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

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME11

This Page Intentionally Left Blank

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.

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK3, N. Y.

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

Library of Congress Catalog Card Niiinber 52-5203

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.

This Page Intentionally Left Blank

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

This Page Intentionally Left Blank

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-

10

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.

12

K. KUROSUMI

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

ELECTRON MICROSCOPIC A N A L Y S I S OF SECRETION

13

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,

14

K. KUROSUMI

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

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

15

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

16

K. KUROSUMI

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-

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

17

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

18

K. KUROSUMI

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-

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

19

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.

20

K. KUROSUMI

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.)

ELECTRON MICROSCOPIC A N A L Y S I S OF SECRETION

21

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.

22

K. KUROSUMI

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.)

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

23

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.

24

K. KUROSUMI

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.

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

25

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-

26

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

27

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

28

K. KUROSUMI

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-

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

29

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.)

30

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

31

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-

32

K. KUROSUMI

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.)

34

K. KUROSUMI

( 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.

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

35

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

36

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

37

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

38

K. KUROSUMI

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.

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

39

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.

40

K. KUROSUMI

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.

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

41

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.)

42

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

43

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

44

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

45

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.)

46

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

47

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

48

K. KUROSUMI

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-

ELECTRON MICROSCOPIC A N A L Y S I S OF SECRETION

49

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

50

K. XUROSUMI

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.

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

51

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

52

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

53

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

54

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

55

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.)

ELECTRON MICROSCOPIC ANALYSIS OR SECRETION

57

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. ’

58

K. KUROSUMI

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,

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

59

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

60

K. KUROSUMI

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.)

62

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

63

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,

64

K.

KUROSUMI

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-

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

65

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.)

66

K. KUROSUMI

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.

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

67

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-

68

K. KUROSUMI

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.)

70

K. KUROSUMI

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

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

71

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

72

K. KUROSUMI

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

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

73

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.)

74

K. KUROSUMI

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.)

76

K. KUROSUMI

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 ‘
80

K. KUROSUMI

FIG.24. A diagrammatic illustration showing the fine structure of the secretory portion of the human apocrine sweat gland. I-VZ indicate the successive stages of the process of formation and -the following disintegration of dark secretory granules, while 1-3 indicate the maturation of light secretory granules. (K. Kurosumi r t al., 1959a.)

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

81

outline. The content of this granule is not homogeneous, but much denser particles of various sizes and shapes are unevenly scattered within the granule. The over-all density of this granule is strikingly similar to that of fat droplets of adipose cells, but the presence of far more opaque particles within this granule is in contrast. Small dense particles contained

FIG.25. Supranuclear zone of an apocrine sweat gland cell. A large dark secretory granule in the course of disintegration ( d D S C ) is observed at the center. This is surrounded by a thin delicate membrane and contains many fatty droplets and small dense particles. Dark (DSC) and light ( L S G ) secretory granules are observed neatby. L, lumen of the gland tubule; N, nucleus. ( X 10,000.) (K. Kurosumi rt al., 1959a.)

may probably be of pigment or iron as suggested by Minamitani (1941) with the use of the light microscope. 'It is very frequently observed that dense granules similar in density and in shape to this type of secretory granule are enclosed within smooth membraned vacuoles. Such encapsulated granules are most frequently observed in and near the Golgi apparatus. Thus, we presumed the site

82

K. KUROSUMI

of the formation of this granule as either Golgi vacuoles or smoothsurfaced endoplasmic reticulum. At the apical part of the cytoplasm, various grades of the disintegration of the dark secretory granules are observed. The initial step of the change may be noted as an occurrence of a clear spot within the granule (Fig. 13A). That may be enlarged gradually and lessen the density more and more, forming a ring-shaped body. The next step is the granulation of the dark shell, i.e., grains or droplets appear at the dark rim of a changing granule. In large secretory granules, the clearing up may occur from multiple loci, and the lipid substance is divided into many droplets. A thin membrane surrounds the total area of the disintegrating granule (Fig. 25). Finally it converts into a large vacuole bounded by a delicate membrane, the internal substance of which is exceedingly clear, with small dark droplets sparsely attached to the inner surface of the limiting membrane. Such vacuoles are not always observed. If the limiting membrane has been ruptured earlier, the vacuole may not be formed and the content of the granule may be dispersed in the apical cytoplasm. In some cases, the light secretory granules, in fact, contain some dense material. A possibility is left undetermined that some of the light secretory granules may transform into dark ones absorbing the lipid probably derived from the disintegrated dark granules. As none of the dark granules or similar substance can be observed at the uppermost layer nor in the glandular lumen, it is evident that the dark secretory granules are all dissolved away before the extrusion. Huge apocrine secretory projections were observed on the luminal surface of this gland.

2. The Eccrine Sweat Gland The human eccrine sweat gland is lined with a pseudostratified epithelium which consists of two cell types, superficial and basal cells. Between two neighboring basal cells appear intercellular secretory canaliculi (Toshio Ito, 1949). Electron microscopic observations on human eccrine sweat glands were first performed by Laden et d. (1955), but their description did not exceed the old light microscopic knowledge. More detailed studies by Kurosumi et al. (195&), Miyake and Yamada (195S), Hibbs ( 1958), and Iijima ( 1959) have subsequently been published. Both the light and dark secretory granules found in the apocrine sweat gland are alsQ detected in both the cell types of this gland (Kurosumi et al., 1 9 5 8 ~ )(Fig. 2 6 ) . However, gland cells without secretory granules are frequently encountered. The authors other than from our laboratory did not refer to the secretory granules. Perhaps they observed only such

ELECTRON MICROSCOPIC A N A L Y S I S OF SECRETION

83

gland cells without granules. In both cell types we very often perceived polyv.esicular fat droplets (lipochondria) (cf. p. 2 8 ) , which were never found in the axillary apocrine glands. Immediately beneath the luminal free surface, many vesicles with quite clear contents were observed by Hibbs (1958) and Iijinia (1959). These vesicles are gathered also in the vicinity of intercellular secretory canaliculi. Iijima considered that these might be concerned with water transport as in the case of the apocrine glands.

FIG.26. Secretory granules of the human eccrine sweat gland. T w o distinct types of the granule are evident. Light secretory granules ( S G P ) , probably of protein nature, possess a double limiting membrane and internal double membranes, whiqh may be the residue of cristae mitochondriales. Dark secretory granules (SGL) are extremely dense and somewhat irregular in outline. These are considered to be of lipid nature. A t the mid-lower margin, a dense mass surrounded by smooth membrane is observed as shown by an arrow, indicating the probable immature form of the dark secretory granule. ZC, intercellular boundary. ( x 24,000.) (K. Kurosumi et ai., 1958c.)

84

K. KUROSUMI

The superficial cell occasionally extends tonguelike or balloonlike projections, which are thought to represent. the apocrine-type secretion. This observation agrees with light microscopic findings by Ito and Iwashige (1951) that the eccrine sweat gland may release the secretion in two ways, eccrine and apocrine, and hence may be called an “amphicrine” gland. The carpal organ is a skin gland localized in the palmar side of the carpal or metacarpal region of pig’s forelegs. Kitamura (1957, 1958) ascertained by light and electron microscopy that this organ is one of the eccrine sweat glands. The secretory portion of the carpal organ also consists of two cell types, dark and clear cells, but they align in a single layer. The dark cell is filled with a bulk of large empty and clear secretory vacuoles, while the clear cell contains relatively small and dense granules. Kitamura (1958) asserted that the secretory granules of the dark cell might be formed from Golgi apparatus or vesicular microsomes (one of the smooth-surfaced endoplasmic reticulum), whereas those of the clear cell might originate from mitochondria. Microvilli of the clear cell are well developed and often take the form of the “polypoid border,” whose expanded tips may be pinched off and turned into the secretion. This is a good example of the submicroscopic apocrine secretion, which has been visualized with the electron microscope exclusively.

3. The Mammary Gland The mammary gland is known by light microscopy as a specifically modified apocrine sweat gland. Therefore, the ultrastructure of the mammary cells has a resemblance in some respects to that of the apocrine sweat gland. But some fundamental differences between the two glands were detected by electron microscopy (Bargmann and Knoop, 1959; Hollmann, 1959). The most conspicuous characteristics of the mammary gland in which it differs from the apocrine sweat gland are (1) that the ergastoplasm is extensively developed in a form of parallel lamellae, ( 2 ) that the secretory granules and droplets are released without dissolution or transformation prior to the discharge, and ( 3 ) the straight course of the lateral and basal cell surfaces. The secretion of the mammary gland, viz., the milk, contains two kinds of formed elements: the lipid droplets and the protein or lipoprotein granules, both of which can be traced in the cytoplasm of the gland cell to have the same appearance as those found in the glandular lumen. Protein secretory granules are small round bodies 100-300 mp in diameter with a high electron density. In lactating cells, these granules

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

85

are always contained within a smooth-membraned vacuole, which is apparently coincident with the Golgi vacuole because it is closely applied or continuous with the Golgi lamellae. It is usual that three or more granules are contained in a single vacuole, and that clear space is left around and between the granules within a vacuole. From the result of this observation may be justified an assumption that the Golgi apparatus participates directly or indirectly in the formation of milk protein. However, Bargmann and Knoop (1959) confessed that the question of whether the protein generation is performed in the Golgi apparatus from the earliest beginning, or whether the protein is produced elsewhere and is discharged into the space of Golgi vacuoles, could not be determined by electron microscopy. According to Hollmann ( 1959), Haguenau obtained an electron micrograph of human breast cancer cells showing the existence of secretory granules within the space of ergastoplasmic sacs. Therefore, Hollmann suggested the secretory granules of the mammary gland appearing in Golgi vacuoles might be initially elaborated in the ergastoplasm. Although the mechanism of the extrusion of protein granules was not entirely clarified, Bargmann and Knoop presented a picture depicting a granule-containing vacuole closely applied to the surface plasma membrane, through which a small perforation occurred. They did not decide whether the opening is an artifact or a physiological exit for protein granules, but the latter possibility seems to be strongly suggested since the protein granules in the gland lumen are the same in appearance as the intravacuolar granules in the cell. The second type of secretion product, the lipid droplets, are much larger than the protein granules and measure one to several microns. They are widely variable in shape, in texture, and in density, i.e., some are regularly round, homogeneous, and intensely osniiophilic, but others are notched and the content has been partially extracted, leaving a small amount of reticular or granular clot. According to Bargmann and Knoop, mature lipid droplets approach the cell surface and come into contact with it. They ascend more and more over the level of the cell surface, and the junction of the droplet with the cell body becomes constricted until the droplet entirely surrounded by a plasma membrane is conlpletely detached. Therefore, the extrusion of the lipid droplets from the cell is performed without rupture of the apical plasma membrane or the cytoplasm. Hollmann stated, however, that the passage of the droplets was achieved by a localized rupture of the cell membrane, without noticeable loss of cytoplasmic material. These concepts of secretion release are quite different from that asserted

56

K. K C R O W M I

by us (Kurosumi et al., 1959aj on the apocrine sweat gland. Yet the discrepancy may be reasonable, since the. mammary gland eliminates the secretion without change in form, but the secretory granules of the apocrine sweat gland are always dissolved away prior to the discharge. In pregnant rats whose mammary gland is not yet loaded with protein granules, lipid droplets have already appeared in a close relationship with the ergastoplasm (Bargmann and Knoop) . Both authors who described the secretion phenomena of the mammary gland found no direct relation between the lipid production and the Golgi apparatus, although the droplets often appeared in the proximity of the Golgi apparatus. Hollmann suggested the participation of mitochondria in the lipid formation, based upon the result of observations on estrogen-stimulated maminary glands, in which vacuoles with double limiting membranes probably identical to the lipid droplets were found. A striking difference of mammary cell structure from that of sweat glands was described in connection with its morphology of the cell surface by Bargmann and Knoop. In rats’ mammary glands neither interdigitation on the lateral intercellular boundaries nor basal infolding was observed. Thus, the problem of how the water transport through the cell is carried out for the building of the highly watery milk has been left unsettled. The form of microvilli on the free cell surface and the involution or disappearance of these at the site of secretion release is reconciled with the case of sweat glands.

4. The Sebaceous Gland The sebaceous gland is the best example for the holocrine gland, the secretion of which is made by the breakdown of the mature gland cells. Sebaceous glands of infant mice were observed with the electron microscope by Rogers (1957). Secretory granules or lipid droplets are seen to be empty except for a small amount of debris. Rough-surfaced endoplasmic reticulum is very scanty. It was remarked that mitochondria contain dense granules 400-800 A. in diameter, and Rogers suggested that these intramitochondrial granules might be in a close relationship with the process of the formation of lipid in the sebaceous cells. The human sebaceous gland is not fundamentally different (Kitamura and Kurosumi, 1959) from that of the mouse reported by the above author. The youngest cell situated at the peripheral zone of an acinus contains no secretory granules, but small numbers of round or rod-shaped mitochondria and clear vesicles of various sizes are scattered through the cytoplasm (Fig. 27A). The latter element is apparently part of the smooth-surfaced variety of endoplasmic reticulum, and markedly multi-

ELECTRON MICROSCOPIC A N A L Y S I S OF SECRETION

s7

FIG.27. Electron micrographs of the cells from the human sebaceous gland. A. Two adjacent glandular cells of relatively immature stage. The cell on the left hand side is slightly younger than the cell on the right, in which several lipid droplets (L), as well as many vesicles, are contained. N , nucleus; M,mitochondria. ( X 9000.) B. Mature sebaceous gland cells. Lipid droplets ( L ) are eiiormously enlarged and the content is almost extracted. The cytoplasm is filled with a bulk of vesicles belonging to the smooth-surfaced endoplasmic reticulum. ( x 7000.) (T. Kitamura and K. Kurosumi, 1959.)

88

K. KUROSUMI

plies as the cell grows to produce the secretory droplets. Although the rough-surfaced endoplasmic reticulum is almost absent in the sebaceous cell, freely scattered R N P granules are abundant especially in the slightly grown cells. Delicate filaments probably identical to the tonofilaments of epidermal cells are randomly distributed in the youngest cells but diminish in number in accordance with the aging of the cell. The cells in the intermediate zone acquire a few small secretory (lipid) droplets whose structure is quite the same as that reported by Rogers (1957). Most of the mitochondria are rounded and swollen. Between such expanded mitochondria and young lipid drbplets, probable transitional forms are observed. Smooth-surfaced vesicles are crowded in the vicinity of the growing secretory droplets (Fig. 27A). W e tentatively assumed that the secretory droplets of the sebaceous cells might be produced by the swelling of mitochondria, which might absorb some material from vesicles of the smooth-surfaced endoplasmic reticulum. The cell in the central region is ready to break up and looks like sponge. Lipid droplets become larger and fill almost all of the space in the cytoplasm. Furthermore, narrow interstices among the droplets are filled with vesicles (Fig. 27B). Mitochondria are very few. Along the surface of the droplets as well as along the cell periphery, vesicles are forcibly pressed to form spurious parallel lamellae. The nucleus is markedly irregular in outline and filled with very dense contents. No typical Golgi apparatus has been observed in the sebaceous cell. Lamellae-like membranes surrounding the mature lipid droplets are actually collapsed vesicles, but not the Golgi apparatus. I n younger cells no corresponding feature is observed. Palay (1958) studied the Meibomian gland of the rat, which is a modified sebaceous gland. His result is a little different from ours and those of Rogers. H e observed the Golgi apparatus composed of parallel tubular elements and vesicles, in the peripheral cells, although the development of these is weak. According to Palay, the lipid droplets of the Meibomian gland may be produced in the space enclosed by the “agranular reticulum.” Each droplet is surrounded by a husk of agranular reticulum in the form of circularly arranged tubules or cisternae. H e considered that the “agranular reticulum” corresponds to the Golgi apparatus. H e also detected a crystal-like pattern on the husk of lipid droplets, which is composed of interlaced tubule-formed cisternae of agranular reticulum. Our results agree with his on the point that the vesicles belonging to the smooth-surfaced endop!asmic reticulum increase in amount as the cell grows, and finally surround the lipid droplets with collapsed sacs. However, we neither consider that the smooth-surfaced endoplasmic I

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

89

reticulum in this cell is completely identical to the Golgi apparatus, nor do the secretary droplets arise directly from the agranular vesicles. GLAND D. THETHYROID 1. The Ultrastructure of the Normal Thyroidal Cells The thyroid may find its position, in structural characteristics, between the typical endocrine and exocrine glands. The unit of the thyroid is a sac lined with a simple epithelium called “the follicle,” which is akin in all respects to the acinus of exocrine glands, the only difference being that the follicle is closed, having no excretory duct. Therefore, the cell structure as well as the secretory mechanism is also similar to those of exocrine cells. With the use of electron microscopy, many investigations have been reported on human and animal thyroids, which were either normal, experimentally changed, or pathological (Monroe, 1953 ; Braunsteiner, et al., 1953; Dempsey and Peterson, 1955; Wissig, 1956; Ekholm and Sjostrand, 1957; Ichikawa and Irie, 1957b; Fujita et al., 1958a,b; Wang, 1958; Yoshimura and Irie, 1959a,b; Irie, 1960; etc.). The follicular cell is either cylindrical, cuboidal, or flat, depending on its functional state and on the repletion of the follicle. The follicular lumen with the colloid is of medium density. The free luminal surface is provided with many microvilli (Monroe, 1953; Braunsteiner et d.,1953) except for the surface of apocrine secretory projections (Braunsteiner et al., 1953; Yoshimura and Irie, 1959a). Interdigitations at the lateral intercellular boundary were found by Ekholm and Sjostrand ( 1957), and a number of adhesion plates (nodes of Bizzozero) were observed especially in its apical part by Ichikawa and Irie (1957b). The basal plasma membrane is most complicated : the infolding is not so extensive as compared with that of sweat glands, but small footlets are irregularly extended toward the basement membrane. Irie (1960) suggested that the basal infolding might be concerned with the iodine uptake. The endoplasmic reticula are relatively abundant in this cell type, but the arrangement is considerably irregular and dilatation of the space in the cisternae is remarkable (Fig. 28). In general, rough-surfaced types are numerous in the basal cell territory, but smooth-surfaced ones are found in the supranuclear region. The Golgi apparatus of the thyroid cell was first observed by Dempsey and Peterson (1955), and is usually situated immediately above the nucleus. The ultrastructure is not different from that of the other cell types. Centrioles were found by Irie (1960) at the Golgi region (Fig. 12). Mitochondria are pleomorphic and distributed

90

K. KUROSUMI

FIG.28. A part of the thyroidal cell of a snake (Ela+he quadrivirgata). The nucleus (N) seen at the bottom possesses marked pores in its envelope as indicated by arrows. Endoplasmic reticulum (Er) is very well developed. Secretory granules are widely variable in size and density; the smallest granules appear to be transitional from the vesicles in the Golgi apparatus (G). M , mitochondria. ( X 10,000.) (N. Watari.)

ELECTRON MICROSCOPIC AN..ZLYSIS OF SECRETION

91

at random throughout the cytoplasm. Occasionally, mitochondria and cisternae of the endoplasmic reticulum are closely related topographically to each other; a part of a mitochondrion is often enclosed by a roughsurfaced membrane. Secretory granules of the thyroid cells are often called “colloid droplets.” Ekholm and Sjostrand (1957) divided the big granules found in the apical cell zone into two distinct types. The first type is a regularly round granule 0.15-0.8 p in diameter, with a homogeneous dense content. This granule is surrounded by a single smooth membrane about 50 A. thick, and frequently contains a core of aggregated small dense particles. The second type of granule is characterized by its wavy outline and less dense and unhomogeneous content. The surface membrane of the granule is also single and smooth. The size ranges from 0.15 p to 2.2 p. Both types of granules appear simultaneously in the same cell in the case of the mouse thyroid. Irie (1960) reported that the round dense granules (the first type of Ekholm and Sjostrand) are found in the thyroid cells of Basedowian women, rats, mice, and cats, but none of these are detectable in oxen, pigs, and rabbits (Figs. 12 and 29). Yoshimura and Irie ( 1959a) and Irie (1960) referred to the second granules of Ekholm and Sjostrand as dilated cisternae of the smooth-surfaced endoplasmic reticulum. Granules with varying grades of density suggestive of intergrades between the dark round granules and the large less dense granules are observed. In addition to these big granules, small granules, and vesicles containing substances of varying density are found in a specific layer immediately beneath the free cell surface (Ekholm and Sjostrand, 1957; Irie, 1960) (Fig. 12). Dempsey and Peterson (1955) suggested that the colloid might be accumulated within the sacs of the ergastoplasm, from which the colloid droplets might be produced. Fujita et al. (1958a) and Wang (1958) agreed with this assumption. In contrast to the notion of Dempsey and others, Wissig (1956) postulated that the colloid droplets arise from the Golgi vesicles. Watari (personal communication) found, in the thyroidal cells of the snake (Elaphe quudrivirgata) , round dense granuIes of various sizes and densities, smaller ones of which are very similar in appearance to the Golgi vesicles (Fig. 28). According to Yoshimura and Irie (1959a), various modes of release of secretion may be observed in the thyroid gland cells : apocrine secretion is prominent in Basedowian thyroid, a free communication between the colloid in the follicular lumen and the content of the granule immediately underlying the free surface through an opening may be present. The disappearance of the limiting membrane of the less dense granules is

92

K. KUROSUMI

occasionally observed. In the latter case, the content of the granule may be released into the cytoplasm and then extruded either by apocrine type secretion or by diffusion on the molecular level through the intact apical plasma membrane. To small vesicles underlying the free cell surface as well as large dilated cisternae of the smooth and rough endoplasmic reticulum, Wissig (1956) ascribed the morphological manifestation of resorption of the colloid. According to him, the colloid may be absorbed from the follicular lumen by a process akin to pinocytosis and then transported through the space of the endoplasmic reticulum to the base of the cell for discharge into the circulation (cited from Palay, 1958). By light microscopy, however, Kano (1952) and Toshio Ito (1958) established a theory of intercellular passage of the colloid through the small intercellular canaliculi. By electron microscopy, such canaliculi were detected in Basedowian thyroids and chicks 5 days after birth (Irie, 1958, 1960), and in human fetuses (Sawano and Kanaya, 1959). The confused problem of the transportation of colloid from the follicular lumen to the blood stream must be resolved in the future after the accumulation of further knowledge by combined studies with the electron microscope, radioactive isotopes, etc.

2. Experimentally Induced Changes in the Thyroidal Ultrastructure a. The Influence of Hypophysectomy. According to Braunsteiner et al. (1953) and Dempsey and Peterson (1955), the hypophysectomy induces a strong flattening of the follicular cells and the consequent compression of the nuclei. The substance of the follicular cells becomes remarkably dense. The ergastoplasmic system has become reduced in amount and the sacs, where present, are collapsed together so that their lumina are nearly obliterated. The small dense granules (RNP granules) of the ergastoplasm are also reduced in number. Microvilli are less noteworthy than in the cells under normal conditions. In a word, the thyroid cells of the hypophysectomized animals show a marked hypofunction or degeneration clearly detectable by electron microscopy. The same morphological changes could be obtained after treatment with thyroxine (Braunsteiner et al., 1953), which may inhibit the production of TSH by the anterior pituitary. b. The Influence of T S H . The thyroid cells of the animals injected with thyroid - stimulating hormone (T’SH) show remarkable changes. Braunsteiner et al. (1953) performed a series of experiments with TSH in two ways, one of which was an administration of low doses of TSH (10 I.U.). After one hour, the cell showed cytoplasmic droplets and blebs

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

93

at the free surface, and diminution of the ergastoplasm as well as of the microvilli was noted. After 6 hours, columnar cells with numerous microvilli were seen. The cytoplasm remained clear and the ergastoplasm was again present. After 12 hours, microvilli were again clearly discernible. The cytoplasm had a washed-out appearance. The second experiment with high doses of TSH (25 I.U.) represented a breakdown of follicular cells, and blood appeared in the follicle. Yoshimura and Irie (1959a) carried out a similar experiment on rats and chicks. By 3 hours after TSH injection, large oval dense granules and expanded endoplasmic reticula were found in the supranuclear region of the cell, and moreover, extrusion of large granules by a mechanism of the apocrine type secretion was observed. During 6 to 12 hours, there were many colloid droplets with huge dimensions, whose density was not so high. They found a peculiar crystalloid in the cytoplasm of chicken thyroid cells in this time and thereafter (Fig. 29). Such a crystalloid had not been reported previously by electron microscopy. The crystalloids are generally prismatic in shape and enveloped by a smooth membrane (Yoshimura and Irie, 1959b). After 24-36 hours, the crystalloids became more distinct, being detectable with the light microscope. The development of the endoplasmic reticulum is best at this time, and the Golgi apparatus is also enlarged. It is probable that the function of the thyroid may be most vivid in this stage. At 48 hours the ultrastructure of the thyroid cells was restored to its normal condition. c. The Influence of Thiouracil. Braunsteiner et al. (1953) reported that 10 days after subcutaneous implantation of 25 mg. of thiouracil, the height of cells increased and the canalicular cytoplasmic structure (endoplasmic reticulum) appeared more marked. According to Dempsey and Peterson (1955), in the glands from animals to which thiouracil had been orally administered, the ergastoplasmic sacs were greatly increased in number and in dilatation, so that the cells assumed the characteristic lacy appearance. The contents of the sacs and the colloid in the follicular lumen were less dense than in the normal glands. The mitochondria were enlarged and the homogeneous mitochondrial matrix was less dense. Yoshimura and Irie (1959a) pointed out that, in thyroidal cells of the thiouracil treated rats, large dense granules were almost undetected and dilated cisternae of the smooth-surfaced endoplasmic reticulum were crowded in the apical cell zone. The rough-surfaced variety of the endoplasmic reticulum was also well developed in the basal cell territory. The cells of the thyroids from the animals being administered thiouracil may have the ability to concentrate iodine, but thiouracil blocks the organic

94

K. KCROSUhII

FIG.29. A part of the thyroidal cell of the chick 6 hours after the administration of TSH. Cisternae of the endoplasmic reticulum ( E ) are dilated. Besides the secretory granules ( S ) , peculiar crystalloids (C) occur in the cytoplasm. These are long and prismatic and sheathed with a smooth delicate membrane. N , nucleus. ( x 25,000.) (Courtesy of F. Yoshimura and M. Irie, 1959b.)

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

95

binding of iodine. The electron microscopic feature of the cell treated with thiouracil is very interesting in comparison with its physiological or biochemical characteristics (Yoshimura and Irie, 1959a). d. The Influence of Cold Stress. Rats maintained in a cold room kept at 5" C. for 7 days showed increased height of the follicular cells. The ergastoplasmic sacs were dilated so that the cell appeared like a lacework (Dempsey and Peterson, 1955). The lumina of the sacs are not empty, but contain a homogeneous, colloid-like substance of slight to moderate density. In cold-exposed animals (2" C. for 5 days), Ekholm and Sjostrand (1957) described that the (secretory) granules were generally more numerous and larger than in animals kept at ordinary laboratory temperature. e. The Influence of Adrenaline Stimulation. Fujita et al. (1958a) examined the thyroid cells of domestic fowls 2 hours after the injection of adrenaline. The endoplasmic reticulum was markedly increased in number and in size, coming to occupy about 60440% of the whole area of the cytoplasm, but the R N P granules on the outer surface of them were reduced. From this experiment, they assumed that the colloid might be produced in the endoplasmic reticulum. E. THEENDOCRINE PANCREAS AND

THE

ADENOHYPOPHYSIS

As examples for the other type of endocrine glands, the endocrine pancreas (islets of Langerhans) and the adenohypophysis may be presented in this review. 1. The Endocrine Pancreas

The organ is composed of three cell types in most animals, i.e., alpha, beta, and delta cells, or C-cell instead of delta cell. The electron optical morphology of these cells, especially of the alpha and beta cells, was examined by P. E. Lacy (1957a,b), Ferreira (1957), Stoeckenius and Kracht ( 1958), Bencosme and Pease ( 195S), Munger ( 195Sb), Yokoh et al. (1959), and A. Watanabe (1960). Lacy established the identification of cell types in islets of various mammals (dog, rabbit, guinea pig, and rat). According to him, the Golgi apparatus is smaller and the amount of the ergastoplasm is less in the alpha cells than in the beta cells ; and the concentration and density of the granules in the alpha cells are greater than that of the beta granules. The shape and size of the granules are variable in various animal species. For example, in dog's islets, the alpha granules are round, but the majority of the beta granules have a rectangular profile, being contained in a clear area bounded by a distinct membrane. Individual granules occasionally

96

K. KUROSUMI

appear club-shaped or assume the form of a V or T (Lacy, 1957b). Bencosme and Pease (1958) found that the beta granule of the cat is also angular in outline, and suggested its semicrystalline organization. In the carp’s endocrine pancreas (Brockmann’s body) A. Watanabe ( 1960) observed a rodlike, horseshoe-shaped or ring-shaped crystalloid in the alpha granules, but not in the beta granules. H e demonstrated the youngest granules of the alpha cell of this fish as a solid spherical or oval granule with high electron density. It is likely that these round granules may change their form into ones with a ring-formed larnellated shell, which in turn is ruptured at some points becoming horseshoe in shape, rods, or plates (Fig. 30). Such peculiar crystalline granules have not been observed in other animals such as mouse, rat, rabbit, guinea pig, and human being, in which both alpha and beta granules are all round, although the size range is variable. Palade (1956a) first found that the beta granule is enveloped by a smooth membrane, which is not directly applied on the granule but remains separated from it by a narrow space. This feature is reminiscent of the “intracisternal granule’’ of the exocrine part of the pancreas reported by the same author, but the membrane surrounding the endocrine granule is not studded with R N P granules. This situation of the beta granules was also found by Ferreira (1957) and Munger (1958b), and they suggested that the granules might arise within the Golgi vesicles. The alpha granule was also found to be enveloped within a smooth-membraned vesicle by Stoeckenius and Kracht (1958) and Bencosme and Pease (1958). These also may originate from Golgi vesicles (A, Watanabe, 1960). C-cells which are characteristic of the guinea pig islets are pale in cytoplasmic density and devoid of distinct granules. Only a small amount of ergastoplasm was observed, and the Golgi complex and mitochondria were usually indistinct in these cells (Lacy, 1957b). The delta cell of the cat was described by Bencosme and Pease (1958). These are either nongranulated or granulated. This cell type contains numerous vacuoles or vesicles, which are of the same general size as granules, and it was thought that the latter may be derived from the former. The third type of the carp islet cells, the D-cell, is usually nongranulated, but in rare cases a small granulemay be contained within a vesicle in the Golgi region. In alpha and D-cells, vesicular smooth-surfaced endoplasmic reticula are abundant, while in beta cells lamellated rough-surfaced ones are well developed (A. Watanabe, 1960).

ELECTRON MICROSCOPIC ANALYSIS

OF SECRETION

97

FIG.30. Alpha granules of the carp’s endocrine pancreas. Several varieties of the granule form which may represent a serial transformation are clearly observed. The most initial form is a dense granule surrounded by a delicate limiting membrane (1). At the next step, a dark lamellated shell may appear at the peripheral zone and the electron density of the internal substance is reduced (2). At the third step, the internal substance is extremely cleared up and the shell may be ruptured resulting in a horseshoe-shaped pattern (3). The last step may be represented by the thinning and partial disappearance of the shell as well as the limiting membrane, from which the internal substance may flow out ( 4 ) . ( X 30,000.) (A. Watanabe, 1960.)

98

K. KUROSUMI

Mitochondria of islet cells are generally slender filamentous bodies, and none of the authors assumed that the specific granules originated from the mitochondria. 2. The Adenolaypophysis

Since the first observation by electron microscopy of Rinehart and Farquhar ( 1953), many investigations on the anterior pituitary cells under either normal or experimental conditions have been published ( Farquhar and Rinehart, 1954a,b ; Farquhar and Wellings, 1957 ; Farquhar, 1957 ; Peterson, 1957 ; Ichikawa, 1959). According to the pioneer work of Rinehart and Farquhar (1953), as well as the most recent study of Ichikawa (1959), both on albino rats, three main cell types are identified as follows. Chromophobes are characterized by the absence of secretory granules and by the poverty of the cytoplasm containing less-developed organelles. The size of this cell is consequently small. The acidophile contains relatively large, round or oval secretory granules with high electron density, which are about 400 mp for the maximum diameter. The Golgi apparatus, mitochondria, and endoplasmic reticulum are well developed, but vary considerably in size, shape, and number according to the functional state of the cell. The roughsurfaced endoplasmic reticulum of this cell is most conspicuous among various cell types of the adenohypophysis, and takes a typical arrangement of parallel lamellae chiefly at the peripheral parts of the cell. The basophile cells possess relatively small granules which are round or oval dense bodies less than 200 my in diameter. The Golgi apparatus is well developed. The rough-surfaced endoplasmic reticulum takes a form of irregular vesicles of various sizes. Recognition of the Golgi apparatus in the acidophile and basophile cells by Rinehart and Farquhar (1953) might be of notable merit in that time when none of the detailed ultrastructure of the Golgi apparatus had been known. Farquhar and Wellings ( 1957) subsequently demonstrated evidence suggesting secretory granule formation within the Golgi apparatus in rat acidophiles. It was found that secretory granules were often surrounded by Golgi membranes, and hence acidophile granules may be formed within the vesicular or lamellar components of the Golgi complex. This possibility has been recently reappraised by Ichikawa (1959), who elucidated the fact that the basophile granules also possibly arise at the Golgi apparatus, as well as the acidophile granules (Fig. 31A). According to him, the acidophile granules may appear as an accumulation or condensation of secretory substance within the Golgi vacuoles or the terminal blebs of the Golgi lamellae, whereas the basophile granules may be trans-

ELECTRON MICROSCOPIC ANALYSIS

'

bF

SECRETION

99

formed from the Golgi vesicles. Mitochondria situated in the Golgi region appear to be swollen or partially destroyed, being suggestive of indirect participation of mitochondria in the mechanism of secretory granule formation. Mature secretory granules may leave the Golgi apparatus, migrate toward the cell periphery, and finally attach to the surface plasma membrane. After the complete fusion of the membrane surrounding the secretory granule and the surface plasma membrane, a small opening may occur at the site of membrane fusion. Granules may emigrate passing through the opening to the intercellular space or the space between the basement membrane and the parenchymatous cell surface, in which the granules may lose their normal density or become amorphous (Ichikawa, 1959) (Fig. 31B). As an example of the adenohypophysis of lower vertebrates, A. Watanabe and Kurosumi (unpublished) observed the organ of the carp (Cyprius carpio). The three cell types are distinguished in this teleost pituitary, too (Fig. 32A). The morphology of the cell organelles generally agrees with that in mammalian hypophyses hitherto reported, i.e., the prevalence in the ergastoplasm of acidophiles over any other cells is marked, and chromophiles are characterized by the absence of granules and poorly-developed organelles. However, specific granules are somewhat different from those of mammalian cells. Acidophile granules are markedly pleomorphic ; in addition to round regular granules, elongated profiles of granules are observed. These look either rodlike, dumbbell-shaped or tadpole-like (Fig. 32B). The electron density of acidophile granules is greater than that of basophile granules. The basophile cells contain two types of granules, one of which is a small round granule resembling those of mammalian cells, but the other is a large globular body up to about 2 p in diameter which is reminiscent of zymogen granules of the exocrine pancreas. This large granule corresponds to the colloid droplet of light microscopists. This cell also contains many vacuolar spaces of various sizes. The results of castration and thyroidectomy. Farquhar and Rinehart (1954a,b) stated that the basophiles might be further classified by electron microscopy into gonadotrophs and thyrotrophs, based upon a series of experiments of castration and thyroidectomy on rats. They stated that in the normal pituitary, the thyrotrophs are angular, are not usually located on a sinusoid, and contain fine ill-defined secretory granules ; while the gonadotrophs are rounded, are typically located on a sinusoid or larger portal vessel, and contain somewhat larger, denser rounded granules. The castration experiment performed by Farquhar and Rinehart

100

K. KUROSUMI

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

101

FIG.31. Electron micrographs of cells of the rat adenohypophysis. A. Golgi area of an acidophile cell. Secretory granules (9)are enveloped with a limiting membrane, some of which are continuous with the Golgi lamellae, being suggestive of the granule formation from the Golgi apparatus. Mitochondria (111’) in the Golgi region are somewhat deformed as compared with outside ones (m). 11, nucleus. ( x 60,000.) B. Peripheral zone of a basophile cell (B) and adjacent sinusoid containing an erythrocyte ( E ) . Basement membranes (bm) are intercalated in the perisinusoidal space. Arrows indicate the feature cf extrusion of secretory granules through openings across the plasma membrane. ( x 35,000.) (Courtesy of A. Ichikawa, 1959.)

102

K . KUROSUMI

FIG.32. Electron micrographs of the adenohypophysis of the carp. A. Survey picture. Ac, acidophile; Bs, basophile; ~ c h , chromophobe. ( X 9000.) B. An acidophile cell ‘containing specific granules of variable shape. G, Golgi apparatus. (x 14,000.) (A. Watanabe and X. Kurosumi.)

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

103

(1954a) revealed a possibility of the presence of two subvarieties in gonadotrophs, one of which may secrete FSH, while another may produce LH. In the early time of castration, the former showed an increase in granular and mitochondria1 content, enlargement of the Golgi apparatus, and pronounced vesiculation of the cytoplasm. I n later stages the vesiculation proceeds and the granule content diminishes, and finally the cells became a signet-ring shape with a fringe of cytoplasm surrounding one or several large vacuoles. The latter subvariety of the gonadotroph was seen in the pituitaries of 35- and 75-day castrates. These cells also showed varying degrees of vesiculation but were characterized by denser, more irregular nuclei and bizarre cytoplasmic formations resembling filigree. Two types of acidophiles were also noted. Following thyroidectomy, changes were noted in fine structures of thyrotrophs which were distinguished from normal or castrated gonadotrophs (Farquhar and Rinehart, 1954b). A vesiculation of the cytoplasm occurred in thyrotrophs after thyroidectomy, too. The size of the vesicle may increase with time following thyroidectomy. They have few or no secretory granules, and if present, the granules are small and ill defined. As early as 10 days after the operation, uniformly dense ovoid or round bodies are found in these cytoplasmic vesicles and increase in size and number as the time increases. In contrast to the description of Farquhar and Rinehart, Ichikawa (1959) argued that such a classification of pituitary cells into further subvarieties might be impossible based only upon the electron microscopy of thin sections, even though experiments such as castration were carried out. H e described the degranulation and cytoplasmic vacuolization in both basophiles and acidophiles as the result of bilateral ovariectomy, but unilateral ovariectomy showed only degradation. The cytoplasmic vacuoles of castrated basophiles may originate from various parts of cell elements such as Golgi vacuoles, swollen mitochondria, endoplasmic reticulum, and the outer nuclear membrane, while the vacuoles in acidophiles may be derived from the rough-surfaced variety of endoplasmic reticulum.

IV. Discussion of the Secretory Mechanism

A. INGESTION OF THE SECRETORY MATERIAL The first step of the secretory activity is the ingestion of raw material for the production of secretory substance. This mechanism, however, has not been discussed so extensively by light and even electron microscopists, because no or very little morphological evidence is available. As the raw material is composed of rather small molecules, the electron den-

104

K. KUROSUMI

sity is very low, resulting in a difficulty of identification even by the high resolution of the electron microscope. Radioactive isotopes may be useful for this sort of study, but the combination of electron microscopy and tracer research with labeled isotopes is still unexploited, although such a trial has already begun (O’Brien and George, 1959). We found elaborate infoldings of the basal plasma membrane in sweat gland cells, and first ascribed them as features of the uptake of secretory material included in water (Kurosumi and Kitamura, 1958; Kurosumi et al., 195&, 1959a). The presence of infoldings in various gland cells such as pancreas (Weiss, 1953), salivary glands (Pease, 1956a), thyroid gland (Ekholm and Sjostrand, 1957), and gastric gland (Kurosumi et ad., 1958b) had previously been noticed, but the interpretation of their functional significance was unsettled at that era. The invagination of the plasma membrane may increase by a hundredfold the surface area of the basal cell surface available in the absorption of raw material from the extracellular tissue fluid or from the blood. Not only the invagination, but also the sending out of irregular processes or footlets into the intercelluIar or pericapillary space is observed in some glands, for instance the thyroid (Ekholm, 1957b) and the parotid (Scott and Pease, 1959). This is also effective for the increase in the absorptive surface area. It may be an important problem whether or not the raw material dissolved in water passes through the intact plasma membrane without any morphological changes. Besides such irregularities in structure of the basal cell surfaces, membrane vesiculation is observed at the base of the cell, especially where continuous to the tip of the infolded plasma membrane. In glandular tissues, Kurosumi and his collaborators ( 1959a) discovered this structure in a study of human apocrine sweat glands. But in other tissues, Palade (1953b) found it in the capillary endothelium and assumed it as a sign of fluid transportation across the capillary wall. Similar features were observed in macrophages (Palade, 195613)) in frog nephron tubules (Bargmann et al., 1955), and in the epithelium of stria vascularis of the inner ear (Smith, 1957). Ingestion of liquid material known as “pinocytosis” or “cell drinking” has been repeatedly found in living cells with the technique of phase microscopic cinematography (Pomerat et al., 1954). Palade ( 1956b) described that the vesiculation of the plasma membrane at the tip of infolding might represent pinocytosis at the submicroscopic level of dimension. This phenomenon may be correlated with phagocytosis and its related cell function in the reticuloendothelial cells (splenic macrophages, Palade, 1956b; Kupffer cell and sinusoidal endothelium of the liver,

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

105

Yamagishi, 1959), and with the function of absorption in some absorptive epithelia such as intestinal (PaIay and Karlin, 1959) and renal tubular cells (Fawcett, 1958). In both examples of the latter case, at the bottom of the intermicrovillous space occur tubule-like invaginations of surface plasma membrane, from which the pinocytotic vesicles filled with absorbed material may originate. Although examples hitherto elucidated have been few in number, this phenomenon at the base of the glandular cell may be reasonably associated with the uptake of raw material for secretion production. However, it should be strongly emphasized that there are many glandular tissues without manifestation of any pinocytotic vesicles and even of infoldings at all. In these cases, at the present state of morphological knowledge, transfer of raw material at the molecular level across the intact plasma membrane must be affirmed. I t seems likely that larger molecules may be ingested through the mechanism of pinocytosis, whereas smaller molecules pass without morphological changes in the plasma membrane. Fawcett (1958) disputed the opinion that the infoldings might accomplish an increase in available cell surface for the fluid exchange, and suggested that certain membrane-associated enzymes might be involved in the transport mechanism, being brought into close proximity with mitochondria, from which the energy for the transport might be generated by their oxidative enzyme system. This may be the case in convoluted tubules of the nephron, but in glandular tissues no mitochondria is associated topographically with the infolding. It should be noticed that the direction of water flow is reversed in the nephron compared with the gland. According to Solomon (1952), the amount of energy spent to transport the inorganic components of one liter of pancreatic juice may be calculated as about 329 calories, which is less than 8% of the 4300 calories available. The figure presented may not strictly represent the amount of energy spent for only the ingestion, but may include some of the calories for extrusion of secretion. Thus the uptake of raw material by glandular cells may probably show very little energy requirement. Close packing of mitochondria among basal infoldings in nephron cells and the presence of mitochondria in the tips of microvilli of Malpighian tubule cells (Beams et al.; 1955) may suggest higher energy consumption for the extrusion of substances out of the cell, as compared with the case of absorption into the cell.

106

K. KUROSUMI

B. SYNTHESIS OF

THE

SECRETORY SUBSTANCE

The most important and energy-spending process of the secretory activity is the synthesis or production of secretory substances, which are widely variable in chemical and biological nature, depending on the types of gland and conditions of animal life. Generally speaking, the secretion substances found in human and higher animal glands may be classified in view of the biochemical standpoint as follows : ( 1) the protein, either simple, enzymic, or mucous (polysaccharide-containing), ( 2 ) the lipid, either simple or with protein (lipoprotein) or pigment (lipopigment), and (3) the electrolytes (salts or acids). The production of these secretory substances may be more or less correlated functionally with the three main organelles of the cytoplasm, the ergastoplasm, the Golgi apparatus, and the mitochondria, although the predominance of any one of these is not uniform among various secretory cells.

1 . The Ergastoplasm (Microsome Fraction) An important role of the ergastoplasm in the secretory activity was postulated long ago by Garnier (1897) and has been discussed repeatedly since then. Cytochemical studies of Brachet (1950) and Caspersson ( 1950) including the results of microspectrophotometry indicated that ribonucleic acid (RNA)is mainly localized in the basophilic parts of the cytoplasm, i.e., the ergastoplasm, and that ribose polynucleotides participate in the process of protein synthesis of the cytoplasm. On the other hand, Claude (1943) isolated submicroscopic particles, the microsomes, by differential centrifugation and determined that these particles contain a high amount of R N A and phospholipid. Identification of the microsome fraction with the ergastoplasm (rough-surfaced variety of the endoplasmic reticulum) in situ was established by Kuff et al. (1956) and Palade and Siekevitz (1956a,b), and moreover, it was known that most of the RNA of the microsome fraction is loaded in small particle components ( R N P granules) of the ergastoplasm. Biochemical studies using radioactive amino acids apparently indicated that the microsome fraction is the part showing the highest early concentration of radioactivity, and it was considered suggestive of the primary participation of the microsome (endoplasmic reticulum) in the protein synthesis of the gland celk (Alfrey et d.,1953; Siekevitz and Palade, 1958b). Electron microscopic morphology of albuminous glands offered two different views on the site of production of secretory granules; for example, in exocrine pancreas, Weiss ( 1953), Siekevitz and Palade ( 1958a), and I. Suzuki (1958) argued that the zymogen granules might be pro-

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

107

duced in the ergastoplasm, while other authors such as Sjostrand and Hanzon ( 1954b), Haguenau and Bernhard ( 1955), Farquhar and Wellings (1957), Palay (1958), I. Suzuki (1959), and Y. Watanabe et al. (1959) ascribed the origin of zymogen granules to the Golgi apparatus. In studies of the secretion of colloid from the thyroid, a similar situation exists: Dempsey and Peterson (1955), Fujita et al. (1958a), and Wang (1958) attributed the origin of colloid droplets to the ergastoplasm, but Wissig (1956) attributed it to the Golgi apparatus. I n the albumin-secreting gland of the hen oviduct, Hendler et al. (1957) showed well-developed ergastoplasm, the cavity of which contained an amorphous precipitate possessing a similar appearance to the secreted material in the gland lumen, but the Golgi apparatus remained clear and empty. The gastric body chief cell looks in morphology very similar to the pancreatic exocrine cell and was thought to produce the secretory substance within the sacs of ergastoplasm (Kurosumi et a,l., 195813). In a more recent study by Shibasaki (1959), however, it has been elucidated that the secretory granules primarily appear at the Golgi region, although the ergastoplasm displays marked changes during an active phase of secretion synthesis at poststarvation feeding. It is probable that the synthesis of protein may be initiated usually in the ergastoplasm, and the product may be transported to the Golgi region where the concentration and segregation of the material manifests itself by the formation of secretion granules. When the latter phase of the production is hindered by a certain unknown reason, secretory granules may unveil themselves in the sacs of the ergastoplasm. Thus the active participation of the ergastoplasm (cytoplasmic ribonucleoprotein) in the synthesis of protein is not left in doubt, wherever the secretory granules appear morphologically. In mesenchymal cells other than glands, active synthesis of protein is also accompanied by a tremendous development of the ergastoplasm, such as in plasma cells producing antibodies (Amano, 1958) and in fibroblasts secreting collagen (Kajikawa and Hirono, 1959).

2. The Golgi Apparatus and the Smooth-Surfaced Endoplasmic Reticulum The Golgi apparatus has been repeatedly assumed as the most significant organelle in the formation of secretory granules according to light microscopic research (Bowen, 1924 ; Hirsch, 1939). Under electron microscopy also, the Golgi apparatus has been known as the site of the first appearance of secretory granules in most of the

108

Ii. KUROSUMI

protein-secreting cells, as mentioned above. Especially in many endocrine glands, such as the islets of Langerhans .(Ferreira, 1957 ; Stoeckenius and Kracht, 1958 ; Munger, 195813 ; A. Watanabe, 1960), anterior hypophysis (Farquhar and Wellings, 1957 ; Ichikawa, 1959), adrenal medulla (Fujita and Kano, 1959), and neurosecretory ganglion (Sano and Knoop, 1959), the Golgi apparatus was assumed as the origin of specific (secretory) granules. Furthermore, in mucous gland cells, almost all of the investigators postulated that the mucous droplets might derive from the Golgi apparatus. The 6-cytomembrane which was thought to be closely related to mucus production by Schulz and de Paola (1958) is the only exception, but is readily considered to belong to the smooth-surfaced endoplasmic reticulum in a broader sense and may be in nature very akin to the y-cytomembrane of the Golgi apparatus. It must be emphasized, however, that the facts of prevailing localization of secretory granules in the Golgi area, and of the absence of attached particles on the limiting membrane surrounding the secretory granule, are not convincing factors of granule formation by the Golgi apparatus. I t can be easily presumed that the granule which first appears in the cavity of rough-surfaced sacs may grow enormously larger and expand the sac, resulting in a very sparse distribution of particles on such an espanded limiting membrane, which is apt to be recognized as a smooth membrane by a cursory inspection of electron micrographs of considerably thin sections (Kurosumi ct al., 1959b). I t may be the most dependable evidence for believing the Golgi apparatus origin of secretory granules that the most immature, small granules are contained in the Golgi vesicles or vacuoles, and a complete series of transition from these youngest granules to the typical mature granules is found. I n pancreas cells, such evidence is still meager, but in fact some gland cells are provided with sufficient evidence. It is not settled which of the three main components of the Golgi apparatus becomes the practical source of the secretory granule. This may be different from cell to cell. The commonest feature of granule formation in the Golgi apparatus is an accumulation of dense substance within the Golgi vacuole or terminal blebs of Golgi lamellae. The second is the transformation of Golgi vesicles into the secretory granules. Hally (1958) states that the granules of Paneth cells may develop from the Golgi vacuole which incorporates Golgi vesicles through a deficiency in its limiting membrane. This is a unique hypothesis on the granule formation. It may be that the problem should be left to future studies on how the secretory material is transported from the original site of production (the ergastoplasm) and incorporated into the Golgi apparatus, and how the

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

109

material is condensed to form the granule in the interior of cavitary Golgi complex. Some secretory cells have no typical Golgi apparatus, such as the gastric parietal cells and the sebaceous gland cells, in which numerous vesicles bounded with a single smooth membrane are scattered throughout the cell instead of a circumscribed Golgi apparatus. These vesicles apparently belong to the smooth-surfaced endoplasmic reticulum, and show a marked variation in amount proportional to the intensity of secretory activity. Secretions probably of 'electrolyte (quite empty in electron density)-for example, hydrochloric acid or its precursor, a sort of chloride, in the case of parietal cells-may accumulate in the vesicles of smooth-surfaced endoplasniic reticulum. It may be suggested tentatively that these vesicles are equivalent in function to the Golgi apparatus.

3. The Mitochondria Light microscopic studies postulating that the mitochondria might be converted into secretory granules have been repeatedly presented ( Nicolas et al., 1914; Eklof, 1914; Minamitani, 1941). Some of the recent electron microscopic studies also ascribed the origin of secretory granules to the mitochondria (Challice and Lacy, 1954 ; Kitamura, 1958; Kurosumi et al., 1958c, 1959a; Lever, 1955, 1956, 1957; Rogers, 1957; Kitamura and Kurosumi, 1959). There are many more reports on the mitochondrial origin of secretory granules in the case of fatty secretion than in that of protein secretion. Challice and Lacy (1954) are the only advocates of mitochondrial origin of pancreatic zymogen granules, but their study was not sufficient to persuade all opponents. W e argued that in sweat glands one type of secretion granules, probably of protein nature, might originate from mitochondria, but another type, from the Golgi apparatus (Kurosumi et al., 1958c, 1959b). The former type of granules, light secretory granules, are large oval bodies with moderate density possessing a double limiting membrane and some residue of cristae mitochondriales. In granules of intermediate steps, cristae are localized at the periphery and shaped in the form of an arch, assuming that some substance may be accumulated and push away the cristae against the mitochondrial wall. Takagi et al. (1959) demonstrated a very similar deformation of mitochondria in liver cells of the mouse fed with Pelzicillium islandicum. Mitochondria of the latter case are apparently degenerated. It is of interest whether the light secretory granules of sweat glands might be a degenerated form of mitochondria. Even if this is the case, the gland cell is not degenerated but normal, and the mitochondria under assumed degeneration may be expelled from the

110

K. KUROSUMI

cell, forming a part of the secretion. Lipochondria (polyvesicular fat droplets) of eccrine sweat glands were also regarded as fatty degenerates from the Golgi apparatus. Pathological degeneration of mitochondria has been described by Gansler and Rouiller (1956) as having many variants. Fatty degeneration is a common type, as well as protein or hyaline degeneration, which is probably the case in sweat gland cells. It must be noted that in some secretory cells, mitochondrial degeneration may occur as a part of morphological changes accompanying the normal secretory cycle. Many reports have been published, that the lipid or steroid secretion granules are derived from mitochondria, for example, in sebaceous glands (Rogers, 1957 ; Kitamura and Kurosumi, 1959) , in adrenal cortex (Lever, 1955, 1956), in brown adipose tissue (Lever, 1957), and so on. However, this assumption was denied by some authors such as Belt (1958) regarding adrenal cortex and Napolitano and Fawcett ( 1958) regarding brown fats. De Robertis and Sabatini (1958) pointed out a peculiar phenomenon of mitochondrial change in the adrenal cortex of the normal hamster, which leads to a flattening and lamination of mitochondria, and then to a formation of concentric lamellar chondriospheres, at the centers of which lipid substances may occur. A similar deformation of mitochondria and production of lipid droplets therefrcm are observed in apparently degenerating gastric parietal cells from a patient with stomach cancer, which is an unpublished observation made in our laboratory. In any event, it is impossible to negate completely mitochondrial transformation into secretory substance, whether the change may be unusual and degenerative or not. Biochemical data have been accumulated concerning oxidative enzyme systems, which may be contained within the mitochondria (Claude, 1954). Junqueira and Hirsch (1956) stated that the role of mitochondria in the processes of cell secretion appeared to be mainly that of energy suppliers. Such an indirect participation of mitochondria in the mechanism of secretion synthesis may be visualized in some protein-secreting cells, in which the Golgi apparatus may play the most significant role. Ichikawa (1958, 1959) on exocrine pancreas and adenohypophysis and Shibasaki (1959) on gastric body chief cells pointed out that mitochondria may be moved into the Golgi region in the most active stage of synthesis, and some of them display a considerable deformation in their ultrastructure.

4. Concludiag Remarks Although the precise conclusion on mechanisms of the synthesis of secretory substances has not been established, a vast area of electron

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

111

microscopic knowledge reviewed in this paper may strongly suggest the following assumption : In protein-secreting cells including mucous glands, the ergastoplasm may initiate the synthesis of protein, which in turn is transported to the Golgi apparatus where the formed secretory granules appear as the result of condensation of material. In exceptional cases, however, the transportation of half-made secretion is hindered, and secretory granules may appear within the ergastoplasmic cavities. Electrolyte secretion may be produced and accumulated in the vesicles of smooth-surfaced endoplasmic reticulum, which is the substitute for the Golgi apparatus in these cells. Lipid secretion may be elaborated either in the Golgi apparatus or in the mitochondria. Protein substances may also be produced in mitochondria in rare cases. Such a direct participation of mitochondria in secretory granule formation may be regarded in part as a degeneration of mitochondria. But in all secretory cells, the mitochondria may play an important role in secretion synthesis as energy suppliers, in an indirect way of participation.

C. EXTRUSION OF T H E SECRETORY PRODUCT The mode of extrusion of secretion from the gland cell has already been discussed in detail on the basis of results by light microscopy. Ranvier (1887) classified glands into holocrine and wzerocrine glands. In the former, gland cells perish and are converted into secretion; while in the latter, the cell survives throughout the cyclic changes of secretion activity with loss of a part of the cell body or in some cases with loss of the secretory products only. The merocrine glands were further divided into two, the apocrine and eccrine glands, as the results of studies on skin glands by Schiefferdecker (1917, 1922). The apocrine gland makes the secretion through decapitation of a cytoplasmic projection extended from the free surface of the glandular cell, and hence a part of the cytoplasm is lost. I n the case of eccrine glands, however, the secretion may leak out without loss of the cytoplasm. The definitions mentioned above are now widely adopted among Japanese investigators, and are thought to be more suitable in the historical and etymological sense than those presented in most textbooks published in English-speaking countries (Maximow and Bloom, 1957 ; Ham, 1953) since the latter use the term “merocrine” synonymously with “eccrine,” and thus simply classify the mode of extrusion into three types as: holocrine, apocrine, and merocrine. The high resolution of the electron microscope helped to point out that

TABLE I MODESOF EXTRUSION OF SECRETORY SUBSTANCE

Typel Examples

I

Sebaceous gland

111

IV

-

~

~~

*

Breakdown of the whole cell which converts into the secretion

Electron microscopy

Light microscopy

Holocrine secretion

Holocrine secretion

Apocrine sweat gland Decapitation of a stout The secretion conprojection extended tains more or Mammary gland* from the apical free less the debris of Thyroid* surface cytoplasm and Apocrine Gastric parietal cell* Eccriiie sweat gland* plasma memsecretion brane besides the Choroid plexus subof microvilli Carpal organ stance Bile duct epithelium

I1

V

Extrusion mechanism of the secretory products

Release of the secrePancreas tory substance through Adenohy pophysis an opening occurring Gastric surface a t a junction between The secretion does epithelium not contain any the plasma membrane Above glands marked parts of cytoand a secretory vacwith asterisks Eccrinc plasm or plasma uole (granule) secrction membrane other Body chief cell of the Transfer of the secrethan the secretory substance through stomach tory substance the intact plasma Salivary gland ineinbrane (serous cell?) ~~

~

The glands marked with asterisks appear again under Type IV

Macroapocrine secretion

Apocriue secretion

Microapocrine secretion

Opening niechanisni Eccriiie secretion

Not detectable with the electron niicroscope (niolecular level)

secretion

FIG.33. A diagrammatic illustration of various types of the extrusion mechanism of secretion observed with the electron microscope. The number of each figure corresponds to the type number of extrusion mechanism shown in Table I and the text. Type I. Holocrine secretion found in the sebaceous gland. Type 11. Macroapocrine secretion in the apocrine sweat gland. Type 111. Microapocrine secretion in the choroid plexus. Type IV. Eccrine secretion through an opening in the exocrine pancreas. Type V. Eccrine secretion under molecular level in the gastric body chief cell. (K. Kurosumi, original.)

