, Oxyntic cells, etc.

Mucus glandular, origin of, 108 Muscle phase contrast cine time-lapse records for, 310

N Neuroglia rhythmically active, response to drugs, 320 Neurons radioresistance, 318 sensory, of insects, 128ff., 156 Nucleic acids cartilaginous calcification and, 299 distribution in developing human retina,

182 in osseous tissue, 290-291 Nucleolus duplication, 278 experimental changes in shape and density of, 329, 333 as site of R N A synthesis, 278 Nucleus membrane, cinematographic studies on,

324-329 effect of irradiation on, 321, 325, 328,

329, 330 pore system of, 322 nuclear rotation, 323-324, 329 of secretory cells, electron microscopy of, 3-12 karyoplasm (nucleoplasm) , 8-9 morphology of, 3-5 nuclear inclusions, 12

SUBJECT INDEX

position of, 5 shape of, 3-5 structure of nuclear envelope (karyotheca), 5-7 structure of nucleolus, 9-12 of nucleoplasm (karyoplasm), 8-9 0 Ocelli of dragonfly, structure, 151 Ommatidia insect, 151, 152, 153, 207 of the housefly, 152-153, 154, 155 structure, 207 Ossification enchondrial, enzymes in, 300 histochemistry of, 283-306 endocrine disturbances and, 303 glycogen and, 287-288 materials and methods in study of, 284-286 types of, 286-287 Osteoblasts enzymes in active, 299, 300 glycogen in, 288 Otic complex chick embryo, development, cine records of, 320 Oviduct hen, albumin-secreting gland of, 107 Oxyntic (acid-secreting) cells, of gastrointestinal mucosa, 67-70 electron microscopy of, 67, 69 mitochondria of, 68 secretory mechanism of, 68

P PAS-positive granules in osseous tissues, 289-290 Pancreas endocrine, cytology of, 95-98 C-cells, 95, 96 cell types, 95 morphological differences in aand p-cells, 95 secretory granules of, 95-97 of carp, %, 97 origin of, 98, 108 species differences in shape and size of, 95-96

353

exocrine, acinar cells of, 58-63 effect of chemicals on, 60-62, 63 of starvation and refeeding on, 59-60 normal structure, 58-59 secretion, mode of extrusion, 112 Paneth cell of gastrointestinal mucosa, 66-67 secretory granules of, 66, 67 Paramecium cell growth rate during growth-duplication cycle, 265, 266 effect of trypsin on mating reactivity, 250 Pertussis antigens cytopathogenic effects, 324, 326 Phnseolus vulgaris chlorophyll holochrome of, 204 Phosphatase acid, in bone tissue, 294-295 alkaline, in bone tissue, 292294,299 Phosphorylase in bone tissue, 297, 300 Photoreception, rod outer segment and, 190 Photoreceptors, 195-218 animal, 205-215 fine structure, 205 invertebrate, 205-210, see also Eyespot, Cells, sensory, Ocelli and Compound eyes electron microscopy of, 195-196 function, possible requirements for, 215 molecular dimensions, 215 nomenclature, 197 photosensitive pigments of, 195, 215 plant, 196-205 structure, 215 Photosynthesis, 196 during cellular growth duplication cycle, 261 in plants, chloroplast structure and, 204 role of carotenoids in, 197 Pinocytosis, 104-105 in antigen-antibody reactions, 324 Planaria sensory cells of, 207

354

SUBJECT INDEX

Plants chloroplasts of, development, 203 as photoreceptors, 195, 196-204 photoreceptors of, 195, 196-205 Plasma membrane, 42-43 invagination, 16, 17 of basal, 47-50 ingestion of secretory material and, 104 water transport and, 48, 49, 50 structure, 43 thickness, 24 Porphyropsin, 210 Poteriochromonas chloroplast, molecular structure, 200 Protein synthesis ergastoplasm and, 106, 107 Proteins role in stabilizing cell properties, 309

R Reticulum “agranular,” 26, 32 endoplasmic, 15, 17-18 electron micrograph of, 22 iormation, 24 microsomes and, 19 protein synthesis and, 106 rough-surfaced, see Ergastoplasm smooth-surfaced (cytoplasmic vacuoles), 31-32 occurrence, 17, 31-32 size of, 31 synthetic role of, 18, 19 Retina, 179ff. developing, enzymes in, 184, 185 mucopolysaccharides in, 183 nucleic acid in human, 182 neural, 180-190 cytogenesis, 181- 184 development, 180-181 glycogen in, 183-184 synaptic patterns in, 184 origin of, 179 pigmented epithelium of, 179-180 function of, 179, 180 rods and cones of, 210-214 as adult visual elements, 185 development, 185, 207 ellipsoid of, 186 eyespot and, 207