114

I<. KUROSUhII

the last (eccrine or merocrine) could be further divided into several types. Thus the classification of- the extrusion mechanism appears to be becoming confused. The author proposes, in the light of electron microscopy, a classification with noncommittal numerals. Table I and Fig. 33 shorn this classification.

1 . Extrusion T y p e I This is an extrusion known as holocrine, and is represented by the sebaceous gland. Death of the cells accompanying the pycnosis of nuclei was observed with the electron microscope (Kitamura and Kurosumi, 1959), and a complete agreement between light and electron nlicroscopy is established in this type. 2. Extrusion T y p e 11 This type corresponds to the apocrine secretion described by light microscopists. The free luminal surface of the gland cell sends out a stout projection which is destined to be pinched off. This mechanism is known from light microscopy in the apocrine sweat gland, the mammary gland, the thyroid (Toshio Ito, 1958), the intrahepatic bile duct epithelium (Mizutani, 1944), etc. However, Montagna et al. (1953) denied a decapitation of the projection in the apocrine sweat gland. But in many electron micrographs of the apocrine sweat gland, thyroid gland, gastric parietal cells, duct of salivary gland, bile duct epithelium, and eccrine sweat gland, distinct cytoplasmic projections were observed (cf. p. 57), and these are believed not to be artifacts. Although Bargmann and Knoop (1959) also denied the apocrine mechanism in the rat’s mammary gland, some electron micrographs suggestive of this mechanism are included in their illustrations. Secretory granules may be either contained (the thyroid) or not contained (the sweat glands) within the apocrine projections. Vl’hen these are not contained, the dissolution of granules may occur prior to the entering of secretory substance into the projection. In all the cases, important organelles such as the mitochondria and Golgi apparatus do not enter the projection, making the least sacrifice of cell components. It is noted that the microvilli have disappeared on the surface of the apocrine projection, and it may be reasonable to consider that the surface plasma membrane is strongly distended at that portion.

3. Extrusion T y p e III Electron ~ i c r o s c o p yfirst elucidated this mechanism, which had been classified in the eccrine (merocrine) mechanism by most light microscopists with only one exception, the opinion of Montagna et al. (1953),

ELECTRON MICROSCOPIC ANALYSIS OF SECRETION

115

upon the human apocrine sweat gland, which may correspond to this. This type is represented by the pinching ofjF of expanded tips of microvilli, and may be called “microapocrine secretion,” which was reported in the choroid plexus, the carpal organ of the pig, and the intrahepatic bile duct. (cf. p. 54). The case of the Malpighian tubule reported by Beams et al. (1955) is a specific example, because the expanded tips of microvilli contain mitochondria. The reality of the shedding of the tips laden with mitochondria in this organ is still doubted. Kada (1959) claimed that the gastric parietal cell may release the secretion by this mechanism, but was not totally in agreement with our findings on rats.

4. Extrusion T y p e IV Most of the eccrine (merocrine) glands release their secretion by this mechanism, through which only the secretory substance is extruded without loss of any part of the cytoplasmic components, nor of the plasma membrane. A vacuole bounded with a distinct membrane containing either one or several secretory granules, or filled up with secretory substance, approaches and then attaches to the surface plasma membrane, and finally a small opening occurs through the plasma and vacuolar membrane leading from the interior of the vacuole out to the lumen of the gland. Secretory granules may go out with or without dissolution, but the surrounding membrane does not disappear except for the point of the small orifice. Yasuzumi and Tanaka (1958) first noted this mechanism in a study of the snail spermatogenesis, during which granules originated from nuclei might be discharged in this way from the atypical spermatid. It may not be called “secretion” in the strict meaning in that material, but the mechanism is totally the same as that found later in various types of eccrine glands. Kurosumi et al. (1958b) then suggested a similar mechanism of secretion release in parietal cells and surface epithelium of rat’s stomach. Y . Watanabe et al. (1959) on exocrine pancreas and Ichikawa ( 1959), on adenohypophysis, displayed obvious features of extrusion process by this mechanism. In mammary glands, protein granules are expelled in this way, as reported by Bargmann and Knoop (1959). They argued that lipid droplets might be shed into the lumen in a manner similar to the eccrine mechanism. However, their description showed that the lipid droplet pushes out the surface of the cell, being enveloped by the plasma membrane, and that the constriction at the base of lipid droplet detaches the droplet covered with a part of the plasma membrane. This mechanism cannot be called pure eccrine secretion because some

116

K . KUROSUMI

part of the cellular components, at least the plasma membrane, is apparently extruded. - The present author considers that this case must be classified into Type I1 (apocrine secretion), although this may be somewhat different from other examples of that type. ,

5. Extrusion T y p e V This type is the most vague of all in electron microscopic morphology. The surface plasma membrane is always intact throughout the cycle of secretion. The product may be diffused away through the plasma membrane. When the disappearance of membranes surrounding the secretory granules or vacuoles is apparent and no evidence of apocrine type extrusion accompanies it, it may be most probable that the secretory substance diffused into the apical cytoplasm may pass through the intact plasma membrane. The secretion of gastric body chief cells may be released by this mechanism (Kurosumi et al., 195813). Yoshimura and Irie (1959a) suggested that colloid of the thyroid might be transmitted partly in this way into the follicular lumen. Direct ascertainment by electron microscopy, however, is impossible because such substances transferable through the intact membrane may be of far smaller molecules, below the limit of resolution of the electron microscope. Since absorption at the molecular level is reasonably considered (cf. p. l05), extrusion in a similar way is also to be accepted.

6 . Combination of Two or More Types of Extrusion This occurs in the same cell either simultaneously or in different phases of cell function. For example, parietal cells of the stomach (Kurosumi et ul., 1958b), eccrine sweat glands (Iijima, 1959) the thyroid gland (Yoshimura and Irie, 1959a), and the mammary gland (Bargmann and Knoop, 1959) display two mechanisms of secretion release, one of which is mostly apocrine (Type 11) and another is eccrine (Type I V ) . Type I1 extrusion in these glands, at least partially, might be a stressed secretion after enormously intense stimulation or under a sort of abnormal cellular condition. The fact that apocrine projections are frequently observed in Basedowian thyroid as well as stomach parietal cells after a long period of starvation may suggest this possibility. Using light microscopy, Ries (1935) presented the fact that fragments of the cytoplasm were eliminated from pancreas cells after very intense stimulation.

ACKNOWLEDGMENTS The author is much obliged to the workers who have kindly offered their electron micrographs and drawings for this paper, and whose names are mentioned in each legend of the illustration with cordial thanks of the author.

ELECTRON MICROSCOPIC ANALYSIS OP SECRETION

117

The author also wishes to express his deep gratitude to Professor Toshio Ito and other colleagues of the Department of Anatomy, Gunma University, for their collaboration and useful suggestions given him during the progress of each of the studies which have become the skeleton of this review.

REFERENCES Afzelius, B. A. (1955) Exptl. Cell Research 8, 147. Afzelius, B. A. (1956a) Exptl. Cell Research 10, 257. Afzelius, B. A. (1956b) Exptl. Crll Research 11, 67. Alfrey, V.,Daly, M. M., and Mirsky, A. E. (1953) J. Gen. Physiol. 37, 157. Amano, S. (1958) Allergy (Tokyo) 6, 409. Amano, S., and Tanaka, H. (1957) Acta Haematol. Japon. 20, 319. Anderson, E., and Beams, H. W. (1956) J . Biophys. Biochem. Cytol. 2, SUPPI.439. Arai, M. (1956) Acta Anat. Nippon. (Tokyo) 31, 103. Bairati, A., and Lehmann, F. E. (1952) Experientia 8, 60. Baker, J. R. (1944) Quart. J. Microscop. Sci. 86, 1. Baker, J. R. (1951) Nature 168, 1089. Bargmann, W., and Knoop, A. (1959) Z . Zellforsch. u. mikroskop. Anat. 49, 344. Bargmann, W., Knoop, A., and Schiebler, Th. H. (1955) Z . Zellforsch. 14. mikroskop. Anat. 42, 386. Barter, R., and Pearse, A. G. E. (1953) Nature 172, 810. Beams, H. W., and King, R. L. (1932) Anat. Record 63, 31. Beams, H. W., and Tahmisian, T. N. (1953) Cytologiu 18, 157. Beams, H. W., and Tahmisian, T. N. (1954) Exptl. Cell Research 6, 87. Beams, H. W., Tahmisian, T. N., and Devine, R. L. (1955) J . Biophys. Biochem. Cytol. 1, 197. Beams, H. W., Anderson, E., and Press, N. (1956) Cytologia (Tokyo) 2l, 50. Belt, W. D. (1958) J . Biophys. Biochem. Cytol. 4, 337. Belt, W. D., and Pease, D. C. (1956) I.Biophys. Biocltem. Cytol. 2, Suppl. 369. Bencosme, S. A., and Pease, D. C. (1958) Endocrinology 63, 1. Benda, C. (1898) Cited from G. Hertwig (1929) in “Handbuch der mikroskopische Anatomie des Menschen” (von Mollendorff, ed.), Vol. 1, Part 1, p. 232. Springer, Berlin. Bennett, H. S. (1956) J. Biophys. Biochem. Cytol. 2, Suppl., 99. Bennett, H. S., and Porter, K. R. (1953) Am. J. Anat. 93,61. Bernhard, W., and Rouiller, C. (1956) J . Biophys. Biochem. Cytoi. 2, Suppl., 73. Bernhard, W., Gautier, A., and Oberling, Ch. (1951) Compt. rend. soc. biol. 145, 566. Bernhard, W., Haguenau, F., and Oberling, Ch. (1952a) Experientia 8,58. Bernhard, W., Haguenau, F., Gautier, A., and Oberling, Ch. (1952b) Z . Zellforsch. u. mikroskop. Anat. 37, 281. Bernhard, W., Bauer, A., Gropp, A., Haguenau, F., and Oberling, Ch. (1955) Exptl. Cell Research 9, 88. Borysko, E., and Bang, F. B. (1952) J . Appl. Phys. 23, 163. Bowen, R. H. (1924) Am. J . Annt. 99, 197. Brachet, J. (1950) “Chemical Embryology.” Interscience, New York. Bradfield, R. G. (1953) Quart. J . Microscop. Sci. 94, 351. Braunsteiner, H., Fellinger, K., and Pakesch, F. (1953) Endocrinology 63, 123.

118

K. KCROSUMI

Brettauer, J., and Steinach, J. (1857) Sitzber. Akad. Wiss. W i e n , Math.-rtaturw. K1. 23, 303. Callan, H. G., and Tomlin, S: G. (1950) Proc. R o y . Soc. B137, 367. Carlson, J. G. (1952) Chromosoma 6, 199. Caspersson, T.0. (1950) “Cell Growth and Cell Function.” Norton, New York. Challice, C. E., and Lacy, D. (1954) Nature 174, 1150. Challice, C. E., Bullivant, S., and Scott, D. B. (1957) Exptl. Cell Research 13, 488. Charles, A. (1959) J . Anat. 93, 2 6 . Chauveau, J., MOUE,Y., and Rouiller, Ch. (1957) Exptl. Cell Research 13,398. Chou, J. T. Y., and Meek, G. A. (1958) Quart. J . Microscop. Sci. 99,279. Christie, A. C. (1955) Quart. 1. Microscop. Sci. 96, 295. Clark, S. L., Jr. (1957) J . Biophys. Biochem. Cytol. 3, 349. Claude, A. (1943) Science 97, 451. Claude, A. (1954) Proc. R o y . Soc. B142, 177. Claude, A.,and Fullam, E. F. (1946) J . Exptl. Med. 89, 499. Coman, R. D. (1954) Cancer Research 14, 519. Dalton, A. J. (1951a) Nature 168, 244. Dalton, A. J. (1951b) Am. J. Anat. 89, 109. Dalton, A. J. (1951~) J . Natl. Cancer Inst. 11, 1163. Dalton, A. J., and Felix, M. D. (1953) Am. J . Anut. 92.277. Dalton, A. J., and Felix, M. D. (1954) Am. J . Anat. 94, 171. Dalton, A. J., and Felix, M. D. (1956) J. Biophys. Biochem. Cytol. 2,Siippl., 79. Dalton, A. J., Kahler, H., Striebich, M. J., and Lloyd, B. (1950) J . Natl. Cancer Inst. 11, 439. Dalton, A. J., Kahler, H., and Lloyd, B. (1951) Anat. Record 111, 67. De Harven, E., and Bernhard, W. (1956) 2. Zellforsch. u. mikroskop. Anat. 46,378. Dempsey, E. W., and Peterson, R. R. (1955) Endocrinology 66, 46. De Robertis, E., and Bennett, H. S. (1955) J. Biophys. Biochem. Cytol. 1,47. De Robertis, E., and Sabatini, D. (1958) J . Biophys. Biochem. Cytol. 4,667. Eichenberger, M. (1953) Exptl. Cell Research 4, i 7 5 . Ekholm, R. (1957a) J. Ultrastruct. Research 1, 26. Ekholm, R. (1957b) 2. Zellforsch. u. mikroskop. Anat. 46, 139. Ekholm, R., and Edlund, Y. (1959) J . Ultrastruct. Research 2, 453. Ekholm, R., and Sjostrand, F. S. (1957) 1. Ultrastruct. Research 1, 178. Eklof, H. (1914) Anat. Hefte 61, 1. Engstrom, H., and Wersall, J. (1958) Exptl. Cell Research, Suppl. 6,460. Erspamer, V. (1953) Arch. exptl, Pathol. Pharmakol. Nauityn-Schniedeberg’s =a, 92. Estable, C., and Sotelo, J. R. (1951) Piibl. Inst. Invest. Cieitcias Biol. 1, 105. Farquhar, M. G. (1957) Anat. Record 127, 291. Farquhar, M. G., and Rinehart, J. F. (1954a) Endocrinology 64, 516. Farquhar, M. G., and Rinehart, J. F. (1954b) Endocrinology 66, 857. Farquhar, M. G., and Wellings, S. R. (1957) J . Biophys. Biochem. Cytol. 3, 319. Fawcett, D. W. (1954) d n a t . Record 118, 422. Fawcett, D. W. (1955) J. Natl. Cancrr Inst. 16, Suppl., 1475. Fawcett, D. W. (1958) I n “Frontiers in Cytology” (S. L. Palay, ed.), p. 19. Yale Univ. Press, New Haven, Connecticut. Fawcett, D. W., and Porter, K. k. (1954) J . Morphol. 94, 221. Fawcett, D. W., and Selby, C. C. (1958) J . Biophys. Biochem. Cytol. 4, 63.

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

119

Ferreira, D. (1957) J. Ultrastruct. Research 1, 14. Freeman, J. A. (1956) J. Biophys. Biochem. Cytol. 2, Suppl., 353. Fujita, H., and Kano, M. (1959) Kagaku ( T o k y o ) 29, 149. Fujita, H., Kano, M., and Okamoto, S. (19%) Arch. Histol. Japan. 14, 157. Fujita, H., Kano, M., and Kido, T. (1958b) Arch. Histol. Japan. 14, 567. Fujiwara, T. (1957a) A c t a Anut. Nippon. ( T o k y o ) 32, 91. Fujiwara, T. (1957b) A c t a Anat. Nippon. ( T o k y o ) 32, 658. Gander, H., and Rouiller, C. (1956) S c h w e i z 2. allgem. Pathol. u. Bakteriol. 19, 21 7. Gamier, Ch. (1897) Bibliographie anut. 5, 278. Gaule, J. (1881) Zentr. &ed. Wiss. 19, 561. Glauert, A.M., Rogers, G. E., and Glauert, R. H. (1956) Nature 178, 803. Goldschmidt, R. (1905) Zoologische Jahrbiicher 21, 41. Golgi, C. (1898) Arch. ital. biol. 30, 60, 278. Granger, B., and Baker, R. F. (1950) Anat. Record 107, 423. Grass&, P. P., and Carasso, N. (1957) Nature 179, 31. Haguenau, F. (1958) Intern. R e v . Cytol. 7, 425. Haguenau, F., and Bernhard, W. (1955) Arch. anat. microscop. et morphol. exptl. 4, 27. Hally, A. D. (1958) I. Anut. 92, 268. Hally, A. D. (1959a) Nature la,408. Hally, A. D. (1959b) J. Anat. 93, 217. Ham, A.W. (1953) “Histology,” 2nd ed. Lippincott, Philadelphia, Pennsylvania. Hamperl, H. (1952) Arch. pathol. A i d . u. Physiol., Virchow’s 321, 482. Hartmann, J. F. (1953) I . Conzp. Neurol. 99, 201. Hartman, R. E., Smith, R. B. W., Hartman, R. S., Butterworth, C. E., Jr., and Molesworth, J. M. (1959) I. Biophys. Biochem. Cytol. 5, 171. Hay, E. D. (1957) Anat. Record 127, 305. Hay, E. D. (1958) I . Biophys. Biochem. Cytol. 4, 583. Heidenhain, R. (1858) Arch. med. Zentralzeit. 14, 105. Hendler, R. W., Dalton, A. J., and Glenner, G. G. (1957) J. Biophys. Biochem. Cytol. 3, 325. Hertwig, R. (1902) Arch. Protistenk. 5, 1. Hibbs, R. G. (1958) Am. J. Annt. 103, 201. Hillier, J. (1950) Conzpt. rend. congr. intern. microscope ilectron 1st Congr., Paris, p. 592. Hirsch, G. C. (1939) “Form- und Stoffwechsel der Golgi-Korper.” Protoplasma Monographien, Bd. 18. Springer, Berlin. Hirschler, J. (1927) 2. Zellforsch. u. mikroskop. Anat. 5, 704. Hollmann, K.-H. (1959) J. Ultrastmct. Rescarclz 2, 423. Holmberg, A. (1956) In “Electron Microscopy” Proceedings of the Stockholnl Conference (F. S. Sjostrand and J. Rhodin, eds.), p. 139. Academic Press, New York. Holmgren, H., and Wilander, 0. (1937) 2. mikroskop.-anat. Forsch. 42,342, Honjin, R. (1956) Okajimas F o l k Annt. Japon. 29, 117. Honjin, R., and Yamato, K. (1958) Acfa Anat. Nippon. ( T o k y o ) 33,225. Honjin, R., Izumi, S., Yamato, K., and Okumura, T. (1957) Jzizen IgakuRai Zasshi 59, 1093. Horstmann, E., and Knoop, A. (1957) 2. Zellforsch. u. mikroskop. Anat. 46, 100.

120

K. KUROSUMI

Ichikawa, A. (1958) Acta Anat. Nippon. ( T o k y o ) 99 (Abstr. 15th Regional Meeting of Anatomists in Kanto District, p. s ) , Ichikawa, A. (1959) Acta Anat. Nippon. ( T o k y o ) 34, 460. Ichikawa, A., and Irie, M. (1957a) Acta Anat. Nippon. ( T o k y o ) 32 (Abstr. 13th Regional Meeting of Anatomists in Kanto District, p. 8 ) . Ichikawa, A., and hie, M. (1957b) Acta Anat. Nippon. ( T o k y o ) 32, 93. Iijima, T. (1959) Acta Anat. Nippon. ( T o k y o ) 34, 649. Irie, M. (1958) Proc. Regional Conf. East Jap. SOC.Endocrinol. 2, 7. hie, M. (1960) Arch. Histol. Japan. 19, 39. Ishimaru, S., and Kada, K. (1956) Acta A n d . Nippon. ( T o k y o ) 31, 86. Ito, Toru (1958) Actn Haematol. Japan. 21, 631. Ito, Toshio (1943) Okajimas F o l k Anat. Japon. 22, 273. Ito, Toshio (1949) “Histology and Cytology of the Sweat Glands” (Japanese Text), Igaku no Shintpo, Ser. 6, p. 106, Tokyo. Ito, Toshio (1958) “The Physiology of the Thyroid Gland” (Japanese Text). Kyodo-Isho, Tokyo. Ito, Toshio, and Iwashige, K. (1951) Okajimas Folia Anat. Japon. 23, 147. Ito, Toshio, and Watari, N. (1958) Arch. Histol. Japon. 14, 369. Ito, Toshio, Takahashi, T., and Kitagawa, T. (1956) Arch. Histol. Japan. 11, 317. Iwashige, T. (1952) Arch. Histol. Japan. 4, 75. Junqueira, L. C. U., and Hirsch, G. C. (1956) Intern. Rev. Cytol. 6, 323. Kada, K. (1959) Kanazawa Daigaku Igakuhu Kaibogaku Kyoshitsu Gyoseki (Kanazawa) 66, 1. Kajikawa, K., and Hirono, R. (1959) Proc. 15th Congr. Electron Microscopy Japan p. 74. Kakinuma, T., Abe, H., and Nomura, Y. (1955) Okajimas Folia Anat. Japon. 27, 97. Kanda, S., and Tanaka, A. (1959) Denshi Kembikyo ( T o k y o ) 7, 92. Kano, K. (1952) Arch. Histol. Japan. 4, 245. Karrer, H. E. (1956) J. Biophys. Biochem. Cytol. 2, Suppl., 237. Kitamura, T. (1957) Arch. Histol. Japan. 13, 299. Kitamura, T. (1958) Arch. Histol. Japan. 14, 575. Kitamura, T., and Kurosumi, K. (1959) Proc. 6th Meeting of Kitakanto Med. SOC. (Maebashi) p. 1. Kite, G. L. (1913) Am. J. Physiol. 32, 146. Kolliker, A. (1855) Wzirzburger Verhandl. 6, 253. Kolster, R. (1913) Verhandl. anut. Ges. 27 Vers. (Greifswald) p . 124. Kopsch, Fr. (1902) Sitzber. preuss. Akad. Wiss. Physik.-math. Kl. 40,929. Kopsch, Fr. (1926) 2.mikroskop.-onat. Forsch. 6, 221. Kuff, E. L., Hogeboom, G. H., and Dalton, A. J. (1956) J . Biophys. Biochcm. Cytol. 2, 33. Kull, H. (1912) Arch. mikroskop. Anat. u. Entwicklungsmech. 81, 185. Kurosumi, K. (1954) Acta Anat. Nipporz. ( T o k y o ) 29, 82. Kurosumi, K. (1956) Acta Awt. Nippon. ( T o k y o ) 31, 90. Kurosumi, K. (1957a) Acta Anat. Nippmz. ( T o k y o ) 32, 91. Kurosumi, K. (1957b) Acta Anat. Nippmz. ( T o k y o ) 32, 175. Kurosumi, K. (1957~) Kitakanto Igaku (Maebashi) 7, 835. Kurosumi, K. (1957d) Denshi Kembikgo (Tokyo) 6, 101. Kurosumi, K. (1958) Protoplasma 49, 116. Kurosumi, K., and Akiyama, Y. (1958) Arch. Histol. Japan. 14, 291.

ELECTRON MICROSCOPIC ANALYSIS O F SECRETION

121

Kurosumi, K., and Akiyama, Y. (1959) Proc. 15th Congr. Electron Microscopy, Jnpan p. 60. Kurosumi, K., and Kitamura, T. (1958) Nature 181, 489. Kurosumi, K., and Watari, N. (1960) Proc. 21st Regional Meeting of Anatomists in Kanto District, p. 9. Kurosumi, K., Yamagishi, M., and Nagakawa, T. (1958a) Okajimus Folia Anat. Japon. 90, 369. Kurosumi, K., Shibasaki, S., Uchida, G., and Tanaka, Y. (1958b) Arch. Histol. Japan. 15, 587. Kurosumi, K., Iijima, T., and Kitamura, T. (1958~) Verhandl. 4te Intern. Kongr. Elektronenmikroskop. (Berlin), p. 361, Springer, Berlin (published in 1960). Kurosumi, K., Kitamura, T., and Iijima, T. (1959a) Arch. Histol. Japan. 16, 523. Kurosumi, K., Takahashi, Y., and Watanabe, A. (1959b) Denshi Kembikyo ( To k y o ) 8, 48. Lacy, D. (1953) J . Roy. Microsrop. SOC.73, 179. Lacy, D. (1954) Quart. J . Microscop. Sci. 96, 163. Lacy, P. E. (1957a) Anat. Record 128, 255. Lacy, P. E. (195713) Diabetes 6, 498. Laden, E. L., Linden, I., and Erickson, J. 0. (1955) A.M.A. Arch. Dermatol. 71, 219. Lever, J. D. (1955) Am. J. Anat. 97, 409. Lever, J. D. (1956) J. Biophys. Biochem. Cytol. 2, Suppl., 313. Lever, J. D. (1957) Anat. Record 128, 361. Lim, R. K. S., and Ma, W. C. (1926) Quart. J. Exptl. Physiol. 16, 87. Low, F. N. (1956) J. Biophys. Biochenr. Cytol. 2, Suppl., 337. Low, F. N., and Freeman, J. A. (1958) “Electron Microscopic Atlas of Normal and Leukemic Human Blood.” McGraw-Hill, New York. Luft, J. H. (1956) J. Biophys. Biochem. Cytol. 2, 799. Masson, P. (1928) Am. J. Pathol. 4, 181. Maximow, A. A., and Bloom, W. (1957) “A Textbook of Histology” 7th ed. Saunders, Philadelphia, Pennsylvania. Maxwell, D. S., and Pease, D. C. (1956) J . Biophys. Biochem. Cytol. 2, 467. Melczer, N. (1931) Dennutol. Wochschr. 93, 1101. Meves, F. (1904) Ber. deut. botan. Ges. 22, 284. Meves, F. (1908) Arch. Zellforsch. 1, 612. Millen, J. W., and Rogers, G. E. (1956) I. Biophys. Biochem. Cytol. 2, 407. Minamitani, K. (1941) Okajimas Folia Anat. Japon. 9, 61. Miyake, S., and Yamada, H. (1958) Symposium on Sweat Secretion, May 29, 1958, Kyoto, Japan, p. 11. Mizutani, Y. (1944) Okajimus Folia Anat. Japon. 22, 597. Monnk, L. (1945) Arkiv 2001.36A, 28. Monnk, L. (1948) Advances in Enzymol. 8, 1. Monroe, B. G. (1953) Anat. Record 116, 345. Montagna, W. (1956) “The Structure and Function of Skin.” Academic Press, New York. Moptagna, W., Chase, H. B., and Lobitz, W. C., Jr. (1953) Am. J . Anat. 92,451. Morita, Sh. (1931) J . Chiba Med. SOC.(Chiba) 9, 1069. Morita, Sh. (1958) Acta Anat. Nippon. ( T o kyo ) 33, 95. Miiller, E. (1898) 2. wiss. 2001.64, 624. Munger, B. L. (19%) Am. J . Anat. 103, 1. Munger, B. L. (1958b) A m . J . Anat. 103, 275. Nagamitsu, G. (1941) Keib Zgaku 21, 1011.

122

R. KUROSUMI

Nagano, T. (1959) Arch. Histol. Japan. 16, 311. Nakanishi, T. (1959) J . Ehiba Med. SOC.(Chiba) 35, 474. Napolitano, L., and Fawcett, D. W. (1958) J. Biophys. Biochenz. Cytol. 4, 685. Nassonow, D. (1923) Arch. mikroskop. Anat. u. Entwicklungsmech. 97, 136. Nicolas, J., Regaude, C., and FavrC, M. (1914) 17th Intern. Congr. Med., Secf. 13, Dermatol. Syphil. p. 105. Nilsson, 0. (19%) J. Ultrastrurf. Research 1, 375. Nilsson, 0. (1958b) J . Ultrastruct. Research 2, 73. Nilsson, 0. (1958~) J. Ultrastruct. Research 2, 200. Nussbaum, M. (1882) Arch. mikroskop. Anet. u. Entwicklungsmech. 21,296. O’Brien, R. T., and George, L . A., Jr. (1959) Nature 189, 1461. Odland, G. F. (1958) I . Biophys. Biochem. Cytol. 4, 529. Ogiso, K. (1959) J. Chiba Med. SOC.(Chiba) 36, 434. O n d , T., Hashimoto, M., Umetani, K., and Otsuka, R. (1957) Dertshi Kentbikyo (Tokyo) 6, 111. Ottoson, D., Sjostrand, F. S., Stenstriim, S., and Svaetichin, G. (1953) dcta Physiol. Scand. 29, Suppl., 106, 611. Palade, G. E. (1952) Anat. Record 114, 427. Palade, G. E. (1953a) J. Histochm. and Cytochem. 1, 188. Palade, G. E. (1953b) J . Appl. Phys. 24, 1424. Palade, G. E. (1955a) J . Biophys. Biochem. Cytol. 1, 59. Palade, G. E. (1955b) J . Biophys. Biochem. Cytol. 1, 567. Palade, G. E. (1956a) J. Biophys. Biochem. Cytol. 2, 417. Palade, G. E. (1956b) J . Biophys. Biochem. Cytol. 2, Suppl., 85. Palade, G. E., and Claude, A. (1949) J. Morphol. 86, 35. Palade, G. E., and Porter, K . R. (1952) Anat. Record 112, 370. Palade, G. E., and Siekevitz, P. (1956a) 1.Biophys. Biochem. Cytol. 2, 171. Palade, G. E., and Siekevitz, P. (1956b) J . Bioplzys. Biochem. Cytol. 2, 671. Palay, S. L. (1958) In “Frontiers in Cytology” (S. L. Palay, ed.), p. 305. Yale Univ. Press, New Haven, Connecticut. Palay, S. L., and Karlin, L. J. (1959) J. Biophys. Biochem. Cytol. 6, 373. Palay, S. L., and Palade, G. E. (1955) J . Biophys. Biochem. Cytol. 1, 69. Parat, M., and PainlevC, J. (1924) Compt. rend. mad. sci. 179, 612. Pease, D. C. (1955) Anat. Record 121, 723. Pease, D. C. (1956a) J. Biophys. Biochem. Cytol. 2, Suppl., 203. Pease, D. C. (1956b) Blood 11, 501. Pease, D. C., and Baker, R. F. (1950) A m . J . Anat. 87, 349. Peterson, R. R. (1957) Anat. Record 127, 346. Pomerat, C. M., Lefebre, C. G., and Smith, McD. (1954) Ann. N . Y . A c d Sci. 68, 1311. Porter, K. R. (1953) I . Exptl. Med. 97, 727. Porter, K. R. (1954) J . Histochem. Cytochem. 2, 346. Porter, K. R. (1955) Federation Proc. 14, 673. Porter, K. R. (1956) J . Biophys. Biochem. Cytol. 2, Suppl., 163. Porter, K. R., and Kallman, F. L. (1952) Ann. N. Y. Aced. Sci. 64, 882. Porter, K. R.,and Palade, G. E. (1957) J. Biophys. Biochem. Cytol. S, 269. Porter, K. R., and Thompson, H. P. (1947) Cancer Research 7, 431. Porter, K. R., and Thompson, H. P. (1948) J . Exptl. Med. 88, 15. Porter, K. R., Claude, A., and Fullam, E. F. (1945) J. Exptl. Med. 81, 233. Powers, E. L., Ehret, C. F , and Roth, L. E. (1955) Biol. Bull. 108, 182.

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

123

Powers, E. L., Ehret, C. F., Roth, L. E., and Minick, 0. T. (1956) J . Biophys. Biochem. Cytol. 2, Suppl., 341. Ranvier, L. (1887) I . micrographic 11, 7. Rasmont, R., Vandermeersche, G., and Castiaux, P. (1958) Nature 182, 328. Rebhun, L. I. (1956) J . Biophys. Biochenz. Cytol. 2, 159. Rhodin, J. (1954) “Correlation of Ultrastructural Organization and Function in Normal and Experimentally Changed Proximal Convoluted Tubule Cells of the Mouse Kidney.” Karolinska Inst., Stockholm. Rhodin, J. (1958) Intern. Rev. Cytol. 7, 485. Rhodin, J., and Dalhamn, T. (1956) Z. Zellforsch. u. mikroskop. Anat. 44, 345. Ries, E. (1935) Z. Zellforsch. u. mikroskop. Anat. 22, 523. Rinehart, J. F., and Farquhar, M. G. (1953) J . Histochem. Cytocheni. 1, 93. Robertson, D. (1957a) J. Biophys. Biochem. Cytol. 3, 1043. Robertson, D. (1957b) I . Physiol. (London) 140, 58. Robertson, D. (1958) J . Biophys. Biochem. Cytol. 4, 39. Rogers, G. E. (1957) Exptl. Cell Research 13, 517. Rouiller, C., and Bernhard, W. (1956) J . Biophys. Biochem. Cytol. 2, Suppl., 355. Ruska, H., Moore, D. H., and Weinstock, J. (1957) J . Biophys. Biochem. Cytol. 3, 249. Saguchi, S. (1920) Am. J , Anat. 36, 347. Sano, Y.,and Knoop, A. (1959) Z. Zellforsch. u. mikroskop. Anat. 49, 464. Sawano, J., and Kanaya, T. (1959) dcta Anat. Nippon. ( T o k y o ) 34,45. Schiefferdecker, P. (1917) Biol. Zentr. 37, 534. Schiefferdecker, P. (1922) Zoologica, Stuttgart 72, 1. Schulz, H., and de Paola, D. D. (1958) 2. Zellforsch. u. mikroskop. Anat. 49, 125. Scott, B. L., and Pease, D. C. (1959) A m . J . Anat. 104, 115. Sedar, A. W. (1955) Anat. Record 121, 365. Sedar, A. W., and Rudzinska, M. A. (1956) 1. Biophys. Biochem. Cytol. 2,Suppl., 331. Seki, M. (1959) Proc. 48th Congr. Japan. Pathol. Assoc. (Byori-gakkai Kaishi), p. 75. Selby, C. C. (1955) J . Biophys. Biochem. Cytol. 1, 429. Shibasaki, S. (1959) Acta Anat. Nippon. ( T o k y o ) S4, 19 (Abstr. Associated Anatomical Meeting of Chubu and Kanto District). Siekevitz, P., and Palade, G. E. (1958a) J . Biophys. Biochem. Cytol. 4, 309. Siekevitz, P., and Palade, G. E. (1958b) I . Biophys. Biochcm. Cylol. 4,557. Sjostrand, F. S. (1953) Nature 171, 30. Sjostrand, F. S. (1956) Intern. Rev. Cytol. 6, 456. Sjostrand, F. S., and Hanzon, V. (1954a) Exptl. Cell Research 7, 393. Sjostrand, F. S., and Hanzon, V. (1954b) Exptl. Cell Research 7, 415. Sjostrand, F. S., and Rhodin, J. (1953) Exptl. Cell Research 4, 426. Sjostrand, F. S., Anderson-Cedergren, E., and Dewey, M. M. (1958) J . Ultrastruct. Research 1, 271. Smith, C. A. (1956) Ann. Otol. Rhinol. G Laryngol. 65,450. Smith, C. A. (1957) Ann. Otol. Rhirtol. G Laryngol. 68, 521. Smith, C. A., and Dempsey, E. W. (1957) Am. J . Anat. 100, 337. Solger, B. (1894) Anat. Anz. 9, 415. Solomon, A. K. (1952) Federation PYOC.11, 722. Sorokin, H. (1938) Am. J. Botany 25, 28. Stoeckenius, W. (1956) Exptl. Ccll Rcscarch 11, 656. Stoeckenius, W., and Kracht, C. (1958) Endokrinologie 36, 135. I

124

K. KUROSUMI

Suzuki, I. (1958) Clin. Gastro-Enterology ( T o k y o ) 6, 27. Suzuki, I. (1959) Japan. J.-Gastrocntcrol. 66, 155. Suzuki, Y. (1958) J. Electroninicroscopy (Chiba) 6, 52. Takagi, F. (1959) Proc. 15th Gen. Assembly Japan. Med. Congr. ( T o k y o ) 1, 70. Takagi, F., Sekiguchi, H., Onodera, Y., and Nagao, T. (1959) Proc. 15th Meeting Electron Microscopy, Japan p. 84. Tanaka, H., Uchino, F., Dohi, S., and Amano, S. (1956) Acta Haematol. Jupon. 19, 571. Tanaka, H., Hanaoka, M., and Amano, S. (1957) Acta Haematol. Japon. 20,85. Taylor, J. J., and Hayes, E. R. (1959) J. Appl. Phys. SO, 2033. Tehver, J. (1930) 2. mikroskop.-anat. Forsch. 27, 65. Uchida, G. (1958) Arch. Histol. Japan. 16, 435. Umetani, K. (1957) Sapporo Med. J . 12, 270. Van Breemen, V. L., and Clemente, C. D. (1955) J . Biophys. Biochem. Cytol. 1, 161. Van Weel, P. B. (1937) Z.Zellforsch. 14. mikroskop. Anat. 27, 65. Wang, L. (1958) Denshi Kembikyo ( T o k y o ) 6, 56. Wasserman, F. (1958) 2. Zellforsch. u. mikroskop. Anut. 49, 13. Watanabe, A. (1960) Arch. Histol. Japan. 19, 279. Watanabe, Y. (1955) J. Ekctrmmicroscopy (Chiba) 3, 43. Watanabe, Y. (1957a) Symp. SOC.Cell. Chem. (Tokyo) 6, 35. Watanabe, Y. (1957b) SBgb RinshB (Tokjlo) 14, 649. Watanabe, Y., Takamatsu, M., and Osako, R. (1956) Denshi Kembikyo ( T o k y o ) 4, 146. Watanabe, Y., Arakawa, K., and Yamamoto, J. (1959) Proc. 15th Congr. Electron Microscopy Japan p. 79. Watson, M. L. (1955) J . Biophys. Biochem. Cytol. 1, 257. Weiss, J. M. (1953) J. Exptl. Med. W, 607. Weiss, J. M. (1955) J . Exptl. Med. 102, 775. Welcker, H. (1857) 2. rationelle Med. fN.F.1 8, 239. Wersall, J. (1954) Acta Oto-Laryngol. 44, 359. Wischnitzer, A. (1958) J. Ultrastruct. Research 1, 201. Wissig, S. L. (1956) “The Anatomy of Secretion in the Follicular Cell of the Thyroid Gland.” Yale Univ. Press, New Haven, Connecticut. Cited from S. L. Palay, 1958. Yamada, E. (1955) J. Biophys. Biochem. Cytol. 1, 445. Yamada, E. (1956) Proc. 1st Conf. Electron Microscopy in Asia and Oceania p. 247. Yamada, E. (1958) J. Biophys. Biochem. Cytol. 4, 329. Yamagishi, M. (1959) Arch. Histol. Japan. 18, 223. Yasuzumi, G. (1959) Proc. 15th Congr. Electron Microscopy Japan p. 70. Yasuzumi, G., and Tanaka, H. (1958) J . Biophys. Biochem. Cytol. 4, 621. Yasuzumi, G., Sawada, T., Sugihara, R., Kiriyama, M., and Sugioka, M. (1958) 2. Zellforsch. u. mikroskop. Amt. 48, 10. Yokoh, S., Iizuka, M., and Kano, M. (1959) Arch. Histol. Japan. 17, 429. Yoshimura, F., and Irie, M. (1959a) Hormone and Clinic ( T o k y o ) 7, 367. Yoshimura, F., and hie, M. (1959b) Trans. 1st Asia and Oceania Regional Congr. Endocrinol., Kyoto, Japan, p. 3. Zetterqvist, H. (1956) “The Ultrastructural Organization of the Columnar Absorbing Cells of the Mouse Jejunum.” Karolinska Inst., Stockholm. Zimmermann, K. W. (1898). Arch. mikroskop. Anut. u. Entwicklwngsmech. 62, 552.

The Fine Structure of Insect Sense Organs' ELEANOR H. SLIFER Department of Zoology, State University of Iowa, Iowa City, Iowa Page

I. Introduction

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

IV. Plate Organs

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

A. Thick-Walled Pegs

C. Coeloconic Pegs

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

125

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

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

VIII. Compound Eyes .................................... IX. Summary ...... References ..........................................

I. Introduction During the past one hundred and fifty years a very large number of papers which deal with the sense organs of insects has been published. Often the results obtained by one worker have been contradicted by those reported by others. This is scarcely surprising since the study of insect sense organs presents special difficulties over and beyond those commonly encountered by microscopists. The body wall of the insect serves as its skeleton and is composed in large part of hard and impermeable cuticle. Below the cuticle lies the layer of epidermal cells which produced the cuticle, and beneath this, in turn, lies a basement membrane, the origin of which is still in dispute. Those sense organs which form a part of the body wall consist, basically, of the same elements: (1) specialized cuticle -which may take the form of a hair, peg, plate, tympanum, cornea, etc.produced by ( 2 ) specialized epidermal cells; ( 3 ) from one to many bipolar sensory neurons which arose froin other epidermal cells; and (4) fibers of uncertain origin. Because the cellular parts of many sense organs lie, either entirely or in part, within cavities in the cuticle, penetration of the fixative to them is hindered. The cuticle is extremely difficult to section in paraffin and during fixation, dehydration, and embedding procedures shrinks little, if at all, while the delicate cellular elements which are attached to it tend to 1 Supported in part by a grant (RG-5479) from the National Institutes of Health, United States Public Health Service.

125

126

ELEANOR 11. SLIFER

shrink and to pull away. Even when good sections are obtained for use with the light microscope: details of great interest frequently lie beyond the resolving power of the best oil immersion lens, and those which can be seen often can not be interpreted. The introduction of the electron microscope and the development during the early 1950’s of methods of fixing and sectioning material for examination with it opened the way for a fresh attack on the problem. With these new techniques fixation artifacts have been decreased, but the difficulty of sectioning cuticle still remains as a major obstacle, and only a few papers have been published in which cuticular parts have been included in the study. Despite this, our knowledge of the fine structure of insect sense organs has increased rapidly during the last few years, and the results of one or more investigations of each of the principal types of insect sense organs-mechanoreceptors, chemoreceptors, and photoreceptors-are now available for comparison. The choice of terms to be used in discussing insect sense organs is one not easily made. Many workers, using several languages and studying a wide variety of sense organs in a large number of different species, have given different names to the same structure or applied the same name to different structures. In some cases the name selected had functional connotations which later work showed to be unsuitable, and, recently, to confuse matters further, there has been a tendency to introduce terms used in mammalian histology. So far as possible the names employed here will be those used by Snodgrass in his classical paper on insect sense organs (Snodgrass, 1926) or in his textbook on insect morphology (Snodgrass, 1935). Other terms will be applied, however, when it seems advantageous to do so. During the past ten years reviews of the literature on insect sense organs have been published by Dethier (1953), Hodgson (1955, 195S), Imms, Richards, and Davies ( 1957), and Wigglesworth ( 1953). These may be consulted for additional information on physiology and structure.

11. Tactile Organs Many of the hairs present on the body surface of insects represent the cuticular component of tactile sense organs. Their function has been demonstrated by physiological experiments for a number of species. The structure of these hairs is relatively simple. The hair, which may be long or short, is set in a circle of soft, membranous cuticle so that the hair moves readily-when it is touched. The slender tip of the dendrite of a single sensory neuron is attached just inside the base of the hair, and with the light microscope a delicate cuticular sheath may be seen to enclose

FINE STRUCTURE OF INSECT SENSE ORGANS

127

the dendrite and to extend inwards almost to the cell body of the neuron. This. sheath is shed with the exuviae when the insect mol;:; (Richard, 1952). Not far below2 the point where the sheath is attached to the hair, it is enlarged to form a structure which has been described by earlier workers as a scolopale or rod, The relation between the enlargement and the dendrite is not clear in preparations examined with the light microscope. A t least two modified epidermal cells form part of the sense organ : ( 1 ) the trichogen cell which secreted both the hair and the cuticular sheath, and (2) the tormogen cell which produced the soft membrane surrounding the base of the hair. The dendrite, then, lies within the cuticular sheath, and this is enclosed by the trichogen cell. The tormogen cell, in turn, surrounds the trichogen cell, and on its outer surface a loose layer of fibers forms a boundary between the sense organ and neighboring epidermal cells. No papers have yet been published in which studies of tactile sense organs made with the electron microscope are described. The writer has partially completed an investigation of the tactile hairs of the grasshopper, Melanoplus diferentiulis, and a few of the more interesting observations made so far will be included here. The tactile hairs selected for examination were those on the distal end of the subgenital plate of the male. The subgenital plate in this region is closely set with stout, curved hairs which are a little less than a millimeter long. The dense wall of the hair, as seen with the electron microscope, is bounded externally by a thin edge which is composed of an even denser material. The lumen of the hair is partially filled with a loose network of irregular strands. Granules in the interstices of the net are probably the precipitate from a fluid which was present in life. The membrane by which the hair is attached to the body wall is strengthened by thickenings which radiate as branching and interlacing bars about the hair base. The base of the hair extends downward for a short distance below the body surface, and its inner end is slightly expanded and flares outwards. The cuticular sheath, which contains the dendrite of the sensory neuron, is attached to the inner surface of the hair a short distance above its base. Distally it is less than a micron in diameter. This is the region which contains the slender tip of the dendrite. Below this the sheath widens suddenly to 3 or 4 p, its wall thickens, and ridges appear on its inner surface forming irregular cavities and pockets. This thickened portion is the scolopale or rod of earlier workers. Below this the walls of the sheath become thinner and more uniform in thickness. The open end of the sheath lies a short distance above the cell body 2 For convenience in description each sense organ will be described as if it were oriented with its external surface upward.

128

ELEANOR H. SLIFER

of the single neuron of this sense organ. The cell body is often larger than are the surrounding epidermal cells and may lie either directly below the opening in the cuticle upon which the hair is set or well to one side of it. That portion of the dendrite which lies inside the cuticular sheath contains neurofilaments but no mitochondria. In the region where the sheath expands below the base of the hair the dendrite, likewise, expands and fills

PLATE I FIG.1. A. Tip of dendrite of neuron of tactile hair from subgenital plate of male grasshopper, Melanoplus diflercntialis, reconstructed from electron micrographs of sections. Knoblike structures fit into irregular compartments in wall of cuticular sheath in which dendrite is enclosed. Slender extension a t top lies in terminal portion of sheath which is attached inside base of hair. B. Section of dendrite in knobbed region. Compare with Figs. 2 and 3.

the grooves and cavities in the thickened sheath wall (Figs. l A , lB, 2, 3 ) . The dendrite is thus anchored firmly in place when the hair moves. After secreting the substances which compose the hair and the cuticular sheath, the irichogen cell retracts from the base of the hair leaving a vacuole which, in fixed material, contains a granular residue. In life, presumably, the vacuole is filled with fluid which permits the sheath with

FINE STRUCTURE OF INSECT SENSE ORGANS

129

its enclosed dendrite to move freely when the hair is touched. The tormogen cell which produced the membrane at the base of the hair remains adherent to the wall of the cavity in the cuticle of the body wall (Fig. 2 ) . Its cytoplasm, like that of the trichogen cell, contains numerous mitochondria, and the surface which is in contact with the vacuole is covered thickly with microvilli. Just above the point where the cuticle of the body wall meets the epidermis, the outer surface of the tormogen cell is enclosed by bands of fibers of different widths. These extend downward and separate into smaller fibers as they approach the basement membrane and merge with it. The branching fibers are especially conspicuous in silver preparations of whole mounts of the body wall, and can be identified in electron micrographs of cross sections of the sense organ. Similar fibers were described in the chemoreceptors of the grasshopper antenna (Slifer et al., 1959). It should, perhaps, be noted that delicate fibers, which otherwise resemble these, also pass from the basement membrane up between the unspecialized epidermal cells and end not far from the cuticle. They may be seen both in preparations made for the light microscope and in electron micrographs. 111. Auditory Organs There is general agreement among those interested in insect sense organs, well supported by experimental evidence, that tympanal membranes to which sensory neurons are attached function as receptors for sound waves. Such structures are found in Orthoptera, Hemiptera, and Lepidoptera. The grasshopper's auditory organs are located on the sides of the first abdominal segment. A thin plate of cuticle forms the tympanum, and this is set in a frame or rim of thicker cuticle. Below this lies the delicate layer of epidermal cells which secreted the cuticle. This is bounded on its inner surface by the basement membrane. At several places on the tympanum the cuticle is thickened. Here the groups of sensory neurons are attached by means of modified epidermal cells. The distal tips of the dendrites of the sensory neurons end within small but complex tubular scolopales. With the light microscope a fiber can be traced to the outer end of the scolopale. Here it disappears. As many as ninety sensory neurons may occur in the auditory organ of the grasshopper (Slifer, 1936). Proximally the axons of the neurons join to form the auditory nerve which eventually enters the metathoracic ganglion. 'A note by Gray and Pumphrey on the fine structure of the tympanal organ of the locust,3 Locusta migratoria migratorioides, appeared 3 A distinction is made by some entomologists between grasshoppers and locusts. Both are members of the family Acrididae.

130

ELEANOR H. S L I F E R

in 1958. In it they described the occurrence within the distal end of the dendrite of nine peripheral and, possibly two central fibrils which run longitudinally. This pattern closely resembles that now familiar in electron micrographs of cilia and flagella from many species of plants and animals. A detailed paper on the locust auditory organ has now been published by Gray (1960). The description which follows has been taken from it. The cuticle of the tympanum of Locwtu is, for the most part, from 3 to 4 p thick. The sixty to ninety sensory neurons which are associated with the tympanum are arranged in four groups and their long axes lie in three different planes which are mutually perpendicular. The dendrites of these neurons may be as much as 100 p long. The distal end of each dendrite lies inside a scolopale and is attached to its upper end which is known as a cap or end-knob. Modified epidermal cells (cap cells, attachment cells), containing fine fibrils in their cytoplasm, serve to fasten the scolopales, together with the cells enclosing them, to the inner surface of the tympanum. The cuticle at these several points of attachment is thickened, dark in color, and sclerotized. The most extraordinary of the findings made by Gray with the electron microscope concern the fine structure of the dendrite and that of the scolopale into which it is inserted. The scolopale-except for its cap or end-knob-lies within a scolopale cell of curious shape. This elongate cell is tubular and surrounds a cavity which is thought to be filled with fluid in the living locust. Four to six, or more, fingerlike extensions of its cytoplasm hang down into the cavity from its outer end. These extensions each contain a part of the scolopale. Proximally, they join to form a continuous ring. The material of which the several parts of the scolopale is composed is dense, vesiculate, and, superficially at least, is suggestive of cuticle ; but its composition is unknown. Neighboring rods of the scolopale, together with the cytoplasm containing them, may coalesce, or one rod may branch to form two, so the number present at different levels is not constant. The end-knob or cap of the scolopale, an extracellular body, lies just above the scolopale cell and is embedded in a depression in the attachment cell distal to it. The PLATE11. Electron micrographs of sections through tactile sense organ from subgenital plate of male grasshopper, hrelanoplus differentialis. FIG.2. Dendrite, enclosed in dense-walled cuticular sheath with internal compartments, lies to right of center; cuticular sheath surrounded by vacuole which is enclosed by tormogen cell. Note microvilli on. surface of cell. Cuticle of body wall at periphery. ( x 4800.) FIG.3. Section through cutidular sheath and enclosed dendrite showing irregular cavities in sheath wall with knobs of dendrite fitting into them. Cell membrane and neurofilaments of dendrite show distinctly. ( x 26,000.)

F I N E STRUCTURE OF INSECT SENSE ORGANS

131

132

ELEANOR H. SLIFER

dendrite of the neuron has a structure which is even more remarkable. That portion of it which-lies in the distal half of the space within the scolopale has a fine structure closely resembling that of a typical cilium. Nine pairs of longitudinal fibrils lie in a circle near the periphery (Figs. 4C, 7). The center of one component of each pair is less dense than is its periphery and has the appearance of a tubule (Fig. 17). There is some evidence that a fibril, or pair of fibrils, occupies the center of the circle in the dilated region near the upper end of the dendrite. The peripheral fibrils join proximally to form a tubular ciliary base (Figs. 4D, 9), which rests on the nine arms of a complex structure embedded in the cytoplasm of the dendrite (Figs. 4 E and Figs. 11 and 12 of Plate V ) . These nine arms arise from another tubular structure, composed, apparently, of the same material. Farther down in the dendrite the second tube becomes a solid rod (Fig. 4 F ) , and then branches repeatedly to form an array of rootlets which pass deep into the cytoplasm of the cell body of the neuron. Although these rootlets are visible in good silver preparations and have been seen and figured by earlier workers, their significance has been obscure. The rootlets-f which there may be as many as thirty or forty-show a periodic structure in electron micrographs (Fig. l o ) , and at very high magnifications an asymmetric repeat pattern of at least six bands can be distinguished. The tubular ciliary base, in which the longitudinal fibrils of the cilium terminate, bears a close resemblance to the basal body which is commonly found below the motile cilium of other animals. However, it lies above the general cell surface and not below it as does the basal body described for other cilia. The occurrence of cilia within a class of animals where they have long been thought to be absent is indeed surprising. The surprise, however, is somewhat lessened when it is recalled that insect spermatozoa are provided with flagella and that cilia and flagella, as recent work with the electron microscope has demonstrated, both possess a basically similar pattern of longitudinal fibrils (Gray and Pumphrey, 1958). For purposes of comparison, sections of grasshopper flagella are shown in Figs. 18 and 19 of Plate VIII. The association of cilia with the receptor cells of an auditory organ is also known to occur in the mammalian ear. Engstrom and Wersall (1958) described a single kinocilium which is present, together with some sixty to eighty stereocilia, on each vestibular cell of the guinea pig ear. The cytoplasm of the sensory neuron-of the tympana1 organ of the locust in the region. of the nudeuq contains mitochondria, Golgi bodies, endoplasmic reticulum, and granules of the kinds now familiar in many types of cells. The cell body of the neuron is wrapped in the folds of a

F I N E STRUCTURE O F I N S E C T SENSE ORGANS

133

PLATE 111

FIG.4. A. Tip of dendrite of neuron from auditory organ of locust, Locusta migratoria migratorioidcs, reconstructed from data and figures of Gray (1960) ; portion above enlargement at (Z) lies within scolopale; slender part above ( Y ) modified as cilium; dilated region of cilium at (X). B to H. Diagrams of cross sections through dendrite at different levels. B. Extreme tip of cilium which lies within cap or end-knob of scolopale. C. Dilated region of cilium, central fibrils ( ?) present in some preparations. D. Ciliary base. E. Root apparatus. F. Solid portion of root. G. Root starting to separate into rootlets. H. Rootlets in lower portion of dendrite.

134

ELEANOR H. SLIFER

neurilemma or Schwann cell, while the base of the dendrite is surrounded by a fibrous sheath cell which contains innumerable fine filaments in its cytoplasm. Collagen-like fibrils are also present on the external surface of this cell and, perhaps, are comparable to the fibers described in the preceding section for the tactile sense organ. The cytoplasm of the scolopale cell is finely vesicular, while the attachment cell which lies distal to the cap of the scolopale contains many delicate tonofibrils. The axons of the neurons described above, together with the axons of a few other sense organs which are located on the tympanum, join to form the auditory nerve. IV. Plate Organs The thin, oval plates which cover a large part of the surface of the eight distal subsegments of the antenna1 flagellum of the honey bee, APis mellifera, have attracted the attention of many investigators. Their structure has been studied with great care, but much of the important detail lies beyond the resolving power of the light microscope. Experiments carried out to determine their function have led different workers to various conclusions. Some believe that they serve as chemoreceptors, others as thermoreceptors, pressure receptors, auditory organs, etc. Reviews of the earlier literature are given by von Frisch (1921) and Wacker (1925). Essentially, the plate organ consists of a group of sensory neurons, the dendrites of which converge below a thin, flattened area or plate, set i n the cuticle of the body wall. From the point where the dendrites converge a slender strand composed of a number of filaments extends upwards and seems to end on or close to the edge of the plate which is nearest to the proximal end of the antenna. The plate is attached to the surrounding cuticle of the body wall by a narrow and extremely delicate membrane. Experiments carried out and reported by von Frisch (1921, 1923) led him to the conclusion that the plate organs are olfactory structures. Vogel (1921, 1923) agreed with von Frisch’s interpretation but pointed PLATEIV. Electron micrographs of sections of auditory organ of locust, Locusta migrutoria. migrutm’oides, from Gray (1960). FIG. 5. Cross section of cap of scolopale with cilium lying in cleft in center. FIG.6. Longitudinal section through cap of scolopale with cilium lying in cleft and dilated region of cilium visible below cap. FIG.7. Cross section through dilated region of cilium showing nine peripheral and two or three smaller, central fibrils. Cilium. lies in space surrounded by dense wall of scolopale. FIG.8. Cross section of cilium surrounded by six dense rods of scolopale near base of cilium. Small portion of upper edge of one side of expanded part of dendrite at left. Scolopale lies within scolopale cell.

FINE STRUCTURE OF INSECT SENSE ORGANS

135

136

ELEANOR H . SLIFER

out that certain of the thin-walled hairs which are present in the same regions where the plate organs occur might also be sensitive to odors. If this were so, doubt would be cast on the conclusions drawn from von Frisch’s experiments. Snodgrass (1935, p. 525) commented that the plate organs seemed to be poorly constructed to serve as olfactory receptors. According to him: “-the relatively thick sclerotic plates cannot be supposed to be pervious to odor substance, those of the honey bee being about 1.5 microns in thickness, and the narrowed distal stalk of the receptive cells attached in the groove of the plate presents a vkry restricted area at which stimuli could be effective. If the organs are, nevertheless, olfactory, the plate would appear at least to be an entirely superfluous adjunct. ’’ In 1952 Richards published the results of a series of studies on the plate organs of the honey bee. Some of his work was done with the electron microscope, but modern techniques for obtaining thin sections had not been developed at that time and only whole mounts of hand-cut sections were examined. The most interesting finding described by Richards was the presence of fine, radiating bands of dense material, some of them branched, which cross the thin membrane and pass into the plate. These, although they are much smaller, resemble the branched, thickened bars in the membrane in which the tactile hair is set. Richards also found the region between the radiating bars in the thin membrane to be penetrated by fine holes a few hundredths of a micron in diameter but believed them to be artifacts caused by electron bombardment. After a study of the olfactory sense organs of the grasshopper had been completed (Slifer et al., 1957, 1959), the structure of the plate organs of the bee assumed special interest, since descriptions in the literature showed them to differ so greatly in their basic pattern from those of the grasshopper. Sections of the antenna of the honey bee worker were prepared for study with the electron microscope and a note on the first results

PLATEV. Electron micrographs of sections of auditory organ of locust, Loczlsta migraforia migratorioidcs, from Gray (1960). FIG.9. Cross section through ciliary base; parts of three scolopale rods included. FIG. 10. Longitudinal section through portion of root of cilium showing periodic structure. FIG.11. Cross section of dendrite showing, nine arms of root apparatus; dendrite enclosed by six r o d s of scolopale; scolopale lies in space surrounded by scolopale cell. FIG.12. Cross section of scolopale with enclosed dendrite and root apparatus. FIG.13. Cross section of scolopale with enclosed dendrite and hollow portion of root apparatus.

FINE STRUCTURE OF INSECT SENSE ORGANS

137

135

ELEANOR H . SLIFER

has been published (Slifer and Sekhon, 1960). The investigation is not yet complete but some of the observations made will be included here. According to Kuwabara and Takeda (1956) and Dostal (1955), about 3000 plate organs are present on each antenna of the worker honey bee. In whole mounts of the unstained antenna1 wall the plate organs stand out as oval, transparent areas which contrast sharply with the black or dark brown body wall in which they are set. Vogel (1923) gives the dimensions of a typical plate as 10 x 13 p. There is considerable variation in size, however, and some are twice as long as others. Each is oriented in such a way that its long axis lies parallel, or nearly parallel, to the long axis of the antenna itself. In electron micrographs the rim of the plate is seen to be slightly thicker than is its center (Fig. 16). The membrane by which the plate is fastened to the surrounding body wall is about 1 p in width and less than 0.2 p thick. This is the membrane in which Richards (1952) described radiating bands. In sections these appear as riblike thickenings which extend below the membrane and leave its outer surface smooth. About 1 p beyond the outer edge of the membrane the cuticle is cut by a furrow or cleft which is, itself, about a micron in depth (Fig. 16). According to Richards (1952), the cuticle which lies between the cleft and the thin membrane, like the plate, is incompletely sclerotized. Richards thought that the epicuticle extends over the furrow in a continuous layer, but it is clear in electron micrographs of sectioned material that this is not the case. The furrow is open at the top (Fig. 16), as McIndoo (1922) described it, and it is interesting to note that the width of the furrow varies in different preparations. I t is possible that this variation is not an artifact and that the width of the groove changes in the living insect. The cavity in the cuticle of the body wall below the plate has an unusual shape and might be compared to that of a shoe with the sole of the shoe forming the plate and the toe turned towards the distal end of the antenna. The cavity is almost completely filled by the large cap cell. The nucleus of this cell may lie in the toe of the shoe-shaped cavity or deeper in the narrow portion of the cavity near the epidermis. It is often enveloped by many concentric layers of fine membranes. A few of these membranes-but not the nucleus-may be seen in Fig. 16. According to Vogel (1921), who examined several species of bees and wasps, the number of sensory neurons associated with each plate varies from twelve to eighteen. The cell bodies of these neurons lie in the deeper portions of the epidermis and their dendrites converge as they approach the cuticle. In-this region the dendrites are closely wrapped by the folds of one or several neurilenima 6r Schwann cells so that they form a compact mass. Both mitochondria and neurofilaments are present in the cytoplasm

FINE STRUCTURE O F INSECT SENSE ORGANS

139

of the dendrites. At a point a little closer to the cuticle each dendrite emerges from the mass as a single structure and suddenly narrows to a third, or less, of its former width (Fig. 14). Beyond this point the dendrite assumes the structure of a cilium, and nine pairs of peripheral fibrils, all of which run longitudinally, may be identified in it (Slifer and Sekhon, 1960). Rootlets and a root apparatus such as Gray

PLATE VI

FIG.14. A. Tip of dendrite of plate organ of honey bee, Apis mcllifera, reconstructed from electron micrographs of sections and showing cilium at tip. B. Diagrammatic cross section through cilium.

(1960) describes for Locusta appear to be absent; or, at least, no evidence for their presence has been obtained as yet. The cilia (Fig. 15), now well separated from one another, lie in a space enclosed by the envelope cell. Numerous microvilli are present on that surface of the envelope cell which faces the cavity, and it is probable that secretion droplets produced in the cell are passed into the space. A fine granular material present in this space may represent coagulated substance from a fluid in the living bee. Close to the point where the dendrites enter the space below the plate,

140

ELEANOR H. SLIFER

a mass of tiny, refringent bodies may be seen. These are visible in light niicroscope preparations, and VogeI deseribes each dendrite as having two ; one of which lies a little farther out on the dendrite than the other. They can be seen with relative ease with an oil immersion lens in silver preparations, just as Vogel described them in 1921. From each cilium a fibrous strand extends toward the plate. AS they near it the distance between them decreases and, since sections close to the plate show fewer of them, it is possible that they unite. This strand, or group of strands, lies in a vacuole which is a continuation of that which enclosed the dendrites and their cilia, but the envelope cell does not extend up this far and the cytoplasm of the cap cell now surrounds the vacuole. As yet, no section showing both the terminal portion of the strand and the plate have been found in the same electron micrograph. In good silver preparations, however, it seems very nearly certain that the strand (or strands) ends on the extreme edge of the plate nearest the proximal end of the antenna, and not on the thin membrane which surrounds the plate as von Frisch (1921) believed. The cell bodies of the neurons of the thousands of sense organs which cover the antennal wall lie in a closely-packed layer in the inner part of the epidermis. They are small, similar in appearance, and form an almost continuous mass of cells with little to indicate the boundaries of the group which belongs to a particular sense organ. Often they do not lie beneath the hair, peg, or plate to which they are attached but at some distance from it. Because of these difficulties the cell bodies of the neurons of the plate organ have not been identified with certainty and no description of them can be included here. A comparison of the structure of the plate organ of the honey bee with that of the tympana1 organ of the locust indicates that the two share a number of characteristics. Dendrites which terminate in cilia, or cilia-like PLATE VII. Electron micrographs through plate organs from antennal flagellum of honey bee, Apis mellifera. FIG.15. Section through tips of dendrites in region where they are modified as cilia. Nine double peripheral fibrils can be counted in most of these, and one or two central fibrils appear to be present in several. One of cilia at left has a group of central bodies. Cilia lie in vacuole containing a granular residue; vacuole enclosed by envelope cell with microvilli on surface. ( x 26,000.) FIG. 16. Section through plate of plate. organ showing attachment to body wall by thin membranes at either side. Groove or furrow which encircles plate shown a t left. Space below plate filled with cytoplasm of cap cell; some of membranes (poorly preserved) which are associated with nucleus appear at lower right as a dense oval structure. ( x 11,200.)

FINE STRUCTURE OF INSECT SENSE ORGANS

141

142

ELEANOR H. SLIFER

structures, are present in both. The plate of the bee to which the strand arising from the dendrites is seemingIy attached resembles the several thickened points on the tympanum of the locust. The thin membrane encircling the plate might be compared to the tympanum itself; for, although it is narrow, it is so delicate that it must permit some slight movement of the plate when it is touched. The groove or furrow lying outside the rim to which the membrane is fastened may well increase the response to slight pressure applied externally, Richards’ (1952) finding that the material between the thin membrane and the furrow is pdorly sclerotized suggests that this region may be somewhat flexible. Each plate organ is small, but when it is recalled that several thousands of these structures are present on each antenna, it is apparent that their collective response might be considerable. As yet we possess no study of the fine structure and physical properties of the cuticle of the locust tympana1 organ, and so no comparisons can be made of the plate cuticle with that of the tympanum. However, Richards’ (1952) description of the minute, thickened rods, from 120 to 150 in number, which arise in the region between the furrow and the membrane, traverse the membrane and converge in the center of the plate, assumes new interest when these are compared with the fibers in the human ear drum. In the human tympanum radially-arranged fibers lying in the middle layer of the drum converge at the umbo (Stuhlman, 1950). Moreover, Richards-from evidence obtained with the polarizing microscopedescribed two bands of circumferentially-arranged micelles (presumably chitin micelles), one of which lies in the thin membrane and the other in the ridge of flexible cuticle just outside it. The human tympanum, likewise, possesses a band of fibrils which lies close to its periphery. Another instance of similarity between the two lies in the eccentric point of attachment through which stimuli are transmitted to the sensory elements of the organ. The structure of the plate organ, then, suggests that it may respond to slight pressures applied to it from the outside and so serve to detect sound waves or other vibrations. I t must be emphasized that the information provided by the electron microscope does not disprove von Frisch’s conclusions that the plate organs are olfactory structures, but it does indicate the need for new experiments to discover whether they are not, instead, receptors for vibrations either in the air or from some other source. One of the criticisms made in the past o f attempts to demonstrate the use of sounds for communication in bees has been the fact that no sense organs suitable for the reception of such stimuli had been identified. It would be most interesting, now, to study the reactions of bees, with and without

F I N E STRUCTURE O F INSECT SENSE ORGANS

143

antennae, to recorded sounds made by other bees. Wenner (1959) has beghn an analysis of the sounds produced by bees during their dances. Work of this type should be encouraged.

V. Gustatory Organs Pioneer work in the study of the gustatory organs of the blow fly, Phorinia regina, with the electron microscope was carried out by Dethier and Wolbarsht (1956). The long hairs of these organs are innervated by three sensory neurons; two of which send their dendrites to the distal tip of the hair, while the third seems to end near the base of the hair and may have a tactile function (Dethier, 1955). Hairs of this type are present on the tarsi as well as the labella. The four electron micrographs published by Dethier and Wolbarsht are of the tip of a hair but, since the material was not fixed or sectioned, show little more than the outline of the structure. A large amount of experimental work has been done with these sense organs, and studies in which modern sectioning techniques are used would be welcome.

VI. Olfactory Organs Several years ago the writer found that the distal tips of the long, thickwalled pegs of the grasshopper antenna could be stained when the intact, living insect was immersed for a short time in a solution of acid fuchsin (Slifer, 1954a). This discovery initiated a series of studies which involved the application of a variety of light microscope techniques, the use of the electron microscope, and experimental work on the behavior of the insect. Most of the work was carried out with Romulea microptern and Melanoplus differentialis. At least three different types of sense organs have an olfactory function in the grasshopper. These are the thickwalled pegs, the thin-walled pegs, and the coeloconic pegs. A. THICK-WALLED PEGS Thick-walled pegs are found not only on the antennae of the grasshopper but on almost every other part of the body surface as well (Slifer, 1955a). They serve, apparently, as the receptors for the common chemical sense and are stimulated by strong repellent odors (Slifer, 1954b, 1955b, 1956). Pegs of this type range in length from 20 to 50 p (Slifer et d.,1957). As in the tactile hair the wall is composed of an electron-dense material with a thin outer edge which is even denser (Fig. 20). The tip is rounded and has an opening in it about 2 p in diameter. From this opening a tubular sheath of cuticle is invaginated. This sheath passes down the

144

ELEANOR H. SLIFER

lumen of the peg, narrows sharply before reaching the base, and widens again below the peg. It-ends just above the cell bodies of the neurons which are associated with the peg. The neurons, usually-although not invariably-five in number, send their dendrites into the inner, open end of the sheath. They continue upward to the tip of the peg and are there exposed to the air. Five dendrites cut in cross section may be seen inside the narrow portion of the sheath in Fig. 20. Mitochondria and neurofilaments are present in the cell bodies of the neurons, but only the latter may be seen in the dendrites after they enter the cuticular sheath. The cuticular sheath is enclosed within the trichogen cell which secreted both the peg and the sheath and later withdrew from the peg lumen. The tormogen cell, which produced the membrane at the base of the peg, surrounds the trichogen cell, and a fluid-filled vacuole lies between the two cells. This vacuole extends upward into the peg lumen. Both trichogen and tormogen cell are provided with a thick fringe of microvilli on the surface which is in contact with the vacuole. Large mitochondria and many Secretion droplets are present in the cytoplasm of these cells. The secretion droplets are added to the fluids in the vacuole and, since the cuticular sheath is permeable, the liquid evidently serves to keep the exposed dendrite tips moist. When the insect molts the old sheath is pulled out through the open tip of the new peg and is shed with the exuviae.

B. THIN-WALLED PEGS Thin-walled pegs have been found, so far, only on the antennal flagellum and are structurally the most complex of the olfactory organs. When the antennae are amputated the grasshopper loses its ability to find water and certain foods (Slifer, 195513). Since the thin-walled pegs and the PLATE VIII FIG.17. Electron micrograph of cross section of cilium from dendrite of auditory organ of locust, Locusta migratol-ia migratorioides, showing nine peripheral double fibrils; one member of each double fibril appears to be tubular and the other solid. No central fibrils visible. From Gray (1960). Compare with Figs. 18 and 19. FIGS.18 A N D 19. Electron micrographs of cross sections of flagella of spermatozoa (or spermatids I ) of grasshopper, Melanoplus differmtialis. Courtesy of Dr. T. N. Tahmisian. Compare with Fig. 17. ( X 50,500.) FIG.20. Electron micrograph of cross section of thick-walled peg from antennal flagellum of grasshopper, Romalea microptera ; five dendrites visible inside cuticular sheath in lumen of peg. ( x 14,800.) FIG.21. Electron micrograph of cross section of coeloconic peg from antennal flagellum of grasshopper, Romalch microptera. Longitudinal grooves or flutes in peg wall appear as scallops on periphery of peg. Three or four dendrites present inside cuticular sheath which lies in lumen of peg. ( X 9600.)

FINE STRUCTURE OF INSECT SENSE ORGANS

145

146

ELEANOR H . SLIFER

coeloconic pegs are the only types of chemoreceptors limited to the flagellum, either one or both of-these must be responsible for the sensitivity of the normal grasshopper to food odors and water vapor. The thin-walled peg, since its structure is more complex than is that of the coeloconic peg, seems more likely to be the principal olfactory organ. Perhaps the coeloconic peg serves as a hygroreceptor ; but experimental evidence to support this possibility is lacking. The two types of pegs are scattered together over the antenna1 surface in such a way that no experiment has yet been devised in which they could be tested separately. The thin-walled pegs vary greatly in size but a typical peg is about 16 p long and 3 p wide at its base. The top is smoothly rounded and its surface is pierced by about 150 openings each of which is close to 0.1 p in diameter (Fig. 23). At the base of the peg a single, rounded spot about 1 p across is present. This spot is covered with a material of unknown composition and origin which is readily permeable to dyes when these are applied to the outside surface of the antenna. A cuticular sheath is invaginated from this spot and, almost at once, turns downward and widens. Its inner end lies directly above the large group of neurons which belongs to this sense organ. The neuron cell bodies are closely packed and form a mass which is nearly spherical. Their number varies widely but as many as sixty have been estimated to be present in the larger groups. Numbers close to forty are more common, and fewer are found in many. The dendrites of each of these neurons enter the inner end of the cuticular sheath and, filling it completely, pass upward towards the peg (Fig. 24). Just before reaching the base of the peg (Fig. 23), they leave the sheath through numerous small openings in its wall and enter the fluid-filled lumen of the peg. Here each dendrite branches once or twice and each of its branches terminates in one of the many tiny openings in the peg wall. At high magnifications sections through the openings show that each dendrite tip is composed of about twenty-four fingerlike processes which lie parallel to one another (Figs. 22B,C, 25, 26). A single one of these processes has a diameter of about 0.02 p. The processes resemble microvilli but are smaller than those found elsewhere in the grasshopper (Beams et al., 1955; Beams and Anderson, 1957). The dendrite tips are exposed to the air and there is no cuticular covering over them. It is tempting to speculate, at this point, that the dendrite tips may be extended for a short distance from the opening when stimulated by an odorous material and withdrawn at other times; but evidence to support such a possibility would be difficult to obtain. The branches of the dendrites within the lumen of the peg are bathed in a fluid secreted by the trichogen and tormogen cells into the vacuole ,

F I N E STRUCTURE OF INSECT SENSE ORGANS

147

which lies between them and extends into the peg. The trichogen cell, after producing the peg and cuticular sheath in the nymph preparing to molt, retracts from the peg lumen and then encloses orlly the inner portion

PLATE IX FIG.22. A. Tip of dendrite of neuron from thin-walled peg of antenna1 flagellum of grasshopper reconstructed from electron micrographs of sections. B. Diagram of cross section of extreme tip of dendrite close to exposed surface. C. Longitudinal section through dendrite tip, exposed tips of microvilli at right.

of the sheath. The tormogen cell retains contact with the wall of the cavity in the cuticle in which it lies and, consequently, at its inner end encloses, in turn, the vacuole described above, the trichogen cell, the cuticular sheath, and the dendrites within it (Fig. 24). The surface which

14s

ELEANOR H. SLIFER

faces the vacuole, like that of the trichogen cell, is covered with long, slender niicrovilli. Numerous mitochondria, endoplasmic reticulum, and secretion droplets are present in each of these cells. The dendrites of the neurons, as do the cell bodies, contain many mitochondria and neurofilaments in their basal region, but after entering the cuticular sheath only neurofilaments are present. The basal portions of the dendrites show no additional membranes outside the cell membrane itself and so differ conspicuously from the dendrites of the plate organ of the honey bee where folds of one or more neurilemma cells are wrapped about the dendrites to form a compact mass. The cellular parts of the thin-walled peg of the grasshopper which lie below the base of the peg are enclosed by a number of fibers which lie on the outermost surface of the tormogen cell and extend down towards the basement membrane (Slifer et al., 1959). Similar fibers were described in Section I1 for the tactile organ of the grasshopper. When the grasshopper nymph molts, the old cuticular sheath is pulled out through the permeable spot at the base of the new peg and is shed with the exuviae. So far as is known the principal function of the sheath in these sense organs seems to be a mechanical one. It holds the dendrites together in a group and protects them from sudden movements of the grasshopper’s internal organs, which might tear them loose from the body wall to which they are attached. The fate of the distal tips of the dendrites of the thin-walled peg, when the cuticular sheath is withdrawn through the spot at the base of the peg in the molting insect, is not known. Perhaps they are pulled back, beforehand, into the newly-formed sheath and then move out again, later, to take their place in the small openings in the peg surface ; or, perhaps, they are torn off and then regenerate. PLATEX. Electron micrographs of sections through thin-walled pegs from antennal flagellum of grasshopper. FIG.23. Longitudinal section through peg from Melanoplus differentialis including permeable spot at base and portion of cuticular sheath extending inwards from it. Dendrites may be seen leaving sheath through small holes in its upper wall and entering lumen of peg. Note small holes in peg wall where dendrites terminate. Vacuole at base of peg is partially enclosed by cytoplasm of tormogen cell and extends up into peg lumen. ( x 4000.) FIG.24. Cross section below base of peg from Romaleo microptera including cuticular sheath which encloses from 45 to 50 dendrites of sensory neurons. Sheath surrounded by cytoplasm of trichogen cell. Vacuole lies outside trichogen cell; portion of tormogen cell below. Note microvilli on cell surfaces. ( x 5600.) FIGS.25 A N D 26. Cross sgtions through wall of thin-walled pegs of Romalea microptera. Note openings in wall occupied by microvilli at tip of olfactory dendrite. Cuticle does not extend across these openings. ( x 41,000.)

F I N E STRUCTURE OF INSECT SENSE ORGANS

149

150

ELEANOR H . SLIFER

The olfactory epithelium of several vertebrate animals has been examined during the past few years with the electron microscope and it is interesting to compare the results with those reported here for the grasshopper. According to Bloom (1954), each olfactory dendrite in the toad ends distally in a small vesicle which protrudes beyond the surrounding epithelial cells. From six to twelve cilia are arranged in a circle on the vesicle. De Lorenzo ( 1957) described somewhat similar olfactory neurons in the rabbit but was unable to count the cilia. Clark (1957), in describing the structure of the olfactory dendrites of the rabbit, states that in silver preparations a small spot in the center of the ring formed by the cilia is strongly arginophile, and suggests that this spot, rather than the cilia, may be the ultimate receptor surface. It would be most interesting to know whether or not this central area is composed of microvilli. C.

PEGS COELOCONIC

So far as we know, the coeloconic pegs of the grasshopper are found only on the antenna1 flagellum. As was mentioned in the preceding section, they may function as hygroreceptors, but we have no substantial evidence to support this s ~ g g e s t i o n . ~ The peg of the coeloconic sense organ is about 8 p long and is set in the floor of a relatively large cavity in the body wall. A circular hole in the thin roof of the cavity provides contact with the external environment and-except for a small compact mass which represents the residue left from the molting fluid-the cavity is empty. Any agent which affects the peg must enter through the hole in the roof and traverse the space inside the cavity before reaching the peg. The peg, except for its smaller size, resembles the thick-walled peg described in the first part of this section. It is open at the tip, and a cuticular sheath is invaginated from this opening. The sheath extends inward, and ends above the cell bodies of the three or four sensory neurons which form part of this sense organ. The external surface of the peg is grooved or fluted longitudinally and about sixteen scallops are seen at its edge in cross sections (Fig. 21, Plate VIII). The dendrites of the neurons enter the cuticular sheath and pass upward to the tip of the peg where they are exposed to the air inside the cavity in which the peg is set. As in the thick-walled and thinwalled pegs described above, a trichogen cell, and one or more tormogen cells, form part of the sense organ. These cells contain mitochondria, 4 Before it was discovered that the thin-walled peg is covered with extremely small openings where the tips of the dendrites are exposed (Slifer et al., 1959), the coeloconic pegs appeared to be the better suited of the two for detecting water vapor. (Aziz, 1957.)

FINE STRUCTURE OF INSECT SENSE ORGANS

151

membranes, and secretion droplets and that surface of each which is in contact .with the vacuole lying between them is covered conspicuously with microvilli. The fluid within the vacuole, we assume, compensates for moisture lost through the open peg tip and prevents the dendrites from drying. When molting occurs the old cuticular sheath is pulled out through the open tip of the new peg. It is shed with the exuviae.

VII. Ocelli Edwards and Ruck ( 1958) and Edwards (l%O) have examined the ocellus of an unnamed species of dragonfly with the electron microscope, and report that the distal end of each retinula cell is composed of a large number of microvilli. The cytoplasm of the retinula cell contains rows of mitochondria, and the endoplasmic reticulum near the nucleus is vesicular while that near the axon is tubular. The structure of the outer surface of the retinula cell indicates the occurrence of pinocytosis. Large mitochondria, neurofilaments, and small amounts of endoplasmic reticulum are contained in the axon. Pigment cells, some with dark granules and others with white, as well as epidermal cells containing fat droplets, occur between or surround the retinal cells. ~

VIII. Compound Eyes The soft tissues of the compound eye of various insects have received the attention of several workers ; we now have detailed information concerning the fine structure of the retinal cells for a number of species. No one, however, has yet reported work of this type on the outer cellular parts of the eye or on its cuticle. A typical ommatidium of the compound eye of an adult insect consists of (1) a corneal facet or lens composed of transparent cuticle, (2) a crystalline cone made up of four highly-modified cells which lie below the cornea, a group of eight, or fewer, elongate sensory neurons (retinula cells) arranged around a central axis and with their inner surfaces modified to form, collectively, the parts of an optical rod or rhabdom, and (4) a number of pigment cells. Vestiges of the corneagen cells which produced the lens may persist but are usually inconspicuous in the completely developed eye. Numerous tracheae lie between the ommatidia in some species but are scarce or absent in others. I n 1957 Goldsmith and Philpott reported the results of studies on the fine structure of the retinal cells of a blow fly, Sarcophaga bidlata, and a dragonfly, A m x j u n k . The ommatidium of the eye of the blow fly contains eight retinula cells, one of which is rudimentary. The inner border of each of the other retinula cells is modified as a rhabdomere and consists of many long, tubular compartments. Miller (1957) had earlier

152

ELEANOR H . SLIFER

interpreted similar structures in the eye of Limulus as microvilli. The microvilli of the eye of Sarcophrcga, each with a diameter of about 370 A., are packed closely in regular alignment, and their long axes lie at right angles to the long axis of the rhabdomere. Many mitochondria are present in the retinula cell and small pigment granules occur near the rhabdom. The rhabdomeres of the seven retinula cells do not extend to the center of the ommatidium; the elongated space between them is filled, in fixed material, with a loose precipitate. In Anax junius the rhabdom is tripartite in cross section and lacks a central cavity or matrix. Goldsmith and Philpott suggest, as had earlier workers, that the oriented molecules of a dichroic pigment are located in the rhabdoin and that this may account for the ability of the insect to react to polarized light. The description given by Woken et al. (1957) of the compound eye of Drosophila melanogaster agrees closely with that just summarized for the blow fly. These workers, too, believe that visual pigments may be concentrated in the microvilli of the rhabdom and think that the microvilli may play a part in the detection of polarized light. The retinula cells and rhabdom of more than eight species of Diptera, Lepidoptera, Orthoptera, Hymenoptera, and Odonata were examined with the electron microscope by FernPndez-MorLn ( 1956, 1958). The eyes of the house fly, Musca domestica, a large phalaenid moth, Erebus odora, and a hesperiid moth, Epargyreus sp., are described in detail and some information is included for two grasshoppers, Dissosteira sp. and Schistocerca sp., the honey bee, Apis mellifera, the fruit fly, Drosophila melanogaster, and an unidentified dragonfly. The ommatidium of Musca domestica, which possesses a compound eye of the apposition type, contains eight retinula cells, but one is shorter than the others and does not contribute to the rhabdom. Each of the seven rhabdomeres is from 1 to 1.5 p in diameter and 60 to 70 p long. One is smaller in diameter than the others (Fig. 28). They surround a central matrix which, in fixed material, contains a loose network of fine filaments. The microvilli of which each rhabdomere is composed consist of delicate tubules which extend out from the rest of the cell in close-packed, regular rows (Figs. 27, 29, 30). Each microvillus is about 400 A. in diameter and from lo00 to 15,000 A. long. The boundary line of the microvillus, which is a continuation of the surface membrane of the retinula cell, is from 20 to 30 A. thick, and its contents are finely granular. The distal end of the microvillus is rounded off and closed while its proximal end is open and its granular contents are a continuation of the cell cytoplasm. Just before reaching the crystallhe cone, which lies below the cornea, each rhabdomere loses its microvillar structure and becomes very dense. At this

FINE STRUCTURE O F INSECT SENSE ORGANS

153

point the rhabdomeres converge and are in close contact. If the small rhaljdomere is excluded the remaining six can be grouped into three pairs. The members of each pair are usually located opposite one another (Fig. 28) or diagonally across the cell matrix. The size and configuration of the members of a pair of rhabdomeres and the orientation of their

PLATE XI FIG.27. A. Distal tip of retinula cell (sensory neuron) from ommatidium of compound eye of house fly, Musca domrstica, as reconstructed from electron micrographs of sections published by Fernindez-Morin (1958). B. Diagram of cross section of cell with rhabdomere at right. C. Section through part of rhabdomere showing microvilli.

transverse bands of microvilli are approximately matched. Ommatidia in the same region of the eye show close similarities in orientation pattern so that an over-all orientation pattern also exists. Tracheae and pigment cells which lie outside the retinula are arranged in such a way as to accentuate this over-all orientation pattern. Fernindez-Morin suggests that the over-all orientation pattern may also play a part in the perception of polarized light. Mitochondria, endoplasmic reticulum, and unidentified

154

ELEANOR H. SLIFER

dense particles are present in the retinula cell. A polygonal nucleus lies near the periphery. According to Fernhndez-Morin the retinula cells of Drosophila melanogaster and Apis mellifera are very similar to those of the house fly. The retinula of the moth, Erebus odora, which has a superposition type eye, differs in several respects from that of the house fly. Six wedge-shaped rhabdomeres, plus one which is eccentrically placed, meet in the center of the ommatidium, and there is no central matrix. The microvilli occupy each side of the inner part of the wedge. Each microvillus is from 600 to 1200 A. in diameter. The tapetum consists of tracheae which reflect the light back a second time through the rhabdom. I t is not entirely certain that each retinula cell is associated with a single axon, but this appears to be the case since cross sections in the region of the basement membrane show groups of seven to nine fibers. Edwards (1960) thinks that the occurrence of more than seven axons indicates that the neurons branch proximally. Epargyreus sp. and several other unidentified species of hesperiid moth have eight retinula cells in the ommatidium, and each of these has a Vshaped rhabdomere. The rhabdomeres consist of four matched pairs the members of which face each other across a central matrix containing filamentous material. Double-contoured, striated elements, and large mitochondria are present in the cytoplasm of the retinula cell. Each ommatidium is surrounded by a mass of tracheoles, and Fernhndez-Morhn suggests that the double-contoured elements in the cytoplasm of the retinula cell are continuous with these. The tubules, as Fernindez-Morin interprets them, show striations with a period of 150 to 200 A. FernindezMoran proposes that the tubules be called oltratracheoles and the striations microtaenidia. Gray (1960), however, describes what he believes to be similar structures in the auditory organ of the locust as extracellular clefts which contain a honeycomb of hexagonal compartments. Tracheoles are not present in the locust auditory ganglion. PLATE XII. Electron micrographs of sections of retinula cells from ommatidium of compound eye of house fly, Musra domestica, from Fernindez-Morin (1958). FIG.28. Cross section of rhabdom showing seven rhabdomeres composed of microvilli at innermost tips of seven retinula cells ; one rhabdomere smaller than others. Compare orientation of microvilli in rhabdomeres A , and A,, B , and B,, C, and C,. ( X l2,SOO.) FIG. 29. Lungitudinal section through rhabdomere showing closely packed and regularly aligned microvilli ; cytoplasm of retinula cell at left. ( x lY,SOO.) FIG. 30. Longitudinal section through two rhabdomeres. Note hexagonal array of cross-sectioned microvilli at right. ( x 19,500.)

FINE STRUCTURE O F INSECT SENSE ORGANS

155

156

ELEANOR H . SLIFER

The retinulae of unidentified species of grasshoppers belonging to the genera Dissosteira and- Schistocerca, as figured by FernAndez-Morin, appear to contain five retinula c e k 5 These surround a central matrix containing fine filaments which run lengthwise in the cavity in a regular manner, and so differ from those seen in the central matrix of other forms. The microvilli of the rhabdomeres are from 700 to 900 A. in diameter, and the cytoplasm of the retinula cells contains large numbers of mitochondria and pigment granules. In the discussion with which Fernindez-Mor6n concludes his paper he suggests, as did Goldsmith and Philpott (1957) and Wolken et 01. (1957), that the fine structure of the rhabdomeres is associated with the ability of the insect to analyze polarized light. Recent work by Baylor and Smith (1958) and Smith and Baylor (1960), however, suggests that the search for an analyzer of polarized light within the insect eye may be a futile one. In a series of experiments with honey bees they found that these insects are not able to detect the plane of polarized light but indirectly see the effects of this because the polarization plane influences the apparent brightness of the background from which it is reflected.6

IX. Summary The most spectacular additions to our knowledge of the structure of insect sense organs, which we owe to studies made with the electron microscope and those which, undoubtedly, will be of greatest interest to entomologists, concern the sensory neurons-especially their receptor surfaces-and the modified cuticle with which the neurons are associated. The simplest sensory neuron so far examined is that of the grasshopper tactile hair. Here the dendrite of a single bipolar neuron passes toward the base of a movable hair, and a short distance below the point where it will be attached to it, develops irregular knobs on its surface. Above this region the dendrite narrows suddenly and its delicate tip is fastened inside the base of the hair. The knobs on the surface of the dendrite fit into cavities in the thickened wall of the cuticular sheath enclosing it, so 5

Jorschke (1914) states that distally there are six retinula cells in the ommatidium

of the grasshopper, Cahptenus italicws, while below this there are five. Basally two additional cells seem to be present, but it is difficult to be certain of cell boundaries. 6 While this review was in press Jander and Waterman (1960) reported new experiments from which they concluded that arthropods do possess a “separate sensory input channel” for detecting polarized light. They suggest, as did several previous workers, that the submicroscopic villi of the visual cells “contain oriented layers of visual pigment molecules which might not unreasonably be dichroic.” Their conclusions are supported by electrophysiological experiments of Burkhardt and Wendler (1960).

F I N E STRUCTURE OF INSECT SENSE ORGANS

157

that the dendrite is firmly anchored. The discovery that the distal ends of the dendrites of the neurons of the auditory organ of the locust, and the plate organs of the honey bee are modified as cilia, has occasioned much surprise, for it has long been believed that cilia do not occur in insects. However, there is no evidence that these structures show active movements; it is more probable that they are moved passively by vibrations of the tympanum or plate to which they are attached. The absence of a cuticular covering, and the presence of microvilli at the receptor surface of olfactory dendrites of the grasshopper, could not have been predicted with earlier studies made with the light microscope. The microvilli of the rhabdom of the oiiiniatidium and ocellus, on the other hand, are larger; workers long ago described and figured the rhabdoni as finely striated (Grenacher, 1879 ; Hesse, 1901). The significance of these delicate markings could not, of course, be understood until they were examined with the electron microscope. The work done during the past four or five years will, no doubt, be quickly followed by studies on the fine structure of other insect sense organs. Nothing is known, for example, of the detailed structure of the dendrite of the campaniform organ. Perhaps it ends in a cilium as does each dendrite of the tympana1 organ. Those located on the halter of the fly should prove especially interesting because of the experimental work which has already been done with the halteres (Pringle, 1957). For the same reason more information concerning the structure of the gustatory hairs of the blow fly is desirable. A study of the fine details of the abdominal stretch receptors of insects, which have been described recently by Finlayson and Lowenstein (1958), should not be difficult since there would be no need to include cuticle in the sections. Certain of the scoloparia of the grasshopper-especially the chordotonal organs of the prothoracic and mesothoracic femora (Slifer, 1935)-would have this same advantage. The ampullaceous sense organs of the Hymenoptera offer another attractive field for investigation since their function, with our present limited knowledge, is a matter for speculation. Among the unanswered questions which still confront us the following may be listed: Is a cuticular sheath present in any of the sense organs of holometabolous insects? In their larvae? Is such a sheath associated with sense organs other than those in which the specialized cuticular parts are shed, one or more times, after having become functional? What is the physical nature of the cuticle of the cornea and the tympanum? What is its fine structure as seen with the electron microscope? Studies such as those which were carried out by Richards (1952) on the plate organs of the honey bee might be helpful here. Does the terminal strand of

158

ELEANOR H. SLIFER

filaments by which the cilia of the plate organ are attached to the body wall resemble cuticle or- is it formed of some other material?’ How closely do such structures as scolopales, sense rods, terminal strands, the rootlets and root apparatus of the auditory neurons, and the crystalline cone of the eye resemble one another in their structure, composition, and origin? What materials are present in the vacuoles commonly seen in close association with sensory neurons? Are any of these fixation artifacts as some investigators believe? Are the extreme tips of the dendrites of tactile hairs modified as cilia? Are cilia a common feature of the neurons of mechanoreceptors ? Do they occur in the dendrites of sense organs which are not mechanoreceptors? What is the origin and nature of the conspicuous envelope of branching fibers in which the cellular parts of the sense organ are often enclosed? It will be interesting to compare the fine structure of the sense organs belonging to species from different orders as more information becomes available. No larval sense organ has yet been examined with modern techniques, and few studies on the development of insect sense organs during embryonic and postembryonic life have been made even with the light microscope. Nothing at all is known of the fine structure at such stages8 Finally, as knowledge of the structural details of insect sense organs increases, it should be possible to plan in a more rational manner experiments intended to discover their function.

REFERENCES Aziz, S. A. (1957) Indian J . Entomol. 19, 164. Baylor, E. R., and Smith, F. E. (1958) Anat. Record 132, 411. Beams, H. W., and Anderson, E. (1957) J . dlorphol. 100, 601. Beams, H. W., Tahmisian, T. N., and Devine, R. L. (1955) J. Biophys. Biochem. Cytol. 1, 197. Bloom, G. (1954) 2. Zellforsch. t i . mikroskop. Anat. 41, 89. Burkhardt, D., and Wendler, L. (1960) 2. vergleich. Physiol. 43, 687-692. Clark, W . Le G. (1957) Proc. Roy. SOC.B146,299. Dethier, V. G. (1953) In “Insect Physiology” (K. Roeder, ed.), Chapts. 19, 20, 21. Wiley, New York. Dethier, V. G. (1955) Quart. Rrv. Biol. 90, 348. Dethier, V. G., and Wolbarsht, M. R. (1956) Experientia 12, 335. Dostal, B. (1958) 2. vergleich. Physiol. 41, 179-203. Edwards, G. A. (1960) Ann. Rev. Entomol. 5, 17. Edwards, G. A., and Ruck, P. (1958) N . Y . State Dept. Health Ann. Rept. Div. Lab. Research p. 54. Engstrom, H., and Wersall, J. (1958) Exptl. Cell Research Supfl. 5, 460. Fernindez-Morjh, H. (1956) Nature 177, ‘742. 7 Since this review was written, electron micrographs have been obtained which show that the tips of the dendrites are not attached to the plate. 8 While this paper was in press an article by Waddington and Perry (1960) has provided information on the fine structure of the developing eye of Drosophila

mrlanogastrr:

F I N E STRUCTURE OF INSECT SENSE ORGANS

159

FernPndez-Morhn, H. (1958) Exptl. Cell Rest-arch Suppl. 6, 586. Finlayson, L. H., and Lowenstein, 0. (1958) Proc. Roy. SOC.B148, 433. Goldsmith, T. H., and Philpott, D. E. (1957) J . Biophys. Biochem. Cytol. 3, 429. Gray, E. G. (1960) Phil. Trans. Roy. SOC.London B%3, 75. Gray, E. G., and Pumphrey, R. J. (1958) Nattcre 181, 618. Grenacher, H. (1879) “Untersuchungen iiber das Sehorgan der Arthropoden, insbesondere der Spinnen, Insekten und Crustaceen.” Vandenhoeck und Ruprecht, Gottingen. Hesse, R. (1901) 2. wiss. 2001.70, 347. Hodgson, E. S. (1955) Quart. Rev. Biol. SO, 331. Hodgson, E. S. (1958) A m . Rev. Entomol. 3, 19. Imms, A. D., Richards, 0. W., and Davies, R. G. (1957) “A General Textbook of Entomology.” Methuen, London. Jander, R., and Waterman, T. H. (1960) I . Cellular Comp. Physiol. 66, 137-159. Jorschke, H. (1914) 2. wiss. Zool. 111, 153. Kuwabara, M., and Takeda, K. (1956) Physiol. Ecol. (Japan) 7 , 1-6. de Lorenzo, A. J. (1957) J . Biophys. Biochem. Cytol. 3, 839. McIndoo, N. E. (1922) J . Comp. Nenrol. 54, 173. Miller, W. H. (1957) J . Biophys. Biochem. Cytol. 3, 421. Pringle, J. W. S. (1957) “Insect Flight.” Cambridge Univ. Press, London and New York. Richard, G. (1952) Bull. SOC.zool. France 77, 99. Richards, A. G. (1952) Biol. Bull. 103, 201. Slifer, E. H. (1935) J . Morphol. 68, 615. Slifer, E. H. (1936) Entomol. News 47, 174. Slifer, E. H. (1954a) Biol. Bull. 106, 122. Slifer, E.H. (1954b) Proc. Roy. Entomol. SOC.London AN, 177. Slifer, E. H. (1955a) Entomol. News 66, 1. Slifer, E. H. (1955b) J. Exptl. Zool. Iso, 301. Slifer, E. H. (1956) Proc. Roy. Entomol. SOC.London A31, 95. Slifer, E. H., and Sekhon, S. S. (1960) Exptl. Cell Research 19, 410. Slifer, E.H., Prestage, J. J., and Beams, H. W. (1957) J . Morphol. 101,359. Slifer, E.H., Prestage, J. J., and Beams, H. W. (1959) J . Morphol. 106, 145. Smith, F.E., and Baylor, E. R. (1960) Ecology 41, 360-363. Snodgrass, R. E. (1926) Smithsonion Znst. Publs. Misc. Collections 77(8). Snodgrass, R. E. (1935) “Principles of Insect Morphology.” McGraw-Hill, New York. Stuhlman, H. (1950) In “Medical Physics” (0. Glasser, ed.), Vol. 2, p. 385. Year Book, Chicago. Vogel, R. (1921) Zool. Anz. 65, 20. Vogel, R. (1923) 2. win. 2001.l20,281. von Frisch, K. (1921) Zool. Jahrb. Abt. Allgem. 2001.Physiol. Tiere 38,449. von Frisch, K. (1923) 2001.Jahrb. Abt. Allgem. 2001.Physiol. Tiere 40, 1. von Frisch,.K., Lindauer, M., and Daumer, K. (1960) Experientia 16,289-301. Wacker, F. (1925) 2. Morphol. Okol. Tiere 4, 739-812. Waddington, C. H., and Perry, M. M. (1960) Proc. Roy. SOC.B163, 155-178. Wenner, A. M. (1959) Bull. Entomol. SOC.Am. 6, 142. Wigglesworth, V. B. (1953) “The Principles of Insect Physiology.” Methuen, London. Wolken, J. J., Capenos, J., and Turano, A. (1957) J . Biophys. Biochem. Cytol. 5. 441.

This Page Intentionally Left Blank

Cytology of the Developing Eye ALFRED J. COULOMBRE Department of Anatomy, Yale University School of Medicine, N m Haven, Connecticut

I. Introduction ..................................................... 11. Cornea ......................................................... A. Early Development .......................................... B. Epithelium ................................................. C. Bowman’s Membrane ........................................ D. Stroma .................................................... E. Descemet’s Membrane ....................................... 111. Sclera ........................................................... A. Sclera Proper .............................................. B. Cribriform Plate ............................................. C. Trabecular Meshwork ........................................ IV. Iris ............................................................. V. Choroid Coat .................................................... VI. Ciliary Body ................................................... VII. Lens ........................................................ A. Induction and Early Morphogenesis . . . .............. B. Lens Cells ...................................... C. Lens Capsule .............. ......................... VIII. Retina .............................................. A. Pigmented Epithelium ...................................... B. Neural Retina .............................................. References ......................................................

Page 161 163 163 164 166 166 170 170 170 171 172 172 173 173 174 174 175 178 179 179 180 190

I. Introduction Few organs are equal to the vertebrate eye in its diversity of cell types or in the high degree of differentiation which these cells achieve. The ectoderm contributes such vastly different structures as the corneal epithelium, the lens, the neural retina, the pigmented epithelium, the ciliary epithelium, and even muscular tissue in the iris. The mesodermal derivatives achieve equally startling divergencies in the corneal endothelium, the cells of the filtration angle, the transparent collagenous stroma of the cornea, and the adjacent opaque collagenous sclera. This list, by no means exhaustive, suggests the wide range of end points available in ocular tissues for the study of mechanisms underlying cytogenesis. In addition, these assorted tissues must, as nowhere else in the body, bear precise geometric relationships to one another if optic and photoreceptive functions are to be possible. Thus, at the gross level, the size and curvature of the cornea ; the size, shape, and position of the lens ; and the location and attachments of the ciliary muscles must, within narrow 161

162

ALFRED J. COULOMBRE

limits, bear well-defined spatial relationships both to each other and to the size and curvature of the retina. At the histologic level most ocular tissues are arranged in precise strata. This is seen most strikingly in the posterior eye wall, the neural retina, and the cornea. I n each of these structures this stratification is related to some aspect of optical function. The size, shape, and internal organization of the visual cells illustrate that, even at the cytological level, the geometry of the cellular and subcellular units serves visual function. I n their spatial distribution the organelles of the inner segment (e.g., myoid, paraboloid, ellipsoid, oil droplet) are adaptive in view of the path light takes through the retina. The size and shape of the outer segments are important factors in determining visual acuity, and these dimensions vary appropriately from the fovea to the ora serrata. I n addition to meeting strict geometric specifications, the cells and tissues of the eye are differentiated to serve a wide variety of functions. The outer segment of the visual cell transduces light energy into neural impulses. The tissues of the cornea, lens, vitreous substance, and retina are remarkably transparent to light, and maintain refractive indices in a range suitable for image formation. The ciliary body regulates the composition and flow of aqueous humor so that an appropriate intraocular pressure is maintained. The epithelium and endothelium of the cornea regulate the water content of the stroma within the narrow limits compatible with its transparency. While studies of the adult eye at all levels of structure and function can tell us much concerning its nature, a detailed descriptive and causal analysis of its development is necessary if we are to understand how its cells differentiate and assume their definitive structure, shape, position, and function. This review focuses attention on the cytogenetic aspects of eye development. It is restricted in several ways. Differences in the timing of cytogenetic events in different species are not treated in detail unless they bear directly on the problems of cellular differentiation under discussion. Pathologic or anomalous phenomena are outside the scope of the paper except where they throw light on the normal course of development. Information concerning the adult stage is, in general, excluded. Discussion is restricted to the globus oculi and does not treat of the adnexa. The period covered by the review is, in the main, from 1940 to 1960. Material prior to 1940 has been summarized by a number of authors. An extensive bibliography of historical, descriptive, and experimental work on ocular structure was compiled by Polyak (1941, 1957). Publications dealing with all aspects of eye developrhent are covered by Mann (1950). The careful bibliographic wprk of O’Rahilly and Meyer (1959) covers early developmental changes in the eye. Literature on the developing cornea is covered by O’Rahilly and Meyer (1960) and Meyer and

CYTOLOGY O F THE DEVELOPING EYE

163

O’Rahilly (1959). The work of Detwiler (1943) and Saxen (1954) cover work on some aspects of visual cell development. 11. Cornea The cornea of the vertebrate eye has both mechanical and optical functions. Mechanically it balances the forces generated by intraocular pressure. Its optical properties are of three types. It refracts incident rays of light, it is transparent, and it exhibits a characteristic interference pattern when viewed between crossed polarizers. These properties depend not only on the nature of the cells and of the several extracellular materials of the cornea, but also on the relationships that exist among them. I t is, therefore, of some interest to review the cytogenesis of corneal cells, and the sequence and manner in which intercellular substances are deposited, in order to relate these developmental changes to the functional maturation of the cornea. DEVELOPMENT A. EARLY The epithelium of the cornea is the first of its layers to appear. I t can be delimited immediately after closure of the lens pore as the region of ectoderm overlying the lens vesicle. At this time in the chick embryo it has, in common with the rest of the ectoderm, a superficial layer of squamous cells underlain by one or two rows of cuboidal basal cells. A periodic acid Schiff positive (PAS+) basement membrane adheres to its inner surface, and is continuous with the one underlying the contiguous prospective coiijunctiva (O’Rahilly and Meyer, 1960). An acellular directional membrane appears beneath the basement membrane of the anterior epithelium. I t is along this membrane that mesothelial cells migrate to form the inner epithelium (epithelium of Descemet, corneal endothelium) . Shortly after completion of this simple squamous sheet of cells, a PAS+ basement membrane appears between it and the directional membrane. While this membrane, which is distinct from Descemet’s membrane which appears later, has been seen in the light microscope in stained sections (cf. Meyer and O’Rahilly, 1959), it is not in evidence in electron micrographs (Jakus, 1956). Between the basement membranes of the two epithelia lies the post-epithelial layer, within which the stroma will form. This acellular layer is composed of a feltwork of fine fibrils which are probably collagenous in nature (Jakus, personal communication) . The post-epithelial layer is invaded by stromal cells which arise from mesenchymal elements at its edge. At this stage the major layers of the cornea have been blocked out, with the exception of the anterior limiting lamina (Bowman’s membrane), and the posterior limiting lamina (Descemet’s membrane). It will be convenient to treat each of the corneal layers in turn.

164

ALFRED J . COULOMBRE

B. EPITHELIUM The anterior corneal epithelium serves several important functions. First, its anterior surface is the site of initial refraction of the incoming light rays. Second, like many other epithelia, it has complex interrelationships with the tissue underlying it. Thus, it shares with the posterior corneal epithelium the function of eliminating excess water from the stroma (Harris, 1957). The system within these epithelia which is responsible for stromal deturgescence has been called the “corneal pump mechanism.” When the corneal temperature is lowered (Davson, 1955)‘, or when the cornea is denied access to oxygen (Davson, 1955; Smelser, 1952; Smelser and Ozanics, 1952), the pump is impaired and the stroma begins to hydrate with a consequent loss of transparency. In addition the epithelium controls other events in the stroma. It appears responsible for the initiation of wound healing in the stroma by proteolytic release of a factor which elicits fibroblastic activity from stromal cells ( Weimar, 1959a,b ; Dunnington and Weimar, 1958). Another manifestation of the functional interrelationship between the epithelium and the stroma is evidenced by the fact that only regions of stroma underlying intact epithelium will take up g 1 y ~ i n e - l - C (Herrmann, ~~ 1958). Herrmann further speculates that the epithelium may be a decisive influence in the differentiation of stromal cells from inmigrating mesenchyme.

1. Squamous Cells The anterior layers of the adult corneal epithelium are composed of squanious cells. The entire squamous zone is characterized by: ( a ) strongly PAS+, orthochromatic substances, ( b ) a moderate concentration of sulfhydryl and disulfide groups, ( c ) sudanophilic substances (possibly phospholipids), and ( d ) a blue-white fluorescence (Wislocki, 1952). The PAS+ components are of two types. One is evenly distributed and diastase resistant. O’Rahilly and Meyer ( 1960) summarize current opinions concerning the composition of this component, and it is evident that additional work is needed to identify it critically. The second component is glycogen which occurs in the form of granules. These have been seen by some investigators but not by others (cf. O’Rahilly and Meyer, 1960). The squamous layer appears early in development ( O’Rahilly and Meyer, 1959). O’Rahilly and Meyer (1960) did not detect the characteristic PAS+ reaction in this layer in the chick embryo until stage 40 (14 days of incubation). Further, they demonstrated no glycogen at any stage of development. Yoneyama ,(1932), however, observed glycogen granules in this layer on the seventh day. The granules increased in number until the fifteenth day, decreased abruptly until the nineteenth day, and then

CYTOLOGY OF T H E DEVELOPING EYE

165

increased again until adulthood. Unpublished data from our laboratory reveal a faint but definite PAS positivity in the squanious layer on the fourth day, the earliest day that was studied. In addition to an evenly colored background, glycogen granules are present in the squamous cells throughout the period of observation (4-21 days of incubation). In some cells the granules stain faintly ; however, other cells, which are scattered throughout the layer, stand out because of the number and the intensity of staining of their glycogen granules. By 14 days the squamous layer is intensely PAS+ and contains many glycogen granules. In an electron microscopic study of the post partum mouse Sheldon (1956) reported the cytogenesis of the squamous cells. The cell membrane is about 80 A. thick and comprises a central non-electron-dense layer bounded by two electron-dense layers. The outer surface of the superficial layer of cells has scattered microvilli. Each cell contains a nucleus bounded by a typical two-layered nuclear membrane. Cytoplasmic organelles include the Golgi substance, small, rod-shaped mitochondria, numerous filaments 50 A. in width, and granules (150 A. in diameter) which occur singly or in rosettes. Some of the latter are associated with endoplasmic reticulum. The only notable developmental change in the mouse was a progressive increase in electron density of the mitochondria. The entire picture is one of a living cell. Thus, while some degree of keratinization may have occurred in this layer, there is no evidence of cornification. It must be remembered that the corneal epithelium moves water from the stromal compartment to the corneal surface. Whatever the involvement of the deeper layers of cells in this process, it is probable that the squamous cells, the last living units before the tear envelope is reached, play an active role in corneal deturgescence.

2. Intermediate Cells The intermediate celIs of the corneal epithelium arise relatively late in development. In the post partum mouse they are separated by extensive intercellular spaces (Sheldon, 1956). Later these spaces decrease in number, and there is a concomitant increase in the number of cell membrane interdigitations between adjacent cells. The cells contain a few glycogen granules and stain moderately for disulfide and sulfhydryl groups (Calmettes et al., 1956).

3. Basal Cells The cuboidal or columnar basal cells interdigitate less frequently than the intermediate cells, have an electron-dense cytoplasm, and an indented nuclear membrane studded with discontinuities ( Sheldon, 1956).

166

ALFRED J. COULOMBRE

4 . Basement Membrane Until recently it has not been widely appreciated that the epithelium of the cornea is underlain by a delicate basement membrane. Calmettes et al. (1956) and Offret and Haye (1959) review the literature bearing on this structure, and report its presence in a wide variety of mammals including man. The latter authors report the presence of the membrane from very early stages in development. O’Rahilly and Meyer (1960) summarize recent work on the adult basement membrane of many mammalian forms. They add their own findings on the origin of this .membrane in the chick embryo. The primitive ectoderm is underlain by a basement membrane. After closure of the lens pore, and separation of the lens vesicle from the overlying ectoderm, this membrane completely covers the inner aspect of the prospective corneal epithelium. It is more closely adherent to the epithelium than to underlying stromal structures, and regenerates along with the epithelium following corneal wounding. Calmettes et al. (1956) have shown that the developing basement membrane is PAS+, contains no glycogen, is orthochromatic with toluidine blue, and contains lipid and reticulin. In the post partum mouse the basement membrane of the epithelium is about 600 A. thick and contains banded fibrils (Sheldon, 1956). C. BOWMAN’S MEMBRANE The anterior limiting lamina is demonstrable in relatively few vertebrate corneas, It is characteristic of primate eyes, but has also been described for some submammalian forms. I n its histochemical properties Bowman’s membrane resembles the stroma, with which it is continuous (Calmettes et al., 1956). It differs from the stroma in the absence of cells, the random orientation of its collagenous fibrils in the plane of the cornea, and in the fineness of the fibrils. O’Rahilly and Meyer (1960) report that this membrane appears first at stage 40 (14 days of incubation) in the chick embryo.