inner segment of, 185-187 organelles produced by, 185-186 lentiform body of, 186 oil droplets of, 186-187 outer segment components of, 210 development of, 187 molecular model, 213 photosensitive pigments in, 210 structure of, 210-212, 214 precursor of rod sacs, 187-188 structure, 21Off. vertebrate, photoreceptors of, see Retina, rods and cones vessels, morphology, 185 visual cells, see also Retina, rods and cones cytogenesis of, 185-190 Retinene as photosensitive pigment, 195 Retinenel isolation from insect rhabdomeres, 207 in outer segment of retinal rods and cones, 210 Retinene2 in outer segment of retinal rods and cones, 210 Rhabdomeres of compound eyes, 151-155, 207-210 as photoreceptors, 156, 207 spatial arrangement, 208, 209 Rhodopsin, 210 isolation, 215 molecular weight, 215 rod outer segment and, 189 Rhodospirillum ritbrzinz photoreceptors of, 198 Ribonucleic acid in cytoplasmic membranes, 18, 19 distribution in bone and cartilage tissue during ossification, 302 mitosis and, 277 nucleolar, role in cytoplasm, 322 protein metabolism and, 301 synthesis, during cell life cycle, 274275, 276 nucleolus as site of, 278 Ribonucleoprotein cytoplasmic, identification, 18-19 in developing retina, 182

SUBJECT INDEX

Ribosomes genetic information and, 276 role in protein synthesis, 276 Root ganglia dorsal, cine records of, 314, 316-318 S Salivary gland secretion, mode of extrusion, 112, 114 Sanddollar egg jelly dissolving agent in sperm of, 224 Schwann cells pulsatile activity, 320, 321 Sclera, 170-172 collagen of, 171, 172 transparency and, 171 development of, 170-171 Scurvy effect on bone tissue, 303 Sea urchin eggs, effect of antiserum on, 245-249 cortical damage, 248 inhibition of fertilization, 248 parthenogenetic activation, 246 fertilizing capacity of fertilizin-treated sperm, 222 Sebaceous gland human, cytoplasmic vacuoles in, 31-32 morphology of, 86-89 secretion, mode of, 86 of extrusion, 112, 114 Secretion, cellular extrusion of secretory material, 111116 modes of, 112ff. ingestion of secretory material, 103105 mechanism, electron microscopic analysis of, 1-124 cellular, morphology of, 3 significance of mitochondria in, 38 synthesis of secretory substance, 106111 possible mechanism of, 110-111 Sensory cells phylogenetic development, 206 Skin glands, see also Sweat glands, Mammary gland, etc. secretory cells of, 78-89

355

Sperm acrosomal reaction, 226 fertilization and, 226, 228, 229 antigenicity, 241ff., 250 effect of antibodies on, 241-245 fertilizing activity, effect of fertilizin on, 221, 228 isoagglutinin of, see Fertilizin surface, fertilization and, 220, 221, 223, 229 Sperm receptor substance, see Antifertilizin Spermatogenesis snail, discharge of secretory granules during, 115 Stnphylococcus toxin cinematographic studies on, 322, 324 Stomach body chief cells, electron micrograph of, 20 nucleus of, 3 secretory vacuoles of, 14 Succinic dehydrogenase in bone tissue, 296 in developing retina, 185 Sweat glands animal, secretory granules of, 13 apocrine, 78-82 manimary gland and, 84, 86 secretory granules of, 79, 81-82 secretory portion, morphology of, 78-82 fine structure, 80 hun~an,eccrine, morphology of, 82-84 release of secretion, 84 secretory granules of, 82 electron micrograph, 83 lipochondria of, 30 nuclei of, electron micrographs, 9, 10, 11 nucleoplasm of, 8-9 secretory granules of, 14 structure of lateral cell surface, Mff. nuclear structure, 8-9, 10-11 secretion, modes of extrusion, 112, 114, 115, 116 secretory granules, origin of, 109 Sulfhydryl groups mitosis and, 277

356

SUBJECT INDEX

T Tetralzymena cell growth rate d u r h g growth-duplication cycle, 263, 264 DNA synthesis during growth-duplication cycle, 267, 271 production of division synchrony in, 257, 258 Thiouracil effect on thyroidal ultrastructure, 93, 95 Thyroid gland, 89-95 fine structure, effect of adrenaline on, 95 of cold stress on, 95 of hypophysectomy on, 92 of T S H on, 92-93, 94 of thiouracil on, 93, 95 phase contrast cine time-lapse records for, 310 secretion, mode of extrusion, 91-92, 112, 114, 116 secretory granules of, 91 types of, 91 ultrastructure of normal cells, 89-92 Thyroid-stimulating hormones effect on thyroidal ultrastructure, 9293

Tissue cultures organotypic, 308-322 apparatus for, 309-310, 311 dorsal root ganglia, 314, 316-318 kidney cultures, 310, 312-314, 315 Tissues bone, decalcification, 284-285 demonstration of acid mucopolysaccharides in, 289-290 of enzymes in, 285, 291-297 of glycogen in, 287-288 of PAS-positive substances in, 289 of ribonucleic acid in, 290-291 resorption, histochemistry of, 301, 303 cartilage, calcification, 298-299 histochemical modifications during, 298-299

z Zymogen granules, see also Granules, secretory ergastoplasm and, 23 origin of, 2-3, 28, 63, 106-107, 109 in pancreatic acinar cells, 58 effect of chemicals on, 60, 62-63 nutrition effects on, 60