D. STROMA Following completion of the simple squamous posterior epithelium of the cornea, mesenchymal cells invade the hitherto acellular post-epithelial layer. These cells are fibrocytes (Fornes-Peris, 1948). In common with other fibroblasts the stromal cells have prominent nucleoli and contain cytoplasmic granules which range in diameter from 0.7 to 2.0 p (Jackson, 1955). In tissue culture the granules stain supravitally with neutral red and gentian violet. Their cortex is composed of diastase-resistant PAS+, toluidine blue, and thionine metachromatic mucopolysaccharide. The nature of the core of these granules is not known. The granules exhibit

CYTOLOGY O F THE DEVELOPING EYE

167

alkaline phosphatase activity. Jackson suggests that they are precursors of ,extracellular components. The extracellular compartment of the stroma contains collagen, mucopolysaccharides, water, noncollagenous proteins, inorganic ions, and miscellaneous solutes. The first three account for most of the stromal mass, and the manner in which they develop and interrelate has been studied extensively in recent years. While the post-epithelial layer is probably collagenous in nature, the first coarse, cross-str.iated fibers of collagen in the chick embryo are seen in close association with the stromal fibroblasts following their invasion of the post-epithelial layer (Jakus, personal communication) . A comparable situation obtains in man (Schwarz, 1960). The fibers lie parallel to the surfaces of the stromal cells. By the eighth day of incubation enough collagen has accumulated in the chick embryo stroma so that it can be detected by analysis for hydroxyproline. The amount of hydroxyproline per stroma increases rapidly until hatching, and thereafter more slowly (Herrmann, 1958). Smits ( 1957) made similar determinations with developing cattle cornea, but reports values for hydroxyproline as per cent of dry weight. Relative to dry weight, collagen first increases, and subsequently shows a moderate decrease. The patterns formed by stromal collagen bear directly on several of its optical properties, and is receiving the close study it deserves. Maurice (1957) has concentrated on the fibrillar lattice within the collagen fibers, and suggests that the fibrils must be of uniform diameter and evenly spaced to assure transparency of the cornea. Kikkawa (1955, 1956, 1959) points out that collagen forms a double lattice in the stroma, one within the fibers at the fibrillar level, and that formed by the fibers themselves. He, as well as Naylor ( 1953a,b), Stanworth (1950), and Stanworth and Naylor (1950, 1953), has pointed out that the complex collagen lattice forms the physical basis for the interference pattern which is seen when the cornea is viewed between crossed polarizers. Electron micrographs of sectioned stroma reveal collagenous fibrils of small diameter. Jakus (1954) places the diameter at 250-300 A. for the adult rat, a figure identical with that measured by van den Hooff (1957) in beef. Both authors give a crossband macroperiod of about 640 A., and van den Hooff describes two microperiods spaced at about 210 A. In the developing human cornea, Schwarz (1953) measured the range of fibril diameters as 300-350 A., and pointed out, as did Jakus and van den Hooff, that there is a remarkable uniformity of diameter from fibril to fibril. In addition, Schwarz found the fibril diameter essentially the same at all developmental stages. Bundles of parallel fibrils constitute the collagen fibers, and the latter,

168

ALFRED J. COULOMBRE

in turn, are arranged in a gross lattice in some species. In the chick embryo, the collagen fibers do not show. an orderly arrangement in the stroma prior to the eighth day. At that time, however, serial sections of the cornea, in a plane tangent to its surface, reveal that the fibers within each laniella are arranged in an orthogonal gridwork that lies parallel to the corneal surface (Coulombre and Coulombre, 1958a). Successive lamellae are laid down beneath the anterior epithelium and are not in register with those below. While the detailed geometry of the lamellar grid patterns cannot be treated here, the following facts are of general interest: (1) the fibers forming the gridworks of the laniellae are in the plane of the cornea, ( 2 ) the angular shift of the major grid axes, as one moves from the inner to the outer surface of the cornea, is progressively greater and is clockwise in both eyes, and thus asymmetric around the body midline, and ( 3 ) the interference pattern, seen when the cornea is viewed between crossed polarizers, appears on the eighth day, when the collagenous gridwork is first established, and has a fixed geometric relationship to the collagenous grid pattern. In addition to collagen, an analysis of beef cornea (Meyer et al., 1953) reveals that the adult stroma contains at least three mucopolysaccharides : chondroitin sulfate A, chondroitin, and keratosulfate. Great interest attaches to these compounds for several reasons. Their presence is associated with the notorious avidity with which the cornea absorbs water. I t is also possible that they play a role in the development and maintainance of corneal transparency. In this connection they may, in an appropriate state of hydration, bring the interfibrillar spaces to the same refractive index as collagen. Alternatively, or at the same time, they may be responsible for the equidistant spacing of the collagen fibrils which Maurice (1957) considers essential for transparency. Since these acid mucopolysaccharides are PAS+ and stain metachromatically with appropriate dyes, their distribution in the stroma can be easily followed during development. Such investigations have been carried out in the chick embryo by van Walbeek et al. (1950), van den Hooff (1951), Coulombre (1956a), Ghiani and Bergamini (1957), and Coulombre and Coulombre (1958b). These authors are in essential agreement that intense toluidine blue metachromasia appears at the endothelial border of the stroma on the thirteenth or fourteenth day of incubation and spreads toward the epithelial margin of the stroma during the ensuing week. O’Rahilly and Meyer (1960) observed, in agreement with these findings, that the posterior layers of the stroma become intensely PAS+ on about the fourteenth day. As this change occurs the cornea begins to increase in transparency, and achieves the adult value shortly before hatching. While the stroma is increasing in transparency and exhibiting

CYTOLOGY OF THE DEVELOPING EYE

169

an ever more widespread intense metachromasia, it undergoes a relative dehydration. The decrease in amount of water per unit dry weight is very rapid between the fourteenth and the nineteenth day. On about the nineteenth day the cornea achieves its adult transparency and its adult level of hydration simultaneously (Coulombre and Coulombre, 195813). A similar series of changes occurs during the development of the mammalian stroma. I n an extensive study of man, guinea pig, rat, mouse, and rabbit, Aurell and Holmgren (1953) first detected intense metachromasia in the inner lamellae of the stroma, and later throughout the stroma. This developmental sequence was confirmed by later authors (man : Gemolotto and Patrone, 1955; rat: Alagna, 1954; Seo, 1955; rabbit: Smelser and Ozanics, 1956, 1957, 1959). Thus the time and place of first appearance of intense metachromasia in the corneal stroma has been well established for several species. Smelser and Ozanics (1959) have carried the analysis a step further. They find that pretreatment of early rabbit corneas with testicular hyaluronidase removes the metachromatic substances. Since keratosulfate is metachromatic, but is not attacked by hyaluronidase, they conclude that it is not present at earlier developmental stages and must appear later in development. The significance of the appearance and spread of intense metachromasia in the developing stroma is by no means clear. A faint metachromasia with toluidine blue is present from early development in both the rabbit (Smelser and Ozanics, 1959) and the chick (Coulombre, unpublished data). Smits (1957) observed that beef stroma, like other connective tissues, showed an initial drop in niucopolysaccharide (measured as hexosamine HCl), but that it later increased in amount. Until quantitative determinations are made of the individual mucopolysaccharides in the developing stroma, it is impossible to say whether the advent of intense metachromasia represents an increase in the rate of synthesis, or the appearance of a new mucopolysaccharide, or simply an increase in the local concentration of pre-existing acid mucopolysaccharide. I t may well be the latter, since as the cornea loses water and becomes thinner, its inner lamellae are the first to become more tightly packed. This change would tend to bring pre-existing mucopolysaccharide into higher concentration locally, and might account for the increase which is observed in metachromatic intensity. In any case, the appearance of intense metachromasia at the time the cornea begins to become transparent must reflect stromal changes which are intimately connected with the development of transparency.

170

ALFRED J . COULOMBRE

E. DESCEMET’S MEMBRANE The internal limiting lamina of the cornea lies between the stroma and the endothelium. This PAS+, orthochromatic membrane ( Wislocki, 1952) has been thoroughly investigated with the electron microscope by Jakus (1956). She followed its development in the chick embryo. It forms from the endothelium for which it is a basement membrane. If injured it can be regenerated. I t is radially isotropic in polarized light, and shows positive form and negative intrinsic birefringence with respect to the plane of its surface. I t contains a collagenous protein atid probably a mucopolysaccharide. In the chick embryo its presence cannot be detected with the light microscope during the prehatching period (cf. Meyer and O’Rahilly, 1959). Electron micrographs, however, reveal its presence on about the tenth day of incubation (Jakus, 1956). At first only isolated pads of material are in evidence, but these soon fuse laterally to form a continuous membrane. The membrane has a lamellar structure. The lamellae are disposed parallel to the surfaces of the membrane, and are about 270 A. apart. Electron-dense nodes are distributed 1070 A. apart, in a regular array within each lamella. The nodes are interconnected, in tangential sections, by fine electron-dense lines. The functional significance of this membrane remains to be explored. It is situated just inside the endothelium, and water and solutes moved out of the stroma must traverse it. It appears during development of the chick cornea at the onset of the period of corneal dehydration and just prior to the functional maturation of the cornea. The role of this layer can best be assessed, not in the adult, but in the embryo, since experimental intervention is possible before, during, and after the development of this membrane. 111. Sclera A.

SCLERA PROPER

The sclera is foreshadowed by a condensation of mesenchyme surrouriding the optic cup. These cells subsequently differentiate into fibroblasts and, in most submammalian eyes, into chondroblasts. Their fate is fixed some time before any visible differentiation. Thus, in the chick embryo, Weiss and Moscona ( 1958) showed that explanted precartilagenous mesenchyme from the tail bud and from the scleral region differentiated into cartilage similar in shape and internal structure to that found in their sites of origin. This is so even after dissociation and reaggregation of the cells, demonstrating conclusively that the formation of flattened scleral cartilage is not dependent on efrtrinsic mechanical factors, but upon factors indigenous to the differentiating tissue itself. Mechanical factors do, however, play some important roles in the dif-

CYTOLOGY O F THE DEVELOPING EYE

171

ferentiation and in the details of shaping of scleral cartilage. Thus estreme tensions inhibit the differentiation of cartilage from prescleral mesenchyme (Weiss and Amprino, 1940). On the other hand, moderate tensile forces, such as those normally generated in the eye wall during development, do influence the growth and morphogenesis of the scleral cartilage (Weiss and Amprino, 1940 ; Amprino and Pansa, 1955-1956). If the eye of the chick embryo is pricked at four days, the vitreous humor escapes, and the eye wall is not brought under tension. During subsequent development the scleral cartilage of such eyes continues to grow, as evidenced by an increase in its volume and by the uptake of radiosulfate. However, the growth rate is depressed and the sclera conies to have a smaller area and volume, and a greater thickness than normal. This depression is attributable to a decrease in appositional growth as distinguished from internal growth, and is reflected by a lowered mitotic index. In the collagenous sclera, typical of mammals, the cells of prescleral mesenchyme differentiate into fibroblasts. The young sclera, like the cornea, has a high concentration of mucopolysaccharide, as evidenced by its metachromasia and by measurements of its hexosamine content. As in cornea the concentration of mucopolysaccharide decreases as collagen is deposited. Unlike cornea, but like tendon and skin, the scleral hexosamine continues to decrease until the adult level is achieved (Smits, 1957). The collagen fibrils of human sclera continue to increase in diameter as development progresses, and eventually achieve a much larger mean diameter, and a much wider range of diameters, than those of the corneal stroma (Schwarz, 1953). Schwarz points out that, in this respect, the corneal collagen becomes arrested in the embryonic condition, and suggests that this is a necessary condition for transparency. The irregular thickening of scleral fibrils, he believes, accounts for scleral opacity.

B. CRIBRIFORM PLATE The lamina cribrosa is continuous with the sclera across the head of the optic nerve and is pierced by the axons of the retinal ganglion cells. These fibers grow out to form the optic nerve prior to the time at which the sclera differentiates. As the sclera forms around the exit of the nerve, a series of changes are initiated which provide strong structural continuity across the head of the optic nerve without compression of its axons. Redslob (1956) demonstrated that these changes are initiated during the second half of the third month in human fetuses when glial elements orient transversely to the nerve fibers. The neuroglial fibers of these cells constitute the primitive cribriform plate. A t the end of the fifth month fibroblasts invade from the adjacent sclera, become arranged in palisades, and lay down collagen which anchors the cribriform plate to the scleral canal.

172

ALFRED J. COULOMBRE

Toward the seventh month elastic fibers appear, and by birth the plate is predominantly elastic. During postnatal .life collagen achieves a higher concentration than elastin and the adult plate is predominantly collagenous. The factors responsible for this complex of events await identification and analysis by experimental cytologists and embryologists.

C . TRABECULAR MESHWORK The trabecular meshwork, through which aqueous humor drains from the anterior chamber into the canal of Schlemm, contains both weal and scleral contributions. In the human fetus both the uveal and scleral meshworks are predominantly cellular and are traversed by relatively few pores (Speakman, 1959a). During subsequent development the number and size of the pores increases and the collagen fibers become thicker and more numerous. The fibers of both meshworks anchor anteriorly in the margin of Descemet’s membrane, and posteriorly in the connective tissue of the iris and ciliary muscle. The fibers surrounding the canal of Schlemm orient parallel to its long axis, but become more transversely arranged as the anterior chamber is approached. At maturity passageways 3 to 1 2 p in diameter, and 30 to 5 0 p in length, open into Schlemm’s canal. The cells of the meshwork are cytologically similar to the cells of the corneal endothelium ( Speakman, 1959a ; Berggren, 1957). They contain numerous vacuoles and react strongly for nonspecific alkaline phosphatases, acid phosphatases, esterases, and succinic dehydrogenase. They may, like the corneal endothelium, take an active part in fluid transport ( Speakman, 1959b). IV. Iris The iris diaphragm arises as an anterior extension of the optic cup and uveal tract from the cup margin. Vrabec (1952) has studied the development of its anterior epithelium in man. This simple epithelium derives from mesoderm which migrates across the anterior surface of the iris to form a continuous squamous sheet. One year after birth the cells of this layer become fibroblastic and deposit reticular fibers. At this time the epithelial discontinuities known as crypts begin to appear. The pigmentophores of the iris migrate in from the more posterior portions of the weal tract. Barden (1942), by means of reciprocal transplantation of optic vesicles between Amblystoma tigrinurn and Triturus torosus, demonstrated that while the time of appearance and the numbers of xanthophores and guanophores in .the iris are host dependent, the patterns formed by thesecelIs are under the influence of the transplanted optic vesicle.

CYTOLOGY O F T H E DEVELOPING EYE

V.

173

Choroid Coat

Recent work on the embryology of the choroid coat deals with the pigment cells. These make their appearance sometime after the onset of pigmentation in the pigmented epithelium of the retina: at 8 to 10 days in the chick embryo (Nordmann and Stoll, 1947; Brini, 1949) ; at 5 months gestation in man (Igo, 1956, 1957) ; and at 22 days gestation in the rabbit (Guttes, 1953a). They appear first at the posterior pole of the eye and migrate forward. Sondermann (1950, 1951) postulates that the pigment of uveal melanoblasts arises from the nuclei of fetal erythroblasts circulating in the blood. This hypothesis seems unlikely in view of the work of Moyer (1960) which clearly demonstrates the development of melanin granules within the Golgi complex.

VI. Ciliary Body The ciliary body appears relatively late in development from extensions of the embryonic retina and uveal tract at the margin of the optic cup. It consists of a highly vascularized mesodermal core in which the ciliary muscles develop, and a complex epithelium which faces the lens and folds to form the ciliary processes. Minute zonular fibrils connect the ciliary folds to the lens capsule and, in the aggregate, constitute the suspensory ligament of the lens. Prior to the appearance of the ciliary body the cup margin is smooth and adheres tightly to the lens capsule at the equator. In the chick embryo the ciliary processes begin to appear on the eighth day of incubation (Coulombre and Coulombre, 1957). The tips of the processes remain adherent to the lens capsule for a time, but the troughs between the processes separate from the capsule, beginning at a point just nasal to the choroid fissure, and quickly proceed around the cup margin in both directions. Intraocular pressure keeps the developing ciliary zone under tension and contributes to the regular geometry of the developing processes. The time at which the ciliary body begins to secrete aqueous humor was determined in the developing rabbit by studies of ascorbic acid concentration and changes in fluid volume (Kinsey et al., 1945). Secretory function begins at 7 to 9 days post partum. The ultrastructural correlates of this functional maturation were followed in the electron microscopic study of Holmberg (1959). At 1, 5, and 30 days post partum the Golgi apparatus of the rabbit ciliary epithelium was in the basal cytoplasm. The nuclei, which were apically situated until 5 days, had, by the thirtieth day, assumed the basal position characteristic of the adult. Numerous vesicles were present in the cytoplasm at all ages. Pappas et al. (1959), in a similar study, focused attention on the first 2 weeks of extrauterine life.

174

ALFRED J. COULOMBRE

They found that the complex interdigitations of the cell membranes of adjacent epithelial cells made their first appearance at 7 days, and thereafter became progressively more complex. They suggest that these interdigitations are associated in an obligatory way with secretory function in this tissue. Later the apical surfaces of the ciliary epithelial cells begin to infold. Lines of minute vesicles near the bases of the infoldings suggest that the folds are budding vesicles into the cytoplasm. Pappas et ul. feel that surface infolding is associated with a selective resorption which gives the aqueous humor its final composition. The appearance of myoblasts, with a highly granular cytoplasm and some myofibrils, on the thirteenth day of incubation, signals the differentiation of chick embryonic ciliary muscle (Woolf, 1956). Cross-striations begin to appear on the fourteenth day. The Q and J bands extend completely across the cells in some instances on the fifteenth day. On the sixteenth and seventeenth days the nuclei move to the periphery of the cells, and fibrillar cross-banding becomes more prominent. By the fourth day after hatching the 2 lines are present. Thus, the ciliary musculature matures toward the end of incubation at a time when the cornea is achieving adult transparency, the retina has become photoreceptive, the pupillary reflex is being established, and the eye as a whole is assuming adult function. VII. Lens

A. INDUCTION AND EARLY MORPHOGENESIS The vertebrate lens arises from competent ectoderm when it is contacted by the developing optic vesicle. I t is not intended to review the vast literature concerning the broader aspects of lens induction. W e will focus attention on the cytologic changes which occur in the cells of the optic vesicle and the contiguous lens ectoderm during the period of induction. Intimate contact between the optic vesicle and the presumptive lens ectoderm is necessary for lens induction to occur (McKeehan, 1951 ; de Vincentiis, 1954). In the chick embryo the optic vesicle contacts the presumptive lens ectoderm at the 9 somite stage. Between the 10 and 20 somite stages the 2 layers adhere tightly, but are again separable at the 26 somite stage. During the period of intimate association, when lens induction is in progress, the cells of the optic vesicle are separated from the overlying ectoderm by a discrete interval which is occupied by PAS+ material. O’Rahilly and Meyer (1960) have demonstrated that this PAS+ layeP results from the fusion of the basement membranes which cover the inner surface of the primitive ectoderm on the one hand, and the outer surface of the early neural tube on the other. Any transfer of substances between the optic vesicle and the lens ectoderm must occur

CYTOLOGY O F THE DEVELOPING EYE

175

across this membrane. There are several indications that substances may move from the optic vesicle to the lens ectoderm during induction. Thus, a sequence of events which suggests the transfer of RNA has been reported for amphibia (de Vincentiis, 1954) and for birds (McKeehan, 1956). Shortly after contact, and only in the area of contact, the cells of the optic vesicle show an increase in RNA (estimated photometrically in stained sections by McKeehan). Subsequently, the amount of RNA decreases in the optic vesicle and increases in the cytoplasm of the overlying lens ectoderm cells. While this may suggest a transfer of material, it does not establish it. A more direct approach was used by Sirlin and Brahma (1959) who l labeled the eye vesicles of Xenopus Zaevis larvae with ~ ~ - 3 - p h e n y(alanine-2C14). The label was incorporated into protein. When such vesicles were confronted with competent ectoderm, and subsequently sectioned and radioautographed, the label was found in high concentration in the cells of the induced ectoderm but not in adjacent ectodermal cells. The cytoplasm of the lens ectoderm cells was more heavily labeled than the nucleus. Whatever the extent of transfer of materials from the optic vesicle to the lens ectoderm may be, the striking feature is its apparently cell-sharp restriction to the area of immediate contact. Contact with the optic vesicle initiates a sequence of changes in the lens ectoderm. McKeehan (1951) has detailed these for the chick embryo. Prior to the 12 somite stage the cells of the lens ectoderm have a vacuolated cytoplasm. Between 12 and 19 somites the vacuoles disappear, the cell number is doubled, the cells palisade (become columnar), and the oval nuclei orient perpendicularly to the cell base. Throughout this period cell volume is constant. The lens placode which is thus formed persists from the 19 to the 24 somite stage. Toward the end of this period acidophilic fibers appear in the apices of the lens placode cells, as the placode invaginates to form the lens vesicle (Langman, 1959a). During the 28 to 32 somite interval the lens fibers of the posterior wall of the vesicle elongate to form the “lens nucleus,’’ and basophilic granules appear in the basal cytoplasm of the lens cells.

LENSCELLS The elongation of the cells of the posterior wall soon obliterates the cavity of the lens vesicle. A t this stage the lens is composed of a “lens nucleus” posteriorly, comprising the primary lens fibers, and a cuboidal lens epithelium anteriorly. The lens epithelial cells are continuous with the fibers of the “lens nucleus” at the equatorial margin of the lens. It is in this marginal zone that the secondary lens fibers form, and proceed to enclose the lens nucleus in concentric layers of fibers, known in the agB.

176

ALFRED J. COULOMBRE

gregate as the lens cortex. In the chick embryo this process commences between 42 and 45 somite€ (Langman, 19S9a). The addition of secondary lens fibers to the cortex continues for an indefinite period. 1. Lens Epithelium, Margin, and Cortex

The regional cytology of the 20-day chick embryo lens reflects this continued differentiation (Hertl, 1955). As one proceeds from the center of the lens epithelium toward the marginal zone and on into the region of the cortical lens fibers, the following sequence of changes is observed. Cell height begins to increase part way toward the lens margin. This is accompanied by an increase in nuclear volume. As the margin is approached there is an increase in both the number of nucleoli and in the number of feulgen-positive granules attached to the nucleoli and to the nuclear membrane. As the cells rotate around the marginal zone they continue to elongate and form cortical fibers. In the process the feulgenpositive granules and the nucleoli decrease in number and the nucleus becomes smaller. The latter observations were confirmed for bovine and human lenses by van den Heuvel (1957). The sequence of changes in lens cell mitochondria shows a parallel behavior (Stroeva, 1959). The early lens vesicle has many rod-shaped mitochondria. These decrease in number and size as the lens fibers develop and become granular as the latter move beyond the marginal zone. Mitochondria are lacking in the central portion of the mature lens. These morphologic changes suggest that the forming lens fibers are in an active synthetic phase at or near the lens margin, and that further differentiation of the lens fibers (e.g., elongation of the cells) is dependent on changes in cell constitution which are initiated near the margin. In addition the involution of mitochondria is reflected in the predominantly anaerobic metabolism of the mature lens fibers.

2. Lens Antigens The types of organ-specific substances synthesized by lens tissue at all stages of development have been investigated by a variety of inmunochemical techniques. Complement fixation and precipitin methods (Burke et al., 1944) detect lens antigen as early as the seventh day of incubation in the chick. The early antigens appeared to differ from those of the adult lens. The capillary precipitin method used by ten Cate (1949), and ten Cate and van Doorenmaalen (1950), revealed substances which reacted to adult lens antiserum in lens vesicles of the chick and frog (60 hours in the chick). Beloff (1959), utilizing the agar diffusion method of Oudin, detected chick lens substances reacting with adult lens antiserum on the fifth day of incubation (stages 24 and 25). An additional band appeared between the eighth and the tenth days, another at 18 days, and

CYTOLOGY OF T H E DEVELOPING EYE

177

a fourth between hatching and adulthood. Langman (1959b) also studied the sequential appearance of lens antigens in the chick, using the sensitive double diffusion technique of Ouchterlony. H e detected no reaction prior to the 19 somite stage. Thereafter additional reacting substances appeared serially. The temporal correlations between the appearance of each new antigen and cytogenic events is interesting and suggests further study. During the lens placode stage (19 to 24 somites), and at the time when acidophilic fibers appear in the cytoplasm, Langman detected one or two bands. At 28 to 32 somites, as the acidophilic fibers enlarge, and as basophilic granules appear in the cytoplasm, three to four bands are in evidence. Between 35 and 37 somites, when the fibers of the lens nucleus are elongating, there were four bands. A t 42 to 45 somites, as the cortical fibers begin to appear in the marginal zone, a fifth band is detected. A sixth band is added at 10 days and a seventh at hatching. The adult yields seven bands. Langman (1959a) used a technique, more sensitive than those already mentioned, to demonstrate the early presence of lens antigen. H e cultured lens ectoderm of different embryonic ages in normal media and in media containing lens antiserum. Outgrowth occurred from all specimens in the normal medium, but failed to take place in the lens antiserum medium in specimens derived from 11 to 17 somite embryos. Lens ectoderm from embryos, younger than 11 or older than 17 somites, grew well even in anti-lens medium. When optic vesicles and their overlying ectoderm were explanted to anti-lens medium the cells of the presumptive lens ectoderm degenerated in specimens from embryos younger than 18 somites. The effect was specific, since adjacent head ectoderm survived the treatment, and the area of destruction was cell sharp at the edge of the presumptive lens area. Langman concludes that the first specific lens antigen makes its appearance at the 11 somite stage, just after contact between the optic vesicle and the overlying ectoderm. Fowler and Clarke (1960) have demonstrated that rabbit antisera to adult chick lens can produce specific defects during development. Pieces of filter paper which had been soaked in the antiserum were applied to the punctured vitelline membrane over the cranial region of 1 to 25 somite chick embryos. Animals treated prior to 10 somites developed forebrain and optic cup abnormalities. Treatment initiated between 11 and 19 somites resulted in eye cup defects and the absence or reduction in size of the lens. Treatment begun from 20 to 25 somites resulted in minor eye cup defects, but a lens was always present. These effects were, there‘fore, region and time specific, and correlate well within the findings of Langman. Some light has been shed on the localization of lens antigens. Clayton (1954), applying lens antisera labeled with fluorescent dyes to sections of

178

ALFRED J . COULOMBRE

the mouse eye, demonstrated lens antigens in the lens, ciliary processes, retina, and in traces in other ocular tissues. Using a similar technique, van Doorenmaalen (1958) could demonstrate lens antigen only in the lens of the chick embryo. The first specific reaction was detected at 5 days of incubation. The antigens were most concentrated in the cytoplasm of cells in the marginal zone. The distribution within the lens cells is the same as that of the acidophilic fibers and the basophilic granules seen when a hematoxylin-azophloxin stain is used. The nuclei showed no reaction. Clarke and Fowler ( 1960) applied fluorescein isocyanate conjugated anti-adult lens globulin to sections of chick embryo eyes between 7 somites and 5 days of incubation. Fluorescence was strongest in the optic vesicle prior to the inductive period, became equal in intensity in the optic vesicle and lens ectoderm during induction, and became stronger in the lens following induction. The substances in the optic vesicle which bind antiadult lens serum are apparently necessary for induction. When vesicles of 4 to 7 somite embryos are explanted for 18 hours to rabbit antiserum to adult chicken lens and subsequently challenged with competent ectoderm, lenses form in only a small percentage of cases. Controls treated with normal rabbit serum induced lenses in a high percentage of cases.

C. LENSCAPSULE The primordium of the lens capsule is derived from the basement membrane which underlies the primitive ectoderm of the embryo (O’Rahilly and Meyer, 1960). During the formation of the lens vesicle the basement membrane is pushed in ahead of the invaginating lens placode. During closure of the lens pore this portion of the membrane closes over the lens vesicle to form a complete capsule. At the same time the continuity of the basement membrane is maintained under the prospective corneal epithelium which is left behind. During subsequent development the capsule thickens, though the mechanisms involved have not yet been adequately studied. Cohen (1958) used the electron microscope to study the capsule of the neonatal mouse lens. At this time it is about 2.5 p in thickness, and is composed of lamellae which are arranged parallel to the lens surface. At the equator there are about forty lamellae, each from 300 to 400 A. thick. I n the adult monkey Wislocki (1952) distinguishes an inner layer, the lens capsule proper, and a thinner outer zone, the pericapsular membrane. The lens capsule is PAS+ and orthochromatic whereas the outer zone is metachromatic and contains considerable reticulin. While adequate developmental +dies are lacking, Wislocki’s observations suggest that the capsule thickens by addition to, its outer surface. I t is at the outer surface of the capsule, near the equator of the lens, that the zonular fibers make their attachment.

CYTOLOGY O F THE DEVELOPING EYE

179

VIII. Retina The two portions of the retina, its pigmented layer and its neural portion, have common origin from the optic vesicle, an outpocketing of the primitive brain wall. These two layers are intimately associated structurally and functionally.

A. PIGMENTED EPITHELIUM Structurally the processes of the pigmented epithelial cells interdigitate with the rods and cones. Functionally the pigmented epithelium is a versatile tissue. Interposed as it is between the neural retina and the richly vascularized choroid coat, it serves as a common avenue of exchange between the vascular compartment and the highly active neural retinal tissue. This takes on added significance when it is recalled that the retinas of submammalian forms (the eel excluded) are avascular, and that even among mammals only the inner layers contain blood vessels. The pigmented epithelium serves vision in several ways. In some forms (chiefly diurnal) it contains melanin and absorbs light which has already traversed the neural retina, thus preventing back-scatter, and improving visual acuity. In other species (chiefly nocturnal) a tapetum is formed in this layer, which reflects light back through the neural retina, thus increasing sensitivity at the expense of acuity. I n certain urodeles this layer is capable of regenerating the neural retina (Stone, 1950). I n view of its obvious importance the developmental cytology of this simple squamous layer has been given little attention. Glycogen accumulates in the pigmented epithelial cells prior to their differentiation. Janosky and Wenger ( 1956) detected moderate deposits of glycogen in the pigmented epithelium of Amblystoma maculatum during the early nonmotile stage (Harrison stage 30) and during the early flexure stage (H-33). The amount of glycogen decreases during the coil stage (H-36). Zimmerman and Eastham ( 1959) demonstrated a nonsulfated acid mucopolysaccharide in the pigmented epithelium of the mouse. It appears during the first week post partum and is secreted onto the surface of the rods as they develop. The early appearance of melanin granules in the chick embryo (Smith, 1920) is correlated with the first detectable tyrosinase activity in this layer (Harrison, 1951) . Smith made the observation that the precursors of the granules were, at first, unpigmented. On the basis of Janus green staining of embryonic chick eyes, Sin-ikk ( 1939) had tentatively identified mitochondria as the organelles which gave rise to melanin granules. This view has not been substantiated by recent work. The detailed light microscopic studies of Giittes (1953a), on rabbit, sheep, and chick embryos reveal that the unpigmented precursors of the melanin granules are

180

ALFRED J. COULOMBRE

associated with the Golgi apparatus. The observation that the Golgi complex, and not the mitochondria, gives rise to the melanin granules was strikingly confirmed by X-irradiation of early chick embryos (Giittes, 1953b). A dose of 1200 r destroys the mitochondria of the pigmented epithelium leaving the Golgi complex intact. Melanin granules appear subsequent to such treatment. Both the mitochondria and the Golgi bodies are destroyed by 1500 r, and no melanin granules appear after such treatment. Spherical melanin granules appear first on the side of the cell away from the neural retina, become progressively darker as melanin is deposited, and elongate into rods as they mature. Albino rabbits have nonpigniented granules similar in time-space distribution and in size to the melanin granules of pigmented forms (Guttes, 1953a). Koecke ( 1959) made similar observations in duck embryos. Moyer (1960) has recently given striking confirmation of these findings in an extensive and elegant study of the developing pigmented epithelium of the mouse. By applying electron microscopy to the developing pigmented epithelia of several mouse strains with different pigment genotypes, he showed that the granules arise in ovoid regions within the Golgi compartment. These regions are bounded by membranes 70 A. thick and contain small vesicles which increase in number and coalesce. The membranous lamellae which result are arranged spirally around the long axis of the ovoid and in successive stages of maturation become studded with electrondense material (melanin ?) which eventually obscures the membranous structure. Subtle differences in the sequence of events characterize the different genotypes. Moyer’s work provides a new and exciting context within which to evaluate the roles of genetic, chemical, and physiologic factors in cytodifferentiation. It should be emphasized that the function of the pigmented epithelium is not restricted to melanogenesis. It must certainly be active in a two-way transport of substances between the outer layers of the retina and the blood stream. The identification of its specific activities, and investigations of how these interlock with the activities of the neural retina, demand attention. There are few areas which promise to yield as much in terms of understanding the complex interrelationships which exist between epithelial sheets and adjacent tissues. B.

NEURAL RETINA

Invagination of the optic vesicle results in the progressive obliteration of the vesicle cavity. Eventually the inner layer (prospective neural retina) becomes approximated to the outer layer (prospective pigmented epithelium). When the vitreous humor begins to accumulate between the lens

CYTOLOGY O F T H E DEVELOPING EYE

181

and the neural retina, the margin of the optic cup retains its connection with the lens equator. Thus, following closure of the choroid fissure, the vitreous chamber becomes a closed space, and the accumulation of vitreous humor is responsible for the subsequent increase in the size of the eye (Coulombre, 1956b). While the sclera, choroid coat, and pigmented epithelium will not expand in the absence of an enlarging vitreous body, the neural retina continues to increase in area when vitreal expansion is prevented. This can best be understood in terms of the mitotic patterns of the embryonic retina.. In sections of immature retinas mitotic figures are confined to the outer surface of the tissue and to the margin of the optic cup. Their location next to the now-obliterated ventricular space of the primary optic vesicle is analagous to that seen in the rest of the central nervous system (Da Costa, 1947). When the nuclei are labeled with tritiated thymidine at appropriate stages in development their subsequent fate can be followed. If, after a discrete interval, such retinas are fixed, sectioned, and radioautographed, it can be shown that cells produced in the mitotic zone migrate deeper into the retina to take up appropriate positions in the layers which are differentiating internally (Sidman, 1960). In this, too, the neural retina resembles the embryonic brain wall. The mitotic activity at the external surface of the retina, then, adds to its thickness. The mitotic activity at its margin adds to its area. The oldest portions of the retina differentiate first. Thus the fundic region is in advance of the margin. Further, the inner layers differentiate before the more superficial zones. The latter concept must now be somewhat modified since it is clear from Sidman’s work that migration from the superficial layers into the deeper zones can occur even after these are discrete and their cells well differentiated. Once the visual cells begin to differentiate mitosis ceases in the outer retinal zone. Mitotic activity continues longest at the cup margin, but eventually ceases there, also. Thereafter the retina expands in area to keep pace with the growing eye (Coulombre, 1955). The forces responsible for this late expansion are generated by the growing vitreous body (Coulombre, 1956b). Most of the cytologic studies of the developing neural retina fall into two groups. The first deals with the application of existing histochemical and cytochemical techniques to the developing retina as a whole. The second group is concerned with observations of the structure and function of the rods and cones during development.

1. Developmental Retinal Histochemistry The developing retina affords a unique context within which to study the sequence of events in chemical maturation. I t contains a diversity of welldefined cell types : the glial elements (Miiller’s fibers), the photoreceptive

182

ALFRED J. COULOMBRE

rods and cones, and the several types of neurons. During development these are separated into discrete strata according to kind. The strata differentiate in turn, in an orderly temporal and spatial sequence. The structural and functional interrelations among the cells of the different layers are established one after another, and, although in time they become exceedingly complex, they remain of a highly ordered nature. Thus it becomes possible not only to determine the sequence of events in the cytogenesis of each cell type, but also to see how the timetable of differentiation of each cell type fits into the pattern of interrelationships which is evolving at the supracellular level. Initially the cells of the presumptive neural retina are indistinguishable one from another. As each layer differentiates its cells undergo a characteristic sequence of changes. The most striking changes common to all of the cellular types concern nucleic acids. Nakayama (1957a) followed the distribution of nucleic acids in the developing human retina, using feulgen, thionine, and methyl green-pyronine stains. Miiller’s fibers, which undergo an early differentiation, are strongly pyronine positive until the fourth month, when the intensity of staining decreases. The ganglion cells are moderately pyronine positive until they begin to elaborate the Nissl bodies at about 4 months, when there is a marked increase in cytoplasmic pyronine positivity. The cells of the outer mitotic zone are strongly pyronine positive until mitosis ceases at 4 months. The protoplasmic buds (presumptive inner segments of the rods and cones) are positive from the outset. The outer segments are positive during their period of most active differentiation ( 6 to 7 months). Da Costa (1947) confirms the early appearance of basophilia in the cytoplasm of human ganglion cells. Rickenbacher (1952) obtained comparable data in similar studies of salamander and chick embryos. H e notes, in addition, that the nucleoli of retinal neuroblasts become prominent during the maturation phase and that R N A appears first in a cap overlying the nucleus. The increase in cytoplasmic ribonucleoprotein during maturation is accompanied by a progressive decrease in nuclear volume (Genis-Gilvez, 1956), and by an increase in dry weight and protein concentration (Brattgsrd, 1952). It appears from these studies that ribonucleoprotein increases in the cells of the retinal layers in sequence, starting with the ganglion cell layer, before or during protein elaboration. In the frog the differentiation of each retinal stratum is accompanied by a wave of cell deaths (Gliicksmann, 1940). The significance of this is discussed by Giicksmann ( 1951) in a general review of the topic. Following these early developmental events, further changes are seen in late development in some forms. In the chick embryo retinal protein content rises to peaks on the twelfth, sixteenth, and eighteenth days

CYTOLOGY OF T H E DEVELOPING EYE

183

(Kataoka, 1955). Following these peaks Nissl bodies appear in ganglion cells at 17 days, in amacrine cells at 18 days, and in bipolar cells at 19 days. Retinal cytogenesis is characterized by other changes. There is general agreement that in the retina, as in many other embryonic tissues, demonstrable alkaline phosphatase is in high concentration during cellular differentiation and decreases after this process is complete (Moog, 1944; Osawa, 1951 ; Yoshido, 1958a). I n a more extensive study of the developing chick retina, Rebollo (1955) found that stains for alkaline phosphatase activity become intense in the ganglion cells at 17 days and increase progressively in intensity until 20 days of incubation. The increase in phosphatase activity appears successively in the retinal layers, in correlation y i t h the time of appearance of the Nissl substance and neurofibrils. This complex of events in the retina corresponds in time with the first detectable electroretinogram (ERG) (Garcia-Austt, 1953). The localization of mucopolysaccharides in the developing retina has yielded information that correlates with the changes already discussed. Bembridge and Pirie ( 1951) localized PAS+, Best’s carmine-positive, water-soluble granules in all layers of the rabbit retina. The granules appear at 8 days post parturn and disappear after the third week. They are most concentrated in the inner retinal layers, and are present during the time of terminal differentiation of the retinal cells. Koishikawa and Kuroki (1957a) studied these granules in more detail in the ganglion cells of the postnatal rabbit. The granules were most abundant between 10 and 20 days post partum and are probably glycolipid in composition. These authors identified a second, diffuse PAS+ substance in the central portions of the ganglion cells. It appears 28 days after fertilization, and increases to a maximum at about 10 days post partum. They believe it to be a glycolipid-protein combination. Essentially the same sequence of changes occurs during the terminal maturation of the developing cat retina (Koishikawa, 1957). I n an amphibian (Amblystoma maculatum) Janosky and Wenger (1956) found glycogen granules throughout the neural retina, but particularly in its inner layers during the period of maturation. They suggest that it is utilized as an energy source during the period of rapid cellular differentiation. Koishikawa and Kuroki ( 1957b) found that the peak level of glycogen in the developing visual cells of the Japanese toad occurred during active cytogenesis, and suggest that the glycogen depot is used in lipid synthesis. In the frog (Rana temporaria) SaxCn (1955) found glycogen granules in the visual cells (in addition to those amassed in the paraboloid) near the time of metamorphosis. In man, too, the glycogen granules are present in the visual elements at about the time they undergo terminal differentiation (Nakayama, 1957b). In the chick Rebollo and Casas (1956) found that mucopolysaccharides appear

184

ALFRED J. COULOMBRE

in the retinal cells at the times when and in the places where an increase in ribonucleic acid and aikaline phosphatase signals cellular differentiation. These authors also detected a concentration of glycogen in the paraboloid on the nineteenth day of incubation. The outer segments of the visual cells are PAS+ from the time they first appear in the kitten (Sidman and Wislocki, 1954) and in the chick embryo (Rebollo and Casas, 1956). The synaptic patterns in the neural retina are complex, but orderly because of the separation of different neuronal types into discrete strata. Polyak (1941), using adult material, took advantage of this in his studies of retinal synaptic interconnections. The use of the developmental parameter gives the added advantage that, from relatively simple beginnings, the synaptic connections of different types are established one after another in a definite sequence. Thus, studies of the developing retina afford a temporal as well as a spatial separation of the several neuronal types which facilitates morphologic and functional analyses. In the chicken, for esample, the synapses which occur in the inner plexiform layer are not randomly scattered but are marshaled into discrete layers which lie parallel to one another and are circumferential with respect to the eye wall. During development these synaptic mats appear one after another in an orderly sequence (Coulombre, 1955). This sequential appearance of circumferential planes, representing the terminal expansions of neuronal processes, suggests an orderly development of synaptic connections between the bipolar and amacrine cells on the one hand, and the ganglion cells on the other. Shen et al. (1956) confirmed this by use of silver stains. They extended the study by localizing acetylcholinesterase in the retina of the chick embryo. This enzyme appears first in the cytoplasm of the ganglion cells on the ninth day. Thereafter it appears in turn in the amacrine cells, the horizontal cells, and the itiner segments of the rods and cones. The latter localization holds for the frog (Yoshido, 1958b). The chick embryo bipolar cells and the outer plexiform layer are relatively free of acetylcholinesterase. Within the inner plexiform layer the endynie is localized in the synaptic mats and appears sequentially as they appear. I t is to be hoped that these studies will be extended to increase our knowledge, not only of the cytoarchitecture of the neural retina, but also of those factors which govern the development of specific neuronal patterns and the eniergence of function. Several other substances have been localized in the developing retina. Hellstrom (€956) found that succinic dehydrogenase was demonstrable in the outer layers (presumptive inner segments) of the rat retina on the first day following birth. By the ninth day the plexiform layers showed detectable levels of the enzyme. Kojima et al. (1957) found that succinic

CYTOLOGY O F T H E DEVELOPING EYE

185

dehydrogenase appears during the twelfth to the fifteenth day of incubation in the chick embryo. Inami (1953) found that thiamine appeared in the chick embryo retina on the thirteenth day, and that its localization paralleled that in the postnatal rat. While the neural retina remains avascuiar throughout the life of most vertebrates, mammalian retinas become partially vascularized during development. The grosser morphological patterns of the retinal vessels in the adults and embryos of many mammals are described by Michaelson (1948a,b, 1954). The vessel patterns in the optic nerve and lamina cribrosa are recorded by Franqois and Neetens (1954). I n human retinas vessel outgrowth from the head of the optic nerve into the nerve fiber layer begins at 12 weeks of gestation (Serpell, 1954; Nilausen, 1958). At first there are four separate, radially arranged vascular complexes, but these later fuse to form a continuous advancing edge. This is surrounded by a fringe of radially oriented, spindle shaped cells. Their nuclei exhibit dustlike chromatin, and their cytoplasm contains glycogen granules. The zone associated with the growing tips of the vessels reacts positively for alkaline phosphatase.

2.

Visual Cells

The visual cells are composed of inner dendritic processes which synapse in the outer plexiform layer, of cell bodies in the outer nuclear layer, and of the visual elements protruding through the external limiting membrane. The nuclei of the visual cells appear similar at first in the post partum rat, but later differentiate into two types, one of which is characteristic of cone cells and one of rod cells (Brockhoff, 1957). The adult visual elements, the rods and cones, comprise an inner segment, located just outside the external limiting membrane, and an external segment, which is photosensitive and which interdigitates with the processes of the pigmented epithelial layer. The comparative adult morphology, and the details of histogenesis of these elements, has been ably summarized in a number of works (cf. Detwiler, 1943 ; Rochon-Duvigneaud, 1943 ; Walls, 1942; Wolff, 1958). W e shall be concerned here only with the recent additions to our knowledge of the cytogenesis of the visual cells. a. Inner Segment. As mitosis ceases in the outer layers of the developing retina, protoplasmic buds (blebs) of the visual cells begin to protrude through the external limiting membrane. These are the precursors of the inner segments of the rods and cones. Shortly after a bleb forms the diplosome migrates into it (Tokuyasu and Yamada, 1959), and a dense aggregation of mitochondria appear at the bleb apex (Lewis, 1923). The prospective inner segments of the rods and cones will elaborate several specialized organelles : myoid, ellipsoid, paraboloid, arid oil droplet. The

186

ALFRED J. COULOMBRE

presence or absence of each of these elements in the rods or cones of different species is summarized by a niiniber of authors (cf. Detwiler, 1943; Prince, 1956), and need not be detailed here. The development of the contractile inner region of the inner segment (myoid) has been given scant attention. Shen et al. noted that it was rich in acetylcholinesterase activity in the chick embryo. This enzyme is present also in the adult frog inyoid (Yoshido, 1958b). Other components of the neural retina have been given more attention. The ellipsoid or lentiform body is a remarkably constant feature of vertebrate rods and cones. It is localized at the outer end of the inner segment, and begins to form shortly after the blebs push through the external limiting membrane. The blebs appear in the chick embryo at about 10 or 11 days of incubation (Coulombre, 1955). At 11 days Lewis (1923) detected a scattering of Janus green positive bodies at the apical pole. At about 15 days these aggregate to form a compact body. Carasso (1958) observed the same sequence in amphibia ( A l y t e s obstetricans, Rana temporaria) in an electron microscopic study. I n keeping with its structure the ellipsoid is a focus of high succinic dehydrogenase activity in the rat from the ninth day after birth (Hellstrom, 1956). The paraboloid lies proximal to the ellipsoid. It makes its appearance relatively late in development (SaxCn, 1955 ; Rebollo, 1955), and is characterized by a high concentration of glycogen. Carasso (1960) followed the details of its development in the Pleurodele, using the electron microscope. Initially endoplasmic reticulum studded with Palade granules appears proximal to the ellipsoid. Subsequently aggregations of granules appear in the spaces within the reticulum, and increase in number. The diameter of these granules in sections ranges from 150 to 250 A. Their core is more electron dense than the cortex, and Carasso suggests that the core may be protein in nature and that the cortex may contain glycogen. As the number of granules increases they form a tightly packed mass, the paraboloid. Eventually the membranes of the endoplasmic reticulum disappear from the center of the paraboloid. The oil droplets are clear, spherical bodies located distal to the ellipsoid. In many species these are colorless. I n the cones of some reptiles and birds they are colored due to the presence of carotenoids. In the hen retina four carotenoids have been identified : astacene, xanthophyll, carotenoid hydrocarbon, and galloxanthin (Wald and Zussman, 1937, 1938). Of these at least astacene must by synthesized, presumably by the retina, since it appears before hatching even though it is absent in the hen’s egg. In the chick embryo the dr4plets begin to appear at the distal end of the inner segment on the thirteenth or fourteenth day of incubation (Coulombre, 1955). By the.fifteenth day, these droplets begin to assume their

CYTOLOGY OF T H E DEVELOPING EYE

187

adult hues (red, yellow green, or yellow). It is first possible to demonstrate astacene photometrically in retinal extracts on the fifteenth day. The amount of this substance in each retina increases rapidly until hatching, and thereafter more slowly. This increase is attributable to a progressive increase in volume of the red droplets rather than to an increase in their numbers. It has been noted by many (e.g., Carasso, 1958) that the oil droplet arises in the midst of the mitochondria which will form the ellipsoid. While attempts to critically evaluate the function of these little droplets are urgently needed, they deserve attention for yet another reason. In those forms with oil droplets of different colors each cone apparently concentrates only one species of carotenoid in its droplet. Thus the droplets indicate a differential distribution among the cones of carotenoid synthetic abilities. This invites attention to the mechanisms underlying the development of such differences, and to the factors which govern the spatial distribution of the different types of cones on the surface of the retina. b. Outer Segment. The outer segments of the rods and cones are the last elements to differentiate in the vertebrate retina. While it has long been suspected that they take their origin from cilia arising from the developing inner segments, it has remained for recent electron microscopic studies to establish this point and to reveal something of the manner in which the differentiation occurs. Sherman ( 1951) examined formalinfixed sections of tadpole and adult frog retinas cut at 0.2 p and concluded that the cilium appeared at the 9 mm. stage, became “cross-striated” by the 15 mm. stage, and contained a longitudinal filament at all stages. De Robertis (1956) examined the fine structure of the retinal rods of white mice from birth to the sixteenth day post partum, and describes in more detail three stages in the formation of the outer segment. In the first stage the basal body (outer centriole of the diplosome), located in the inner segment, gives rise to a cilium containing nine pairs of peripheral filaments but lacking central filaments. The cilium grows out from the inner segment in an envelope of cytoplasm bounded by the plasma membrane. This cytoplasm contains a number of small, membrane-bound vesicles which De Robertis refers to as “morphogenetic material,” and which he believes to be precursors of the rod sacs. In the second stage, larger membrane-enclosed spaces, the rod sacs, appear at the outer tip of the developing external segment. Each is connected by a tubular stalk to the region of the peripherally placed cilium (axial filament). In the third stage, the sacs, at first vesicular and irregular in shape, flatten down to form double membrane discs that are arranged in a regular stack. The plane of each disc is perpendicular to the axis of the outer segment. This rearrangement proceeds from the center of the outer segment toward each

188

ALFRED J. COULOMBRE

end. A parallel study of the process in amphibia (Pleurodele) revealed a similar series of changes ihere. Carasso (1959) confirmed the presence of small vesicles in the flagellar process, and noted that the rod sacs take their origin from infoldings in the plasma membrane at both ends of the outer segment. The rod sacs flatten and stack at the center of the outer segment, and additions to the stack account for increase in the length of the outer segment. Carasso noted that, in axial sections, the rod sacs were separated from the cilium by rows of vesicles, and suggests that coalescence of these vesicles with the sacs accounts for their progressive increase in area, and coiisequently for the increase in diameter of the outer segment. In sections the profiles of osmium-fixed rod sacs become more electron dense as development proceeds, a change which invites farther study since it suggests a progressive internal differentiation of the membrane. An elegant study of the developing fine structure of the kitten and mouse rod outer segment (Tokuyasu and Yamada, 1959) adds further detail. As the primitive inner segment begins to protrude through the external limiting membrane the diplosome moves into it. The more distal centriole sends out ciliary tubules. When these achieve a height of 1.0 to 1.5 p the enveloping cytoplasm swells apically and becomes clublike. From the tip of each ciliary tubule rows of minute vesicles (ciliary vesicles) stretch toward the apical plasma membrane. At the points where the vesicles contact the plasma membrane large infoldings occur. These will become the rod sacs in the manner described above. In the meanwhile the basal body has undergone changes. It puts out several radial filaments (60 to 100mp long and 15 to ZOmp in diameter) at right angles to its axis. Each of these is capped by a spherule 60 to 8 0 m p in diameter. It is clear that the rod sacs form from the plasma membrane. It is attractive to assign to the ciliary vesicles the role of eliciting this local response. Support for this notion derives from observations of abnormally developing rods. Tokuyasu and Yamada (1960) found that in some instances the connection between the inner and outer segments (ciliary tubule) is not normally constricted, but is wide, allowing extensive continuity between the contents of the inner and outer segments. In such cases the ciliary tubules are not aligned in a parallel bundle, but tend to become splayed out. When this is the case, the ciliary vesicles can impinge on the plasma membrane of the base of the outer segment or the apex of the inner segment. Rod sacs form at these points of contact. This not only suggests that the vesicles are the stimulus for rod sac formation, but also indicates thal the plasma membrane of the inner segment is competent to respond. These observations bring into sharp focus the existence of interrelations among cell organelles during development that are analogous to inductive interactions between cell aggregates in the embryo.

CYTOLOGY OF T H E DEVELOPING EYE

189

There are several functional correlates of these events. Tansley (1933) demonstrated that visual purple could first be demonstrated in the rat at the time the outer segments began to develop. Thus, the synthesis of rhodopsin appears to occur as soon as the rod sacs begin to appear. Contact between the neural retina and the pigmented epithelium is not essential for either the differentiation of the outer segments or the elaboration of rhodopsin, since both appear in the developing neural retina when it is isolated in tissue culture. The retinal degeneration which occurs when the developing retiw is separated experimentally from the pigmented epithelium in vivo (Coulombre, 195Gb) is, therefore, probab!y the result of removing the retina from its nutritional supply. The appearance of rhodopsin may be the terminal step in the functional maturation of the rods in some species. Thus, Detwiler (1932) noted the light-avoiding reaction in young rats just as the outer segments began to appear. In the rabbit the electroretinograni ( E R G ) can first be elicited at 8 days post partum, when the outer segments appear. At this time the outer segments are only fifteen per cent as long as their adult counterparts. One must conclude that the entire chemical and physical apparatus necessary to trigger a response to light is already present in the young outer segments, and that further growth of these elements probably effects quantitative changes without qualitatively altering the functional properties of this structure. The dog (several breeds) also shows a synchronous development of the outer segments and the ERG. Parry (1953) first detected the a-wave at about 80 days post coitus, when the outer segments were first seen. The b-wave appeared at 83 days and the adult ERG was established by about 100 days. In the predominantly cone retina of the chick embryo the situation differs. The outer segments are present as cilia on the twelfth day (Rebollo, 1955) and begin to develop rapidly on the fifteenth (Cou!ombre, 1955). It is not, however, until the eighteenth day that the embryonic E R G is first detected, and the nineteenth day that the adult ERG appears (Rebollo, 1955 ; Garcia-Austt, 1953). Rebollo notes that the outer segments are metachromatic with the Nissl stain up to the eighteenth day. This might reflect a continued maturation of the outer segments, until, at this time, they become functional. On the other hand, the limiting steps in retinal differentiation may occur in other layers of the retina since, as noted above, the ganglion cells and amacrine cells of this species are undergoing notable changes in the 17- and 18-day chick. Lindeman (1947) used the pupillary constrictor reflex of the chick embryo as a test for the onset of function and elicited the first response on the nineteenth day of incubation. Since central stimulation could elicit pupil constriction prior to this time, he concluded that the retina could not respond to light until

190

ALFRED J. COULOMBRE

about 19 days. A similar delay between the advent of the outer segments and the onset of function-was noted in the groundling. The predominantly cone retina of this form has all of its layers by one month, but does not show a distinct reaction to light until the end of the second month (Krainisheva, 1956). The available evidence points to a possible difference between predominantly rod and predominantly cone retinas. It is clear that the rod outer segment can play its role in photoreception as soon as it begins to form and before it is fully grown. The delay between the. appearance of the cone outer segment and the onset of visual function suggests that components of the cone outer segment are immature when they first form. Alternatively, it is possible that it is the deeper portions of the neural retina that have not differentiated to a functional state.

REFERENCES Alagna, G. (1954) Arch. ottalmol. 68,259-271. Amprino, R., and Pansa, E. (1955-1956) Wilhelm Roux' Arch. Enhwicklzingsmech. Organ. 148, 179-194. Aurell, G., and Holmgren, H. (1953) Acta Ophthalmol. 31, 1-28. Barden, R. B. (1942) J . Exptl. Zool. 90, 479-519. Beloff, R. H. (1959) J . Exptl. Zool. 140, 493-518. Bembridge, B. A., and Pirie, A. (1951) Brit. J . Ophthalmol. 35, 784-789. Berggren, L. (1957) Acta S O C Med. . Upsaliensis 62,157-175. Brattgird, S. (1952) Acta Radiol. Suppl. 96,44-50. Brini, A. (1949) Bull. soc. opthalmol. France 3, 474. Brockhoff, V. (1957) Anat. Anz. 104, Suppl., 162-166. Burke, V., Sullivan, N. P., Peterson, H., and Weed, R. (1944) I . Infectious Diseases 74, 225-233. Calmettes, L., Deodati, F., Planel, H., and Bec, P. (1956) Arch. ophtalmol. ( P a r i s ) 16, 481-506. Carasso, N. (1958) Compt. rend. acad. sci. 247, 527-531. Carasso, N. (1959) Compt. rend. acad. sci. 248,3058-3060. Carasso, N. (1960) Compt. rend. mad. sci. 260, 600-602. Clarke, W. M.,and Fowler, I. (1960) Develop. Biol. 2, 155-172. Clayton, R. M. (1954) Nature 174, 1059. Cohen, A. I. (1958) A m . J . Anat. 103, 219-245. Coulombre, A. J. (1955) A m . J. Anat. 96,153-189. Coulombre, A. J. (19%) Anat. Record 124, 278. Coulombre, A. J. (1956b) J . Exptl. 2001.139, 211-226. Coulombre, A. J., and Coulombre, J. L. (1957) A m . J . Ophthalmol. 44, No. 4, Part 11, 85-92. Coulombre, A. J., and Coulombre, J. L. (1958a) A m . J . Ophthalmol. 45, 291. Coulombre, A. J., and Coulombre, J. L. (1958b) J . Cellular Comp. Physiol. 51,

1-12. Da Costa, A. C. (1947) Acta Anat. 4,79-86. Davson, H. (1955) Biochem. J . 59, 24-28. De Robertis, E. (1956) J. Biophys. Biochem. Cytol. 2, Suppl., 209-218.

CYTOLOGY OF T H E DEVELOPING EYE

191

Detwiler, S. R. (1932) J. Comp. Neurol. 55, 473-492. Detwiler, S. R. (1943) “Vertebrate Photoreceptors.” Macmillan, New York. de Vincentiis, M. (1954) Riv. biol. (Perugia) 46, 173-192. Dunnington, J. H., and Weimar, V. (1958) Am. J. Ophthalmol. 46, No. 4, Part 11,

89-95. Fornes-Peris, E. (1948) Arch. SOC. oftalmol. hispano-am. 8, 821-826. Fowler, I., and Clarke, W. M. (1960) Anut. Record ls6, 194-195. Francois, J., and Neetens, A. (1954) Brit. J. Ophthulmol. 98,472-488. Garcia-Austt, E. (1953) “Trabajo de Adscripcion de Fisiologia,” Facultad de Medicina, Montevideo ; cited in Rebollo (1955). Gemolotto, G., and Patrone, C. (1955) Giorn. ital. oftalmol. 8, 42-52. Genis-Gilvez, J. M. (1956) Arch. SOC. oftalmol. hispano-am. 16, 59-87. Ghiani, P.,and Bergamini, G. (1957) Atti accad. ligure sci. e lcttere (Genoa) 14, 298-301;(1959) Chem. Abstr. 63 (22378),abstr. Gliicksmann, A. (1940) Brit. J. Ophthalmol. 24, 153-178. Gliicksmann, A. (1951) Biol. Revs. Cambridge Phil. SOC. 26,59-86. Giittes, E. (1953a) Z. Zellforsch. u. mikroskop. Anat. SS, 168-202. Giittes, E. (1953b) Z. Zellforsch. u. mikroskop. Anat. SS, 260-275. Harris, J. E. (1957) Am. J . Ophthalmol. 44, No. 5, Part 11, 262-280. Harrison, J. R. (1951) J. Exptl. 2001. 118, 209-241. Hellstrom, B. E. (1956) Acta Pathol. Microbiol. S c u d . 39,8-14. Herrmann, H. (1958) I n “A Symposium on the Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), pp. 329-338. Johns Hopkins Press, Baltimore, Maryland. Hertl, M. (1955) Z. Zellforsch. u. mikroskop. Anat. 43, 228-242. Holmberg, A. (1959) A.M.A. Arch. Ophthalmol. 6!2, 935-948. Igo, Y. (1956) Acta SOC. ophthalmol. Japan. 60, 658-673; (1956) Ophthalmic Lit. 10 (1445), abstr. Igo, Y. (1957) Japm. J. Ophthalmol. 1, 124-130; (1957) Ophthalmic Lit. 11 (1554), abstr. Inami, E. (1953) Acta. SOC. ophthalmol. Japan. 67, 346-349; (1953) Ophthalmic Lit. 7 (1157), abstr. Jackson, S. F. (1955) Nature 176, 39-40. Jakus, M. A. (1954) Am. J. Ophthalmol. 98, No. 1, Part 11, 40-52. Jakus, M. A. (1956) J. Biophys. Biochem. Cytol. 2, Suppl., 243-252. Janosky, I. D., and Wenger, B. S. (1956) J. Comp. Newrol. 106, 127-150. Kataoka, S. (1955) Acta SOC. ophthalmol. Japan. 59, 1603-1609,1676-1677;(1955) Ophthalmic Lit. 9 (2392), abstr. Kikkawa, Y. (1955) Japan. I . Physiol. 5, 167-182. Kikkawa, Y. (1956) Japan. J. Physiol. 6, 300-312. Kikkawa, Y. (1959) J. Clin. Ophthalmol. ( T o k y o ) 13, 1409-1421. Kinsey, V. E., Jackson, B., and Terry, T. L. (1945) A.M.A. Arch. Ophthalmol.

a,415-417.

Koecke, H. U. (1959) Z. Zellforsch. u. mikroskop. Anat. 60,238-274. Koishikawa, S. (1957) Okajimas Folia Anut. Japon. SO, 139-154; (1958) Excerpta . Med. Sec. I, l2 (3812), abstr. Koishikawa, S.,and Kuroki, S. (1957a) Acta Anat. Nippon. 32, 395-398; (1958) Excerpta Med. Sec. I, l2 (3503), abstr. Koishikawa, S.,and Kuroki, S. (1957b) Acta Anut. Nippon. 32, 406-415; (1958) Excerpta Med. Sec. I , 12 (3502), abstr.

192

ALFRED J . COULOMBRE

Kojima, K., Okada, S., and Majima, Y. (1957) Acta soc. ophthalmol. Japan. 61, 925-935 ; (1957) Ophthalmic Lit. 11 (1625), abstr. Kramisheva, V. N. (1956) Doklady Akad. Nauk. S.S.S.R. 109, 1219-1221 ; (1959) Intern. Abstr. Biol. Sci. 14 (1853), abstr. Langman, J. (1959a) J . Embryol. Exptl. Morphol. 7 , 264-274. Langman, J. (1959b) I. Embryol. Exptl. Morphol. 7 , 193-202. Lewis, M. R. (1923) Anat. Record 25, 110-111. Lindeman, V. (1947) Am. J. Physiol. 148, 40-44. McKeehan, M. S. (1951) J . Exptl. 2001.117, 31-64. McKeehan, M. S. (1956) A m . J. Anat. 99, 131-155. Mann, I. (1950) “The Development of the Human Eye.” Grune and Stratton, New York. Maurice, D. M. (1957) J . Physiol. (London). 136,263-286. Meyer, D. B., and O’Rahilly, R. (1959) J . Embryol. Exptl. Morphol. 7 , 303-315. Meyer, K., Linker, A., Davidson, E. A., and Weissmann, B. (1953) J. Biol. Chem. 206, 611-616. Michaelson, I. C. (1948a) Trans. Ophthalmol. SOC.United Kingdom 68, 137-180. Michaelson, I. C. (1948b) J. Anat. 82, 167-174. Michaelson, I. C. (1954) “Retinal Circulation in Man and Animals.” C. C Thomas, Springfield, Illinois. Moog, F. (1944) Biol. Bull. 86, 51-80. Moyer, F. H. (1960) Anat. Record 136, 248. Nakayama, K. (1957a) J . Clin. Ophthalmol. (Tokyo) 11, 1024-1032. Nakayama, K. (1957b) J . Clin. Ophthulmol (Tokyo) 11,635-644. Naylor, E. J. (1953a) Brit. J . Ophthalmol. 37, 77-84. Naylor, E. J. (1953b) Quart. J . Microscop. Sci. 94, 83-88. Nilausen, K. (1958) Acta Ophthalmol. 36, 65-70. Nordmann, J., and Stoll, R. (1947) Ophthalmologica 114,99-102. Offret, G.,and Haye, C. (1959) Arch. ophtalmol. (Paris) 19, 126-159. O’Rahilly, R., and Meyer, D. B. (1959) Acta Anat. S6, 20-58. O’Rahilly, R., and Meyer, D. B. (1960) Z . Anat. u. Entwicklungsgrschichte l2l, 351-368. Osawa, S. (1951) Embryologia 2, 1 ; cited in Yoshido (1958). Pappas, G. D., Smelser, G., and Brandt, P. W. (1959) A.M.A. Arch. Ophthalmol. 62, 959-965. Parry, H. B. (1953) Brit. J . Ophthalmol. 37, 385-404. Polyak, S. (1941) “The Retina.” Univ. Chicago Press, Chicago, Illinois. Polyak, S. (1957) “The Vertebrate Visual System” (H. Kliiver, ed.), Univ. Chicago Press, Chicago, Illinois. Prince, J. (1956) “Comparative Anatomy of the Eye.” C. C Thomas, Springfield, Illinois. Rebollo, M. A. (1955) Acta neurol. latino-am. 1, 142-147. Rebollo, M. A., and Casas, M. (1956) Acta nrurol. latino-am. 2, 310-314. Redslob, E. (1956) Ann. oculist. (Paris) 189, 749-759. Rickenbacher, J. (1952) Wilhelm Roux’ Arch. Entwicklungsmech. Organ. 146, 387-402. Rochon-Duvigneaud, A. (1943) “Les Yeux et la Vision des Vertebrks.” Masson, Paris. Saxen, L. (1954) Ann. Acad. Sci. Fennicae Ser. A . IV 23, 1-93. Saxen, L. (1955) Acta. Anat. 26, 319-330.

CYTOLOGY OF T H E DEVELOPING EYE

193

Schwarz, W. (1953) Z. Zellforsch. u. mikroskop. Anat. 38, 78-86. Schwarz, W. (1960) Anat. Record 136,275. Seo, S. (1955) Kytishti Menz. Med. Sci. 6, 169-182; cited in O’Rahilly and Meyer (1960). Serpell, G. (1954) Brit. J . Ophthalmol. 38, 460-471. Sheldon, H. (1956) J. Biophys. Biochem. Cytol. 2, 253-262. Shen, S.-C., Greenfield, P. C., and Boell, E. J. (1956) J. Conzp. Neurol. 106, 433-461. Sherman, J. K. (1951) Anat. Record 109,383. Sidman, R. (1960) Anat. Record 136, 276-277. Sidman, R., and Wislocki, G. B. (1954) J. Histochem. and Cytochem. 2, 413-433. Sin-ik6, T. (1939) Okajimas Folia Anat. Japon. 18, 371-377; (1940) Brit. Chem. and Physiol. Abstr. A. 111, 86, abstr. Sirlin, J. L., and Brahma, S. K. (1959) Develop. Biol. 1, 234-246. Smelser, G. (1952) A.M.A. Arch. Ophthalmol. 47, 328-343. Smelser, G., and Ozanics, V. (1952) Science 116, 140. Smelser, G.,and Ozanics, V. (1956) Anat. Record 124, 362. Smelser, G.,and Ozanics, V. (1957) Am. 1. Ophthalmol. 44, No. 4, Part 11, 102-110. Smelser, G., and Ozanics, V. (1959) A m . J. Ophthalmol. 47, 100-101. Smith, D. T. (1920) Bull. Johns Hopkins Hosp. 31, 239-246. Smits, G. (1957) Biochim. et Biophys. Acta 26, 542-548. Sondermann, R. (1950) Arch. Ophthalmol. Graefe’s 160, 580-591. Sondermann, R. (1951) Ophthalmologica 122, 166-171. Speakman, J. (1959a) Proc. Roy. SOC.Med. 62,72-74. Speakman, J. (1959b) Brit. J . Ophthalmol. a,129-138. Stanworth, A. (1950) Acta 16th Concilium Ophthalmol. (Britannia) pp. 1368-1376. Stanworth, A., and Naylor, E. J. (1950) Brit. J . Ophthalmol. 34, 201-211. Stanworth, A., and Naylor, E. J. (1953) J. Exptl. Biol. 30, 160-163. Stone, L. (1950) A n d . Record 106,89-110. Stroeva, 0.G. (1959) Doklady Akad. Nauk. S.S.S.R. 126, 461-464; (1959) Intern. Abstr. Biol. Sci. 16 (3979), abstr. Tansley, K. (1933) Proc. Roy. SOC.B114,79-103. ten Cate, G. (1949) Be1g.-Ned. Cyto-embryol. Dagen (Ghent), p p . 92-95. ten Cate, G., and van Doorenmaalen, W. J. (1950) Proc. Koninkl. Ned. Akad. Wetenschap. SS, 894-909. Tokuyasu, K., and Yamada, E. (1959) J. Biophys. Biochem. Cytol. 6, 225-230. Tokuyasu, K., and Yamada, E. (1960) J . Biophys. Biochem. Cytol. 7, 187-190. van den Heuvel, J. E. A. (1957) Ophthalnzologica 133, 440-447. van den Hooff, A. (1951) Ned. Tijdschr. Geneesk. 96, 2491-2494. van den Hooff, A. (1957) Electronenmicroscopisch onderzoek ten behoeve van cytologie en histologie, Thesis, Univ. of Amsterdam. Excelsior, The Hague. van Doorenmaalen, W. J. (1958) Acta morphol. neerl. scand. 2, 1-12. van Walbeek, K., Neumann, H., Winkelman, J. E., Ruyter, J. H. C., and van den Hooff, A. (1950) Koninkl. Vluam. Acad. Gen. Belg. 12,226-238. Vrabec, F. (1952) Ophthalmologica l23, 20-30. Wald, G., and Zussman, H. (1937) Nature 140, 197. Wald, G.,and Zussman, H. (1938) J . Biol. Chem. 122, 449-460. Walls, G. (1942) “The Vertebrate Eye and its Adaptive Radiation.” Cranbrook Inst. Sci., Bloomfield Hills, Michigan. Weimar, V. (1959a) Exptl. Cell Research 18, 1-14.

194

ALFRED J. COULOMBRE

Weimar, V. (1959b) Trutis. N . Y . Acad. Sci. 21, 582-584. Weiss, P., and Amprino, R. (1940) Growth 4, 245-258. Weiss, P., and Moscona, A. (1958) J . Embryol. Exptl. Morphol. 6, 238-246. Wislocki, G. (1952) A m . I . A m t . 91, 233-261. Wolff, E. (1958) “The Anatomy of the Eye and Orbit.” McGraw-Hill, New York. Woolf, D. (1956) Anat. Record 124, 145-163. Yoneyama, K. (1932) Kaib6 2. ( T o k y o ) 6, 507-588; (1934) Japan. J. Mrd. Sci. I. 6, 74-75, abstr. Yoshido, M. (1958a) Japan. J . Physiol. 8, 31-40. Yoshido, M. (1958b) Juputc. J . Physiol. 8, 155-159. Zimmerman, L. E., and Eastharn, A. B. (1959) A m . ./. Ophthalmol. 47, No. 1, Part 11, 488-499.

The Photoreceptor Structures’ J. J. WOLKEN Uiopltgsical Rrsearch Laboratory, E y e and Ear Hospital, Univrrsity of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

I. Introduction ..................................................... 11. The Plant Photoreceptors ...................................... A. Chloroplasts ................................................ B. Molecular Weight .......................................... C. Structure and Function ...................................... 111. The Animal Photoreceptors ...................................... A. Visual Structures ........................................... B. Molecular Weight .......................................... IV. Summary ....................................................... References ......................................................

Page 195 196 196 204 205 205 205 215 215 216

I. Introduction Photoreceptors are structures containing photosensitive pigments (eg., chlorophyll and retinene) that upon light absorption initiate phototropisms, photosynthesis, and vision. The photoreceptors in plant cells are the chloroplast for photosynthesis ; in animal cells they are the retinal rods and cones for vision. The structure of these photoreceptors will be described in a variety of plant and animal species, and an attempt will be made to indicate, from these studies, a molecular structure for both the chloroplast and retinal rod. Reference should be made to recent Symposia (Gaffron, 1957; St. Whitelock and Wolken, 1958; Brookhaven Symposia in Biology, 1959 ; Reviews of Modern Physics, 1959 ; Smelser, 1961), reviews (Wolken, 1958a, 1959a ; Wald, 1959 ; Crescitelli, 1960), and current research literature because of the increased research interest in the photoreceptors to photosynthesis and vision. The techniques of microscopy, polarization, fluorescence, phase and interference microscopy are the most direct methods for obtaining information on the structure of photoreceptors. These methods have been applied to the study of the structure of the chloroplast (Frey-Wyssling, 1948; Goedheer, 1957 ; Greenblatt et al., 1960), and to the retinal rod (Schmidt, 1935 ; Sidman, 1957). However, the electron microscope, utilizing newer techniques for tissue preparation, has helped clarify these structural studies and has added fine structure details of the photoreceptors (Sjostrand 1 Aided in part by the U.S. Public Health Service Institute of Neurological Diseases and Blindness (b-397-C6), the National Council to Combat Blindness (G-199-C8), and the McClintic Endowment Eye and Ear Hospital, Pittsburgh, Pennsylvania.

195

I 96

J . J . WOLKEN

1953a,b, 1956). The methods for electron microscopy require that the tissue be fixed with a metabcontaining fixing agent, thereby increasing the electron density of the structure. The most successful fixative used is 1% osmium tetroxide buffered with 0.028 M acetate-Verona1 pH 7.0-8.0 ; controlling the osmotic conditions, e.g., with added sucrose (0.1 5 M ) , helps prevent cellular distortion during fixation. Other fixatives such as potassium permanganate, potassium chromate and dichromate, uranyl nitrate, and combinations of these and other metals have been used. The fixed tissues are then embedded in a resin which when polymerized can be easily sectioned with a glass or diamond knife. The embedding of the fixed material is usually carried out in an acrylic monomer (n-butyl methacrylate, methyl methacrylate, and mixtures of these) or in epoxide resins which when polymerized possess the right properties of hardness and ductility for sectioning. Almost all the published electron micrographs of the chloroplast and retinal rod structures have been fixed with osmium tetroxide. The methods of tissue preparation have been summarized in several recent texts (Ivan Sorvall, Inc., 1959; Pease, 1960). In attempts to overcome the inadequacies in these methods, techniques in freeze-drying have been investigated by Muller (1957) for the chloroplasts and, more recently, by rapid freezing, using liquid helium I1 for the retinal rods ( Fernindez-Adoran, 1960).

11. T h e Plant Photoreceptors A.

CHLOROPLASTS

The structure of the chloroplast, its biochemistry and genetics were reviewed by Weier and Stocking (1952). Since then, improved techniques for microscopy, particularly in electron microscopy, have resulted in numerous research papers and reviews on chloroplast structure and photosynthesis (Miihlethaler, 1955 ; Frey-Wyssling, 1957 ; Granick, 1955 ; Thomas, 1955 ; Wolken 1959a,b,c ; Hodge, 1959; Calvin, 1959a,b,c).

1. Composition Techniques in the isolation of chloroplasts have been described by Clendenning (1957). However, precise chemical data on the chloroplasts’ composition are difficult to obtain (Lumry et al., 1954). The chemical analyses of isolated chloroplasts from a variety of plants are summarized by Rabinowitch (1945, 1956) and Frey-Wyssling (1948, 1957). For example, the chloroplasts of spinach (Spinucia oleracea), according to Menke (1938), consist of 48% protein and 35% lipid. A range of values for the protein of from 41 to 55% and for the lipids of from 18 to 3776, on a dry weight basis, is given for a variety of plant chloroplasts. The chlorophylis and carotenoids represent about 5 to 10% of the chloroplast; the chloro-

PHOTORECEPTOR STRUCTURES

197

phyll concentration is of the order of 5 to 6%. The chloroplast pigments have been reviewed by Strain (1944, 1951), the bacterial pigments by Stanier (1959, 1960), and the carotenoids by Goodwin (1955). Chlorophyll a occurs in all plant chloroplasts, but other isomers b, c, and d are also found. In the higher plants, the two main chlorophyll isomers are a and b in a ratio of about 3:l. The carotenoids also are intimately linked with chlorophyll in the chloroplast. There are the xanthophylls, lutein and zeaxanthin, and the carotene, p-carotene. Phycoerythrin and phycocyanin are also present in the red and blue-green algae. The carotenoids’ function has been a debatable one, although they appear necessary for the chloroplast function in photosynthesis (Lynch and French, 1957). Fujiniori and Livingston (1957) indicate from studies of flash photo!ysis that the carotenes play a direct part in the primary events of photosynthesis. However, Sager and Zalokar (1958) indicated from their studies of a mutant strain of the alga Chlamydomonas reinhardi that the carotenoids are not essential for photosynthesis, except in catalytic amounts, and that the carotenoids are necessary for protection against photodynamic destruction. The chloroplast proteins have not as yet been purified. However, the amino acids have been assayed in chloroplasts from various plant species by Sissakian (1957). The synthesis of the peptide bond by chloroplasts has been studied by Sissakian (1958) using C14 and S35,and he found that this was linked to the enzyme activity of the chloroplast. I t has also been indicated that a cytochrome system is a part of the photosynthetic complex (Hill and Whittingham, 1955; Hill and Bendall, 1960). Such cytochromes ( f , ba, and modified c ) have been isolated from chloroplast fractions of higher plants and algae, and from the chromatophores of photosynthetic bacteria. These cytochromes may constitute as high as 20% of the chloroplast proteins. In actively photosynthesizing cells, one molecule of cytochrome f or modified c was found present to every 300-400 chlorophyll molecules (Kamen, 1956 ; Gross and Wolken, 1960). The nucleic acids, both RNA and DNA, were also found in the chloroplasts by enzymic staining technique (Metzner, 1952). Sissakian ( 1957) has also shown that there is 0.3-3.5% nucleic acids on a dry weight basis in the chloroplast.

2. Structure The photosynthetic structure exists in a variety of shapes and sizes, ‘from the bacteria to the higher plants. These have been referred to as chromatophores, plastids, free grana, megaplasts, and chloroplasts. This nomenclature was intended to help in classifying the structure phylogenetically, and is based on the degree of internal organization as seen with

198

J. J. WOLKEN

the electron microscope. In this discussion, chloroplast is used for all of these structures unless otherwise defined. Two kinds of organized chloroplasts have been described : the continuous lamellar chloroplasts found in most of the algae, and the grana-containing chloroplasts of the higher plants, which are also lamellar (Figs. 1 and 2). These higher plant chloroplast lamellae have been differentiated as the grana lamellae containing chlorophyll and the nonpigmented stroma’s lamellae. Structures intermediate between both types of chloroplasts were observed in Spirogyra (Butterfasz, 1957) and in some Desmidicae by Chardard and Rouiller (1957). In the photosynthetic bacteria, Rhodospirillum rubrum, the photosynthetic structures are referred to as chromatophores, which contain bacteriochlorophyll as well as carotenoids, and are small by comparison with the chloroplasts of the algae and higher plants. The chloroplast of the algal flagellate, Euglena, for example, is two to three times the size of the whole bacterium Rhodospirillum rubrum. In the blue-green algae, lamellae were observed (Niklowitz and Drews, 1955, 1956), but these structures lacked the chloroplast membrane and the stroma. Frey-Wyssling ( 1948) proposed, from his and Menke’s previous studies on birefringence of the chloroplast, that the chloroplast consists of twenty to thirty parallel lipid layers which are separated from layers of aqueous protein by monomolecular films of chlorophyll molecules, the hydrophilic porphyrin “head” of each chlorophyll molecule extending into the aqueous protein complex, and the lipophilic phytol “tail” reaching into the lipid layer. I t was further postulated that each lipid layer is 50 A. thick, and that one layer is separated from another by aqueous protein. The electron microscope has clearly established proof of this picture (Wolken and Palade, 1953; Wolken and Schwertz, 1953). Data has been collected for the average thickness of these dense chloroplast lamellae from a variety of species by Leyon (1956) ; from electron micrograph measurements, they were found to measure of the order of 100-300 A. In some chloroplasts, low angle X-ray diffraction techniques, as demonstrated for the fine structure of Aspidistra (Finean et al., 1953; Finean, 1954), have corroborated the electron microscopic measurements showing a repeating unit of the order 250 A. However, it was difficult to get good results with freshly isolated chloroplasts, and only chloroplasts fixed with osmic acid ( 0 ~ 0showed ~ ) good X-ray diffraction patterns. There is no chemical analyses yet of these layers. The location of the lipids, lipoproteins, and proteins has- been assumed from the chemical reactions of the fixing agents and various stains with the biological material (Thomas et aZ., 1954). The electron-dense layers are referred to as either lipid and/or lipoprotein because of their affinity for osmium tetroxide used in fixation ;

FIG.1. Chloroplast, higher plant, Elodea canadensis. a. Proplastid, Chlorophytum. The arrangement of the structure is similar to a crystal lattice. (From Miihlethaler and Frey-Wyssling, 1959.) b. Lamellae of the Euglena chloroplast. FIG.2. Chloroplast, Euglena granulufa. (From Wolken, 1959.)

200

J. J. WOLKEN

the areas of least density are aqueous proteins including enzymes and dissolved salts. Frey-Wyssling and Steinman (1953) pointed out the fact that the lamellae can be seen, in good electron micrographs, to consist of spherical particles, of the order of 65 A., that represent globular protein macromolecules. There is experimental evidence to indicate that the chlorophyll molecules are preferentially oriented within the lamellae (Goedheer, 1957). The pigment molecules are not within the resolution of the electron microscope at present.

3. Molecular Structure In order to determine more precisely the meaning of the lamellar structure and to test the pigment monolayer hypothesis, the diameter, length, number, and thickness of the lamellae of the chloroplasts were measured and statistically evaluated (Wolken and Schwertz, 1953). For example, the chloroplast of E . gracilis consists of twenty-one electrondense layers -250 A. in thickness with less dense interspaces of 300 to 500 A. in thickness. Each dense layer appears to be covered on both sides by a thinner and denser layer (lamellae), 50 to 100 A. in thickness. The average thickness for these lamellae, measured for a variety of plant chloroplasts, ranges from as low as 20 A. to an average of 100 A. (Leyon, 1956; Sager and Palade, 1957; Sager, 1959). From the geometry of the chloroplast, the number of dense layers, and the chlorophyll concentration per chloroplast, the cross sectional area occupied by each chlorophyll molecule was calculated to be 222 A.2 for the Euglena chloroplast, and 246 A.2 for the Poteriochromonas chloroplast (Wolken and Schwertz, 1953). Elbers et al. (1957) have since collected data on chlorophyll concentrations and geometry of chloroplasts in a variety of plant species, and they calculated the mean area which is available for the chlorophyll molecule in the monolayer to be of the order 200 A.2 in cross sectional area. Studies of the dichroism, birefringence, and polarization of fluorescence in Mougeotia chloroplasts also indicate that chlorophyll resides as a monolayer on the lamellar surface, and the area available per chlorophyll molecule was calculated to be 250 A.2 (Goedheer, 1955, 1957). Since the cross sectional area of the porphin head of the chlorophyll molecule is known from X-ray studies to be about 225 to 242 A.2, these results indicate that all the available chlorophyll molecules could be packed into the interfacial atea and could cover the whole surface of the lamellae as a monolayer. On the basis of these calculations; a simplified schematic molecular model was proposed (Fig. 3,). The suggestion of Bass-Becking and Hanson that four chlorophyll molecules are united to form tetrads, in which the reactive isocyclic rings turn toward each other, was employed (Wolken

PHOTORECEPTOR STRUCTURES

20 1

FIG.3. Molecular model for the chloroplast. (From Wolken and Schwertz, 1953.) a. Interfacial plane showing orientation oi chlorophyll and carotenoid molecules. b. Molecular organization in the layers of the chloroplast as visualized. (From Calvin, 1959.)

202

J. J. WOLKEN

and Schwertz, 1953). Interaction between the phytol tails was eliminated in the model by arranging the tetrads in such a way that one, and only one, of the phytol tails was located at each virtual intersection in the rectangular network. If the chlorophyll were packed as a monolayer as shown in the schematic molecular network in Fig. 3a, there would still be space available at the interstitial positions, between the chlorophyll molecules, for the carotenoid molecules. If these spaces are occupied as shown, there will be one carotenoid molecule for at least every three chlorophyll molecules in the network. This kind of close packing of the chlorophyll and carotenoid molecules in the pigment monolayers would also permit energetic interaction between them. Calvin ( 1959c) has recently suggested a similar model (see Fig. 3b) in which one aqueous protein layer has COz-reducing enzymes and another aqueous protein layer has OZ-evolving enzymes. Other models, slightly modified from the one presented here, indicate that the chlorophyll molecules are also turned inward as well as being oriented on the surface (Hodge et al., 1955). There are several possible ways in which the chlorophyll molecules could be oriented in the lamellae. If the porphyrin heads of the chlorophyll molecules lay at 0" as flat plates, as indicated in Fig. 3a, their greatest cross section would be available. However, if they were oriented within the lamellae at increasing angles up to 90°, the cross sectional area available would be decreasing. Studies of chlorophyll monolayers on various liquid surfaces suggested that the chlorophyll molecules would probably lie at an angle of 35" to 55" within the chloroplast, thus reducing the above calculation for the cross section of the chlorophyll molecule to 100 A.2 (Trurnit and Colmano, 1958). Chlorophyll a has been shown to crystallize out in thin sheets of the order of 50 A., corresponding to about two molecular layers of chlorophyll. The crystallized chlorophyll molecules occupied an area of 106 A.2 (Jacob et al., 1954). It is very likely that the absorption oscillators of these pigment molecules are arranged with an orderly orientation within the chloroplast lamellae in a way that a maximum absorption will occur for an incident light polarized in a given direction.

4. Development The chloroplast must be derived from some pre-existing chloroplast (a proplastid) ; therefore the question of how the chloroplast evolves from a rudimentary proplastid into an organized structure is a difficult experimental problem. According to Strugger (1950), the proplastid is a protoplasmic-particle that always contains a primary granum, while Heitz and Maly (1953) find that ,this granum is lacking in the youngest stages and appears only as a first step in the process of differentiation. This primary granum has a crystalline structure as demonstrated by Heitz

PHOTORECEPTOR STRUCTURES

203

( 1954, 1955, 1956), Leyon (1953), and Miihlethaler and Frey-Wyssling (1959). It is not known whether such crystalline structures are enhanced by techniques for electron microscopy. Some preliminary studies indicate that the granum contains nucleoproteins, hence suggesting a self-duplicating unit. Euglena, grown in darkness for long periods, are no longer green and become almost colorless. This is accompanied by obvious changes in the chloroplasts ; they fragment and their lamellar structure is destroyed. Such dark-adapted euglenas, when brought back to the light, fixed at regular time intervals, and studied by electron microscopy, showed that after as little as four hours of light exposure, elongated bodies with characteristic laminations of the chloroplasts were recognizable. At the beginning, their lamellae were very thin, less numerous, and not tightly and regularly packed ; with continuous light, the lamellae increased in number along with the synthesis of chlorophyll. The chloroplasts had the form and organization described for actively photosynthetic euglenas by 72 hours of light exposure (Wolken and Palade, 1953). As soon as we could detect chlorophyll spectrophotometrically, the chloroplasts were already lamellar. The alga Chlamydomonas, whether grown in light or in darkness, shows a chloroplast organization of lamellae, pyrenoid, and eyespot within a limiting membrane. In the absence of chlorophyll, the lamellae are not formed, although the eyespot, starch grains, and the pyrenoid are still found (Sager and Palade, 1957 ; Sager, 1959). In the development of the chloroplast in flowering plants, the first stages to be seen lack all structural properties which are typical of the fully developed chloroplast. The crystalline structure of the primary granum, as found in Chlorophytum (Fig. l a ) , Aspidistra, and in other monocotyledons, is also present in the dicotyledons. This suggests that some connection must exist between the crystalline lattice and the development of the lamellae, and that the lamellae develops from “tubules” or “cristae” analogous to the mitochondria (Heitz, 1955 ; Miihlethaler and FreyWyssling, 1959). With continuous illumination the crystalline structure gives rise to the lamellar system as seen in the full-grown chloroplast of Elodea canadensis (Fig. 1). These studies also indicate that the formation of the lamellae proceeds parallel with pigment synthesis. Von Wettstein (1957a,b, 1959) investigated the gene action on the submicroscopic structure of the chloroplasts by employing chlorophyll lethals of barley. In the development of the barley chloroplast, there is an undifferentiated proplastid with a center core ; starch grains accumulate, lamellae are formed, and the plastid as a whole becomes transversed by lamellae. In an albino type, normal early proplastids enclosed by a double membrane, were found. Differentiation did not proceed beyond the

201

J. J. WOLKEN

earliest stages, but the plastid increased in size to that of a full-grown chloroplast. The gene change had blocked the development of the chloroplast lamellae at an early stage, but not the growth of the plastid as a whole. Instead of lamellae forming, pigmented globules accumulated in large masses, and their ability to photosynthesize appeared arrested. The pigment concentration in the mutant indicated that there was a greater accumulation of chlorophyll b in the globules. The study on chloroplast development still remains inconclusive and more experimental work is necessary. However, for the plant to carry on photosynthesis, an ordered lamellar structure appears necessary for the chloroplast (Wolken, 1961a). B.

MOLECULAR WEIGHT

The chlorophylls and carotenoids in their natural state are bound to proteins or lipoproteins. Such pigment-complexes, chloroplastins, have been prepared by digitonin (1-276) extraction. The molecular weight of spinach and Aspidistra chloroplastin has been calculated from its sedimentation in the analytical ultracentrifuge to be of the order of 265,000 by Smith and Pickels (1940) and Smith (1941a,b,c). This is a high estimate for chloroplastin due in part to the contribution of the digitonin micellesince Miller and Anderson (1942) and Smith and Pickels (1940) have shown that digitonin forms micelles of minimal molecular weight equal to 75,000, and Hubbard (1954) has demonstrated that three such micelles are associated to form a unit with an average molecular weight of 225,000. Takashima ( 1952) crystallized a chlorophyll-lipoprotein complex with a-picoline from a leaf extract at low temperature; from diffusion studies its molecular weight was found to be 19,200, and it was postulated that the complex would contain two molecules of chlorophyll per molecule of lipoprotein. These crystals are probably contaminated with protein and other substances (Krasnovsky and Brin, 1954). The chlorophyll holochrome isolated by Smith (1959) and collaborators from bean seedlings, Phaseolits vulgaris, in glycine-KOH ( p H 9.6) suggests a molecular weight of the order of a million for the macromolecule. The average molecular weight calculated from the geometry of the chloroplast and from the analytical data of the pigment concentration, nitrogen, and dry weight of the complex from Euglena and Poteriochromonas is of the order of 21,000 to 40,OOO (Wolken, 1956b; Wolken and Schwertz, 1956). The analytical data also indicated that there would be one chlorophyll molecule to one proteiii molecule. Frey-Wyssling (1957) predicts that there would be sixteen molecules of chlorophyll to one protein macromolecule calculated on the basis that the macromolecule is 65 A. in diameter; therefore, the chlorophyll complex would have a molecular weight of 68,000. These

PHOTORECEPTOR STRUCTURES

205

values are, for the most part, in agreement as to the order of magnitude. However, there are differences in the proteins in various plant species and, therefore, there would be differences in the molecular weights.

C. STRUCTURE AND FUNCTION Does the molecular structure tell us about how the chloroplast functions as an energy capturing, storage, and transferring device? Is the efficiency (photosynthetic) related to the degree of internal molecular organization as, e.g., found in the higher plant chloroplasts, and less so for the photosynthetic bacteria? Such questions are yet to be resolved. The chloroplasts’ ability to function is related to the synthesis of chlorophyll and to its lamellar structure (Wolken, 1956a ; von Wettstein, 1957a; Sager, 1959), for any physical or chemical force that affects the synthesis and chemistry of chlorophyll will disrupt the lamellar structure of the chloroplast, and hence its ability to carry on photosynthesis. From the chloroplastordered or crystalline structure (see Figs. 1 and 2) , resonance properties (Calvin, 1959a,b,c) and photoconductivity (Arnold and Maclay, 1959 ; Arnold and Clayton, 1960) appear to indicate that we are dealing with an electron-transfer phenomenon by a conduction band mechanism, and that the chloroplast functions analogously to a semiconductor.

111. T h e Animal Photoreceptors A. VISUAL STRUCTURES Animals, in the course of evolution, have developed various kinds of eyes for light perception and image formation. I n the invertebrates, for example, eyespots, sensory cells, ocelli, and compound eyes have developed with differences in physical organization. The invertebrate photoreceptors and their physiology have been reviewed by Milne and Milne (1956) and Wulff (1956). In the vertebrates the photoreceptors are the retinal rods and cones. The invertebrate and vertebrate photoreceptors’ fine strzictirre have been described by Sjostrand (1953a,b, 1959), De Robertis ( 1956), Ferilindez-Morin ( 1959), and Wolken ( 1958a, 1961) . The photoreceptor process in vision has recently been reviewed by Wald (1959) and Crescitelli (1960). Let us then see what has been revealed on the structure of these photoreceptors from a phylogenetic point of view-this is illustrated schematically in Fig. 4A-E.

1. Eyespot In the protozoan flagellate, Euglena, the eyespot is a photoreceptor which directs the organism by phototropic reactions. It was speculated, a long time ago, that the eyespot functions analogously to the retinal cells of higher animals (Mast, 1911) . However, no photosensitive pigment has

A

/'

FIG.4. Schematic phylogenetic development of the photoreceptor structures. A. Eyespot and flagellum, pigment granules (9). B. Sensory cells (re), surrounded by pigment gcanules (9). C. Insect compound eye, lens ( a ) , pigment granules (9). D. Mollusc cephalopod eye shpwing compound retina. E. Vertebrate eye, iris (i), lens ( a ) , pupil ( p ) , ciliary muscle (c), sclera (s) and retina (r), showing an enlarged rod (re) outer segment (0s) and inner segment (is). It is to be noted that the structure connecting the inner with the outer segment (f) of the retinal rod (re of E) is similar to that of the eyespot with flagellum of A. (Modified from Wolken, 1959a.)

PHOTORECEPTOR STRUCTURES

207

so far been isolated, from the eyespot, that resembles the visual complex (k,rhodopsin) , although carotenoids such as p-carotene and lutein are indicated from the Eulgena eyespot absorption spectra (Strother and Wolken, 1960). Willmer ( 1955) suggested that structurally the most interesting feature in the development of the rods and cones is the flagellum-like fibers that connect the outer and inner segments. FaurC-Fremiet and Rouiller (1957, 1958) indicate that the second internal flagellum associated with the eyespot (stigma) of Chronzulina has a lamellar structure similar to other photoreceptors. The flagellum is a sensory structure which is structurally similar to the fibril that penetrates from the outer through the inner segment of the vertebrate retinal rod (De Robertis, 1956). In Euglena the eyespot is intimately linked with the flagella. It is about 2 X 3 p and consists of the order of fifty tightly packed pigment granules (orange-red) forming a mosaic (Wolken, 1956a). Each granule is of the order of 0.1 p in diameter, and indications are that these granules have an internal organization similar to other photoreceptors. Euglena phototaxis and photokinesis (rate of swimming irrespective of direction) indicates that selective absorption by the eyespot is related to its photomotion (Wolken and Shin, 1958). The eyespot flagellum act as a receptoreffector system, directing the organism towards light. The eyespot can then be considered structurally and functionally analogous to a retinal rod.

+

2. Sensory Cell In the flatworm, Planaria, the two eyes consist of pigment granules that shade the sensory visual cells from light, in all directions but one, and so enables the animal to respond in a negative way to the direction of light. The sensory cells are about 5 p in diameter, with a more variable length of approximately 35 p, and each sensory element appears as packed tubules of the order of 400 A. in diameter, or lamellae of 100 A. in thickness (Wolken, 1958a). The appearance of tubes or lamellae depends on the angle at which the sensory cells are cut for electron microscopy.

3. Coiizpound Eye The structure of the compound eye is of considerable interest in the organization of its visual cells. The arthropod compound eye consists of ommatidia ; in the insect each ommatidium has a distal cone and lens, and a sheath of pigment cells which extends throughout its entire length. Each ommatidium consists of retinular cells, of which the differentiated photoreceptor structures are the rhabdomeres. The rhabdomeres contain the photosensitive pigments and are the photoreceptors (retinal rods) in which the visual process is initiated. For example, the eye of Drosophila is com-

208

J. J. WOLKEN

posed of approximately 700 ommatidia ; each ommatidium contains seven retinular cells with seven rhabdomeres that are radially arranged to form a cylinder (Fig. 5 ) . The rhabdomeres are of the order of 60 p in length and 1 p in diameter ; they consist of tubules from 200 to 400 A. in diameter, whose walls are of the order of 100 A. in thickness (Wolken et al., 1957). A similar arrangement of the rhabdomeres within the ommatidium with similar fine structure is found for the housefly, dragon fly, honeybee, and spider ( Fernindez-Morin, 1958 ; Goldsmith and Philpott, 1957 ; Khalaf, 1958). Retinenel has been isolated from the rhabdomeres of the housefly (Wolken et al., 1960) and the honeybee (Goldsmith, 1958a,b). This would indicate that the insects, too, contain the visual complex, a rhodopsin. A number of insects also show photosensitivity to the ultraviolet, and have been shown to have color discrimination. That they may possess other photosensitive pigments cannot be excluded (Bowness and Wolken, 1959). The eyes of molluscs resemble those of the vertebrates. However, the eye of the cephalopods, Sepia and Octopus, is a single lens eye, provided with a mechanism for accommodation. The lens is formed out of two halves joined together; the photoreceptors of the retina are not inverted as in the vertebrate eye, and are directly exposed to the incident light. The retinas of Sepia and Octopus are made up of rhabdomes in which there are four rhabdomeres radially arranged (see Fig. 6 ) (Wolken, 1958b). A central space separates the rhabdome, which contains pigmentscreening granules that migrate, depending on the light intensity. Each retinal rhabdomere (rod) is of the order of 1 p in diameter and of 60 p in length, and is made up of densely packed tubules of the order of 200 A. in diameter. I t will be noted in Fig. 4D how the retina resembles the insect compound eye, Fig. 4C. In the king-crab, Limitlus, and in the mollusc, Pecten, a similar fine structure, a lamellar arrangement of tightly packed tubules, has been observed (Miller, 1958). When one looks more closely at the spacial arrangement for the rhabdomeres within the omniatidia of the arthropod compound eyes, two distinct patterns are observed, a “closed”-type in which the rhabdomeres are in close proximity forming a star-shaped structure (e.g., cockroach, Octopus and Sepia), and an “open”-type in which the rhabdomeres project systematically through neck-like projections into a comparatively large axial cavity (e.g., Drosophila and housefly). The closed-type belong to a large number of insects described i o have “superposition” eyes. The open-type is characteristic of dipterous and hymenopterous insects which are provided with “apposition” eyes. This is of interest since Autrum (1958) suggests that two physiological types of eyes may be distinguished I

FIG.5. Orientation of rhabdomeres within the ommatidium of Drosophila melanogarter. a. Enlarged cross section of a rhabdomere. (From Wolken et al., 1957.) FIG.6. Arrangement of the Sepia rhabdomeres in the retina. a. A cross section X - X of a three-dimensional view of a single rhabdom made up of four rhabdomeres ( a ) , ( b ) , ( c ) , and ( d ) . Y-Y represents an oblique section through a rhabdom. b. Lamellar structure of Sepia rhabdomere. (From Wolken, 195813.)

210

J. J. WOLKEN

by their electrical responses : a “slow”-type characterized by negative monophasic wave, the mgnitude of the .potential being markedly affected by light and dark adaptation; and a “fast”-type of a di- or polyplasniic potential with a positive on- and a negative off-effect that is not effected by light- or dark-adaptation. Many of the arthropods exhibit orientation relative to the direction of vibration of polarized light; such sensitivity to the plane of polarization suggests the existence of a polarized light analyzer within the eye.

4.

Vertebrate Retinal Rods and Cones

The photoreceptors of the vertebrate retina are rods and cones, that lie side by side forming a mosaic of light-sensitive elements. Each is composed of an inner segment and a rod- or cone-shaped outer segment. These outer segments are packed plates or discs, of the order of 200 A. in thickness, containing as their photosensitive pigments either retinenel or retinenea (the aldehydes of vitamin A1 or vitamin Az), linked with a protein or lipoprotein opsin. Such extracted pigment complexes are identified by their absorption spectra as rhodopsin (retinenel rod opsin) or rod opsin), for the rods, and iodopsin (retporphyropsin (retinenez inenel cone opsin) or cyanopsin ( retinenez cone opsin) , for the cones (Wald, 1959). The principal components of the retinal rod outer segments are pigments, proteins, and lipids. In terms of wet weight, the pigment-protein rhodopsin accounts for &lo%, the lipids for 20-40%, and the proteins To be more specific, cattle rhodopsin constitutes 3.6% of for +SO%. its wet weight and 13% of its dry weight. However, frog rhodopsin constitutes 10% of its wet weight and more than 35% of its dry weight. I t is interesting to note that there were no metallic ions found in rhodopsin (Fukami et al., 1959). The precise locations of the pigment, lipid, and protein within the outer segment of the rod can only be assumed from the chemical reactions of the tissue with the fixing agent or with various stains. Experimental evidence indicates that the pigment molecules most probably reside in the (osmium-fixed) dense lamellae. In order to reconstruct a structural model for the retinal rod, it was necessary to visualize a geometric structure that would resemble the in vivo rod. One of the largest retinal rods is that of the frog which is about 6 x 55 p and can be easily observed by light microscopy prior to fixation for electron microscopy. It was previously demonstrated, using polarized light, that the freshly isolated rods are ordered systems of lamellae (Schmidt, 1935). The retinal rod outer segments of the frog can be easily isolated in dim red light and suspended in physiological saline. ‘L‘pon ex-

+

+

+

+

PHOTORECEPTOR STRUCTURES

21 1

amination in red light, they appear made up of longitudinal fibers 1 p in diameter and extend throughout the length of the entire outer segment. However, upon continued observation, the outer segment swells and begins to break transversally, rather than longitudinally ; when “bleached” the whole outer segment then appears as plates which are falling apart (Wolken, 1961b). For the geometry of the retinal rod we have taken the measurements from numerous electron micrographs of fixed retinal rod outer segments. The photoreceptor geometry (length, diameter, thickness, and number of dense layers), as determined from the electron micrographs (Table I), can be used together with the rhodopsin concentration to calculate the size of the rhodopsin molecule. The dense layers consist of double layers of lipids and lipoproteins. A double layer is then structurally represented as lipoprotein macromolecules ; the low molecular weight lipids would then occupy the interstitial spaces. Monomolecular layers of pigment molecules occur at the interfaces between these layers. This is shown by the electron micrograph of the cattle rod, Fig. 7, and is illustrated schematically in Fig. 8. The cross sectional area A, which would be associated with each macromolecule, and therefore with each pigment molecule is

A = -

XD2

4P where D is the diameter of the photoreceptor and P is the number of pigment molecules in a single monolayer. The maximum cross sectional area A with each rhodopsin molecule can be derived from the above equation, where P is replaced by N/2n, in which N is the pigment concentration in molecules per retinal rod and n is the number of dense layers per outer segment, then

The cross sectional areas calculated for cattle and frog rhodopsin were found to be 2500 A.2 and 2620 A.2, respectively. The diameter of the rhodopsin molecule would then be of the order of 50 A. This would be about the right order of magnitude since a rhodopsin molecule (cattle, frog), if symmetrical, would have a diameter of the order of 40 A. (Wald, 1954). A small area is greatly enlarged in Fig. 8, to show the molecular packing of retinene with opsin in the retinal rod dense layers in which there would be one retinene molecule per opsin molecule. The area available, therefore, indicates that there would be sufficient space for all of the pigment molecules to cover all of the electron-dense surfaces. The orientation of retinene to the opsin follows from the kinetic studies of rhodopsiii by Hubbard and Kropf (1959).

212

J. J. WOLKEN

FIG.7. Retinal rod outer segment, cattle. a. An enlarged area. (From Woken, 1961.)

PHOTORECEPTOR STRUCTURES

213

FIG.8. Molecular model for the outer segment of the retinal rod. (From Wolken, 1961a.) The relation of retinene with opsin in rhodopsin. (From Hubbard and Kropf, 1959.)

!2

P

TABLE I PHOTORECEPTORS : STRUCTURAL DATA

Retinal rod outer segment Frog

Cattle

n

N

(P)

Thickness of dense layers (A.1

Number of dense layers per photoreceptor

5.0 1.o

200 200

1000 180

u Diameter

T

Number of rhodopsin molecules per photoreceptor

A Cross sectional area of rhodopsin (A.2)

dm of Diameter rhodopsin mo1ecu1e

3.8 x 109 4.2 x 106

2620 2500

51 50

(-4.1

4 ?

2r 27

PHOTORECEPTOR STRUCTURES

215

MOLECULAR WEIGHT The rhodopsin molecular weight M can also be calculated from B.

nD2TsLn (3) 4N where D is the diameter of the photoreceptor; T is the thickness of the dense layers ; s is taken as 1.3 the density of the protein ; L is Avogadro’s number, 6 x loz3; n is the number of dense layers ; and N is the number of pigment molecules per photoreceptor. The molecular weights calculated from this equation for frog and cattle rhodopsin are 60,OOO and 4O,OOO, respectively (Wolken, 1957, 1958a). The visual complex, rhodopsin, is prepared from the retinal rods by extraction with digitonin, a nonionic detergent (C5sH90029). Rhodopsin possesses physiological activity in solution analogous to that of the intact retinal rod. The sedimentation of frog and cattle rhodopsins was studied in the analytical ultracentrifuge. The molecular weight calculated from its sedimentation was found to be 67,000 for frog rhodopsin (Wolken, 1956b) and 40,000 for cattle rhodopsin (Hubbard, 1954). The molecular weight calculated from the geometrical consideration is 60,OOO (data from Table I ) . There is agreement between the rhodopsin molecular weight calculated from the geometry of the retinal rod and that determined directly of the extracted rhodopsin in solution. The analytical data (dry weight, pigment, nitrogen) show that there is one retinene molecule per protein (opsin) in rhodopsin ( Hubbard, 1954 ; Wolken, 1956b).

M =

IV. Summary These studies on the molecular anatomy of the plant and animal photoreceptors show that they are all ordered lamellar structures ; they are plates or discs for the chloroplasts and vertebrate retinal rod outer segments, and tubes or rods for the invertebrate photoreceptors, all having molecular dimensions of the order of 50-200 A., and whose integrity and functioning depend on their photosensitive pigments. The lamellar structure may be an efficiency mechanism for light capture, rather than a critical functioning device ; however, this does not exclude the probability that a crystalline-type of matrix is necessary for function. Further evidence from resonance and photoconductivity studies of the photoreceptors suggest that we are dealing with an electron transfer phenomenon by a conduction band mechanism, as in a semiconductor. It is unlikely that living systems would build different structures at a molecular level for the initial event-“light-trapping.” The end results, of course, photosynthesis and vision, are very different. W e are, however, beginning to bridge the gap in which the anatomy and the chemistry of photoreceptors are becoming one with their physiology at a molecular level.

216

J. J. WOLKEN

REFERENCES Arnold, W., and Clayton, R. C. (1960) Proc. Natl. Acad. Sci. U.S. 46, 769. Arnold, W., and Maclay, H. K. (1959) Brookhaven Symposia in Biol. No. 11. Autrum, H. (1958) Exptl. Cell Research Suppl. 6, 426. Bowness, J. M., and Wolken, J. J. (1959) J . Gen. Physiol. 42,779. Brookhaven Symposia in Biol. (1959) No. 11. Butterfasz, T. (1957) Protoplastno 48, 368. Calvin, M. (1959a) Revs. Modem Phys. 31, 147. Calvin, M. (1959b) Revs. Modern Phys. 31, 157. Calvin, M. (1959~) Brookhaven Symposia in Biol. No. 11, 160. Chardard, R., and Rouiller, C. (1957) Rev. cytol. et biol. vigktales 18, 153. Clendenning, K. A. (1957) Ann. Rev. Plant Physiol. 8, 137. Crescitelli, F. (1960) Ann. Rev. Physiol. 22, 525. De Robertis, E. (1956) J . Biophys. Biochem. Cytol. 2, 319. Elbers, R. F., Minnaert, K., and Thomas, J. B. (1957) Acta Botan. N e e d 6,345. FaurC-Fremiet, E., and Rouiller, C. H. (1957) Compt. rend. acad. sci. 244, 2655. Faurk-Fremiet, E., and Rouiller, C. H. (1958) Exptl. Cell Research 14, 47. FernLndez-Morin, H. (1958) Exptl. Cell Research Suppl. 6, 586. FernLndez-Morin, H. (1959) Revs. Modern Phys. 31 (2), 319. Fernindez-Moran, H. (1960) Ann. N.Y. Acad. Sci. 86. 689. Finean, J. B. (1954) Exptl. Cell Research 6,283. Finean, J. B., Sjostrand, F. S., and Steinman, E. (1953) Exptl. Cell Research 6,

557. Frey-Wyssling, A. ( 1948) “The Submicroscopic Morphology of Protoplasm and its Derivatives,” p. 243. Elsevier, New York. Frey-Wyssling, A. (1957) “Macromolecules in Cell Structure,” p. 49. Harvard Univ. Press, Cambridge, Massachusetts. Frey-Wyssling, A., and Steinman, E. (1953) Vierteljahresschr. nafurforsch. Ges. Ziirich Beih. 98,20. Fujimori, E., and Livingston, R. (1957) Nature 180, 1036. Fukami, I., Vallee, B. L., and Wald, G. (1959) Nature 183,28. Gaffron, H., ed. (1957) Gatlinburg Conference, “Research in Photosynthesis.” Interscience, New York. Goedheer, J. C. (1955) Biochirn. et Biophys. Acta 16, 471. Goedheer, J. C. (1957) Optical properties and in vivo orientation of photosynthetic pigments. Thesis, State University of Utrecht, Netherlands. Goldsmith, T. H. (1958a) Ann. N.Y. Acad. Sci. 74, 223. Goldsmith, T. H. (1958b) Proc. Natl. Acad. Sci. U.S.44, 123. Goldsmith, T. H., and Philpott, D. E. (1957) J . Biophys. Biochem. Cytol. 3,429. Goodwin, T. W. (1955) Ann. Rev. Biochern. 24,497. Granick, S. (1955) “Handbuch Der Pflazenphysiologie” (W. Ruhland, ed.), Vol. I, p. 507. Springer, Berlin. Greenblatt, C. L., Olson, R. A,, and Engel, E. K. (1960) J . Biophys. Biochem. Cytol. 7, 235. Gross, J. A., and Wolken, J. J. (1960) Science 132,357. Heitz, E. (1954) Exptl. Cell Research 7, 606. Heitz, E. (1955) Nova Acto Leopoldina 17, 517. Heitz, E. (1956) Experientia 12, 476. Heitz, E., and Maly, R. (1953) 2.Natrirforsch 8b, 2430.

PHOTORECEPTOR STRUCTURES

217

Hill, R., and Bendall, F. (1960) Nature 186, 136. Hill, R., and Whittingham, C. P. (1955) “Photosynthesis,” p. 164. Methuen, England. Hodge, 4.J. (1959) Revs. Modern Phys. 31, 331. Hodge, A. J., McLean, J. D., and Mercer, F. V. (1955) J . Biophys. Biochem. Cytol. 1, 606. Hubbard, R. (1954) J. Gen. Physiol. 37, 381. Hubbard, R., and Kropf, A. (1959) Ann. N . Y . Acad. Sci. 81,388. Ivan Sorvall, Inc. (1959) “Thin Sectioning and Associated Technics for Electron Microscopy.” Norwalk, Connecticut. Jacob, E. E., Vatter, A. E., and Holt, A. S. (1954) Arch. Biochem. Biofihys. 68,

228. Kamen, M. D. (1956) “Enzymes: Units of Biological Structure and Function” (0.H. Gaebler, ed.), p. 483. Academic Press, New York. Khalaf, K. T. (1958) Mikroskopie 13,206. Krasnovsky, A. A., and Brin, C. P. (1954) Doklady Akad. Nauk. S.S.S.R. 96, 611. Leyon, H. (1953) Exptl. Cell Research 6, 497. Leyon, H. (1956) Svensk Kem. Tidskr. 68, 70. Lumry, R., Spikes, J. B., and Eyring, A. (1954) Ann. Rev. Plant Physiol. 6, 271. Lynch, V.H., and French, C. S. (1957) Arch. Biochem. Biophys. 70, 382. Mast, S.0. (1911) “Light and the Behaviour of Organisms.” Wiley, New York. Menke, W. (1938) 2. physiol. Chenz. a67, 43. Metzner, H. (1952) Biol.Zentr. 71, 257. Miller, G. L., and Anderson, K. J. T. (1942) J . Biol. Chem. 144, 475. Miller, W.H. (1958) Ann. N . Y . Acad. Sci.74, 204. Milne, L. J., and Milne, M. J. (1956) Radiation Biol.3, 621. Miihlethaler, K. (1955) Intern. Rev. Cytol. 4, 197. Miihlethaler, K., and Frey-Wyssling, A. (1959) J . Biophys. Biochem. Cytol. 6, 507. Miiller, N. R. (1957) J. Ultrastructure Research 1, 109. Niklowitz, W., and Drews, G. (1955) Arch. Mikrobiol. 23, 123. Niklowitz, W., and Drews, G. (1956) Arch. Mikrobiol. 24, 134. Pease, D. C. (1960) “Histological Techniques for Electron Microscopy.” Academic Press, New York. Rabinowitch, E. I. (1945) “Photosynthesis and Related Processes,” Vol. 1. Interscience, New York. Rabinowitch, E. I. (1956) “Photosynthesis and Related Processes,” Vol. 2, pt. 2. Interscience, New York. Rev. Modern Phys. (1959) 31, No. 1 and 2. Sager, R. (1959) Brookhaven Symposia in Biol. No. 11, 101. Sager, R., and Palade, G. E. (1957) J. Biophys. Biochem. Cytol. 3,463. Sager, R., and Zalokar, M. (1958) N a t w e 182,98. St. Whitelock, O.V., and Wolken, J. J., eds. (1958) “Photoreception.” Ann. N . Y . Acad. Sci. 74, 161. Schmidt, W. J. (1935) 2. Zellforsch. u. mikroskop. Anat. 22, 485. Sidman, R. L. (1957) J. Biophys. Biochem. Cytol. 3, 15. Sissakian, N. M. (1957) In “The Origin of Life on the Earth,” p. 235. Acad. Sci. U.S.S.R., Moscow. Sissakian, N. M. (1958) Advances in Enzymol. a0, 201. Sjostrand, F. S. (1953a) J. Cellular Comg. Physiol. 42, 15. Sjostrand, F. S. (1953b) J. Cellular Comp. Physiol. 42,45. Sjostrand, F. S. (1956) In “Physical Techniques in Biological Research” (G. Oster and A. W. Pollister, eds.), Vol. 3. Academic Press, New York.

218

J. J . WOLKEN

Sjostrand, F. S. (1959) Revs. Modern Phys. 31 (2), 301. Smelser, G. K., ed. (1961) “The Structure of the Eye.” Academic Press, Kew York. Smith, E. L. (1941a) J. Gen. Physiol. 24,753. Smith, E. L. (1941b) J. Gen. Physiol. 24,565. Smith, E. L. (1941~) J. Gen. Physiol. 24,585. Smith, E. L., and Pickels, G. E. (1940) Proc. Natl. Acad. Sci. U.S. 26, 242. Smith, J. H. C. (1959) Brookhaven Symposia in Biol. No. 11,296. Stanier, N. Y. (1959) Brookhaven Symposia in Biol. No. 11, 43. Stanier, R. (1960) Harvey Lectures Ser. 64, 219. Strain, H. H. (1944) Ann. Rev. Biochem. 13, 591. Strain, H. H. (1951) “Manual of Phycology,” p. 293. Chronica Botanica, Waltham, Massachusetts. Strother, G. K., and Wolken, J. J. (1960) Nature 188, 601. Strugger, S. (1950) Natumissenschaften 37, 166. Takashima, S. (1952) Nature 169, 182. Thomas, J. B. (1955) P r o p . in Biophys. and Biophys. Chem. 6, 109. Thomas, J. B., Post, L. C., and Vertregt, N. (1954) Biochim. et Biophys. Ac fa 19, 20. Trurnit, H. J., and Colmano, G. (1958) Biochim. et Biophys. Acta 90, 435. von Wettstein, D. (1957a) Exptl. Cell Research 12,427. von Wettstein, D. (1957b) Hereditas 43, 303. von Wettstein, D. (1959) Brookhaven Symposia in Biol. No. 11, 138. Willmer, E. N. (1955) Ann. Rev. Physiol. 17, 339. Wald, G. (1954) Science 119, 887. Wald, G. (1959) “Handbook of Physiology,” Vol. 1, p. 671. Am. Physiol. SOC., Washington, D.C. Weier, T., and Stocking, C. R. (1952) Botan. Rev. 18, 14. Wolken, J. J. (1956a) J. Protozool. 3,211. Wolken, J. J. (1956b) J . Cellular Comp. Physiol. 48, 349. Wolken, J. J. (1957) Trans. N . Y . Acad. Sci. [11] 19,315. Wolken, J. J. (1958a) Ann. N. Y . Acad. Sci. 74, 164. Wolken, J. J. (1958b) J . Biophys. Biochem. Cytol. 4, 835. Wolken, J. J. (1959a) Ann. Rev. Plant Physiol. 10, 71. Wolken, J. J. (1959b) Am. Scientist 47, 202. Wolken, J. J. (1959~) Brookhaven Symposia in Biol. No. 11, 87. Wolken, J. J. (1961a) In “Origin and Role of Complex Macromolecular Aggregates in Development” (M. E. Edds, ed.). Ronald Press, New York. Wolken, J. J. (1961b) In “The Structure of the Eye” (G. K. Smelser, ed.), p. 173. Academic Press, New York. Wolken, J. J., and Palade, G. E. (1953) Ann. N.Y. Acad. Sci. 66, 873. Wolken, J. J., and Schwertz, F. A. (1953) 1. Gen. Physiol. 37,111. Wolken, J. J., and Schwertz, F. A. (1956) Nature 1 , 136. Wolken, J. J., and Shin, E. (1958) J. Protozool. 6, 39. Wolken, J. J., Capenos, J., and Turano, A: M. (1957) J. Biophys. Biochem. Cytol. 3, 441. Wolken, J. J., Bowness, J. M., and Scheer, I. J. (1960) Biochim. et Biophys. Acta 43, 531. Wulff, V. J. (1956) Physiol. Revs. 36,145.

Use of Inhibiting Agents in Studies on Fertilization Mechanisms' CHARLES B. METZ Occanographic Iitstitute, Florida State University, Tallahassee, Florida

I. Introduction ..................................................... 11. Fertilization-Inhibiting Action of Sperm and Egg Extracts ......... A. Effect of Fertilizin on the Fertilizing Capacity of Sperm ....... B. Effect of Antifertilizin on the Fertilizability of Eggs. ........... 111. Fertilization Inhibitors of Fortuitous Origin ....................... A. Dermal Secretion from Arbacia .............................. B. Fertilization-Inhibiting Extracts of Fucus ...................... C . Other Inhibiting Agents ..................................... IV. Fertilization-Inhibiting Action of Antibodies ...................... A. Action on Sperm ............................................ B. Action on Eggs ............................................. V. Conclusions ...................................................... References ......................................................

Page 219 220 221 228 229 229 237 239 240 241 245

248 251

I. Introduction Since the classic studies of F. R. Lillie on sea urchins, many if not all of the initial steps in fertilization have been considered to result from the interaction of sperm and egg substances. Attention has been directed especially toward the specific sperm isoagglutinin, fertilizin, obtained from the eggs, the complementary sperm substance, antifertilizin, and certain egg membrane lysins obtained in sperm extracts. These lysins have a rather self-evident role in fertilization, namely to dissolve a path through the egg jelly or other extraneous egg membranes for the fertilizing spermatozoan (Tyler, 1948 ; Metz, 1957b ; Brookbank, 1958). Such lysins, then, perform a secondary but essential function in fertilization. For the mechanism of attachment of sperm to egg, especially with relation to its specificity, there is evidence of interaction of surface substances although their mode of action is not fully understood. The mechanism of penetration of the egg surface (engulfment) ; initiation of the cortical reaction of the egg ; the block to polyspermy ; the initiation of development of the egg and the acrosomal reaction of the sperm have not been demonstrated unequivocally to result from interaction of known, well-defined substances. In fact, their relation to surface or other substances is largely obscure, but 1 Contribution No. 158 from the Oceanographic Institute. The author's studies are supported by grants from the National Science Foundation, The National Institutes of Health, and the Florida State University Research Council.

219

220

CHARLES B. METZ

knowledge is accumulating concerning these intriguing problems. Some of this information will be reviewed here. ' For earlier reviews along similar and also along somewhat different lines see : Metz, 1957a,b ; Allen, 1958 ; Rothschild, 1956; Runnstrom et a]., 1959; Runnstrom, 1958; and Tyler, 1959. One of the means for examining the role of known substances in fertilization and perhaps revealing as yet unsuspected substances and investigating their role in fertilization is through an examination of the mode of action of fertilization-inhibiting agents. This approach fo the problem is not new. Indeed, F. R. Lillie (1919) based an important part of his Fertilizin Theory upon studies using an inhibitor which he believed was present in the blood of the sea urchin, Arbaciu. More recently, this approach has gained added interest. Studies have been conducted on several kinds of fertilization-inhibiting agents. These include specific interacting substances obtained from the gametes themselves, certain naturally occurring fortuitous substances, and, finally, antisera obtained in rabbits or other animals by active immunization with eggs, sperm, or extracts of these cells. It is the purpose here to review these studies on fertilization inhibitors, examine their contribution to an understanding of the mechanisms of fertilization, and consider possible avenues of future investigation. In so doing, it is assumed that fertilization involves the interaction of specific surface and perhaps subsurface substances of eggs and sperm, and that premature blocking of these by combination with the same or other substances should prevent the egg and sperm substances from engaging in essential reactions upon union of the gametes. 11. Fertilization-Inhibiting Action of Sperm and Egg Extracts

If specific complementary surface substances are involved in essential interactions in fertilization, these substances should inhibit fertilization under certain conditions (Tyler, 1941, 1959 ; Metz, 1957b). Thus, premature saturation of sperm surface substances by complementary substances extracted from eggs should block the sperm substances and reduce the fertilizing capacity of these gametes. Corresponding treatment of eggs with complementary substances from sperm should reduce the fertilizability of eggs. The only well-established complementary egg and sperm substances are fertilizin from eggs and antifertilizin from sperm (see Metz, 1957b ; Tyler, 1948, 1956; Minganti and Vasseur, 1959 for physical and chemical properties of these substances). Solutions of fertilizin (egg jelly substance) dramatically agglutinate the sperm of the species. This agglutination re-

INHIBITING AGENTS IN FERTILIZATION STUDIES

221

action is highly tissue and species specific, and is found in many, but not all, species in several phyla. By use of special methods, fertilizins that do not ordinarily agglutinate sperm have been demonstrated in a number of species (Metz, 195713). Accordingly, fertilizin occurs widely, if not universally, among metazoa. These facts suggest that fertilizin may perform an essential function in fertilization. The specific agglutination of sperm by fertilizin implies a “complementary surface substance” or combining site on the sperm surface. This specific sperm surface substance is called antifertilizin. Attempts to extract antifertilizin from sperm (Frank, 1939; Tyler, 1939 ; Tyler and O’Melveny, 1941) have resulted in solutions that have certain of the properties required of antifertilizin ; they neutralize the sperm agglutinating action of fertilizin, agglutinate eggs and precipitate the egg jelly. However, the specificity of action of the extracts is less than the spermagglutinating action of fertilizin. This and certain other considerations have led some investigators (Hultin, 1949; Metz, 1957b, 1959a; Kohler and Metz, 1959) to question whether the egg-agglutinating agent in sperm extracts is actually the sperm surface antifertilizin. In any event, the sperm extracts do react with fertilizin and neutralize its sperm agglutinating action. Accordingly, the sperm extracts can serve to block egg surface fertilizin and thereby provide a tool for evaluating the role of fertilizin in fertilization. A.

EFFECT OF FERTILIZIN O N T H E FERTILIZING CAPACITY OF SPERM

Tests of the fertilizing capacity of fertilizin-treated sperm present two difficulties. The mechanical trapping of sperm by agglutination should interfere with the fertilizing action of the sperm quite apart from any blocking of sperm surface substances. In sea urchins this is not a serious problem because the agglutination reverses spontaneously permitting fertilizing capacity tests independently of any mechanical trapping. However, in species other than sea urchins the agglutination does not reverse spontaneously. In these the most practical approach is to convert the fertilizin to a nonagglutinating, univalent form. Such fertilizin combines with sperm surface antifertilizin but does not agglutinate the sperm. Univalent sea urchin fertilizin is readily prepared by heating, ultraviolet irradiation, or oxidation with H202 (Metz, 1957b). Univalent fertilizin has been prepared in only one group of organisms (starfish; Metz, 1945, 1957b) in which the fertilizin-agglutination reaction does not reverse spontaneously. The second difficulty concerns the degree of saturation of the sperm surface with fertilizin. The experiments cannot be considered critical

222

CHARLES B. MET2

unless tests demonstrate complete saturation of the sperm with fertilizin or unless elaborate quantitative measurements are made on the spermfertilizing capacity. Fortunately, reversed sperm can readily be tested for complete saturation by further addition of fertilizin. If the sperm fail to reagglutinate, they may be considered to be saturated. Similarly, failure to agglutinate with normal fertilizin after treatment with univalent fertilizin demonstrates saturation of the antifertilizin receptor sites. When these precautions are taken, it is frequently found that fertilizintreated sperm show marked reduction in fertilizing capacity. For example, this is the case in the sea urchins, Strongylocentrotus Purpuratus (Tyler, 1941, 1959), Hemicentrotus pulcherrimus, Anthocidaris crassispinu and Pseudocentrotus depressus (Dan, 1954). On the other hand, certain species do not show a significant reduction in fertilizing capacity even under the critical conditions outlined above. Notable examples are Arbacia punctulata and Mellita quinquiesperforata (Tyler and Metz, 1955). However, if a sufficiently sensitive test system is employed, namely trypsintreated eggs, then clear-cut differences in the fertilizing capacity of the fertilizin-treated and control sperm are observed even in Arbacia (Table I). I n the above experiments the “sperm dilution’’ method was employed to obtain a quantitative measure of the fertilizing capacity of the treated sperm. This method has the theoretical disadvantage that it ~

TABLE I FERTILIZING CAPACITY OF FERTILIZIN-AGGLUTINATED A N D REVERSED SPERMTESTED ON TRYPSIN-TREATED EGGS OF Arbaciaa Percentage fertilization of Control eggs inseminated with Dilution of 1/3% semen Undiluted 3-fold %fold 27-foId 8l-fold 243-fold 7sfold

Trypsin-treated eggs inseminated with

Control sperm

Fertilizintreated sperm

Control sperm

100

95 98 99 50 80 20 3

100 98 99 35 7 20 5

100 100 99.5 90 95 80

Fertilizintreated sperm 4 3-4 2.5 2 1.5 0.2 0.2

5 Trypsin treatment of eggs took place for 3 hours in 0.05% solution in sea water. For fertilizin treatment 4 ml. of 1% sperm was added to 8 ml. of a solution of titer 256. At the same time 4 ml. of 1% sperm was added to 8 ml. of sea water. After 20 minutes serial &fold dilutions of 4 ml. of the sperm suspensions were prepared as indicated (column 1) and 2 drops of washed trypsin-treated or control eggs (ca. 500) added immediately after each dilution. (From Tyler and Metz, 1955.)

INHIBITING AGENTS I N FERTILIZATION STUDIES

223

fails to cont.ro1 for an effect on the longevity of the sperm. However, fertilizin prolongs the life of sperm (Rothschild, 1956). Reduction in fertilizability in the presence of fertilizin in solution has also been reported using the “fertilization rate” method. This method depends upon killing the sperm but not the eggs in a suspension at intervals after insemination (Rothschild and Swann, 1949 ; Hagstrom and Hagstrom, 1954). It has the disadvantage that it may not distinguish between factors affecting sperm motility and absolute fertilizability. Nevertheless, and in spite of the fact that fertilizin normally increases the motility of sea urchin sperm, fertilizin reduced the fertilization rate in Paracentrotus lividus (Hagstrom, 1956a). In a subsequent study Hagstrom ( 1959) largely eliminated the possibility that the fertilization rate depression may have resulted from temporary agglutination of the sperm. In view of the difficulties involved in testing the fertilizability of irreversibly agglutinated sperm, it is not surprising that few species other than sea urchins have been examined for an effect of fertilizin on the fertilizing capacity of sperm under the critical conditions outlined above. Most of the studies have been performed on species in which the “egg water” (supernatant from egg suspension) preparations do not agglutinate sperm significantly under ordinary conditions. Fuchs ( 1914, 1915) found no reduction in fertilizing capacity of egg water (nonagglutinating) treated sperm of Ciona i n t e s t i d i s . However, Tyler (1941) reported that egg water treatment lowered the fertilizing capacity of Patiria miniata and Urechis caupo sperm ; Metz and Donovan ( 1949) using Mactra (Sp~sula) solidissma, and Wada (Dan, 1956) using Mytilus obtained similar results. In the last instance the effect was observed only if the sperm had been exposed to the egg water for several minutes. I n these experiments the egg water preparations did not agglutinate the sperm. However, in two of the species (Patirk, Mactra) subsequent study revealed conditions for agglutination. I n Patiria a metal binding agent is required (Metz, 195713). From the foregoing it is clear that in the species examined thoroughly fertilizin treatment lowers the fertilizing capacity of the sperm. These species include representatives from several phyla. Evidently then, the effect is a general, if not universal one. This reduction in the fertilizing capacity of fertilizin-treated sperm has been interpreted as evidence for an essential role of fertilizin and antifertilizin in fertilization (Tyler, 1948, 1959). According to this view, the initial attachment of the sperm to egg involves a- binding through an interaction of sperm surface antifertilizin with egg surface fertilizin. Fertilizin pretreatment of the sperm blocks the sperm surface antifertilizin. The

224

CHARLES B. METZ

blocked sperm surface antifertilizin is then unable to combine with egg surface fertilizin with resulting reduction in fertilizing capacity of the sperm. The main difficulty with this explanation is that the fertilizing spermatozoan must pass through a layer of fertilizin (the egg jelly layer) in its most concentrated natural form. During this passage the sperm surface might be saturated with fertilizin and rendered incapable of fertilizing the egg. On the contrary, fertilizin in the form of an egg jelly layer is actually an aid to fertilization, for removal of the jelly reduces the fertilizability of the egg (Tyler, 1941; Tyler and Metz, 1955).2 This difficulty is resolved (Tyler, 1948) by assuming that the combining sites of fertilizin in the gel form are masked by cross-linkages and are not available for combination with the sperm. This view is supported to the extent that fertilizin in sea water solution and in the gel form stain differently with metachromatic dyes (Monroy et al., 1954) and that specific combining sites of fertilizin in the gel form are not destroyed by Arbuciu dermal secretion (Metz, 1959a). It may still be objected that the fertilizing spermatozoan dissolves a path (e.g., by local acidity from metabolic COZ) through the jelly and ruptures cross-linkages to expose fertilizin-combining sites. However, in the sanddollar, Mellitu quzkquiesperforata, Brookbank ( 1958) has described a heat-labile agent which dissolves the egg jelly and antifertilizin-egg jelly precipitation membranes (Fig. 1). Possibly, this agent could dissociate fertilizin-antifertilizin complexes at the sperm surface during its passage through the jelly. However, the same mechanism should interfere with fertilizin agglutination of sperm (especially strong in Mellitu) and an essential fertilizin-antifertilizin interaction at the egg surface. In other sea urchins claims of an egg jelly dissolving action of sperm 2 When the fertilizability of jellyless (acid-treated) eggs is measured using the “sperm dilution” method, it is found that more sperm are required to fertilize the jelly-free eggs than the controls. However, Hagstrom (1956b) failed to obtain such reduction, and when the fertilization rate method was used a t sperm concentrations at least sufficient to yield 1% fertilization, the jellyless eggs fertilized faster than the controls.

FIG.1. Effect of Mellita quinquiesperforafa sperm extract on Mellita egg jelly. The extract was prepared by freeze-thawing sperm. a. Unfertilized egg showing antifertilizin-jelly precipitation membrane produced by sperm extract that had been heated (70” C.;lO minutes) to destroy the jelly dispersing agent. b. Unfertilized egg after 30 minutes in heated sperm extract followed by unheated extract for 15 minutes. The precipitation membrane has disappeared and the jelly has dispersed. Echinochrome granules are embedded in the jelly. (From Brookbank, 1958.)

INHIBITING AGENTS I N FERTILIZATION STUDIES

225

226

CHARLES B. METZ

extracts have been questioned (Krauss, 1950a,b ; Monroy and Tosi, 1952 ; Monroy et al., 1954). Hathaway and Tyler ( 1958) and Hathaway ( 1959) have recently shown that Arbaciu sperm can split a sulfate-rich fraction from fertilizin. However, this action is associated with reversal of agglutination, rather than an effect on the egg jelly or combination of fertilizin with the sperm surface. In the above discussion the fertilization-inhibiting action of fertilizin on sperm is explained in terms of an essential fertilizin-antfertilizin interaction at the egg surface. Variations of such a mechanism ‘can be formulated, such as a masking of a neighboring essential group by fertilizin when the latter saturates the sperm surface. Another possible explanation of the fertilization-inhibiting action of fertilizin on sperm centers about the acrosomal reaction (Dan, 1956). As first described by J. C. Dan (1952), the sperm acrosome undergoes a striking elongation (Fig. 2) under certain conditions (Dan, 1956 ; Colwin and Colwin, 1957 ; Metz, 1957a), and morphological studies indicate that the resulting acrosomal filament assists in penetration of egg jellies (e.g., Holothuria and Asterius; Colwin and Colwin, 1955) and actually activates the egg in normal fertilization. Conditions which cause the acrosomal reaction include treatment of sperm with egg water. Therefore, it is possible that combination of the fertilizin in egg water with the sperm surface antifertilizin initiates the acrosomal reaction. It is further possible that a “premature discharge” of the acrosomal filament by pretreatment with fertilizin renders the sperm incapable of fertilizing the egg. Dan (1954, 1956) has presented considerable evidence in support of this view, including a correlation of failure of both the lowered fertilizability effect and the acrosomal reaction when sperm are agglutinated by fertilizin in a calcium-deficient medium. However, additional evidence is needed to establish whether fertilizin is the acrosomal-reaction-initiating agent in egg water. More study is also desirable concerning whether the acrosomal reaction must occur at or near the egg surface and in a definite temporal sequence with other events of fertilization. Finally, more evidence that FIG. 2. Electron photomicrographs of Aster& forbesii sperm fixed in 5% formalin, dialyzed against distilled water, air dried to the collodion membrane and shadow cast with chromium. a. Control sperm from sea water suspension. Sperm nucleus and middle piece are compact and closely applied to each other; no trace of acrosomal filament. b and c. Spermatozoa with acrosomal filaments from a suspension treated with 0.01 M Versene and egg water. The long acrosomal filament projects upward from the nucleus in b, and to the left in c. In both b and c the entire sperm head region appears to have undergone partial breakdown with “loosening” and displacement of the midpiece. (Metz, 1957a.)

INHIBITING AGENTS I N FERTILIZATION STUDIES

227

228

CHARLES B. METZ

the acrosoiiial reaction is an absolute requirement for fertilization is needed. Clearly, several possible explanations are available to account for the loss of sperm-fertilizing capacity resulting from fertilizin treatment. Whatever the final mechanism may prove to be, this inhibiting effect is indicative of a close association of fertilizin and antifertilizin with the early events in the union of sperm and egg. OF ANTIFERTILIZIN ON B. EFFECT

THE FERTILIZABILITY O F

EGGS

As described above, certain sperm extracts have properties expected of the sperm receptor substance, antifertilizin. The agent in the extracts may not be the actual antifertilizin from the sperm surface (Metz, 1957b). In fact, it is possible that more than one such agent may be obtained, depending on the extraction procedure (Kohler and Metz, 1959). Nevertheless, the extracts do act on eggs. The effects include neutralization of the sperm-agglutinating action of fertilizin, agglutination of eggs, and precipitation of egg jellies in the form of a pronounced membrane (Fig. 1) . In view of this last action, it is not surprising that treatment with sperm extracts reduces the fertilizability of sea urchin eggs (Tyler and Metz, 1955). The precipitation membrane evidently acts as a mechanical barrier to fertilization and only in Mellita is there a known mechanism for penetration of the precipitation membrane (Brookbank, 1958). A number of other agents are known to precipitate egg jellies in the form of a membrane. These include basic proteins (Metz, 1949) and antisera (Perlmann, 1956). Since the fertilization-inhibiting action in question is explained as a mechanical barrier to the sperm at the jelly periphery, it can reveal little concerning the events of fertilization at the egg surface proper. However, if the egg jelly is removed, except possibly for a layer bound at the egg surface, the sperm extracts can be examined for a more direct inhibitory action. When jellyless eggs are treated with sperm extract (antifertilizin) , they agglutinate, and their fertilizability is roughly proportional to their agglutinability (Frank, 1939; Tyler, 1948; Tyler and Metz, 1955). Finally, treatment with sperm extracts (antifertilizin) markedly reduces the fertilizability of the jellyless eggs (Tyler and Metz, 1955). This inhibitory action is most readily interpreted as a blocking of egg surface fertilizin sites (Tyler and Metz, 1955.). However, the inhibitory activity could result from blocking some other egg surface substance. This reservation is made in view of the rather broad specificity of the sperm extracts and of the fact that the usual preparations obtained by freeze-thawing

I N H I B I T I N G AGENTS I N FERTILIZATION STUDIES

229

sperm can contain at least four different substances complex enough to have antigenic activity (Metz and Kohler, 1960). The studies reviewed here show that extracts of sperm and eggs which react with the surfaces of eggs and sperm, respectively, also reduce the fertilizing capacity of these gametes. This inhibiting action is most readily explained as a blocking of specific receptor sites of antifertilizin at the sperm surface and fertilizin at the egg surface. This indicates that fertilizin and antifertilizin perform an important, if not essential, function in fertilization. This function may be primarily a mechanism for attachment of the sperm to, and penetration of, the egg surface (Tyler, 1959), or it may be concerned with the acrosomal reaction (Dan, 1956). In any event, the formation of the acrosomal filament suggests an increase in surface area of the acrosome. This probably involves formation of a new and possibly different acrosomal surface in the vicinity of the egg. A very small area of contact (approximately 0.002 p2 in Asterias) with this surface is sufficient to activate the egg (Metz, 1957a).

111. Fertilization Inhibitors of Fortuitous Origin Certain agents of origin unrelated to eggs and sperm have fertilization inhibiting action. Among these the dermal secretion from Arbacia and certain extracts of the brown alga, Fucus, have been examined in greatest detail. These agents are of added interest because they inhibit fertilizin agglutination of sperm as well as fertilization. The investigations on these two agents are outlined below.

A. DERMAL SECRETION FROM Arbacia

F. R. Lillie (1914) first described a fertilization-inhibiting action of fluids from Arbacia. H e and Just (1922) ascribed the effect to blood from the animals. However, Oshima (1921) and Pequegnat (1948) clearly showed that the inhibiting agent is released from certain cells of the integument under conditions of stress. Dermal secretion solutions are readily obtained from Arbaciu by osmotic shock. The simplest procedure is to immerse an animal in distilled water for 60-90 seconds, rinse in sea water, and collect the yellowish fluid that is released. Such dermal secretion preparations are not toxic to the gametes. They markedly enhance the motility and respiration of sperm (Pequegnat, 1948; Metz, 1959a) and do not affect the development of eggs placed in the solutions some minutes after fertilization. Clearly then, the dermal secretion does not inhibit sperm agglutination and fertilization by a general cytotoxic effect. Its inhibitory action must involve a more direct interference with fertilization mechanisms.

230

CHARLES B. METZ

1 . Inhibition of Fertilizin Agglutination of Sperm Pequegnat ( 1948) first- reported that Arbacia dermal secretion inhibits fertilizin agglutination of sperm. More recently Metz (1959a) has investigated the mechanism of this inhibition. This analysis showed that the dermal secretion inhibits fertilizin agglutination of Arbacia sperm not by action on the sperm but by an inactivation of the combining sites of the fertilizin. Thus upon washing in sea water dermal-secretion-treated sperm regained agglutinability. Sperm did not absorb or neutralize the agglutinationinhibiting activity of dermal secretion. This shows that the dermal secretion does not have an irreversible inhibiting action on the agglutinability of sperm. To test for action on fertilizin, advantage was taken of the fact that dermal secretion is heat labile whereas fertilizin is stable. Nonagglutinating mixtures of dermal secretion and fertilizin were heated sufficiently to destroy the dermal secretion but not the fertilizin. Such heated mixtures failed to agglutinate sperm, indicating an action of dermal secretion upon fertilizin. Furthermore, treatment with such mixtures failed to affect the agglutinability of sperm (Table 11). This means that the fertilizin in the heated mixtures is incapable of blocking the sperm surface. Apparently upon mixing dermal secretion and fertilizin, the combining sites of fertilizin are inactivated. The heating subsequently destroys any escess dermal secretion. In a complementary experiment it was found that unheated dermal secretion-fertilizin mixtures also failed to block the sperm surface. In this case sperm washed from unheated mixtures was found to agglutinate on addition of control fertilizin. Again it is concluded that the dermal secretion inactivates the combining sites of the agglutinin. 2. Fertilization-Inhibiting Action of Arbacia Dermal Secretion Investigation of the fertilization-inhibiting action of the dermal secretion night reveal information concerning normal fertilization. Indeed, the parallel inhibiting action of dermal secretion on fertilization and on fertilizin-agglutination of sperm suggests that inhibition of fertilization as well as agglutination may result from inactivation of fertilizin-combining sites. The dermal secretion has no irreversible inhibitory action on sperm, for sperm washed from inhibiting solutions showed undiminished fertilizability (Metz, 1959b, 1960). However, action on eggs appears to depend to some extent upon quantitative factors. Pequegnat (1948) reported at least partial recovery of fertilizability upon washing eggs from dermal secretion, whereas Harvey (1956, p. 57) states that treated eggs remain

TABLE I1 EFFECT OF HEATING ON SPERM-AGGLUTINATING ACTION OF FERTILIZIN-DERMAL SECRETION MIXTURES" Heated mixtures 2 3

Unheated mixtures 6 7

4

5

0.5 ml.

D.S.

+

D.S.

+

S.W.

+

S.W.

+

D.S.

+

D.S.

+

S.W.

S.W.

0.5 ml.

fertilizin

S.W.

fertilizin

S.W.

fertilizin

S.W.

fertilizin

S.W.

A. Agglutination on addition of sperm to mixtures

-

-

++++

-

-

-

++++

-

1

B. Inhibition tests (agglutination on addition of control fertilizin to samples tested in A, above)

*

+

z

3 bm 5

v)

s

-I

M PJ c3

+++

+++

-

+++

-

-

-

+++

The heated mixtures were held at 100°C. for 4 minutes. In A, the mixtures were tested for agglutinating action on sperm. The heating failed to restore agglutinating action to the D.S.-fertilizin mixture (L4-1). In B, the mixtures were tested for agglutination-inhibiting action by adding control fertilizin to the mixtures in A following spontaneous reversal of the initial agglutination in A-3 and A-7. The heated mixture fails to inhibit agglutination (B-1 and B-5). Both the inhibitor in the dermal secretion and the fertilizin have been destroyed in the heated mixture. (Metz, 1959a.) 5

2 ! E

Y

r I;

%

C

B v)

El

232

CHARLES B. METZ

unfertilizable even after repeated washings. The quantitative factors concern sperm concentration and developmental rate of treated eggs. As seen in Table 111, Experiment 1, dermal-secretion-treated eggs failed to fertilize at reasonable sperm concentrations giving 100% fertilization of control eggs, but when the sperm concentration was increased 25-fold, 89% of the treated eggs fertilized. This quantitative feature of inhibition can be interpreted in terms of a reduced number of essential reactive sites at the egg surface (Metz, 1960). The second quantitative aspect of inhibition is developmental delay. In some experiments dermal-secretion-pretreated eggs failed to cleave synchronously with controls, but finally divided with a one to two division delay. This delay phenomenon has not yet been investigated in detail. a. E f e c t of Dermal Secretion on Eggs. Treatment with dermal secretion has no pronounced effect upon unfertilized eggs except for a dissolving action on the egg jelly in some cases (Pequegnat, 1948; Metz, 1959a). The dermal secretion does not precipitate the egg jelly as might be expected, in view of its action on the combining sites of fertilizin. Indeed, the combining sites of fertilizin (complementary to sperm antifertilizin) in the gel form are not inactivated by dermal secretion (Metz, 1959a). This is additional evidence (see above) that the combining sites of fertilizin in the gel form are masked. Although the dermal secretion has no readily visible effect, it must have some action on the egg cortex. This is evident from the failure of dermalsecretion-treated oocytes to form blebs or papillae when inseminated (Metz, 1960). As indicated above, eggs placed in dermal secretion some minutes after fertilization develop normally. However, exposure to dermal secretion during the initial stages of fertilization results in dramatic arrest of at least some of the fertilization phenomena. These concern cortical changes and especially those related to fertilization membrane formation. Membrane elevation is stopped virtually instantaneously upon addition of dermal secretion to eggs. This is especially evident in the large, relatively clear eggs of Lytechinus variegutus as seen in Fig. 3. Preliminary examinations with phase optics indicates that the cortical granules fail to break down in eggs treated with dermal secretion shortly after insemination. Dermal secretion may even inhibit propagation of the cortical reaction from the site of sperm-egg interaction. Regardless of the outcome of these possibilities, the observations show that the dermal secretion inhibits cortical phenomena associated with early events of fertilization. b. E f e c t of Partial Dissection of the Egg Surface on Sensitivity to Dermal Secretion. In an attempt to determine the site of inhibition, eggs were subjected to mild “dissection” and then tested for sensitivity to the

TABLE I11

EFFECT OF PROTEOLYTIC ENZYMES O N SENSITIVITY OF EGGS TO

Experiment l a Trypsin-trypsin inhibitor treated Arbmia eggs

THE

FERTILIZATION-INHIBITING ACTIONOF ArOacia DERMALSECRETION

Enzyme-treated eggs

S.W.-treated eggs

.

Sperm dilution

D.S.-Treated

S.W.-Treated

D.S.-Trratcd

S.W.-Treated

1 1/5 1/25 1/125

9%b ( 149)C 98% (151) 42% (295) 8% (199)

97% (154) 68% (187) 21% (187) 5% (187)

sS% (158) 91 % d (162) 3% (156) <1% (Zoo+)

100%(200+) 100%(200+) l00%(200+) 100%(2oo+)

; 5

g Y

Experiment 2e Protease-treated Lytechinus eggs

1:

93% (97) f (f (0%)’ 1/12% 14% (133) 1%(200+) 0% (200+) <3%(200+) a Experiment 1 : Two-ml. samples of drbacia eggs were added to 4-ml. samples of S.W. and 0.05% trypsin (in S.W.). After 45 minutes the eggs were washed two times in S.W., 1.5 ml. of the eggs were transferred to 10 ml. 0.1% ovomucoid in S.W. Subsequently the eggs were washed again, samples were treated with D.S. and S.W., and washed. Four drops eggs were added to 5 ml. .4rbacia sperm dilution series. A duplicate control experiment omitting the ovornucoid treatment gave similar results. Sperm dilution 1 = 0.1% semen. Degree of polyspermy was not recorded in this experiment. b % = Per cent cleavage. c Figures in parentheses are the total number of eggs counted. d Cleavage was delayed two divisions as compared to controls. e Experiment 2: One-half-ml. samples of unfertilized Lytechinus eggs were placed in 5 ml. 0.1% protease solution in S.W. After 89 minutes 1-nil. samples of protease and S.W. treated eggs were added to 1 nil.-samples of D.S. and S.W. Twenty minutes later the eggs were washed in S.W. and 4 drop samples were added to 5 ml. Lytechinus sperm a t the dilutions indicated. Sperm dilution 1 = 16% of undiluted semen, approximately 2 x 10s sperm/ml. in this case. Percentage of eggs counted showing the abnormal cleavage characteristic of polyspermy. (Metz, 1%0.) 1

94% (107)

*

t,

d

m v1

q

F

-2

Y

Z u1

8

u1

N

w w

234

CHARLES B. MET2

dermal secretion. The first agent used for such dissection was acid sea water ( p H 4-5) which dissolves the jelly layer (fertilizin) from sea urchin eggs. Such removal of the jelly did not affect the sensitivity of the eggs. Their fertilizability was strongly inhibited by dermal secretion. It follows that the egg jelly is not the site of inhibitor action. This also suggests

FIG.3. Effect of Arbocia dermal secretion on fertilized Lytechinus vuriegatus egg. a. Three drops eggs placed in 1 ml. dermal secretion 55 seconds after insemination, photographed 15 minutes after insemination. b. Control eggs in sea water, photographed 11 minutes after insemination. The dermal secretion has arrested fertilization membrane elevation of the treated eggs.

that fertilizin is not involved in the inhibitory effect since fertilizin constitutes the jelly. However, as noted above, the possibility remains that an essential acid-resistant layer of fertilizin remains bound to the egg surface. A further dissection of the egg surface was achieved by treatment with proteolytic enzymes (crystalline trypsin ; crystalline “protease”). As is

INHIBITING AGENTS I N FERTILIZATION STUDIES

235

well known, trypsin-treated sea urchin eggs lack jeilies, fail to raise fertilization membranes, and become polyspermic readily when inseminated. Following exposure to dermal secretion, trypsin-digested eggs fertilized as readily as trypsin-treated control eggs (Metz, 1959b, 1960). In fact, trypsin treatment rendered the eggs insensitive to the dermal secretion whether it preceded or followed the exposure to the inhibitor (Table 111). These results indicate that proteolytic enzymes destroy or remove some dermal-secretion-sensitive site of the egg which is essential in normal fertilization. In so doing, the enzyme must expose some alternative pathway for fertilization. This alternative pathway lacks the normal fertilization specificity and the block to polyspermy (Bohus Jensen, 1953a,b). However, this last relationship is not causally related to loss of dermal secretion sensitivity, for removal of the block to polyspermy with nicotine did not alter the sensitivity of eggs to the inhibiting action (Metz, 1960). The alternative pathway is more “refractory” to fertilization than the normal pathway since trypsin-treated eggs require more sperm than control eggs to achieve a given level of fertilization. This is interpreted by Tyler and Metz (1955) as a reduced number of available combining sites at the surface of trypsin-treated eggs. However, if the reduction in fertilizability resulted from a simple reduction in available combining sites, trypsin treatment should produce increased rather than decreased sensitivity to the inhibiting action of dermal secretion. Therefore, it is concluded that qualitative factors are also involved. One possibility is that fertilizin is the dermal secretion sensitive substance removed by trypsin. This view is especially attractive because dermal secretion inactivates the combining sites of fertilizin. However, evidence concerning this is conflicting. Thus the fertilization-inhibiting action of dermal secretion is not diminished by addition of fertilizin, as might be expected if dermal secretion inhibited by an irreversible combination with fertilizin (Metz, 1960). On the other hand, Tyler and Metz (1955) have shown that treatment with fertilizin reduces the capacity of sperm to fertilize trypsin-treated eggs. This suggests that fertilization through the “alternative pathway” in trypsin-treated eggs involves antifertilizin or a neighboring sperm surface substance. Specificity requirements dictate that the sperm antifertilizin combine with fertilizin at the surface of the trypsin-treated egg. The fertilization membrane is readily removed mechanically from Lytechinus eggs. The resulting demembranated eggs may be considered to have suffered additional surface dissection. They develop polyspermically ( refertilize) if subsequently reinseminated (Tyler et al., 1956). Such demembranated eggs refertilized as readily as controls after

,

236

CHARLES B. METZ

treatment with dermal secretion (Table IV) . Evidently, refertilization also follows an alternative route to that obtaining in normal fertilization. The failure of dermal secretion to affect the fertilizabilitg of trypsintreated eggs and refertilizability of demembranated eggs is explained above in terms of removal of a dermal-secretion-sensitive site or substance essential in normal fertilization. Removal of this substance must coincide with exposure of an alternative dermal-secretion-insensitive pathway. TABLE IV PERCENT NORMALCLEAVAGE OF DEMEMBRANATED Lytechinus vuriegatus EGGSAFTER REINSEMINATION FOLLOWING TREATMENT WITH DERMALSECRETION^ Eggs in D.S.

Eggs washed from D.S.

Eggs in

S.W.

Eggs washed from S.W.

77% (129) 80% (118) 82% (137) 82% (86)

3%(123)O 3% (107) 45% (110) 84% (129)

3% (132) 2% (103) 53% (88) 84% (76)

0% (106) 3% (114) 49% (93) 85%(112)

Reinseminating sperm dilution Ib

1/ 5 1 /25 Not reinseminated

(1 Eggs were demembranated 2 minutes after the initial insemination. Thirteen minutes later 10 drops eggs were placed in 2.5ml. D.S. These were washed in S.W. 11 minutes later. Forty-two minutes after the initial insemination these eggs, as well as S.W. controls and eggs that had been in D.S. for 14 minutes, were reinseminated. The eggs that failed to cleave normally showed abnormalities characteristic of polyspermy. b Sperm dilution 1 = approximately 1.5%. c Numbers in parentheses = number of eggs counted.

This interpretation must be qualified in one respect. Proteolytic enzymetreated eggs fertilize, and demembranated eggs refertilize, as readily as controls after washing from dermal secretion. However, if the insemination, or reinsemination, is carried out directly in the dermal-secretionsolution the eggs fail to fertilize or refertilize (Table IV). Thus, the action of dermal secretion on normal eggs is essentially irreversible, whereas the action on trypsin-treated or demembranated eggs is readily reversed by washing. Attempts to demonstrate two inhibitors, one acting reversibly on sperm or on both normal and trypsin-treated eggs, and a second acting irreversibly only on normal eggs, have so far failed. Evidently, further study will be required to obtain a complete understanding of the action of the dermal secretion. Nevertheless, the reversibility of inhibitor action on trypsin-treated eggs as compared to control eggs shows that dermal secretion can be used to probe for sites that function in fertilization. c. ChemicaJ Nature of the Dermal Secretion. The dermal secretion

I N H I B I T I N G AGENTS I N FERTILIZATION STUDIES

237

has not been subjected to a systematic chemical characterization. Howevef, the active material for both agglutination and fertilization inhibition is heat labile, precipitated by ammonium sulphate, and is not diffusible through cellophane. The inhibitor solutions contain a substance which auto-oxidizes in air to a brown color and apparently is a large molecule since it also precipitates with ammonium sulphate and fails to diffuse through cellophane. The dermal secretion is evidently a fairly complex mixture since it contains at least three antigens in common with Arbacia sperm (Metz, 1959a). It remains to be determined if the pigment and antigens have any relation to the agglutination and fertilization-inhibiting action of dermal secretion.

B. FERTILIZATION-INHIBITING EXTRACTS OF Fucics Inhibition of fertilization in the sea urchin by extracts of the alga, Fucus, was first reported by Harding (1951). The mechanism of action of the inhibiting agent or agents has since been studied, especially by Wicklund ( 1954), Runnstrom and Hagstrom ( 1955), and Branham and Metz (1959). Inhibiting preparations can be obtained by extraction of triturated algae in distilled water, concentration of the ethanol-soluble material, and dialysis of the final product against sea water. The material apparently is rather inhomogenous even after considerable purification (Esping, 1957a). According to the earlier studies (Wicklund, 1954), the extracts consist largely of carbohydrate and are called fertilization inhibitor (Fucus) or FeInh ( F u ) . Fertilization-inhibiting material was also obtained by lead acetate precipitation of methanol extracts. This was considered to consist mainly of polyphenols. Subsequently, Esping ( 1957a,b), using inhibition of hyaluronidase and other enzymes as an assay, concluded that the inhibiting material of Fucus extracts, including FeInh( Fu) , was probably one or more polyphenolic substances. Information concerning the inhibitory action of the methanol extracts is limited. I t includes the report that the agent(s) is not inactivated by periodate oxidation, and apparently acts reversibly since eggs washed from the solutions regain fertilizability ( Wicklund, 1954). The action of the FeInh(Fu) has been examined more extensively. It should be borne in mind, however, that the preparations are inhonJogenous. The extracts do have physical effects upon unfertilized sea urchin eggs. These include an inhibition of jelly swelling that normally occurs when sea urchin eggs are placed in sea water (Runnstrom and Hagstrom, 1955) and an increase in the rigidity of the egg, as demonstrated by a reduced stretching in the centrifuge (Kriszat, 1953 ; Runnstrom, 1957a).

238

CHARLES B. METZ

Runnstrom and Hagstrom (1955) report that the Fucus extract does not inhibit fertilizin agglutination of sperm in Parcentrotus lividus and Arbacia lixula. The only effect of the agent was a retardation of the normal reversal of agglutination. In an independent study, Branhain and Metz ( 1959) observed strong inhibition of fertilizin agglutination of sperm in Arbacia punctulata. The agent (s) in the extract apparently inactivated the antifertilizin groups of the sperm surface. Sperm washed from Fucus extracts failed to agglutinate with fertilizin and did not absorb the agglutinin from the solution. The Fucus agent apparently does not inactivate fertilizin-combining sites, for the active agglutinin could be recovered from nonagglutinating fertilizin-Fucus extract mixtures by preferentially adsorbing the inhibiting agent on activated charcoal. The fertilization-inhibiting activity of the aqueous extract is heat stable (100°C.; 30 minutes), does not diffuse through cellophane, and is reportedly destroyed by periodate oxidation (Runnstrom and Hagstrom, 1954; Wicklund, 1954). The agent does not inhibit fertilization by irreversible action on the sperm. In fact, Wicklund (1954) found that the extracts enhanced the motility and prolonged the fertilizing life span of sperm. Even in Arbacia punctulata, where the extracts inactivated the antifertilizin sites on the sperm surface, washing restored the fertilizing capacity (but not the agglutinability) of treated sperm suspensions (Branham and Metz, 1959). According to Wicklund (1954) and Runnstrom ( 1957a), eggs washed from inhibiting extracts regained fertilizability, and especially in the presence of albumen. In fact, albumen and sea urchin egg homogenate neutralized the inhibiting action of the extract ( Wicklund, 1954). In Arbaciu punctulata, however, washing failed to restore the fertilizability of Fucus extract-treated eggs (Branham and Metz, 1959). Paracentrotus eggs, pretreated with periodate, or rendered jelly-free by acid treatment, were also inhibited but to a somewhat lesser extent than controls inseminated in the Fucus preparation (Runnstrom and Hagstrom, 1955). Periodate pretreatment also lowered the sensitivity of Psammechinus eggs in fertilization rate experiments (Runnstrom and Immers, 1956). As seen in Table V, normal Arbacia punctulata eggs fail to fertilize but trypsin-pretreated eggs are fertilizable after washing from the extract. Thus, Fucus extract and dermal secretion are similar, at least to the extent that they both fail to inhibit irreversibly the fertilizability of trypsintreated eggs. As seen above, the observations of Wicklund and Runnstrom and Hagstrom on-the one hand, and Branham and Metz on the other, are not in complete agreement either as to the action of Fucus extracts on fertilizin agglutination of sperm or on all points regarding inhibition of ferti-

INHIBITING AGENTS I N FERTILIZATION STUDIES

239

lization. These differing results may stem from differences in sea urchin material or from differences in the Fzicus extracts. Irrespective of the situation with the European material, it is of considerable interest that Fuciis extracts block the antifertilizin of the Arbacia punctulata sperm surface. In this connection, it would be of interest to examine the fertilizing capacity of such sperm employing the more sensitive trypsin-treated TABLE V EFFECT OF Fiicais EXTRACT O N FERTILIZABILITY OF TRYPSIN-TREATED Arbacia EGGS Trypsin-treated eggs FZlCUS-

Sperm

dilution

0.5% 0.05% 0.005%

extracttreated

s.w.treated

S.W.-treated eggs FUCUSextracts.w.treated treated

0% (ZOO+) 100%(200+) 78% (135) 50% (139) 0% (ZOO+) 100%(200+) 0% (ZOO+) 98% (200+) 2% (128) 0% (ZOO+) 54% (111) 0.0005% (ZOO+) 0% (200+) a Arbacin eggs were treated in 0.05% crystalline trypsin solution for 71 minutes, washed in S.W. and in 0.5% trypsin inhibitor (both S.W.- and trypsin-treated eggs). After a final sea water wash, the eggs were treated for 10 minutes in Fucus extract, washed in sea water and inseminated. Controls were employed as indicated in the table. b Percentages of cleaving eggs are recorded; the values in parentheses are number of eggs counted.

93% 94% 99% 0%

(118)b (115) (141)

eggs (Tyler and Metz, 1955), and to test the effect on sperm of a species such as S. purpuratus that show marked loss of fertilizing capacity following fertilizin treatment. The insensitivity of trypsin-treated eggs to the Fucus extract suggests that this agent may operate by blocking or inactivating the same site as that blocked or inactivated by dermal secretion.

C. OTHERINHIBITING AGENTS A number of other agents have been found to inhibit fertilization but their mechanism of action has been examined less thoroughly. The agents which do not kill the sperm or eggs at fertilization-inhibiting concentrations include heparin, human H substance, chitin disulfuric acid, cellulose trisulfuric acid, sialic acid, certain flavonols, and germanin. Of these, heparin has been examined most extensively. In the presence of 0.05% heparin, Ckaetopterus eggs apparently fail to fertilize except at high sperm concentrations (Heilbrunn and Wilson, 1949), and frog eggs showed reduced susceptibility to parthenogenetic activation (D. Harding, 1949). In sea urchins parthenogenetic activation by hypertonic sea water and normal fertilization are inhibited in the presence of some, but not all,

240

CHARLES B. METZ

preparations of heparin (Gagnon, 1950; Harding, 1950, 1951 ; Harding and Harding, 1952; Hagstrom, 1956a, ,1959). The inhibiting action is reversed by washing eggs or simply diluting inhibited sperm-egg mixtures with sea water. As might be expected, the fertilization-inhibiting action of heparin is readily destroyed by periodate oxidation. Harding (1951) also found that A T P counteracted the fertilization-inhibiting action of heparin and suggests that the agent may inhibit fertilization by interfering with phosphate metabolism. Harding ( 1950, 1951) reported that fertilization of Arbacia eggs failed to occur in solutions of human H substance (mucopolysaccharide) . Wicklund (1954) confirmed this observation and showed that this and the other carbohydrate derivatives listed above were rendered tioninhibiting by periodate oxidation. She also reports that eggs washed from H substance, chitin disulfuric acid, and flavonols regain fertilizability. Aside from the agents considered above, interesting fertilization-inhibiting effects of two oxidizing agents, porphyrexid and porphyrindin, have been reported by Runnstrom (1957b). Appropriate concentrations of these agents inhibit fertilization. Eggs regained fertilizability upon washing, although fertilization membrane formation was abnormal. If the pretreatment did not exceed 30 minutes, all eggs cleaved. Longer pretreatment resulted in monaster formation without cleavage. In these the oxidizing agent apparently had the interesting effect of permitting attachment of the sperm to the egg and activation of the egg, but preventing penetration of the egg by the activating sperm. The preparations discussed in this section, insofar as they have been tested, failed to show species specificity. It is clear that only Arbacia dermal secretion and aqueous extract of Fucus have been investigated sufficiently to demonstrate their usefulness as possible probes or labels of active sites that function in fertilization.

IV. Fertilization-Inhibiting Action of Antibodies Sperm, eggs, and extracts of these cells, stimulate the formation of antibodies when injected into an appropriate foreign species. Some of these antibodies are directed against cell surface substances or molecular configurations, as evidenced by agglutinating action on the gametes. If these surface substances engage in essential reactions in fertilization, antibody should combine with and block them at the cell surface, and thereby inhibit fertilization. I n practice, tests for this action are complicated by the possibility of mechanical trapping of cells by the agglutinating action of the antibody and precipitation of accessory jelly layers, etc., to form inipermeable barriers. This difficulty is best overcome by use of univalent, non-

241

INHIBITING AGENTS IN FERTILIZATION STUDIES

agglutinating, and nonprecipitating antibody. It is a particularly important consideration with sperm, for these cells are very susceptible to the agglutinating action of antibodies.

A. ACTION ON SPERM Considerable information has been obtained concerning the antigenic structure of sperm, sperm extracts, and antigenic changes of sperm during passage through the male (mammalian) reproductive tract (see Tyler, 1942, 1949; Landsteine‘r, 1945; Kohler and Metz, 1960; Weil, 1%0; Weil and Finkler, 1958, 1959, for literature). However, only one serious TABLE VI PERCENTFERTILIZATION OF Lytechirtus AND Urechis EGGS BY HOMOLOGOUS SPERM TREATED WITH “UNIVALENT” ANTIB~DIES~ Sperm and eggs of

MI.of serum-treated sperm (0.1%) used

for insemination

Lytechinus pictus L1 N1 U1

0.013 0.025 0.05 0.1 0.2 0.4 0.8

0 10 15 0 30 35 0 85 75 0 95 99 0 100 100 1 100 99

Urechis caupo u1 L1

%

20 55 5 95 12 100 15 99 30 100 2

a Percentage fertilization of ca. 400 eggs in 5 ml. of sea water by sperm treated 15 minutes with photo-oxidized rabbit antisera vs. antifertilizin of Lytechinus ( L l ) and of Urechis ( U l ) and normal rabbit serum ( N l ) , using 1 vol. of 1% sperm to 9 vol. of half-strength serum adjusted to sea water salinity. Photo-oxidation : 352 cu. mm. 0, uptake per ml. serum for L1, U1, N1. Original agglutinative titers: 512 for L1, 1024 for U1. Titers for inhibition of agglutination: 6 for L1 and 8 for U1. (Tyler, 1946a.)

effort has been made to block sperm surface antigen(s) with univalent antibody and to examine for an effect on the fertilizing capacity of the sperm. Following an initial trial using normal antibody (Tyler and O’Melveny, 1941), Tyler ( 1946a) prepared antisera against Lytechinus pictus and Urechis caupo antifertilizin ( p H 3-4 sperm extract), converted these to the univalent form by photo-oxidation (Tyler, 1945 ; Tyler et al., 1954), and tested for effect on the fertilizing capacity of sperm. Two of Tyler’s experiments are given in Table VI. In all of the experiments, sperm treated with homologous, univalent antibody showed marked reduction in fertilizing capacity. In most experiments, treated sperm failed to fertilize at concentrations in excess of 64 times the control sperm concentration required to obtain significant fertilization. The inhibiting action

242

CHARLES B. METZ

was species specific. Evidently, the acid extracts of sperm contain one or more antigens in common with the sperm surface. The extracts used as immunizing antigens by Tyler were purified by (NHa)&04 and isoelectric precipitation, they produced a single electrophoretic peak, and had high sperm-agglutinating titers. O n the basis of these properties Tyler (1946a) concluded that the material contained a single substance, antifertilizin, from the sperm surface. Recently, sperm extracts, including those prepared by acid extraction of sperm, have been found to contain several antigens (Metz and Kohler, 1%0), and some question has arisen concerning the sperm surface origin of the egg agglutinating material in such extracts (Kohler and Metz, 1959). But apart from the identity of the immunizing antigen(s) in Tyler’s sperm extracts, the resulting antisera did reduce the fertilizing capacity of the sperm. The simplest explanation for this action is that the univalent antibody blocks or masks sperm surface combining sites essential in fertilization either by direct interaction or by steric effects through combination with neighboring material. It would be interesting to test the univalent antiserum-treated sperm for agglutination with fertilizin. If they failed to agglutinate it could be concluded that the sperm surface antifertilizin groups were blocked by the univalent antibody. Other experiments concerning sperm surface antifertilizin include the demonstration that sea urchin sperm after fertilizin agglutination and reversal still agglutinate on addition of anti-sperm serum (Metz and Kohler, unpublished). This can be explained by assuming that some, or all, of the sperm-agglutinating antibodies react with surface substances other than antifertilizin. Another possibility is that the antibody combined with the univalent fertilizin to agglutinate the reversed sperm. As a further test for antibody against sperm surface antifertilizin in antisperm rabbit serum, antiserum was absorbed with fertilizin-treated sperm. Such absorbed serum failed to agglutinate control sperm. Although other explanations are not excluded, this result suggests that the fertilizincombining sites of sperm surface antifertilizin are not antigenic. Irrespective of the antigenicity of antifertilizin, the sperm surface possesses a number of antigens. This was demonstrated for mammalian sperm by Henle et al. (1938), who showed that bull sperm had at least three antigens, one restricted to the sperm head, a second confined to the tail, and a third common to both. In the sea urchin, Strongylocentrotus purpuratus, Tyler ( 1949) demonstrated a minimum of two agglutinogens by cross-absorption with the sperm of heterologous, cross-agglutinating species. Using the same method, Kohler and Metz (1960) found a minimum of three such antigens in Arbacia punctulata and Lytechinus vurie-

INHIBITING AGENTS I N FERTILIZATION STUDIES

243

gntus sperm. It is likely that additional antigens might be demonstrated with.more such cross tests. Aside from these, Arbacia sperm, like bull sperm, possess antigens restricted to heads and tails as well as antigen(s) common to both (Kohler and Metz, 1960). Finally, some, but not all, of the sperm surface antigens are insoluble or so firmly built into the cell surface structure that they cannot be extracted by ordinary means, for,

“1 8

6

a w

E 4

2

0

0.5

1.0

I.5

2 .O

ML ANTIGEN

FIG.4. Effect of absorption with increasing amounts of antigen on sperm-agglutinating action of anti-Arbacia puizctzclata sperm serum. Curve ( a ) : Absorbing antigen prepared by heating (100” C., one minute) sperm. Curve (b) : Absorbing antigen prepared by extracting sperm in pH 3.0 sea water. Curve (c) : Absorbing antigen prepared by Mickle disintegration of sperm. Curve (d) : Absorbing antigen : isolated sperm heads. Curve ( e ) : Absorbing antigen: whole intact sperm. Absorbing antigen for curve ( a ) , (b), and (c) centrifuged 3000 x g. Each curve represents a different experiment. Ordinate : Titer = -log2 of highest antiserum dilution giving sperm agglutination. Abscissa: ml. antigen added to 0.5 rnl. antiserum. (Kohler and Metz, 1960.)

as seen in Fig. 4,absorption with Arbacia sperm extracts fails to remove all Arbacia sperm agglutinins from anti-Arbacia sperm serum. This indicates that some sperm surface antigens are not present in the absorbing extract. Likewise, absorption with extracted sperm “ghosts” removed agglutinins from the antiserum. Additional evidence that the soluble and insoluble Arbacia sperm surface antigens are qualitatively different is seen in the observation that Arbacia sperm extract absorbed agglutinins for Arbacia

244

CHARLES B. METZ

but not for Lytechinus sperm, whereas the Arbacia sperm ghosts absorbed agglutinins for both ( F i g . 5 ) . Evidently the Arbacia sperm surface antigens present on Lytechinus sperm are mainly of the “insoluble” type. It is of some interest that these insoluble antigens of the Arbacia sperm are largely confined to the Arbacia sperm head. 12

10

8

K

p I-

4

2

0

.2

0

50

.4 Go

.6

150

.8 ML ANTIGEN (curve a,b) 200 MG DRY WEIGHT (curve c,d)

FIG.5. Sperm-agglutinating action of anti-Arbaciu pultctulata whole sperm serum absorbed with Arbacia antifertilizin and sperm “ghosts.” Curve ( a ) and ( b ) : Absorbing antigen, frozen-thawed sperm extract. ( a ) : Tested on Arbacia sperm. ( b ) : Tested on Lytechiitus variegatus sperm. Abscissa : ml. antigen added to 0.2 ml. antiserum. Curve (c) and ( d ) : Absorbing antigen, frozen-dried “ghosts.” (c) : Tested on Arbacia sperm. ( d ) : Tested on Lytechinus sperm. Abscissa: mg. dry weight. Ordinates for ( a ) , ( b ) , ( c ) , and ( d ) : Titer = -log, of highest antiserum dilution giving sperm agglutination. (Kohler and Metz, 1960.)

Unfortunately, no further localization of Arbacia sperm surface antigens has been achieved (Kohler and Metz, l%O). Further study, possibly using fluorescein or tritium-labeled antibody, should localize more precisely the head and tail as well as the soluble and insoluble antigens. It is possible that antibody specific for acrosome, nucleus, midpiece, and tail may be prepared. Conversion of these to the univalent form followed by investigation of their effects on the fertilizing capacity of the sperm should lead not only to localization of surface substances that function in fertili-

I N H I B I T I N G AGENTS I N FERTILIZATION STUDIES

245

zation, but the specific antibody should provide a label for these substances to be iised in their extraction, purification, and characterization. B. ACTIONO N EGGS The antigenic composition of eggs of several species has been studied rather extensively. Among vertebrates, amphibian and avian eggs have received special attention (see Tyler, 1955 ; Ebert, 1958 for review). However, in view of the difficulty of fertilizing such eggs in vitro, it is hardly surprising that these studies have not been concerned with fertilization. On the other hand, the recent work of Shaver and Barch (1959, 1960) demonstrates the feasibility of experiments with amphibian material. These investigators prepared anti-Rana pipiens egg jelly sera in the rabbit. The antisera precipitated the egg jelly material. Treatment of sperm and of eggs with this antiserum lowered the fertilizing capacity of these gametes. This fertilization-inhibiting action was neutralized by addition of frog egg jelly to the antiserum. Extention of these studies to include antisera against other antigens will be awaited with interest. As might be expected, the effect of antiserum on the fertilizability of sea urchin eggs has been examined more extensively. Sea urchin eggs contain a number of antigens. Using the Ouchterlony agar diffusion technique, Perlmann (1953) has resolved at least ten precipitin bands from Paracentrotus lividus egg homogenates. Similarly, Metz and Kohler ( 1960) have distinguished at least nine bands by immunoelectrophoresis of Arbacia egg extract in preliminary studies. In a recent investigation Went (1959) and Went and Mazia (1959) have identified two of the unfertilized Strongylocentrotus egg antigens with antigens present in the isolated mitotic apparatus. Further identification of sea urchin egg antigens with particular egg extracts or physiological effects have been undertaken by Perlmann (for review see Perlmann, 1959; Runnstrom et al., 1959) and Tyler and Brookbank (see Tyler, 1957, 1959 for review). Tyler and Brookbank ( 1956a,b) have investigated the cleavage-inhibiting action of a variety of antisera. Among these, antisera prepared against egg jelly were especially effective. This property was not markedly altered by absorption of the sperm agglutinating antibodies present in certain of the antisera. It would be of considerable interest to determine if such cleavage-inhibiting antisera precipitate the mitotic apparatus antigens of Went and Mazia (1959). Antisera prepared against fertilizin also increased the respiration of Lytechinus eggs (Tyler and Brookbank, 1956b ; Brookbank, 1959). When sea urchin eggs are treated with antisera prepared against egg homogenates or extracts, a number of morphological effects result. These

246

CHARLES B. METZ

include a precipitation of the egg jelly in the form of a membrane (Perlmann, 1954), a precipitation of the ect6plasmic (hyaline) layer of fertilized, demembranated eggs (Tyler and Brookbank, 1956b), and in strong antisera, wrinkling, blistering, and finally cytolysis (Perlmann, 1954, 1956; Tyler and Brookbank, 1956b). Effects of antisera on eggs that relate more immediately to fertilization include claims of parthenogenetic activation of the egg and inhibition of fertilizability following antiserum treatment. As seen below, Perlmann has attributed several of these effects to separate egg antigens or antigenic complexes. 1. Parthenogenetic Activation Parthenogenetic activation of Paracentrotus lividus and Arbacia lixula eggs was first reported by Perlmann (1954). The activated eggs elevated membranes, although these were not always perfect, underwent changes in the optical properties of the protoplasm and disappearance of the nuclear membrane. Cortical granule breakdown failed to occur. Cell division rarely occurred. Removal of the jelly improved the percentage of activated eggs, although cleavage was rare even in eggs transferred to sea water (Perlmann, 1956). Further improvement was obtained by exposing the eggs to antiserum at low temperature (4”C.). After transfer to room temperature, the eggs frequently cleaved (Perlmann, 1957). Perlmann and Perlmann (1957a) present absorption experiments to show that the jelly precipitating and activating actions result from separate antibodies, reacting with distinct jelly (J) and activating ( A ) antigens, respectively. Chemical studies by these workers indicate that the A-antigen, like the J-antigen, is a polysaccharide. Unfortunately, efforts to confirm these important studies by Perlmann have met with only limited success. In a considerable series of experiments, Tyler (1959, p. 196) observed only formation of a “hyaline (ectoplasmic) layer at the surface of the egg and nuclear dissolution” following treatment of sea urchin eggs with antisera. As he points out, the lack of agreement may be due to species differences or to differences in what is meant by parthenogenetic activation. This latter factor is probably of considerable importance, since Perlmann has emphasized the egg-activating properties of antiserum, whereas Tyler has given special attention to the inhibition of nuclear and cytoplasmic division by antiserum.

2. Inhibition of Fertilization Sea urchin eggs show reduced fertilizability following antiserum treatment. This suggests a blocking of specific receptor sites at the sperm surface. However, two factors invoke caution in interpretation of this

INHIBITING AGENTS I N FERTILIZATION STUDIES

247

action. First, the antisera contain normal, multivalent antibody with agglutinating and precipitating properties. They precipitate the egg jelly (Perlmann, 1954), increase the surface tension of jellyless eggs (Tyler and Brookbank, 1956b), and precipitate the hyaline layer of denuded, fertilized eggs (Tyler and Brookbank, 1956b). Even in the absence of such visible effects, multivalent antibody might form a cross-linked lattice among adjacent antigen molecules which could act as a mechanical barrier to the fertilizing sperm. This difficulty might best be overcome by the use of univalent antibody, or at least antibody rendered nonprecipitating by a procedure such as acetylation (Nisonoff and Pressman, 1958). The second difficulty concerns the site of action of the inhibiting agent. Many steps must intervene between the initial activating reaction and the first visible change in the egg. T o the observer, fertilization is inhibited just as effectively whether the first or the last of these reactions is blocked. Accordingly, the same visible effect may result from blocking any one of several substances involved in a chain of events. These reservations should apply in the study of Shaver and Barch (1959, 1960) on the frog and, as Perlmann (1959) is aware, to the sea urchin studies discussed below. Perlmann ( 1954) initially reported that antiserum-treated sea urchin eggs failed to fertilize even after thorough washing in sea water. In subsequent studies Perlmann and collaborators used the fertilization rate method to evaluate the effect of antiserum treatment on eggs. The eggs are washed prior to insemination. Therefore, any reduction in fertilization rate should be due to an effect on the eggs, not the sperm. In fact, absorption of the anti-egg homogenate serum to remove sperm agglutinins did not affect the fertilization-rate-depressing action of the serum (Perlmann and Hagstrom, 1955). Likewise, anti-sperm serum failed to depress the fertilization rate. Evidently, the antigen(s) involved is not present in sperm. Likewise, the fertilization-rate-depressing antigen is probably not associated with the egg jelly, for anti-jelly sera have little, if any, depressing action, and, when present, such action is not correlated with egg-jelly-precipitating activity (Perlmann, 1957). Absorption with jelly solution had little effect on the fertilization-rate-depressing action of anti-egg homogenate sera, whereas egg homogenate strongly absorbed this activity (Perlmann and Perlmann, 195713). It would be reassuring if an excess of egg jelly had been demonstrated in these absorbing mixtures. However, it seems likely that the fertilization rate (F) antigen( s) is confined to the egg proper. As evidence for separate parthenogenetic-activating ( A ) and fertilization-rate-depressing (F) antigens, Perlmann (1957) cites differences in the activating and fertilization-depressing action of several antisera. However, examination of Perlmann’s (1957, 1959) data suggests a fairly strong correlation be-

248

CHARLES B. METZ

tween the activating and fertilization-rate-depressingactions, Additional evidence for separate A and F antigens is the differential absorbing action of one crude egg jelly preparation. This strongly absorbed parthenogenetic activity (Perlmann and Perlmann, 1957a) of an anti-egg homogenate serum but only weakly absorbed the fertilization-rate-depressing action of a second anti-egg serum (Perlmann and Perlmann, 1957b). It appears that more overwhelming evidence for separate A and F antigens is required before accepting Perlmann’s hypothesis unconditionally.

3. Cortical Dnnulge Eggs that fertilize after treatment with anti-egg serum ordinarily show a variety of abnormalities, including poor fertilization membrane formation and failure of cortical granule breakdown. The same abnormality pattern is not produced by all sera (Perlmann, 1957). This suggests that the effects may be of complex origin and involve several antigens. Perlmann and Perlmaxin (1957b) ascribe these effects to action of antibody on a cortical or C antigen(s). The cortical effects were not produced to any significant degree by the anti-sperm or egg jelly sera tested but were confined to anti-egg homogenate sera (Perlmann, 1957). Absorption of antiegg serum with egg jelly solutions lowered the percentage of damaged eggs only about 25%. These observations indicate that the J and C antigens are distinct and that the C antigen(s) is probably located in the egg proper. Absorption with unheated but not with heated egg homogenate lowered the damaging effect of anti-egg sera drastically (Perlmaxin and Perlmann, 1957b). This heat lability of the C antigen( s) distinguishes it from the heat-stable A antigen and at least in part from the partially heatlabile F antigen. Neither the A, F, or C antigens were inactivated by trypsin (Perlniann and Perlmann, 1957a, b) .

V. Conclusions From the above account it is clear that a number of diverse agents inhibit fertilization. This action of the agents in question is achieved without pronounced toxic action on the gametes, and therefore involves preferential interference with one or more of the reactions that lead to normal development of the egg. Such interference presumably involves actions at the molecular level. For example, this could take the form of blocking a specific substance or receptor at the sperm or egg surface, inactivation of an enzyme, a substrate, or essential product of enzyme action. Inhibiting agents then should serve as probes to reveal such surface or subsurface substances at various steps in the fertilization process. However, except in the simplest and most obvious cases, the inhibiting agents

INHIBITING AGENTS I N FERTILIZATION STUDIES

249

can give precise information only after an elaborate analysis of the mechanism of action. The simplest cases would appear to be inhibition of fertilizing capacity by extracts of the gametes themselves. From the inhibiting action of fertilizin on sperm and antifertilizin on eggs, it appears that a fertilizinantifertilizin interaction performs a significant role in fertilization (see Section 11). But even here the exact nature of the role is obscure. It may be in the attachment of the sperm to the egg (Tyler, 1948), or it may concern penetration of the sperm into the egg by a pinocytosis-like mechanism (Tyler, 1959). Indeed, the possibility has not been ruled out that such interaction constitutes the egg activation initiating mechanism as Lillie (1919) believed. Finally, evidence increasingly indicates that a fertilizin-antifertilizin interaction may initiate the sperm acrosomal reaction, which is probably an essential preliminary to fertilization in many forms (Dan, 1956; Colwin and Colwin, 1957). The resulting acrosomal filament may well have newly exposed and qualitatively different surface groups from those previously exposed on the sperm surface. The relationship of this acrosomal filament surface to fertilization certainly warrants intensive study. Aside from these possibilities, it seems likely that the fertilizinantifertilizin reaction contributes to the specificity of fertilization. Fertilization-inhibiting action of agents of origin unrelated to the gametes have the disadvantage, by virtue of their origin, in that they probably do not normally function in fertilization. Furthermore, agents such as Arbacia dermal secretion and Fucus extract have little species specificity and, since their chemical nature is imperfectly known, their chemical specificity of action, if any, is obscure. Nevertheless, these agents do inhibit fertilization and their action on known groups can be examined. For example, Arbacicl dermal secretion does not irreversibly affect the antifertilizin groups of the intact sperm surface (Metz, 1959a) whereas Fucus extract does inactivate these groups (Branham and Metz, 1959). Both of these agents act irreversibly on eggs, but sensitivity to these agents becomes reversible by washing, following treatment of eggs with trypsin. Perhaps there are two fertilization inhibitors in these solutions, one of which acts irreversibly on an egg surface protein, the Gther, reversibly on some other egg constituent or on the sperm (see Section I11 above). Agents of known and more or less specific chemical action, such as enzymes, oxidizing and reducing agents, and amino acid end group reagents, have value by virtue of their known chemical action, but the use of some of these (e.g., oxidizing and reducing agents) is complicated by the fact that they are applied to living systems which may metabolize or

250

CHARLES B. METZ

otherwise alter the agents. This problem is seen in extreme form in Paramecium. In this organism, trypsin treatment results in a loss of mating reactivity attributable to feeding of the animals on the solution. When Paramecia are killed without destruction of mating reactivity, loss of mating reactivity following trypsin treatment evidently results from enzymatic destruction of the mating-type substances (Metz, 1954). The use of inert systems (e.g., dead sperm which will activate eggs), comparable to those employed with Paramecium studies, would be of great value in studies on metozoan fertilization. Among fertilization inhibitors, antibodies have the virtue of specificity ; they can be used to examine substances independently of living systems by agglutination and precipitation tests, or they can be converted to a nonagglutinating or nonprecipitating form by several means (e.g., Tyler, 1946a ; Porter, 1958, 1959 ; Nisonoff and Pressman, 1958 ; Nisonoff et a,l., 1960). The utility of such univalent antibodies was most effectively demonstrated by inhibiting the fertilizing capacity of sperm (Tyler, 1946a). The sperm surface has several distinct antigens (Henle et al., 1938; Kohler and Metz, l%O) and an examination of each of these for essentiality in fertilization should be rewarding. Finally, fluorescein or tritium-labeled antibodies can be used as a visual means of localizing substances. Interpretation of the action of antisera on complex systems does require caution. For example, antisera against sea urchin eggs are reported to inhibit fertilization of the eggs, parthenogenetically activate the eggs, and inhibit cleavage (see Section I V ) . As Perlmann (1959) and Tyler (1959) have shown, these effects probably result from action of separate antibodies reacting with distinct antigens. It would be of great interest to examine for sequential action of the antibodies and other inhibiting agents. Through their use it should be possible to specify the sequences of some of the fertilization reactions at the molecular level. For example, normal fertilization and parthenogenetic activation of the egg may be initiated through different routes and receptors (Metz and Foley, 1949). If this were the case, eggs rendered unfertilizable by treatment with an appropriate inhibitor might still respond readily to parthenogenetic activation. In the case of heparin inhibition, both fertilization and parthenogenesis are blocked (Harding, 1951), but other inhibitors need to be examined as well. In fact, a sequential action is implicit in Perlmann’s (Perlmann and Perlmann, 1957b) proposal of separate fertilization inhibitors (F) and activating ( A ) antigens. Unexploited sources of potential fertilization inhibitors are the bloods and body fluids of forms which agglutinate sperm and eggs of other species with some specificity (Tyler and Metz, 1945 ; Tyler, 1946b). Undoubtedly,

INHIBITING AGENTS I N FERTILIZATION STUDIES

251

many more fertilization-inhibiting agents will be found and, upon proper analysis, these may yield information which, when combined with that already available, should contribute much toward identification of substances and reactions and their sequences in fertilization.

REFERENCES Allen, R. D. (1958). In “Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), pp. 17-67. Johns Hopkiris Press, Baltimore, Maryland. Bohus Jensen, A. (1953a) Exptl. Cell Research 4, 60-68. Bohus Jensen, A. (1953b) Exptl. CeN Research 6, 325-328. Branham, J. M., and Metz, C. B. (1959) Biol. Bull. 117,392-393. Brookbank, J. W. (1958) Biol. Bull. 116, 74-80. Brookbank, J. W. (1959) Biol. Bull. 116, 217-225. Colwin, A. L., and Colwin, L. H. (1955) J . Morphol. 97, 543-569. Colwin, A. L., and Colwin, L. H. (1957) I n “The Beginnings of Embryonic Development” (A. Tyler, R. C. von Borstel, and C. B. Metz, eds.), pp. 135-168. Am. Assoc. Advance. Sci., Washington, D.C. Dan, J. C. (1952) Biol. Bull. 103, 54-66. Dan, J. C. (1954) Bt‘ol. Bull. 107, 335-349. Dan, J. C. (1956) Intern. Rev. Cytol. 6, 365-393. Ebert, J. D. (1958) I n “The Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), pp. 526-545. Johns Hopkins Press, Baltimore, Maryland. Esping, U. (1957a) Arkiv Kemi 11, 107-115. Esping, U. (1957b) Arkiv Kemi 11, 117-127. Frank, J. A. (1939) Biol. Bull. 76, 190-216. Fuchs, H. M. (1914) Wilhelm Roux. Arch. Ent.rPricklwngsmech. Org. 40, 205-252. Fuchs, H. M. (1915) 1. Genet. 4, 215-301. Gagnon, A. (1950) Biol. Bull. 99, 341. Hagstrom, B. E. (1956a) Exptl. Cell Research 11, 306-316. Hagstrom, B. E. (1956b) “The Role of Jelly Coat and the Block to Polyspermy in the Fertilization of Sea Urchins,” Almqvist and Wicksells, Uppsala. Hagstrom, B. E. (1959) Exptl. Cell Research 16, 184-192. Hagstrom, B. E., and Hagstrom, B. (1954) Ezptl. Cell Research 6,479-484. Harding, C. V. (1950) Biol. Bull. 99, 340. Harding, C. V. (1951) Exptl. Cell Research 2, 403-415. Harding, C. V., and Harding, D. (1952) Arkiv 2001.3, 357-361. Harding, D. (1949) Proc. Soc. Exptl. Biol. Mcd. 71, 14-15. Harvey, E. B. (1956) “The American Arbacia and Other Sea Urchins.” Princeton Univ. Press, Princeton, New Jersey. Hathaway, R. R. (1959) Biol. Bull. 117, 395-396. Hathaway, R. R., and Tyler, A. (1958) Biol. Bull. 116, 337. Heilbrunn, L. V., and Wilson, W. L. (1949) Proc. SOC.Exptl. Biol. h’ed. 70, 179182. Henle, W., Henle, G., and Chambers, L. A. (1938) J. Exptl. Med. 68, 335-352. Hultin, T. (1949) Arkiv Kemi 1, 419-423. Just, E. E. (1922) Biol. Bull. 43, 411-422. Kohler, K., and Metz, C. B. (1959) Biol. Bull. 117, 416. Kohler, K., and Metz, C. B. (1960) Biol. B d l . 118, 96-110.

252

CHARLES B. MET2

Krauss, M. (1950a) J. Exptl. 2001.114, 279-291. Krauss, M. (1950b) Science 112, 759. Kriszat, G. (1953) Exptl. CcEl Rcscarch 5, 420-426. Landsteiner, K. ( 1945) “The Specificity of Serological Reactions.” Harvard Univ. Press, Cambridge, Massachusetts. Lillie, F. R. (1914) J. Exptl. 2001.16, 523-590. Lillie, F. R. (1919) “Problems of Fertilization.” Univ. Chicago Press, Chicago, Illinois. Metz, C. B. (1945) Biol. Bull. 89, 84-94. Metz, C. B. (1949) Proc. SOC. Exptl. Biol. Mcd. 70, 422-424. Metz, C. B. (1954) I n “Sex in Microorganisms” (D. H. Wenrich, ed.), pp. 284332. Am. Assoc. Advance. Sci., Washington, D.C. Metz, C. B. (1957a) In “Physiological Triggers” (T. H. Bullock, ed.), pp. 17-45. Am. Physiol. SOC.,Washington, D.C. Metz, C. B. (1957b) In “The Beginnings of Embryonic Development” (A. Tyler, R. C. von Borstel, and C. B. Metz, eds.), pp. 23-69. Am. Assoc. Advance. Sci., Washington, D.C. Metz, C. B. (195%) Biol. Bull. 116, 472-483. Metz, C. B. (1959b) Biol. Bull. 117, 398. Metz, C. B. (1960) Biol. Bull. 118,439-450. Metz, C. B., and Donovan, J. E. (1949) Biol. Bull. 97, 257. Metz, C. B., and Foley, M. T. (1949) J . Exptl. 2001.112, 505-528. Metz, C. B., and Kohler, K. (1960) B i d . Bull. 119. 202-209. Minganti, A,, and Vasseur, E. (1959) Acta Embryol. ct Morphol. Exptl. 2, 195203. Monroy, A., and Tosi, L. (1952) Experentin 8, 393-394. Monroy, A., Tosi, L., Giardina, G., and Maggio, M. (1954) Biol. Bull. 106, 169177. Nisonoff, A., and Pressman, D. (1958) Scieiice 128, 659-660. Nisonoff, A., Wissler, F. C., Lipman, L. N., and Woernley, D. L. (1960) Arch. Biochem. Biophys. 89, 230-244. Oshima, H. (1921) Science 64, 578-580. Pequegnat, W. (1948) Biol. Bull. 95, 69-82. Perlmann, P. (1953) Exptl. Cell Research 6, 394-399. Perlmann, P. (1954) Exptl. Cell Rescarch 6, 485-490. Perlmann, P. (1956) Exptl. Cell Rescnrch 10, 324-353. Perlmann, P. (1957) Exptl. Cell Research lS, 365-390. Perlmann, P. (1959) Expcrierztiu 16, 41-52. Perlmann, P., and Hagstrom, B. E. (1955) Exptl. Cell Rcsearch Suppl. 3, 274280. Perlmann, P., and Perlmann, H. (1957a) Exptl. Cell Research 19,454-474. Perlmann, P., and PerImann, H. (1957b) E x p f l . Cell Research lS, 475-487. Porter, R. R. (1958) Nature 182, 670-671. Porter, R. R. (1959) Biochem. J. 73, 119-126. Rothschild, Lord (1956) “Fertilization.” Wiley, New York. Rothschild, Lord, and Swaiin, h4. M. (1949) J. Exptl. Biol. 26, 164-176. Runnstrom, J. (1957a) In “Festschrift Arthur Stoll,” pp. 850-868. Sandaz, Basel. Runnstrom, J. (1957b) Expti. Cell Research 12, 374-394. Runnstrom, J. (1958) Exptl. Cell Rcscarch SUpPl, 6, 529.

INHIBITING AGENTS I N FERTILIZATION STUDIES

253

Runnstrom, J., and Hagstrom, B. E. (1954) Exptl. Cell Research 7 , 327-344. Runnstrom, J., and Hagstrom, B. E. (1955) Exbtl. Cell Research 8, 1-14. Runnstrom, J., and Imniers, J. (1956) Exptl. Cell Research 10,354-363. Runnstrom, J., Hagstrom, B. E., and Perlmann, P. (1959) I n “The Cell” (3. Brachet and A. E. Mirsky, eds.), pp. 327-397. Academic Press, New York. Shaver, J. R., and Barch, S. H. (1959) Anat. Record 134, 637-638. Shaver, J. R., and Barch, S. H. (1960) Acta Embryol. et Morphol. Exptl. 3, 180-189. Tyler, .4. (1939) Proc. Natl. Acad. Sci. U.S. 25, 317-323. Tyler, .4. (1941) Biol. Bull. 81, 190-204. Tyler, A. (1942) Western J . Surg. Obstet. Gyncol. 50, 126-138. Tyler, -4. (1945) J . Immunol. 51, 157-172. Tyler, -4. (1946a) Proc. Soc. Exptl. Biol. Med. 62, 197-199. Tyler, -4. (1946b) B i d . Bid/. SO, 213-219. Tyler, ,4. (1948) Physiol. Revs. 28, 180-219. Tyler, A. (1949) Am. Naturalist 83, 195-219. Tyler, r\. (1955) I n “Analysis of Development” (B. H. Willier, P. A. Weiss, and V. Hamburger, eds.) , pp. 556-573. Saunders, Philadelphia, Pennsylvania. Tyler, -4. (1956) Exptl. Cell Research 10, 377-386. Tyler, -4. (1957) I n “The Beginnings of Embryonic Development” (A. Tyler, R. C. von Borstel, and C. B. Metz, eds.), pp. 341-382. Am. Assoc. Advance. Sci. Washington, D.C. Tyler, .4. (1959) Exptl. Cell Rrsearck Suppl. 7 , 183-199. Tyler, .4., and Brookbank, J. W. (1956a) Proc. Natl. Acad. Sci. U S . 42, 304-308. Tyler, .4., and Brookbank, J. W. (1956b) Proc. Natl. Acad. Sci. U.S. 42,308-313. Tyler, .4., and Metz, C. B. (1945) J . Exptl. Zool. 100, 387-406. Tyler, A., and Metz, C. B. (1955) Picbbl. stas. 2001. Napoli 27, 128-145. Tyler, =\.,and O’Melveny, K. (1941) Biol. Bull. 81, 367-375. Tyler, z4., Fiset, M. L., and Coonibs, R. R. A. (1954) Proc. Natl. Acad. Sci. U.S. 40, 736-740. Tyler, .4., Monroy, A., and Metz, C. B. (1956) Biol. Bull. 110, 184-195. Weil, -4.J. (1960) Science 131, 1010-1041. Weil, A. J., and Finkler, A. E. (1958) Proc. Soc. Exptl. Biol. Med. 98, 794-797. Weil, .4. J., and Finkler, A. E. (1959) Proc. SOC.Exptl. Biol. Med. 102, 624-626. Went, H. A. (1959) J . Biopltys. Biorhem. Cytol. 6, 447-455. Went, H. A., and Mazia, D. (1959) Exptl. Cell Research Suppl. 7 , 200-218. Wicklund, E. (1954) -4rkiv Zool. r21 6, 485-502.

This Page Intentionally Left Blank

The Growth-Duplication Cycle of the Cell D. M. PRESCOTT Biology Division, Oak Ridge National Laboratory,l Oak Ridge, Tennessee Page I. Introduction ..................................................... 255 11. Induction of Division Synchrony .................................. 256 A. Temperature Induction of Synchrony ........................ 257 B. Chemical Induction .......................................... 260 C. Mechanical Methods ......................................... 261 111. The Growth-Duplication Cycle ................................... 262 A. Method ..................................................... 262 B. Cell Growth during the Cycle ................................ 262 C. D N A Synthesis and Function during the Cycle ................ 267 D. Respiration and Energy Metabolism .......................... 272 E. RNA Synthesis during the Cell Life Cycle .................... 274 275 F. Enzyme Fluctuations ......................................... G. Reproduction of Structures ................................... 276 IV. Concluding Remarks ............................................. 279 280 References ......................................................

I. Introduction During the last several years the emphasis in the field of cell growth has shifted from a consideration of the growth of cell populations to studies on the increases of structures and materials within the individual cell during the growth-duplication cycle. Current work at the levels of both population and the individual cell is characterized by a constant striving to define growth phenomena in more and more specific and detailed biochemical terms. Through these efforts the subject has moved into a biochemical arena in which it shares information and hypotheses with the fields of physiological genetics, enzymology, cytochemistry, virology, and others. The cell growth-duplication cycle consists of one complete interphase and one cell division, and ordinarily the completion of cytokinesis is the most convenient means of marking the end of one cycle and the beginning of the next. A certain amount of confusion has arisen, however, through the use of several terms to describe this cycle, which carries the cell from division to division. The terms frequently used as synonyms are mitotic cycle, division cycle, life cycle, reproductive cycle, duplication cycle, and growth cycle. The older expressions, mitotic cycle and division cycle, are perhaps less useful since they tend to emphasize the division process 1

Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission. 255

256

D. M. PRESCOTT

itself and frequently have been used intentionally to describe this section of the cycle alone. In the discussions to' follow, growth-duplication cycle and life cycle will be used. Extensive discussions on the subject from several points of view have been presented by S w a m ( 1957, 195S), Anderson ( 1956a,b), Stern (1956), and Campbell (1957). Undoubtedly, some important papers pertinent to the discussions to follow have been missed or overlooked, but because the papers relevant to cell growth are scattered through a large number of journals, such oversights are unfortunately impoksible to avoid. The growth and multiplication rates for a cell are determined by the suitahility of the environmental conditions for the expression, by individual cell types, of genetically prescribed capacities for self-increment and selfduplication. These two sets of factors, environmental and genetic, control the growth rate of the cell over the single life cycle. The rate of proliferation of a cell population must be built on the rate at which the individual cell growth-duplication cycle occurs. Growth at both levels, individual cell and population, is the final net result of the same catabolic and anabolic processes. Measurements of growth in the individual and on the population, however, are usually concerned with different levels of manifestation of the same processes. I n measurements on a population, derived information is largely statistical in nature. The principal concern is with changes in a large number of cells, and for the most part, the particulars of the growth-duplication processes by which each individual cell progresses from division to division are only secondarily a part of the measurements. These growth-duplication processes of the single life cycle form the subject of this review.

11. Induction of Division Synchrony The accurate measurements on single cells needed for the study of the cell life cycle are usually difficult and tedious and too frequently impossible to obtain with techniques available at the present time. A number of methods have been developed in recent years for the synchronization of cell divisions, primarily with the aim of permitting the study of cell life cycle events on a large cell mass with the use of more conventional analytical procedures. For this latter purpose, however, mass induction of division synchrony has several limitations. The induction by any method so far developed is never perfect. By current standards the results are considered to be good when all the cells in a population complete cytokinesis in a time span equivalent to 15 to 30% of a complete generation time. Such synchrony imperfection obscures the finer points of information. The synchrony in these systems is also very rapidly lost over the

GROWTH-DUPLICATION CYCLE O F T H E CELL

257

first few cycles after release from the induction treatment. The loss of synchrony does not stem from any conditions created by the induction treatment, but simply reflects normal variations in the generation times for individual cells (Prescott, 1959a). The induction treatments also change the physiological state of the cells to a lesser or greater degree, and obtained information frequently cannot be assumed to represent unequivocally the events in the normal cycle (Barner and Cohen, 1956; Schaechter et al., 1959). Finally, successful induction of division synchrony may not necessarily mean that the cells are synchronized with respect to the procession of all the events of the cycle. A number of treatments, which can be classified as either temperature, chemical, or mechanical, have been applied for the induction of division synchrony in bacteria, protozoa, algae, yeasts, and vertebrate cells in culture. A single temperature shock or a regime of temperature cycling is the treatment most frequently used.

A.

TEMPERATURE INDUCTION OF SYNCHRONY

One of the earliest and most successful temperature methods was developed by Scherbaum and Zeuthen (1954) for Tetrahymenu. By a cyclic shifting of the incubation temperature between the optimum (29"C.) and a sublethal temperature (34" C.), as much as 85% of a mass culture of Tetrahymena could be brought into division at one time. The temperature cycling prevents any of the cells from entering division but does not inhibit cell growth. By the end of the temperature treatment the average cell size is three to four times normal. The lag between the end of treatment and the first synchronous division is equal to 60% of the normal generation time. Since the normal generation time under the culture conditions used is 140 to 150 minutes, the treatment seems to have caused the population to accumulate at a common stage of early interphase. Studies by Thormar (quoted by Zeuthen, 1958) on the heat shock sensitivity of individual Tetrahymenu of known position in the cell life cycle have given some additional support to this interpretation. During the last half of interphase, heat sensitivity, measured as the effectiveness of a single heat shock to delay division, rises sharply and reaches a maximum at the end of interphase. The closer a cell approaches the completion of division preparations, the more severe is the delay imposed by the temperature shock. In this synchronous system a striking separation between growth and division has been experimentally imposed. During the several synchronous cycles that are produced, the generation time is shorter than normal, the cells do not double in size with each generation, and the average cell size

258

D. M. PRESCOTT

gradually returns to the normal level over several cycles. The divisionless growth of the cells during the treatment includes the production of protein, RNA, and DNA (Scherbaum et al., 1959). In the presence of this presynthesis, the shortened generation time during the first few synchronous cycles may represent an approach to the absolute minimal time in which the purely division-associated activities of the cell can be accomplished. Cells transferred to a non-nutrient medium near the end of the temperature treatment, for example, will still go through several synchronous cycles with no increase in the generation time between the first two synchronous divisions of the population (Hamburger and Zeuthen, 1957). Therefore, it seems that these large cells have been released from metabolic burdens imposed by any imperfections (of unknown character) in the composition of the nutrient medium. It is perhaps pertinent that the very short generation time for these giant cells can be achieved for normal logphase cells by growing them in a medium composed of an untreated homogenate of Tetmhymena (Prescott, 1958a). Division synchrony in Tetrahymena can be produced by cold shock treatment, but the degree of synchrony never approaches that obtained with the sublethal temperature method (Zeuthen and Scherbaum, 1954 ; Prescott 1958b). Zeuthen ( 1958) has suggested that the underlying mechanisms by which synchrony is achieved may be the same in both heat and cold shock methods. It has been shown that sensitivity to both cold and heat shock, measured again as a delay in division, increases through interphase (Thorniar, quoted by Zeuthen, 1958 ; Prescott, 196Oa). Scherbaum (1957) has made a kinetic analysis of synchrony induction (heat shock method) based on the assumption that the effect is produced through the destruction of an enzyme that is necessary for the synthesis of a product critical to division. There is no available information to suggest what the enzyme or the product might be or how the latter might function. It is difficult to visualize how DNA synthesis would fit the model, for example, since the amount of DNA per cell increases continuously during both the induction treatment and the period between the end of treatment and the start of cell divisions (Iverson and Giese, 1957 ; Scherbaum et al., 1959). The completion of preparations for cell division must normally require a high degree of integration of many rates and processes. Because these processes undoubtedly have different temperature coefficients, a sudden temperature change must inevitably have a disintegrative effect on the interactions of predivision preparatory phenomena, and it is not surprising that progress in the growth-duplication cycle is temporarily interrupted. In this sense, the shock recovery process leading to division synchrony

GROWTH-DUPLICATION CYCLE O F T H E CELL

259

should not be considered to be a part of the normal procession of events that, leads to division during the undisturbed cell life cycle. Lark and Maalpre (1954) obtained a good degree of synchrony in DNA synthesis in Salmonella typlaimurium by a single change in temperature from 25" to 37" C., but cell division synchrony could be obtained only by repeated cycling between these two temperatures. In the latter method DNA synthesis and nuclear division occurred directly after the shift from 25" to 37" C. in each cycle, and cytokinesis followed about a half-generation time later. Since the population doubled during each cycle of the treatment, this system is basically different from the heat shock method developed for Tetrahymenu. Synchrony in S. typhimztrium is not produced by an exaggerated separation between growth and division, but apparently results from a phasing of the interphase activities that lead to division. Lark and Maalpre (1954, 1956) postulate that synchrony of DNA synthesis at the beginning of the 37" C. part of the cycle is produced by the development of a larger pool of DNA precursors at 25" C. ; when the temperature is shifted to 37" C., the concentration of DNA precursors in all cells is equal to or above the critical level necessary to initiate synthesis at the new temperature. Synchronization of DNA synthesis evidently leads to synchronization in nuclear division, which may, in turn, be responsible for the production of synchrony in cytokinesis. Padilla and James (1958) have synchronized cell division in the slower growing, flagellated protozoan, Astask longa, by a regime of 10 hours at 5" C. and 14 hours at 25" C. No cell divisions occur at 5" C. Six to eight hours after the cells are released from the low temperature, a sharp doubling in the population takes place. A fair degree of division synchrony has been produced in Bacillus megatherium by a single incubation of a culture at 12" C., followed by incubation at 34" C. (Hunter-Szybalska et al., 1956; Falcone and Szybalski, 1956). No divisions occur during the cold period; synchronous cell divisions begin after 40 minutes at 34" C. The authors express the view that synchrony produced in nuclear divisions is the primary event upon which synchrony in cytokinesis depends. According to the work of Falcone and SzybaIski (1956), upon transfer to cold the synthesis of DNA, RNA, and protein are reduced to an increasing degree in that order. From their data this seems to be true immediately after transfer to the low temperature, but subsequently at this temperature the rate of DNA synthesis seems to fall rapidly, while R N A and protein productions continue at a constant rate. The authors' conclusion that synchrony is produced by a preferential effect of the cold incubation on DNA synthesis is not well supported by their data.

260

D. M. PRESCOTT

With a single shift in temperature from 4” ( 1 hour) to 37’ C., Kewton and Wildy (1959) observed a good amount of division synchrony in HeLa cells. After the change, no DNA synthesis occurred until 14 hours at 37” C. Cell divisions began some hours after the DNA synthesis period. It is quite possible that the synchrony in microorganisms and mammalian cells obtained by low-temperature treatment is produced through effects on the same mechanisms. In the case of either heat or cold treatments alone on microorganisms, the extreme temperature inhibits division and reduces the cell population to a common physiological state. In general, cold treatments bring about synchrony in the synthesis of DNA. No such preindication of division synchrony appears in the Scherbaum-Zeuthen heat shock system ; DNA synthesis is continuous in the population during and after treatment. It does not seem likely that both high and low temperature extremes induce synchrony by affecting the same cellular activities. INDUCTION B. CHEMICAL Barner and Cohen (1956) synchronized cell division in a thymineless strain of Escherichia coli by withholding thymine from the medium for a critical period of time. In the absence of thymine, DNA synthesis and cell division ceased, but turbidity of the culture and R N A both continued to increase. The best synchrony was produced with a deprivation period of 30 minutes. Addition of thymine at the end of 30 minutes of deprivation was followed by a 30- to 35-minute lag and then a period of 10 to 15 minutes in which all cells divided. DNA synthesis in this treatment was also synchronized. Shorter incubations gave poorer synchrony, and thymineless incubations in excess of 30 minutes resulted in a rapid loss of viability. Even cells that had irreversibly lost the power to proliferate could still synthesize some DNA when thymine was added to the medium. The loss of viability in the prolonged absence of thymine (thymineless death) has been attributed to an irreversible disorganization in metabolism as a consequence of the period of “unbalanced growth” (Barner and Cohen, 1956). This interpretation is supported by subsequent observations that thymineless death is impeded by the removal of amino acids or a carbon source from the medium (Barner and Cohen, 1957). Burns (1959) has published a brief account of synchronization of cell divisions and DNA synthesis in a mutant of Lactobacillus acidophilus by the withdrawal and restoration of deoxyriboside. The unicellular alga, Chlorella, has been synchronized by exposure to alternating periods of light and darkness (Tamiya et al., 1953; Nihei, 1955; Sorokin, 1957; Sorokin and Krauss, 1959). When cultures are

GROWTH-DUPLICATION CYCLE OF THE CELL

261

placed in darkness, growth stops for lack of a reduced carbon source but cell division occurs without any further increase in cell size. Daughter cells tend to accumulate in the dark since not only growth but also preparations for a subsequent division are unable to proceed. These are both resumed in the light. Several repetitions of the cycle effectively separate growth (occurs during 9 hours of light) froin division (which takes place during the 4 hours of darkness), and bring the population into a moderate degree of division synchrony. In photosynthetic organisms synchronized by light-darkness changes, it is often difficult to discriminate between rhythmic changes based on the light changes and those based on the cell life cycle. The experiments of Sorokin and Myers (1957), however, suggest that changes in the respiration rate in Chlorella are associated with the cell life cycle. The respiration rate declines through the growth phase of the cell cycle and reaches a low point during cell division. Sorokin (1957) has found evidence that photosynthesis is minimal at the beginning and end of the life cycle and maximal during the middle of interphase.

C. MECHANICAL METHODS Maruyama and Yanagita (1956) have devised a filtration technique for separating bacteria into size classes. The large bacteria retained by a filter paper pile are in the same section of the life cycle; when harvested and incubated, they go through several synchronous divisions. The method, however, is not so simple as it sounds ; several manipulations are involved during which the cells are subjected to mechanical stress and temperature change (Lark and Maruyama, personal communication). Although the division synchrony is quite good, the method contains the element of metabolic shock present in the temperature and chemical induction procedures. Anderson and Pettijohn ( 1960) have simplified the procedures to a considerable extent by collecting the small cells that pass through the filter. The synchrony is apparently very good, and the system appears to be free of environmental changes that might alter cell metabolism. I n the case of cell types of relatively large size, such as Amoeba proteus and Tetrahymena, the selection of a number of dividing cells from a logphase culture by micropipetting and pooling of the daughters has been used to obtain synchrony with a minimum of disturbance to the cells (Prescott, 1955, l%Oa). Because only a few hundred dividing cells can be collected in a short enough interval to give good synchrony, the applicability of the technique is limited to measurements that are possible on small cell numbers. Since the mechanical methods minimize or eliminate metabolic disturbance to the cells, data on life cycle activities obtained on cells syn-

262

D. M. PRESCOTT

chronized in this way are not subject to most of the reservations that apply to information derived in the use of temperature or chemical induction techniques.

111. The Growth-Duplication Cycle A.

METHOD

For reasons already outlined in the preceding section, the usefulness of the currently available methods for the induction of division synchrony in cell masses is limited in the study of the cell growth-duplication cycle. Measurements and experiments on single growing cells, or small groups of cells synchronized by selection, continue to be the most direct and reliable means of analyzing the cycle. As an ancillary approach, a certain amount of information about the manner in which a cell moves from division to division can be derived by a frequency distribution analysis on the state of some process in the individual cells of an asynchronously growing population. Since a number of assumptions about the cell population are necessary (Walker, 1954),and since many of these cannot be made with a high degree of security, the more direct method of using cells of known position in the growth-duplication cycle is preferable.

B. CELLGROWTH RATEDURING

THE

CYCLE

The average cell, in adjustment with a medium that supports proliferation, doubles all its constituents and structures in an average growthduplication cycle. There are certain exceptions to this, notably, early logphase cells, but these are not important in the discussion to follow. The amount or rate of some activity in the individual cells of a clone, however, may deviate appreciably from the value established for the average cell during the average life cycle. Such variations between genetically identical cells proliferating in the same environment probably stem largely from fortuitous inequalities created by the failure of cytokinesis to produce two cells exactly equal in all cytoplasmic respects. Such variations are not of much concern at the level of current experiments because of the low precision in biochemical and other measurements. Individual cell variations, however, are important to the extent that they are reflected in individual cell generation times. If all cells of a clone exposed to the same nutrient medium should proceed from one division to the next at exactly the same rate in each cycle, it would be a simple matter to obtain large numbers of cells in perfect synchrony without the use of induction treatments. Cultures initiated with single cells are ordinarily well synchronized for a few cycles, but the synchrony declines with each doubling of the population. Measurements have been made of the individual vari-

GROWTH-DUPLICATION CYCLE O F T H E CELL

263

ations in generation times for bacteria (Kelly and Rahn, 1932) and Tetrahynaena clones (Prescott, 1959a). The relative deviations from the mean are of the same order of magnitude in both cases and are sufficient to account for the abolishment of all traces of division synchrony in a clone of perfectly synchronized Tetrahymem within four to five generations. In Amoeba proteus, cytokinesis can be experimentally induced to give rise to daughters of different sizes (Prescott, 1956). In such cells the generation times correlate with daughter cell size. Kimball et d. (1959) found no such correlation between daughter cell mass and generation time in Paramecium. The generation time variations in Tetrahymenu cannot be ascribed to volume differences between daughter cells, and it has been assumed that inequalities that have no reflection in cell volume may occur at division. The growth rate of individual bacteria has been correlated with an asymmetry in the distribution of phosphorus (P32)at division by Forro (1957). It is a reasonable assumption that the rate at which an individual cell completes a particular life cycle will be influenced by the endowment it receives at division ; small asymmetries in the distribution of ribosomes, mitochondria, or other materials at cytokinesis may produce proportional changes in generation times of the daughter cells. These variations are sometimes troublesome. The study of cycle events frequently requires that cells be killed at various known time points of the cycle and the course of the event deduced from measurements on a series of individuals. The postdivision age of a cell is ordinarily accepted as an index of its position in the cycle, but this position cannot be unequivocally defined unless the exact proximity to the next division is also known. In some cases, this type of timing error can be reduced by using one sister as a control and the other for experimental measurement (McDonald, 1958 ; Kimball et al., 1959). Division to division growth has been measured with good accuracy for a number of microorganisms by following increases in total volume, protein, or mass. In the early studies of Bayne-Jones and Adolph (1932) on the yeast, Saccharornyces cerevesiae, the new bud showed a constant rate of volume increase during most of the cycle, and a marked reduction in growth during the last 15 minutes before it itself began bud formation. Virtually identical results have since been reported by Lindegren and Haddad ( 1954). Mitchison ( 1958) has confirmed this information on volume and has added studies on mass increase as measured with the interference microscope. Mass increase is linear through the whole cycle, but unlike volume increase, shows no indication of a reduced growth rate before or during budding. The rate of mass increase for the two daughter cells taken together rose sharply shortly after the new bud had appeared.

264

D. M . PRESCOTT

Mitchison (1957) found the same type of linear mass increase in the fission yeast, Schizosacclzaromyces pombe, with again a sharp transition to a new growth rate at the time of fission. Volume increase was close to linear but, similar to the situation found in budding yeast, it entered a plateau of no increase about one hour before cytokinesis. Knaysi (1940) also observed a volume plateau at division in this yeast. Mitchison calculated the density changes that must be assumed to occur during the cycle since volume and mass increases, although linear for most of the cycle, follow different courses, especially for periods that precede and encompass cytokinesis. Cell density rises steadily up to the time of cytokinesis and falls slowly thereafter. The significance of these density changes is not known. Mitchison has discussed the possibility that limitations on the rate of cell wall synthesis may prevent volume increase at the time of cytokinesis. Respiration of a single Tetrahymena and of its synchronously dividing progeny was measured continuously in a Cartesian diver respirometer by Zeuthen ( 1953) over several generations. The respiration rate increases linearly during interphase, ceases to increase or even drops slightly beginning 15 to 30 minutes before cytokinesis, and then rises sharply to a rate double the previous interphase rate just before or at the onset of cytokinesis. Respiration was considered as a quantitative reflection of metabolic machinery, and increases in respiration rate were therefore accepted as a measure of growth. Zeuthen’s conclusion of a constant growth rate during interphase has been confirmed by measurements of C14 methionine incorporation into protein over the cycle (Prescott, 1960a). Division to division growth in Amoeba proteus, measured as volume, protein, and mass increases, occurs at a slightly decreasing rate during most of interphase and ceases altogether about 4 hours before division (24-hour generation time) ( Prescott, 1957). During the predivision period of no growth the amoebae are actively motile and show no cytological signs of the impending division. Nuclear volume increases after telophase, grows slowly during middle interphase, and increases rapidly just before division, a time when the cell as a whole does not grow (nuclear volume is only a few per cent of total cell volume in this cell). Kimball et al. (1959) determined the course of mass increase in Paramecium aurelia during the cycle by interference microscopy and X-ray absorption methods. These experiments revealed a “logarithmic” course of growth and in this respect are markedly different from results on other by microorganisms. Kaudewitz ( 1958) followed the uptake of P3’04--P . aurelia over the cycle and obtained a curve intermediate between a linear and a logarithmic-type curve.

GROWTH-DUPLICATION CYCLE OF THE CELL

265

In the older work on growth of Bacillus megatherium. (Adolph and Bayne-Jones, 1932) the rate of volume increase rose during interphase but not rapidly enough to give a logarithmic type of curve. These results, particularly the more careful studies by Kimball et al. (1959), do not fit into the generalization suggested earlier by measurements on yeasts, amoeba, and Tetrahymena that interphase cell growth occurs at a relatively constant rate. Obviously, growth in Paramecium is controlled in a different manner. It has been suggested that the near-linear growth rate in amoeba is a corkiequence of immediate nuclear control of growth (Prescott, 1957). Possibly in Paramecium, growth is not directly under nuclear control but is more immediately regulated by some cytoplasmic capacity (Kimball et al., 1959), which itself increases in an “exponential manner.” Kimball (personal communication) has re-examined the situation in Paranzecittw and concluded that a growth curve that shows an accelerating growth rate does not necessarily imply an autocatalytic property of cell growth. It might be assumed, for example, that the nucleus synthesizes and releases to the cytoplasm a product (ribosomal RNA, for example) at a constant rate over interphase. If the cell growth rate were proportional to the amount of nuclear product in the cytoplasm, the growth rate would rise unless the product were used up as rapidly as it were produced. If none of the product were used up but all of it repeatedly reutilized, the growth curve under the above assumptions would closely resemble a curve of exponential form. The belief that cell growth or size in some manner acts as a stimulus of cell division has a long history. The earliest serious attempt to state formally the relation between cell growth and division appears in Hertwig’s hypothesis ( 1903) of a critical nuclear-cytoplasmic volume ratio. A principal intention of the recent measurements on single microorganisms has been the attainment of a more precise definition of the relation of cell growth and size to the initiation of division. It is clear from several experiments on amoeba growth that attainment of a particular cell mass does not act as a stimulus to division. First, growth stops completely several hours before the beginning of division. Second, when cell size is limited by removing the source of nutrients during the last half of interphase, the smaller cells divide anyway, although the smaller the cell, the longer is the time required to reach division (Prescott, 1957). Third, cytokinesis can be prevented without affecting mitosis, by immersing dividing amoebae in a protein solution for one hour at the time when cytokinesis is about to begin. From the binucleated cell thus created one nucleus can be removed, producing a division size cell with a nucleus

266

D. M . PRESCOTT

that has just finished telophase (the cytoplasmic to nuclear volume ratio is normally about 5O:l). Cells so constituted grow very little or not at all in total mass and proceed through interphase to enter division again within 60 to 75% of a normal generation time (Prescott, 1959b). Kimball et d.(1959) have succeeded in varying the growth rate and generation time independently of one another. Daughter cell Paramecia transferred from an environment of abundant nutrient to a medium of limited food grew much more slowly but reached the next division with a generation time only slightly longer than normal. Division thus occurred with only a 1.5-fold increase in mass. In the reciprocal type of transfer (starved Paramecia into nutrient medium), the growth rate was rapid but the generation time was still greatly extended, and the cells increased 3.5-fold in mass before dividing. Such cells at division were still well below the size of dividing cells from a well-fed culture. These experiments demonstrate that the relation between growth and division in Paramecium is remarkably flexible. In the synchronization of division in the thymineless mutant of E . coli (Barner and Cohen, 1956) by withdrawal and restoration of thymine, growth (protein and RNA synthesis) continues in the absence of cell division and DNA synthesis. Separation of growth and division processes by this means, however, produces irreversible damage to the cell if allowed to continue beyond a period approximately equal to one generation. In the heat shock method of division synchronization in Tetrahymem (Scherbaum and Zeuthen, 1954), the separation between growth and division is also complete, although this divisionless growth is apparently “balanced” since no irreversible damage is inflicted upon the cells. When the enlarged cells created by this synchronization method are transferred to a non-nutrient salt medium, they go through several life cycles without any growth (Hamburger and Zeuthen, 1957). This situation of division without growth is reminiscent of the complete separation of growth and division in segmenting eggs. Growth is a measure of the total net increase of all the accuniulatory and synthetic processes of a cell, and many of these may be of no direct importance in the preparations for cell division. The adjustment in cell size to different nutrient environments, the completion of division preparation in the absence of cell size increase, and the lack of a rigid relation between the rates of cell growth and division are considered here as evidence that the relatively inflexible syntheses that constitute division preparations take place on a flexible background of cell metabolism. The studies on cell growth, synchronization of cell division, and the relations of cell size or growth to division have each emphasized the

267

GROWTH-DUPLICATION CYCLE O F T H E CELL

necessity for analyzing growth in terms of those processes that are the inflexibIe components of the cycle. What proportion of cell mass constitutes the essentials of division preparation is difficult to estimate, and undoubtedly varies greatly with cell type and with different environmental conditions. This discussion, and especially that to follow, assumes that the growthduplication cycle (or perhaps more precisely, the DNA duplication cycle) has arisen or originated only once and that the same basic core of biochemical events governs the multiplication of all cell types. Direct evidence for such a unitary hypothesis is difficult to obtain because the view is confused by numerous modifications, which are here assumed to be outside the cause-effect chain of life cycle events, and by a diversity of time scales and schedules obeyed by various cell types. Both the absolute time and the time span relative to the complete cycle length required for the accomplishmeiit of a particular step may have widely different values in different cells. This is appropriately illustrated by the difference in the absolute and relative time spans required for DNA synthesis and by the various positions within interphase, which DNA synthesis can occupy. A N D FUNCTION DURING C. DNA SYNTHESIS

THE

CYCLE

In a wide variety of cells, studied by several techniques (usually photometric or autoradiographic), the synthesis of DNA has been shown to occur in the latter part of interphase. There is, typically, a long stretch of interphase between the end of telophase or cytokinesis and the beginning of DNA synthesis. A shorter interphase interval separates the end of DNA synthesis from the first cytologically recognizable sign of prophase. This picture has been observed consistently in vertebrate cells (chicken, rat, mouse, guinea pig, man), and may prove to be a basic and significant similarity among all vertebrate cell types (Firket and Verly, 1957 ; Harris, 1959; Painter and Drew, 1959; Stanners and Till, 1960). In plant root tips, DNA synthesis has been observed to occur in middle to late interphase (Howard and Pelc, 1951 ; Siskin, 1959; Deeley et d.,1957). The position of synthesis in the generative nucleus of the pollen grain, however, is in early interphase (Woodward, 1958; Taylor and McMaster, 1954; Taylor, 1958a). The position of DNA synthesis in the cycle varies considerably in microorganisms. In Tetrahymena, DNA synthesis begins shortly after cytokinesis and ends at or soon after midinterphase (McDonald, 1958; Prescott, 196Oa). In Paramecium, however, the synthesis begins in later interphase and appears to continue up to the beginning of nuclear division (Kimball and Barka, 1959). The same relation obtains in Euplotes

268

D. M. PRESCOTT

(Gall, 1959). In bacteria, the interpretation of the data available so far has not been clear, partly-because the division of the nucleus apparently does not show the same temporal relation to cytokinesis as it does in larger cells. In measurements on bacterial populations in which nuclear division synchrony has been induced, DNA synthesis seems immediately to precede doubling of nuclear bodies (Lark and Maal$e, 1956; Barner and Cohen, 1956), which is similar to the findings for the amitotically dividing macronucleus of Paramecium (Kimball and Barka, 1959). The “normalcy” of the DNA synthesis picture after synchrony induction in bacteria has been questioned by Schaechter et al. (1959) in studies on tritiated thymidine labeling of bacteria in asynchronous multiplication during the logarithmic phase of growth of an untreated culture. After a short pulse of label all cells were labeled, and the authors concluded that DNA synthesis occurs during 80% or more of the cell cycle. It is quite conceivable that the synchrony induction treatment brings about the synchronization in the synthesis of DNA molecules and thereby shortens the time for total DNA doubling. Studies on the incorporation of tritiated thymidine by individual bacteria of known position with respect to nuclear division would clarify the situation. In the rapidly dividing neuroblasts of the grasshopper embryo, DNA synthesis is continuous from middle telophase to very early prophase (Gaulden, 1956). Synthesis in the slime mold is limited to a relatively short space of interphase immediately after mitosis (Nygaard ct al., l%O). It is apparent that the DNA synthesis process occupies widely different proportions of the cycle in different cells and shows a wide variety of different temporal relations to nuclear division. Two generalizations emerge from these studies on such cytologically diverse cell types. DNA synthesis does not occur during nuclear fission itself. The completion of DNA synthesis, although an absolute requirement for successful nuclear and cell division, is usually completed well before nuclear division, and cannot function in the immediate stimulation of division. DNA has received attention from so many quarters that developments concerning its structure, function, and replication have been rapid. I n this emphasis on DNA there has been some tendency to relegate the protein content of chromosomes to a position of minimal importance. Chromosome duplication is sometimes tacitly equated with DNA synthesis. Since basic protein is a constant component of chromosomes, it must be assumed that it takes part in the chromosome replication process. The work of Bloch and Godtnan (1955), Alfert (1958), and Gall (1959) indicates that the basic protein components of chromosomes are synthesized at the same time as DNA, although additional studies are needed. Anderson (1956a)

GROWTH-DUPLICATION CYCLE O F T H E CELL

269

has presented a thorough discussion of the possibility that histones may play an active role in the coiling-uncoiling cycle of chromosomes at mitosis. Little is known about the importance for the structure of the chromosome of nonhistone protein of the nucleus or of the chroniosomeassociated RNA. There is some evidence that one or both may be products of DNA-histone metabolism (see, for example, Gall, 1958). Some examples have been cited in which a relatively long period of interphase separates either the end of the previous telophase from the beginning of DNA synthesis, or the end of DNA synthesis from the beginning of prophase. It must be admitted, however, that neither the actual end of telophase nor the actual beginning of prophase can be timed precisely because chromosome coiling and uncoiling at these points are below the level of detection by light microscopy, and are still undefinable by electron microscopy. The cytological definitions of the beginning and end of mitosis are therefore somewhat arbitrary, depending on ( a ) the visible nuclear characteristics of the particular cell, ( b ) whether the observations are made on living or fixed cells, (c) the type of such things as fixation, staining, and optical system used, and ( d ) the opinion of the individual cytologist. Thus chromosome coiling and uncoiling at a submicroscopic level probably occupy some parts of the “blank periods” between mitosis and DNA-histone synthesis. It seems probable that, even in very rapidly proliferating cells, chromosomes may remain in a completely dispersed state for considerable periods of interphase in addition to the periods of replication, but this point has really not been directly demonstrated. During the growth-duplication cycle, the chromosome participates in at least three separate activities : ( a ) its self-duplication, ( b ) dissemination of information to the rest of the cell, and (c) the coiling-uncoiling cycle associated with the separation of the duplicated chromosome into two independent chromatids. The syntheses of self-duplication ( DNA-histone) occur during some part of interphase. The syntheses concerned with the dissemination of information also occur during interphase and possibly only during this period. In template hypotheses of information transfer, it is possible that information contained in a linear sequence of nucleotides cannot be copied when the linear polymer is condensed by coiling into a mitotic chromosome or chromatid. Both functions of the chromosomes, i.e., duplication and information dissemination, probably occur when the chromosomes are in the dispersed state, but the functions must certainly be separated in time by whatever mechanisms control them. The rapidly accumulating information on DNA structure and synthesis suggests that DNA replication takes place only when the molecule exists

270

D. M . PRESCOTT

in the single strand form (Bollum, 1959). The fact that DNA extracted from cells has apparently- been found only in the double helix form has led to speculation about the function of this form in information dissemination. Unwinding or partial unwinding of the double helix could be the immediate stimulus to DNA synthesis intracellularly. Rapid construction of the complementary helix would permit only a very transient existence of the single strand form. Whether or not this is the manner in which synthesis occurs, replication of DNA must result in some change in the properties of the synthesis system since each molecule replicates only once in the course of one normal cycle. The evidence that all DNA molecules are not synthesized in synchrony with one another suggests that the prevention of a second replication resides in a property of each newly replicated molecule or is a result of the intimate association between the identical members of each new pair. Normally, the next cycle or replication can follow only after the separation of the doubled structure into independent chromatids during metaphase; it is implied here that the separation may return the chromosome into a state that will allow a further replication. I t is perhaps pertinent that this separation of one unit into two at metaphase also immediately precedes the very rapid transition to a doubled growth rate observed in a number of cells (Zeuthen, 1953; Prescott, 1957, 1959b; Mitchison, 1957, 1958; Bayne-Jones and Adolph, 1932). The evidence that all DNA molecules are not synthesized in synchrony comes from several sources. Taylor’s studies (195%) on tritiated thymidine labeling of chromosomes of Crepis indicate that DNA synthesis begins at the ends of the chromosome and proceeds toward the kinetochore. In Vich cells, which apparently had been exposed to labeled thymidine for only a part of the total DNA synthesis period, Woods (quoted by Taylor, 1958a) found an occasional cell that contained completely labeled chromosomes in a complement of labeled and partially labeled chromosomes. In B e l l e v d h , however, Taylor (1958a) could not detect such differential labeling by the autoradiographic method; all chromosomes of a single complement appeared to be labeled simultaneously. I n the ciliate, Euplotes, Gall (1959) has presented a remarkable demonstration of a double wave of DNA synthesis in the ribbon-shaped macronucleus. Synthesis begins at each end of the nucleus and the two waves meet at the middle. DNA synthesis appears to be accompanied by a simultaneous histone synthesis, Finally, Harris ( 1959) has concluded from autoradiographic studies on thymidine inco-rporation by rat heart cells that DNA synthesis starts at the nucleolus and proceeds into the chromosomes. The possibility that R N A may be the product of chromosome metabo-

GROWTH-DUPLICATION CYCLE O F THE CELL

27 1

lism has been widely discussed. Based on this hypothesis and the preceding discussions of the double function of DNA (i.e., self-replication and information dissemination), it has been suggested that a DNA molecule may be incapable of serving simultaneously in its own duplication and in the production of RNA. Taylor and McMaster (1954), Moses and Taylor ( 1955), Taylor ( 1958a,b), Siskin ( 1959), Woodard ( 195S), Prescott (1960a), and Nygaard et al. (1960) have found evidence that RNA synthesis in a number of different cell types is suppressed during the period of DNA synthesis. In Tetrahymena, for example, the rate of incorporation of C14-adenine into R N A during DNA synthesis drops to 15 to 20% of the over-all rate for the rest of interphase (Prescott, 196Oa). Since there is evidence of a sequential synthesis of DNA molecules within the total synthesis period, DNA-directed synthesis of R N A should not be expected to drop to zero during any part of interphase. Such mutual exclusiveness of a temporal character may only reflect a competition for common precursors. The problem is in need of a more direct attack and is additionally complicated by the somewhat confusing data on the intranuclear site of RNA synthesis. An important question to be answered is whether RNA synthesis occurs in or at a DNA-free nucleolus or is produced only in association with dispersed chromosomes. A discussion of the nuclear role in R N A synthesis has been published elsewhere (Prescott, 1960b). Summarizing briefly at this point, the cell growth-duplication cycle consists of a period occupied by chromosome replication, a period for nuclear division (ordinarily followed closely by cytokinesis) , and in most cells, a stretch of interphase that as yet can only be described as a time of protein and R N A syntheses and energy production. This latter period occurs as a continuous stretch in cells in which DNA synthesis and nuclear division (in this or the reverse order) are immediately consecutive events. Without knowing what specific, essential steps occupy this part of the cycle, it is still sometimes possible to examine certain of their properties in a very general way. In Tetrahymena, in the long interval between the end of DNA synthesis and the onset of division, the sensitivity of the cell to temperature shock (measured as the delay imposed on division) rises steadily and reaches a maximum just before division (Thormar, quoted by Zeuthen, 1958 ; Prescott, 196Oa). Arginine deprivation during most of this period prevents cell division ( Prescott, unpublished) ; proteins essential for cell division are apparently synthesized during this time. Foster and Stern (1959) have demonstrated the accumulation of soluble deoxyribosidic compounds in the short space of time just before DNA synthesis in anthers of Lilium. This finding immediately raises the ques-

272

D. M . PRESCOTT

tion of the control or stimulation of such accumulation. Nygaard et al. (1960) have reported that-orotic acid can be converted to thymine nucleotide all during interphase in the slime mold. From their data they conclude that the control of the initiation of DNA synthesis resides close to the final polymerization. D.

RESPIRATION A N D ENERGY METABOLISM

Variations in the intensities of a number of activities or properties besides DNA synthesis have been observed, but the reports are fragmentary in the sense that such findings for the most part cannot yet be tied together causally within the cycle. A number of studies have been made on respiration and energy metabolism over the cycle, and the data present a relatively consistent picture. Zeuthen (1951a,b, 1953, 1955) has repeatedly found that the rate of respiration in cleaving eggs rises to a maximum during early division and falls thereafter to a minimum during telophase. Ericltson (1947) noted a similar depression in respiration during meiotic and mitotic divisions of microspores in lily anthers. Stern and Kirk (1948) have described a rise in oxygen consumption in anthers during prenieiotic stages and a sharp drop immediately preceding and during division. In Tetrahymena, the respiration rate rises steadily throughout interphase, and then remains constant or drops slightly during the early part of macronuclear amitosis, and rises sharply at the beginning of cytokinesis (Zeuthen, 1953). Agrell (1955) observed a decrease in the number of microscopically visible mitochondria in segmenting sea urchin eggs from prophase to metaphase ; the mitochondria1 number increased from metaphase up to the next division. These cytological changes coincide closely with the changes in respiration rate in marine eggs. Related to these findings on fluctuations in oxygen consumption at the time of division are certain experiments designed to yield information about the energy requirements for cell division. Bullough and Johnson (1951) and Bullough (1952) isolated fragments of mouse ear into saline and reported that only glucose was required to bring some of the cells into division. From this Bullough has argued that the build-up of an energy store must occur in the period just before division (antephase) and that the final stimulus to division rests in such an accumulation of energy. As further support for the thesis of an antephase creation of an energy reservoir, Bullough cites the relative independence of cell division from such environmental conditions as oxygen deprivation or lack of carbohydrate once the process of division has begun. As Swann (1957) points out, there is no real evidence in these experiments that the energy

GROWTH-DUPLICATION CYCLE O F T H E CELL

273

storage for division occurs just before division. In a series of papers, Gdfant (1959a,b, l W a , b ) has re-examined the role of glucose and oxygen in the stimulation of division in ear fragments. His work strongly indicates that the stimulus for division is the cutting of the ear; mitoses appear in a concentration gradient from the cut. Energy may be necessary for cells to move from antephase into prophase, but there is no evidence that an energy store or source is acting as a division stimulus. As Hughes (1952) has so succinctly stated, “In a medium completely optimal except for the omission of a single component, restoration of that one limiting factor would raise it to the status of ‘the’ chemical stimulus to cell division. ” Based principally on studies with cleaving eggs and on the observations that respiration inhibitors prevent mitosis if applied during interphase but not after mitosis has begun, Swann (1957) proposed that throughout interphase, cells build an energy reservoir that must be filled before division can be initiated. The scheme implies “a cyclic respiratory mechanism of some sort which controls division” (Swann, 1957). The filled reservoir is presumed to be emptied by the demands of the division activities but immediately begins to refill in preparation for the next division. It is very unlikely that a division-initiating energy reservoir exists in neuroblasts of the grasshopper embryo since an extracellular energy source is indispensable during the early stages of mitosis (Gaulden and Kokonioor, 1955). The experimental observations (see Swann, 1957), demonstrating that cell division requires energy production during interphase, offer no information, however, concerning the time or the manner in which the energy is utilized. Although energy may be fed into a reservoir, as Swann suggests, it seems equally reasonable to suppose that it becomes bound into the interphase syntheses and organization of the macromolecules necessary to produce intracellular division conditions. The relative insensitivity of division to oxygen deficiency and respiration poisons in many cells once the division is under way, or at least past the earlier part of prophase, could be interpreted to mean that either ( a ) division events require very little energy, (b) the needed energy is drawn from a prefilled reservoir (part of Swann’s hypothesis), (c) sufficient energy is stored in macromolecular organization and structure, or ( d ) some combination of these is in operation. Swann has suggested (c) as an alternative to (b) , and Marsland ( 1950) has described evidence that the energy for cytokinesis is already present in the gel of the cell cortex at the time when cytokinesis begins. The decreased demand for energy production during nuclear division could be considered to follow from the observed

274

D. M. PRESCOTT

decrease in respiration during this process. The effect is especially marked in dividing cells of lily anthers (Stern and Kirk, 1948). Nasatir and Stern (1959) have, in addition, noted a drop in the activity of aldolase and ~-glyceraldehyde-3-phosphatedehydrogenase during mitosis, which certainly suggests decreased glycolysis at this time. In amoeba, phosphate uptake from the medium drops to one-third during division (Mazia and Prescott, 1954). These data can be interpreted to reflect a sharp fall in high energy phosphorylations. A similar but less pronounced drop in phosphate uptake has been described for cleaving sea urchin eggs (see Zeuthen, 1958). It seems unlikely that energy production regulates syntheses or cell division even in cleaving eggs, but rather energy metabolism is geared to demand, increasing and decreasing as the physiological state of the cell requires. At present, this thesis cannot be proved by direct demonstration of specific regulatory mechanisms of energy metabolism, but the principle has been solidly established in the detailed investigations of enzyme induction responses and of negative feedback controls of the production of small molecules. The recently described control of adenylic and guanylic acid syntheses, achieved through the intervention of intricate and complex interacting feedbacks on synthetic pathways (Magasanik, 1958), is an exciting example of an efficient and economic arrangement of a type that, in essence, might eventually prove to operate in the metabolism of small molecules generally. This type of stability in precursor pools, maintained through constant readjustments in levels of pertinent enzyme activities (see discussion by Roberts et al., 1959), may very well apply also to the generation of highenergy sources such as A T P through comparable controlling mechanisms. DURING THE CELLLIFECYCLE E. R N A SYNTHESIS

It is apparent from the work of Taylor (1958b), Taylor and McMaster ( 1954), Moses and Taylor ( 1955), Siskin (1959), Prescott ( 1%0a), and Nygaard et al. (1960) that the rate of R N A production fluctuates appreciably over the cycle, at least in the cells studied by these authors. The explanations have been offered that the rate of R N A synthesis falls during periods of DNA synthesis, either because DNA functions in R N A synthesis, and is unavailable during its self-replication, or that the two macromolecules are in competition for common precursors. Depression in the rate of R N A synthesis has also been reported for cells in mitosis (Taylor and McMaster, 1954; Taylor, 1958b; Siskin, 1959). Here too, it has been suggested that DNA acts in R N A synthesis during interphase but cannot do so when the chromosomes are condensed into a mitotic form.

GROWTH-DUPLICATION CYCLE O F THE CELL

275

In Maruyama’s measurements ( 1956) on synchronized cultures of

E. coli, R N A increased in early interphase but leveled off before the onset of protein synthesis. Protein remained almost constant in amount during early interphase and doubled during the last half of the cycle. DNA synthesis occurred shortly before cytokinesis. It would be less difficult to relate these findings to results with other cell types if the time of nuclear division in each study on bacteria were more accurately known. In Bacillus megatherium, nuclear division apparently occurs shortly after cytokinesis, or in effect, almost a full cycle ahead of cytokinesis (HunterSzybalska et al., 1956). This situation is consistent with the observation by MaalZe and Lark (1954) that more than a half-generation period extends between nuclear division and cytokinesis in Salmonella typhimurium. Whether this type of relation between nuclear splitting and cytokinesis holds in all bacteria is apparently not known. Maruyama and Lark ( 1959a), using cultures of Alcaligenes fecalis synchronized by the filtration method, found that the amount of sedimentable R N A stays constant over most of the cycle and doubles close to the time of cytokinesis. Much evidence points to fluctuations in the rate of R N A synthesis as a function of the cell cycle. Mitchison and Walker (1959), however, have measured the R N A contents of a number of individual yeast cells of unknown cycle position from an asynchronously multiplying culture. The histogram record of these data led to the interpretation that R N A is synthesized at an essentially constant rate over the cycle. The method probably would not detect any except very large changes in synthesis rate.

F. ENZYME FLUCTUATIONS Very little information is available as yet on fluctuations of different enzyme activities during the cycle. Decreases in aldolase and D-glyceraldehyde-3-phosphate dehydrogenase ( Nasatir and Stern, 1959) were mentioned in connection with energy metabolism during division. In yeast cultures with synchronous multiplication induced by a preperiod of starvation, Sylven et al. (1959) reported a marked increase in peptidase and proteinase activities during interphase. During budding of the yeast these enzymic levels fell. The authors express the opinion that the “results seem to indicate that cycles of increased proteolysis are forming essential steps during certain stages of cell growth” perhaps by a replenishing of amino acid pools during periods of rapid protein synthesis. Doudney (1960) has measured R N A and DNA syntheses in temperature-synchronized cultures of E. coli in which protein synthesis was inhibited with chloramphenicol for various periods of the cycle. If

276

D. M . PRESCOTT

chloramphenicol was administered just after cytokinesis, subsequent R N A synthesis was inhibited. If administered just before cytokinesis, subsequent DNA synthesis was inhibited. Chloramphenicol treatment at other times was ineffective in both cases. It would seem that proteins required for the syntheses of R N A and D N A are produced at these two parts of the cycle. Possibly these proteins normally appear at the beginning of the respective synthesis periods since, in Maruyama’s synchronized cultures of this bacterium ( 1956), R N A synthesis was restricted to a short interval after division and DNA synthesis occurred in a short period just before cytokinesis. Doudney, however, observed no such stepwise synthesis of either nucleic acid. These differences may be caused by different methods of synchrony induction (temperatures versus filtration). Maruyama and Lark ( 1959b) have studied DNA and R N A syntheses in filtration-synchronized Alcaligenes fecalis cultures in which protein synthesis was reduced to one-third the normal level by chloramphenicol inhibition. In this synchronized system, both nucleic acids double close to the time of cytokinesis (in the absence of chloramphenicol j . In the presence of chloramphenicol, nucleic acids continue to be synthesized according to this same periodicity. The presence of a periodicity of nucleic acid synthesis in this system and its absence in Doudney’s experiments remain to be explained.

G. REPRODUCTION OF STRUCTURES The growth of the single cell over the individual cycle is composed of an approximate doubling in the number or size of most cell structures, and an exact doubling in number can be demonstrated in a few cases (chromosomes, centrioles, nucleoli). The control of increase in size or number is not understood in the case of any structure. Although the demand for energy production during growth may lead to increased size and number of mitochondria, for example, virtually nothing is known of the step-bystep initiating and controlling mechanisms by which the appropriate structural components are synthesized and organized into a mitochondrion. Explanations for increases in other cell parts are at stages of equivalent vagueness at the present time. The productions of all structures must be subject to the rules of integration, however, that guarantee that a balanced metabolism be maintained. It can only be assumed for the present that this is accomplished through interactions that, at least in part, make use of inductions and feedbacks of the type demonstrated for a growing list of enzymes and synthetic pathways. The control-of ribosome production and function is an especially intriguing question because, in their role in protein synthesis, they may represent the cytoplasmic outlet for genetic information. Mitchison ( 1957)

GROWTH-DUPLICATION CYCLE O F T H E CELL

277

speculated that a sudden doubling in ribosome number may be responsible for.the doubling in growth rate that occurs at cell division. There is evidence, however, that the growth rate of a cell is ultimately controlled by the nucleus. The transition, through mitosis without cytokinesis, from a uninucleated to a binucleated state in amoeba, for example, is marked by a sudden doubling in the growth rate of the whole cell (Prescott, 1959b). The key event, as already discussed in an earlier section, may be a reorganization of the one nucleus into two functioning units (two functioning sets of chromosomes). If one of the nuclei is removed from the new binucleate, the growth rate falls to practically zero. Rabinovitch and Plaut (1956) showed with this same cell that a considerable amount of R N A is released from the nucleus during mitosis. The premitotic nucleus is rich in R N A ; the reconstituted post-telophase nucleus is RNA poor. Such a sudden release of R N A from the nucleus into the cytoplasm is reminiscent of Mitchison’s suggestion that niicrosomes double in number at division ; the increased cytoplasmic R N A could conceivably produce the sharp increase in rate of growth. The experiment with binucleated amoebae described previously shows, however, that the continuous presence of the nucleus is required for the maintenance of the new postdivision growth rate. Increases in structures, presumably during interphase, that perform special functions in mitosis must be guaranteed for each cycle. Mazia’s extensive work (1959) on the isolated mitotic apparatus of the sea urchin egg, pointing to an important role of protein sulfhydryl groups in construction of the apparatus, has led to more detailed studies on the cyclic fluctuations in free -SH groups originally observed by Rapkine (1931). The earlier idea that the cycle of free -SH was based on changes in levels of reduced glutathione has been ruled out by the work of Neufeld and Mazia (1957) and Sakai and Dan (1959). The latter authors found evidence that the variations consist of a build-up of -SH groups attached to protein ( a protein soluble in trichloroacetic acid solution) through interphase and a rapid drop during division. It does not seem possible that this TCA-soluble protein is itself the unit of which the mitotic apparatus is built because the -SH protein reaches a maximum level at metaphase, a time when the apparatus is already well developed. Stern (1958) and Nasatir and Stern ( 1959) reported a marked rise in soluble -SH of lily anthers preceding mitosis. During mitosis the soluble -SH level drops slightly, rises again to a maximum at the end of mitosis, and falls steadily in the postmitotic period. The dip in -SH level during mitosis might conceivably be related to the formation of the mitotic apparatus, but it seems that the cyclic fluctuations of -SH are related to more than the direct formation of the mitotic apparatus alone.

278

D. M . PRESCOTT

The cycle timing of centriole reproduction in cleaving sea urchin eggs has been closely studied -by Mazia and associates (1960). The centriole of a daughter cell in telophase was found to be of a dual nature. Each unit of the dual structure contributed to the production of another unit so that a tetrad complex was formed before the beginning of the next division. Splitting of the tetrad into two functional units just before spindle formation left each newly-formed telophase cell with a dual unit centriole. Partly on the basis of Cleveland’s many observations (see for example, Cleveland, 1957), the authors have stressed the “generative” nature of centriolar duplication. Apparently, this self-duplicating, cytoplasmic organelle does not develop the tetrad state by a splitting into equal halves of each unit of the duplex of post-telophase. Each unit gives off a small fragment that grows into a mature unit. Because centrioles and kinetochores of chromosomes show certain analogies in function, it would be interesting to determine whether centriole and kinetochore syntheses occur in synchrony with each other. The basic event for nucleolar doubling presumably takes place at the time of duplication of the organizer region (s) during chromosome synthesis. The principal question at the present time is not the duplication of the nucleolus, but rather its function, especially with regard to R N A synthesis in the nucleus. The problem has been attacked with autoradiography with somewhat conflicting results. The observation of a very rapid labeling of nucleolar R N A suggested that the nucleolus might be a primary site of R N A synthesis (McMaster-Kaye and Taylor, 1958; Goldstein and Micou, 1959a). Two careful time studies on R N A labeling in mammalian nuclei (Goldstein and Micou, 1959b ; Aniano, 1960) have suggested that a rapid transfer of R N A from chromatin to the nucleolus could possibly be responsible for the heavy nucleolar labeling so frequently observed with R N A precursors. There is good evidence that the nucleolus relinquishes R N A to the cytoplasm (Woods and Taylor, 1959), and this phenomenon increases the difficulties in arriving at a clear interpretation of the autoradiographic data. It is also possible that the intensities of nucleolar activities vary from one part of the cell cycle to another. With the short pulse technique of isotope administration, measurements on the rates of R N A and protein labeling of nucleoli at various parts of the cycle might yield significant information on nucleolar physiology and its relation to cell reproduction. On the latter point, Gaulden and Perry (1958) discovered that ultraviolet irradiation of the nucleolus of grasshopper neuroblasts from late telophase to the middle of prophase stopped the progress of the cell in the cycle. Irradiation after middle prophase was without effect on the mitosis already in progress. Whatever the role of the

GROWTH-DUPLICATION CYCLE OF THE CELL

279

nucleolus, its contribution in this cell very rapidly passes from an indispensable to a dispensable status for the division in progress and at a welldefined point of prophase. Finally, a change or discontinuity in nucleolar function most likely accompanies disappearance of the structure as a cytological entity during prophase or metaphase and its reappearance during telophase in most cells. The method by which mitochondrial increases are generally brought about has not been decisively determined. Multiplication by fragmentation of existing mitochondria followed by growth through accretion appears to be the explanation most generally accepted. Whether the breaking of mitochondria occurs continuously during interphase of multiplying cells or only at the time of division is not conclusively decided. Zeuthen’s measurements (1953) of the increase in respiration of individual Tetruhymena over the cycle strongly indicate that the total mitochondrial mass increases continuously by increase in size of existing mitochondria or by increase in number. The proportionality between mitochondrial number and cell volume (see Ernster and Lindberg, 1958) supports the idea of a continuous increase in mitochondrial number during growth. Agrell’s observations (1955) on the interphase number of mitochondria in cleaving sea urchin eggs supports this thesis.

IV. Concluding Remarks In the latter part of this review, I have attempted to examine the growth-duplication cycle of the individual cell in terms of the increase of specific parts or structures. I n no structure do we understand the stepby-step control processes that lead to increase. The integrative mechanisms that maintain the orderly and balanced condition of cell metabolism during any instant must also coordinate the increases of all parts and functions over the time span of one growth-duplication cycle. Implied or stated at various points in this review is the opinion that within the growthduplication cycle a key group of sequential events (DNA synthesis and chromosome coiling-uncoiling are representative) governs the progress of the cell through the cycle. Increase in cell size in this scheme is a response to the changing physiological states of the cell, which states are directly based on the progression of the key series of events. A large part of cell metabolism (e.g., energy generation, precursor production) is considered to subserve the continued progression of the cycle but does not directly participate in cycle regulation. This latter function is a property of macromolecular interaction.

280

D. M. PRESCOTT

REFERENCES Adolph, E. F., and Bayne-Jones, S. (1932) 1. Cellular Comp. Physiol. 1, 409. Agrell, I. (1955) Exptl. Cell Research 8, 232. Alfert, M. (1958) Exptl. Cell Research Suppl. 6, 227. Amano, M. (1960) Amt. Record 136, 153. Anderson, N. G. (1956a) Quart. Rev. Biol. S1, 169. Anderson, N. G. (1956b) Quart. Rev. Biol. 31, 243. Anderson, P. A., and Pettijohn, D. E. (1960) Science 191, 1098. Barner, H. D., and Cohen, S. S. (1956) J. Bacteriol. 72, 115. Barner, H. D., and Cohen, S. S. (1957) J . Bacteriol. 74, 350. Bayne-Jones, S., and Adolph, E. F. (1932) J . Cellular Comp. Physiol. 1, 387. Bloch, D. P., and Godman, G. C. (1955) J. Biophys. Biochtm. Cytol. 1, 17. Bollum, F. J. (1959) J . Biol. C h m . 234, 2733. Bullough, W. S. (1952) Biol. Revs. Cambridge Phil. SOC.27, 133. Bullough, W. S., and Johnson, M. (1951) Proc. R o y . SOC.B138, 562. Burns, V. W. (1959) Science 129, 566. Campbell, A. (1957) Bacteriol. Revs. 21, 263. Cleveland, L. R. (1957) J . Protozool. 4, 230. Deeley, E. M., Davies, H. G., and Chayen, J. (1957) Exptl. Cell Rescnrch 12, 582. Doudney, C. 0. (1960) 1. Bacteriol. 79, 122. Erickson, R. 0. (1947) Nature 169, 275. Ernster, L.,and Lindberg, 0. (1958) Ann. Rev. Physiol. 24,13. Falcone, G., and Szybalski, W. (1956) Exptl. Cell Research 11, 486. Firket, H., and Verly, W. G. (1957) Nature l81, 274. Forro, F. (1957) Exptl. Cell Research 12, 363. Foster, T. S., and Stern, H. (1959) J . Biophys. Biochem. Cytol. 6, 187. Gall, J. (1958) I n “Chemical Basis of Development” (W. D. McElroy arid B. Glass, eds.), p. 103. Johns Hopkins Press, Baltimore, Maryland. Gall, J. (1959) 1. Biophys. Biochem. Cytol. 6, 295. Gaulden, M. E. (1956) Genetics 4, 645. Gaulden, M. E., and Kokomoor, K. L. (1955) Proc. SOC.Exptl. Biol. Med. 90, 309. Gaulden, M. E., and Perry, R. P. (1958) Proc. Natl. Acad. Sci. US.44, 553. Gelfant, S. (1959a) Exptl. Cell Research 16, 527. Gelfant, S. (1959b) Exptl. Cell Research 18, 494. Gelfant, S. (1960a) Exptl. Cell Research 19, 65. Gelfant, S. (1960b) Exptl. Cell Research 19, 72. Goldstein, L.,and Micou, J. (1959a) I. Biophys. Biochem. Cytol. 6, 1. Goldstein, L., and Micou, J. (1959b) J . Biophys. Biochem. Cytol. 6, 301. Hamburger, K., and Zeuthen, E. (1957) Exptl. Cell Research 13, 443. Harris, H. (1959) Biochm. J . 72, 54. Hertwig, R. (1903) Biol. Zentr. !B, 49. Howard, A., and Pelc, S. R. (1951) Exptl. Cell Research 2, 178. Hughes, A. F. W. (1952) “The Mitotic Cycle,” 1st ed. Butterworths, London. Hunter-Szybalska, M. E., Szybalski, W., and Delamater, E. E. (1956) I . Bacteriol. 71, 17. . Iverson, R. M., and Giese, A. C. (1957) Exptl. Cell Research 13, 213. Kaudewitz, F. (1958) Arch. Protistenk. 102, 22. Kelly, C. D., and Rahn, 0. (1932) J . Bacteriol. 23, 147.

GROWTH-DUPLICATION CYCLE OF T H E CELL

28 1

Kimball, R. F., and Barka, T. (1959) Exptl. Cell Research 17, 173. Kimball, R. F., Caspersson, T. O., Svensson, G., and Carlson, L. (1959) Exptl. Cell Research 17, 160. Knaysi, G. (1940) J. Bacteriol. 40,247. Lark, K. G., and Maal@e,0. (1954) Biochim. et Biophys. Acta 16, 345. Lark, K. G., and Maal@e,0. (1956) Biochim. et Biophys. Acta 21, 448. Lindegren, C. C., and Haddad, S. A. (1954) Genetica 27, 45. Maal@e,O., and Lark, K. G. (1954) Proc. Symposium Colston Research SOC.7, 159. McDonald, B. B. (1958) Biol. Bull. 114, 71. McMaster-Kaye, R.,and Taylor, J. H. (1958) J . Biophys. Biochem. Cytol. 4, 5 . Magasanik, B. (1958) In “Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), p. 485. John Hopkins Press, Baltimore, Maryland. Marsland, D. (1950) J. Cellular Comp. Physiol. 36, 205. Maruyama, Y. (1956) J . Bacteriol. 72, 821. Maruyama, Y., and Lark, K. G. (1959a) Exptl. Cell Research 18, 389. Maruyama, Y., and Lark, K. G. (1959b) Bacteriol. Proc. (SOC.Am. Bacteriologists) p. 131. Maruyama, Y., and Yanagita, T. (1956) J . Bacteriol. 71, 542. Mazia, D. (1959) In “Sulfur in Proteins” (R. Benesch et al., eds.), p. 367. Academic Press, New York. Mazia, D., and Prescott, D. M. (1954) Science 120, 120. Mazia, D., Harris, P. J., and Bibring, T. (1960) J . Biophys. Biochew. Cytol. 7, 1. Mitchison, J. M. (1957) Exptl. Cell Research 15, 244. Mitchison, J. M. (1958) Exptl. Cell Research 16, 214. Mitchison, J. M., and Walker, P. M. B. (1959) Exptl. Cell Research 16, 49. Moses, M. J., and Taylor, J. H. (1955) Exptl. Cell Research 9, 474. Nasatir, M., and Stern, H. (1959) J . Biophys. Biochem. Cytol. 6, 189. Neufeld, E. F., and Mazia, D. (1957) Exptl. Cell Research 15, 622. Newton, A. A., and Wildy, P. (1959) Exptl. Cell Research 16, 624. Nihei, T. (1955) J . Biochem. (Tokyo) 42, 245. Nygaard, 0.F., Giittes, S., and Rusch, H. P. (1960) Biochim. et Biophys. Acta 58, 298. Padilla, G. M., and James, T. W. (1958) J . Protozool. 6, Suppl., 22. Painter, R. B., and Drew, R. M. (1959) Lab. Invest. 8, 278. Prescott, D. M. (1955) Exptl. Cell Reseurch 9, 328. Prescott, D. M. (1956) Exptl. Cell Research 11, 86. Prescott, D. M. (1957) In “Rhythmic and Synthetic Processes in Growth” (D. Rudnick, ed.), p. 59. Princeton Univ. Press, Princeton, New Jersey. Prescott, D. M. (1958a) Physiol. 2001.51, 111. Prescott, D. M. (195813) Anat. Record lS0, 359. Prescott, D. M. (1959a) Exptl. Cell Research 16, 279. Prescott, D. M. (1959b) Ann. N . Y . Acad. Sci. 78, 655. Prescott, D. M. (1960a) Exptl. Cell Reseurch 19, 228. Prescott, D. M. (1960b) Ann. Rev. Physiol. B, 17. Rabinovitch, M. P., and Plaut, W. (1956) Exptl. Cell Research 10, 120. Rapkine, L. (1931) Ann. physiol. phjisicochim. biol. 1, 382. Roberts, R. B., McQuillen, K., and Roberts, I. Z. (1959) Ann. Rev. Microbiol. 15, 1. Sakai, H.,and Dan, K. (1959) Exptl. Cell Research 16, 24. Schaechter, M., Bentzon, M. W., and Maal@e, 0. (1959) Nature 185, 1207.

282

D. M. PRESCOTT

Scherbaum, 0. H. (1957) Exptl. Cell Research 13, 11. Scherbaum, 0. H., and Zeuthen, E. (1954) Exptl. Cell Research 6, 221. Scherbaum, 0. H., Louderback, A. L., and Jahn, T. L. (1959) Exptl. Crll Rrsearch 18, 150. Siskin, J. E. (1959) Exptl. Cell Research 16, 602. Sorokin, C. (1957) Physiol. Plantarum 10, 659. Sorokin, C., and Krauss, R. W. (1959) Proc. Natl. Acad. Sci. U.S. 46, 1740. Sorokin, C., and Myers, J. (1957) J . Gen. Physiol. 40, 579. Stanners, C. P., and Till, J. E. (1960) Biochim. et Biophys. Acta 37, 406. Stern, H. (1956) Ann. Rev. Plant Physiol. 7, 91. Stern, H. (1958) J. Biophys. Biochem. Cytol. 4, 157. Stern, H., and Kirk, P. L. (1948) J . Gen. Physiol. 31, 243. Swann, M. M. (1957) Cancer Research 17, 727. Swam, M. M. (1958) Cancer Research 18, 1118. Sylven, B., Tobias, C. A., Malmgren, H., Ottoson, R., and Thorell, B. (1959) Exptl. Cell Research 16, 75. Tamiya, H., Iwamura, T., Shibata, K., Hase, E., and Nihei, T. (1953) Biochim. et Biophys. Acta 12, 23. Taylor, J. H. (19%) Proc. 10th Intern. Congr. Genet., Montreal 1, 63. Taylor, J. H. (1958b) Am. J. Botany 46, 123. Taylor, J. H., and McMaster, R. (1954) Chromosoma 6, 489. Walker, P. M. B. (1954) J. Exptl. Biol. 31, 8. Woodard, J. W. (1958) J. Biophys. Biochem. Cytol. 4, 383. Woods, P. S., and Taylor. J. H. (1959) Lab. Invest. 8, 309. Zeuthen, E. (1951a) Biol. Bull. 98, 144. Zeuthen, E. (1951b) Biol. Bull. 98, 152. Zeuthen, E. (1953) J. Embryol. Exptl. Morphol. 1, 239. Zeuthen, E. (1955) Biol. Bull. 108, 366. Zeuthen, E. (1958) Advances in Biol. and Med. Phys. 6, 37. Zeuthen, E., and Scherbaum, 0. H. (1954) Proc. Symposium Colston Research SOC.7, 141.

Histochemistry of Ossification R ~ M U LL. O CABRINI Department of Pathology, Hospital Ramos Mejia, Buenos Aires, Argentina Introduction ..................................................... Material and Methods ............................................ Types of Ossification ............................................. Histochemical Reactions .......................................... A. Glycogen ................................................... B. PAS-Positive Substances and Metachromatic Substances ...... C. Basophilia and Nucleic Acids ................................ D. Enzymes ................................................... V. Histochemistry of Bone Formation ................................ A. Cartilaginous Tissue ......................................... B. Bone Tissue ................................................ VI. Histochemistry of Bone Resorption ................................ VII. Histochemistry of Ossification in Endocrine Disturbances and Other Experimental Conditions ......................................... Acknowledgments ................................................ References ...................................................... I. 11. 111. IV.

Page 283 284 286 287 287 289 290 291 297 298 299 301

303 303 304

I. Introduction The study of the histochemical distribution of different substances, including a large group of enzymes, has been intensified during the last few years. Nevertheless, its application is still limited, and a large field remains open for future investigations. Numerous histochemical investigations have been carried out on bone tissue and more especially on bone formation. Histochemistry of bone and other calcified tissues was reviewed in 1955, by Sognnaes (1955) : in Bourne’s book (1956a) some problems directly and indirectly related to bone histochemistry were reviewed. Some papers by Italian authors have also appeared on this subject (Monesi, 1958). Since the time these papers were published, new contributions to histochemical methodology have been made, and it has been possible to apply many of them to the study of ossification with some interesting results. It is important to insist on a histochemical study of enzymes, which seem to play an important role in the mechanism of ossification and may eventually explain the process of calcium precipitation and ground substance formation. The presence of calcified areas in bone tissue has been a problem for the use of histological techniques, but in histochemistry this obstacle has proved to be more serious, and the results obtained should be very care283

284

ROMULO L. CABRINI

fully analyzed in accordance with the method being used. These technical problems are compensated for by the particular biology of bone tissue where resorption areas of only a few microns alternate with similar areas of bone formation. This increases the significance of histochemical data, making it an indispensable complement of general biochemical methods.

11. Material and Methods Most of the investigations done up to the present have been performed on mammals, only isolated data being available as to the histochemical behavior of bone tissue formation in other species. The process of ossification is usually quite different in these species and the results cannot be compared. Rats and mice are the mammals most widely used for these studies. There is also data available on other species, such as guinea pigs and man. As a general rule, small animals are preferred as their bones are softer, it being usually unnecessary to decalcify the material when preparing the sections. In our laboratory we prefer the use of small animals (rats and mice 1 to 7 days old), preparing transverse sections of the head, which has areas of direct calcification, and another section which includes femur and tibia, permitting us to observe the area of fast endochondral ossification as well as periosteal ossification. All this material can be easily sectioned after paraffin embedding ; frozen sections can also be prepared. When studying human material, areas of endochondral ossification of ribs and peripheral zones of the endochondral growth of the tibia are preferable. If this material is obtained from newborn human beings, it can be cut under the same conditions as the above, although with more difficulty. Almost all histochemical techniques have been developed for use on soft tissues, and two difficulties arise when calcified areas are present. (1) It is difficult and, in some cases, impossible to obtain acceptable sections for microscopic study. (2) Calcium ions are apt to interfere with the histochemical reactions and care should be taken when interpreting the results (see metachromasia, acid phosphatase) . As a general rule, uncalcified material should be used for histochemical techniques, at least for the first experiment. Later on, decalcified material can be used for evaluating the effect of decalcification on the technique used. Various types of acid solutions are used for the elimination of calcium from the tissues. The action of the acid is directed toward the calcium salts, which become soluble in different proportions according to the acid

HISTOCHEMISTRY OF OSSIFICATION

285

used, pH, concentration, etc. Strong mineral acids, such as hydrochloric acid .and nitric acid, are habitually used when routine stains are required. Nevertheless, the majority of specimens will show some change after decalcification, especially if this has been a long process (Lillie et al., 1951). For example, after 2 or 3 days of decalcification in 7% nitric acid, it is necessary to increase the time the material must remain in routine hematoxylin stain. Buffer solutions which keep the p H within certain limits and which can be controlled better are the best for histochemical investigations. The results obtained by the use of these different decalcifying solutions have been analyzed in detail (Schajowicz and Cabrini, 1955). The introduction of chelating agents capable of eliminating calcium salts at a nonacid p H was received with enthusiasm as a solution to the problem of decalcification in inicroscopic techniques (Dotti ef al.. 1951 ; Freinian, 1951 ; Hahn and Reygadas, 1951 ; Screebny and Nikiforuk, 1951; Birge and Imhoff, 1952; Hilleman and Lez, 1953). However, a careful analysis of the results, when compared with those obtained with buffer solution, showed no noticeable superiority ( Schajowicz and Cabrini, 1956a,b). The above observations for histochemical techniques in general are even more valid where the study of enzymes is concerned because there is always the danger of destruction or inactivation. There is no adequate solution for all enzymes known at the moment. As a rule, each enzyme requires a different method. The majority of enzymes become either totally or partially inactivated during decalcification, and this can be attributed to different factors ; pH, solubility in the decalcifying solution, loss of ions or cofactors, etc.; therefore, it is better to test the decalcification of each enzyme separately. With the use of buffer solutions, the material for alkaline phosphatase demonstration can be decalcified at a p H of not less than 5.2 (Lorch, 1946, 1947; Greep et al., 1947; Zorzolli, 1948). Once decalcification has been performed, it is convenient to activate the enzyme with a p H 9.2 veronal solution prior to its demonstration. Acid phosphatase, on the other hand, easily resists a lower pH, and complete decalcification can be performed with little enzymic loss even with relatively large specimens. These low p H solutions, around 4, are not only decalcifiers but also preserve the enzymic activity of the tissue for a reasonable period,-that is to say, they act as a preservative (Schajowicz and Cabrini, 1959). Other enzymes give a variable response. Succinic dehydrogenase, cytochrome oxidase, and phosphorylase are easily destroyed. 0-Glucuronidase

286

ROMULO L. CABRINI

can resist decalcification at a p H of approximately 5 , for short periods with very little loss. In some cases the presence of calcium salts can present a problem in the interpretation of a histochemical reaction. Calcium salts can mask the response of the rest of the components of the tissue, as in the case of metachromasia of calcified cartilage. Other reactions, especially of those enzymes which act at a low pH, provoke partial dissolution of the calcium salts. This is shown by areas of elevation of p H and a possible precipitation of substances playing a part in the reaction. These examples prove that though, in general, decalcification produces a noxious effect on histochemical techniques, this is not the rule for all reactions. 111. Types of Ossification

It is necessary to determine with a certain amount of accuracy the significance and scope given to different concepts of the mechanism of ossification ; this will facilitate the easy interpretation of histochemical data. Ossification means the formation of bone tissue, that is to say, a mucoprotein matrix which calcifies rapidly, leaving in the interior a characteristic living element, the osteocyte. From the beginning, the bone matrix takes on a fixed and set form. For the organism to modify this form, a complicated process is necessary, and in nearly all cases, this means simultaneous or successive destruction and neoformation. For this reason it is logical that the histochemistry of ossification include both the histochemistry of bone formation and destruction of the calcified mucoproteic matrix. It is necessary to take into consideration two types of ossification: direct or membranous and endochondral. In the first case (direct or membranous), the bone trabeculae are formed in the connective tissue itself; in the second case, there is a stage in which the cartilage participates. Although endochondral ossification is very important, for the long bones especially, there is very little definite bone tissue which can be attributed to this ; it will therefore be seen that endochondral bone has a special histochemistry. Different morphologic images are observed according to the speed of bone growth. In endochondral ossification the height of the growth cartilage is an indication of the speed of growth; the higher it is, the quicker is the growth. With regard to membranous ossification and the formation of subchondral bone trabeculae, the aspect of the osteoblasts and osteogenesis activity are linked together, the most active osteoblasts being those possessing abundant cytoplasm, giving an epithelioid disposition to the cellular

HI S T OC HE M I S T R Y O F OSSIFICATION

287

groups. There is a definite nuclear polarization in these elements; as their .activities diminish, they become less prominent and end by being flat elements with a fibroblastic aspect, indicating an absence of osteogenic activity. IV. Histochemical Reactions W e shall give here the results obtained by the histochemical application of different techniques, special emphasis being given to the distribution of each substance in the different cellular elements taking part in the process of osteogenesis, whether in the formation or destruction of bone trabeculae. W e shall also mention the histochemistry of cartilaginous tissue found in endochondral ossification areas. A.

GLYCOGEN

Old studies on the histochemical distribution of glycogen (Harris, 1932 ; Gendre, 1938; Parvisi, 1938; Glock, 1940) have lost a great deal of their importance owing to the development of new techniques of far greater constancy and sensitivity, such as the periodic acid-Schiff reaction ( P A S ) (McManus, 1946 ; Lillie and Greco, 1947 ; Hotchkiss, 1948). Despite this, great difficulties still exist in the fixation of glycogen, owing principally to its labile qualities. Serious studies require fixation by the Altman-Gersh method (Mancini, 1948), although satisfactory fixations can be obtained with saturated alcoholic solutions of picric acid (such as Rossman’s fixative solution), which at the same time permits weak decalcification (Schajowicz and Cabrini, 1956b, 1958a). Any attempt to effect even a slightly active decalcification causes the loss (in a few hours) of most of the glycogen, only small amounts remaining in the cartilaginous cells. Unfortunately, many investigations carried out with this substance have been performed with material decalcified in solutions which have caused its almost total disappearance. Recently, papers have been written on the distribution of glycogen, where fixation and staining have been effected under good conditions (Bevelander and Johnson, 1950a,b : Heller-Steinberg, 1951 ; Follis, 1952 ; Pritchard, 1952, 1956 ; Schajowicz and Cabrini, 195th). All these works show similar results, with some rare exceptions which we shall comment on later. Cartilage tissue is rich in glycogen. The chondroblastic cell which differentiates from the mesenchymal stroma has little glycogen but acquires an important quantity when the cartilaginous capsules and the mucoproteic matrix differentiate. The same can be said of chordal tissue, the cells of which are characterized by the large quantity of glycogen they contain

288

ROMULO L. CABRINI

(Malinsky, 1959). In growing cartilage the presence of a fair quantity of glycogen in proliferating cells is obsetved ; when these become hypertrophic, glycogen increases in relation to the increase of cytoplasmic volume and not to an increase in the concentration of substance per volume unit. When the matrix becomes calcified, glycogen decreases in the cartilaginous tissue and may even disappear completely ; this loss of glycogen is perfectly evident in cartilage of rapid growth but is not very noticeable in cartilage of slow growth. The presence of calcified sections of cartilage where there is no loss of glycogen is something that should be taken very seriously into account, especially when analyzing the theory that calcification is vinculated with the metabolism of glycogen (Gutman and Yu, 1950 ; Gutman, 1951) . The presence of large quantities of glycogen is observed in membranous ossification. Undifferentiated mesenchynial cells contain no glycogen, but this can be found in abundance in those areas where bone trabeculae will later form. It should be kept in mind that mesenchymal cells in chondroblastic evolution are nearly totally lacking in glycogen. Later on, when the osteoblasts become differentiated, the distribution of glycogen varies according to the speed of growth. In very active hypertrophic osteoblasts, glycogen is found in very large quantities in the mesenchymal cells near the osteoblasts (preosteoblasts) , but the osteoblastic cells themselves are practically devoid of this substance. In cases of less active ossification when quadrangular osteoblasts showing discrete basophilia appear, glycogen can be found in the osteoblasts themselves as well as in the preosteoblasts. When the process of ossification is even slower, glycogen appears only in the osteoblasts. Finally, in zones where there is complete inactivity, osteoblasts show only occasional granules of glycogen in isolated cells. When the osteoblasts are surrounded by ground substance and become transformed into osteocytes, they habitually lose their glycogen content, and only occasionally are some isolated granules found. Later on, the adult osteocyte again acquires glycogen in variable quantities ( Bevelander and Johnson, 195Oa,b). All the foregoing refers to direct ossification ; endochondral bone behaves in a very different manner. When ossification is very active, it is almost impossible to detect glycogen in either the active osteoblast cells or the neighboring cells. In zones where there is less activity, small quantities of glycogen begin to appear. Finally, in zones of very slow ossification, glycogen appears with similar characteristics to that of menibranous ossification.

HISTOCHEMISTRY OF OSSIFICATION

B.

289

PAS-POSITIVESUBSTANCES A N D METACHROMATIC SUBSTANCES

Under this heading we will consider as PAS-positive substances those which give a P A S reaction (periodic acid-Schiff reaction), with the exception of glycogen, which belongs to the group described above. It is possible to discard easily the presence of glycogen with the use of enzymic digestion with ptyalin or other amylolytic enzymes. It is a well-known fact that all cells give a PAS-positive reaction localized almost exclusively in the cytoplasm. This is also true of osteoblastic cells (osteoblasts and preosteoblasts) and of the chondroblast group (Bevelander and Johnson, 1950a,b ; Schajowicz and Cabrini, 1956b, 1958a). Some authors, however, have observed PAS-positive granules characteristic of osteoblasts (Heller-Steinberg, 1951 ; Jackson and Smith, 1955). Heller-Steinberg ( 1951) found abundant granulations in osteoblasts, osteocytes, and osteoclasts. In experimental hyperparathyroidism PAS-positive granules disappear from the osteoblasts and osteocytes when there is resorption of bone tissue but remain in the same proportion in the osteoclasts. When the hyperparathyroid lesions regress, the granulations appear in their usual patterns. Since these granulations are resistant to digestion by ptyalin and methanochlorhydric acid, they can be considered as mucoproteins. It is thought that these granulations are directly related to the formation of ground substance which is rich in mucoproteins. The osteoclasts, however, always show PAS-positive granulations distributed in the cytoplasm in an irregular manner. There is a great deal of variation in the amount of granules and it is possible to identify various types of osteoclasts according to the amount of PAS-positive granules contained, a fact which must be considered as corresponding to different functional stages (Morse and Greep, 1960). Osteoid bands, as a general rule, have a frank PAS-positive reaction, generally of the diffuse type. In those zones where calcification is taking place, the reaction is usually more intense, especially when a granulated form appears. Ground substance in growth cartilage is moderately positive, but this reaction grows in intensity when there is precipitation of calcium salts. The presence of acid mucopolysaccharides can be studied with the use of metachromatic techniques (Lison, 1933, 1935a,b ; Sylven, 1341, 1947a,b, 1948, 1954 ; Atkinson, 1952 ; Akamine et al., 1954 ; Lorber, 1955). Cartilaginous tissue gives an intense metacromasia, due especially to the chondroitin sulfuric acid. This disappears easily with hyaluronidase and can also be eliminated with various other solutions (Schajowicz and Cabrini, 1955).

290

ROMULO L. CABRINI

The behavior of the calcified zone of cartilage is still controversial. While some authors consider that in that area the metachromatic response is lost (Sylven, 1947a,b), others maintain that the loss of response is exclusively due to the calcium salts present. If the precaution is taken to do a prior decalcification, it is possible to observe that the metachromasia reappears (Rubin and Howard, 1950 ; Schajowicz and Cabrini, 1956b). In any case, the zone where calcium is precipitated can be considered rich in mucopolysaccharides, although we must acknowledge that these react in a different manner from the rest of the cartilaginous tissue. The method proposed by Hale (1946), based on the use of colloidal iron, gives similar images to those obtained by metachromasia. This reaction, which at the time was considered to be specific for mucopolysaccharides, has been partially abandoned in purely histochemical studies. The same can be said of the alcian blue method (Steedman, 1950), which gives images similar to those obtained with metachromatic stains and has no chemical basis to favor its use (Lison, 1954). In rats which had been kept on a diet deficient in calcium for a certain length of time, Urist and McLean (1957) observed large quantities of cells with a high proportion of metachromatic granules ; the appearance of these elements coincided with the disappearance of normal osteoblasts. The administration of large quantities of vitamin D caused these elements to disappear; on the other hand, the administration of parathormone caused no modification whatsoever. Urist and McLean identify these cells as mast cells and link metachromatic granulations to the bone ground substance. Similar cells were observed by Riley (1959) in hyperparathyroidism. C. BASOPHILIA AND NUCLEIC ACIDS Purely morphological studies showed that osteoblasts were cells rich in basophilic substance. Since Casperson (1941, 1950) and Brachet (1947) published the results of their studies on the cytology of nucleic acids, relatively reliable methods for its identification have been established, and hypotheses have been put forward as to its functional significance. Cytoplasm nucleic acid (ribonucleic) ( R N A ) has stirred up great interest in histochemical studies, especially as they would appear to be linked with the elaboration of proteic material (Caspersson, 1941 ; Brachet, 1947). Small quantities of R N A are localized in nearly all cartilaginous cells ; the quantity seems to increase in proliferation and hypertrophy but when reaching the calcification zone it tends to disappear

HISTOCHEMISTRY OF OSSIFICATION

29 1

(Schajowicz and Cabrini, 1956b). R N A is related in the osteoblast to its functional state. It increases progressively in the cytoplasm of the osteoblast from the moment of its differentiation to a maximum when the deposition of the bone mucoprotein takes place, and diminishes later when these cells become osteocytes (Cappellin, 1949). This would appear to indicate that the major concentration of R N A coincides with the most active formation of osteoid, which as is known, contains a large proportion of proteins. Good fixation material is necessary for the study of cytoplasmatic basophilia. For this, absolute alcohol ( Pritchard, 1952) and Rossman’s solution (Schajowicz and Cabrini, 1956b) can be used, although acceptable results are also obtained with 80% alcohol. A frank diminution of basophilia, and on occasions its complete disappearance, can be observed in scurvy where there are important changes in the osteocytes. Large quantities of RNA, which is localized in both the cytoplasm and nucleoli (Bhaskar et al., 1956), are found in osteoclasts. There is a diminution of R N A in the cytoplasm of osteoclasts in iu rats, the resorption mechanism of these animals being very much altered. These findings, however, have not been confirmed by other authors, as it would appear that the quantity of R N A is very variable, and only a careful study of R N A in groups of cells with different quantities of RNA will allow useful comparisons to be made (Morse and Greep, 1960). As is well known, Feulgen’s reaction permits the detection of deoxyribonucleic acid (DNA) in the nucleus of preosteoblasts, osteoblasts, osteoclasts, and cartilaginous cells. The distribution of DNA is similar to that of other cells in the organism (Pritchard, 1952; Follis, 1952; Schajowicz and Cabrini, 1956b). Nondecalcified material is required for a serious study of the contents of DNA, and this is specially true of RNA, as decalcification provokes hydrolysis of nucleic acids with relative ease (Schajowicz and Cabrini, 1955). With the use of Feulgen’s staining method, it has been possible to study the mitotic index of the elements which play a part in the process of ossification and in this way demonstrate that the osteoblasts offer a few mitotic figures (Pritchard, 1952). Using this same stain, it has been possible to make microspectrophotometric determinations of the DNA content of cartilaginous cells (Gerzelli and Bottino, 1957).

ENZYMES There have been rapid developments in the techniques for the demonstration of enzymes. However, we wish to stress the fact that although histoenzymic techniques have continued to progress and very accurate D.

292

ROMULO L. CABRINI

information has been obtained, it does not in general seem reasonable to insist, at this stage of fechnical development, on the cytomorphological details of enzyme distribution. It should also be remembered that while the present enzymic techniques show the amount of enzyme that remains in the sections, it is not yet possible to determine the amount of enzyme lost during the process of preparation, whether this be by inactivation or diffusion. In general, histoenzymological methods are only qualitative, with the exception of some attempts which have been made to measure alkaline phosphatase niicrospectrophotonietrically. The judicious use of different incubation times, concentration of substrates, or proportional inhibitions permits, however, the determination of the different enzymic contents of different cells with ease and certainty in many cases. These data are of great value and constitute an important addition to histoenzymological knowledge.

1. Alkaline Phosphatase Robison (1933) showed that there are high quantities of alkaline phosphatase present in bone tissue. Later, when an adequate technique for histochemical demonstration in histological sections was developed (Gomori, 1939 ; Takamatsu, 1939), the first description of this enzyme in bone tissue was made (Gomori, 1943), and large quantities of alkaline phosphatase in active ossification zones, localized specially in osteoblasts and neighboring cells, were found. Once methods had been developed to decalcify bone tissue without too much enzymic loss, it was possible to perform more complete analyses (Lorch, 1946; Greep et al., 1947, 1948; Zorzolli, 1948). Hyaline cartilage, both resting and in proliferation, has a very low enzymic activity ; this is observed also in perichondral zones. Hypertrophic cartilage acquires a great deal of enzymic activity in the hypertrophic chondrocytes, there being also an enzymic reaction in the neighboring ground substance. This more active zone corresponds to that where active precipitation of calcium salts occur. When the calcified hypertrophic cartilage zone is relatively extensive, as can be seen in endochondral growth of the long bones, a band appears in which necrobiosis starts in the cartilaginous cells ; this can also be seen with common staining reactions. This band has a marked loss of activity which shows up quite clearly when all the preparations are observed. Bone trabeculae of endochondral formation show great enzymic activity which is specially localized in osteoblasts and their neighboring cells. Although the observation of cellular details is difficult owing to the great enzymic response, the im-

HISTOCHEMISTRY OF OSSIFICATION

293

pression is that there is little enzymic activity in the chondroclasts and cartilaginous resorption zones, or at any rate, not so much as in the sections in apposition. Most of the investigations carried out up to the present agree, in general, with the above description (Kabat and Furth, 1941; Horowitz, 1942; Gomori, 1943; Bourne, 1948; Lorch, 1946, 1947; Morse and Greep, 1947, 1948 ; Greep et al., 1948 ; Cappellin, 1948 ; Follis, 1949a,b, 1950a,b, 1952 ; Bevelander and Johnson, 1950a ; Heller-Steinberg, 1951 ; Majno and Rouiller, 1951 ; Sibert, 1951 ; Pritchard, 1952; Borghese, 1952; Cessi, 1954). In zones of direct ossification it can be observed how the bone adage is preceded by a zone where the mesenchymal cells acquire large quantities of alkaline phosphatase. When the trabeculae start to form, it can be seen clearly that the osteoblasts have a high alkaline phosphatase content. Osteoclasts show less activity as compared to that of osteoblasts. Some authors (Bevelander and Johnson, 1950a ; Pritchard, 1952) have observed a drop in the enzymic content in osteoblasts when these acquire a hypertrophic type. Young osteocytes have a small enzymic load which is lost almost entirely as they become older. Images of enzyme-carrying bone canalicules (Majno and Rouiller, 1951) are probably due to technical artifacts. It has also been thought that when osteocytes die (onkosis), they again acquire enzymic activity; in this case, they would behave in a manner similar to other cells such as leucocytes (Majno and Rouiller, 1951). These same authors observed a high enzymic content in osteoclasts. Osteoblasts in tissue culture seem to maintain their enzymic activity for a certain time (Henrichsen, 1956). Studies by Lorch (1947) on cartilaginous fish show alkaline phosphatase in zones where chondrocytes start calcification. High quantities of alkaline phosphatase have been found in the shells of mollusks and in the formation of the calcic shell of hens’ eggs. An intense phosphatasic activity appears during bone repair (Bourne, 1943a,b, 1948; Majno and Rouiller, 1951 ; Pritchard, 1952; Rosin, 1952; Utsunomiya and Matsuda, 1953 ; Schajowicz and Cabrini, 1954b). I n our laboratory (in collaboration with Dr. Schajowicz) we have worked on fractures and loss of substance in rats and also on experimental fractures in toads. Our findings agree with the above data, although we recognize the fact that the material used for studies of alkaline phosphatase during consolidation of fractures is not ideal and should only be used as complementary data. In these cases, apart from the bone repair process, there are other changes which complicate the analysis of the histologic picture. Fractures in toads cure in a different way than do fractures in mammals, consolidation being effected by the formation of a large carti-

294

ROMULO L. CABRINI

laginous callus and metaplasia of cartilage into bone. There is not much alkaline phosphatase activity in these areas ; on the other hand, there are large quantities of acid phosphatase. Adult bone shows a variable amount of enzyme on the endostial and periostial surfaces; in some bones, such as the jaws, where structural changes are frequent, the relation between the surfaces in apposition (which are rich in osteoblasts) and the appreciable quantities of alkaline phosphatase can be seen quite clearly (Carranza and Cabrini, 1955; Baratieri, 1955).

2. Acid Phosphatase In spite of the fact that the techniques to demonstrate acid phosphatase and alkaline phosphatase were developed almost simultaneously (Gomori, 1941), technical difficulties have limited the use of the former. Majno and Rouiller (1951), state that with the use of Gomori’s original techniques, the results of studies on acid phosphatase in bone tissue have been unsatisfactory. Schajowicz and Cabrini ( 1957, 1958b,c) and Burstone (1958a, 1959b) verified almost simultaneously the presence of large quantities of acid phosphatase in bone tissue. Burstone (1959b) used material fixed by the freeze-drying method, and demonstrated the enzymes by a method developed by him (Burstone, 1958b). O n the other hand, Schajowicz and Cabrini (1959) insist on the need of previous decalcification of the material and demonstrate the enzyme with Gomori’s technique ; with this procedure, the formation during incubation of areas in which the p H is increased owing to the dissolution of calcium salts is avoided, preventing acid phosphatase from acting at an inadequate pH. Although it would appear that the use of Burstone’s technique makes previous decalcification unnecessary, it is undoubtedly true that this procedure produces only a small enzymic loss and so permits the study of this enzyme in well-calcified bone tissue (Schajowicz and Cabrini, 1959). In endochondral ossification, large quantities of acid phosphatase are observed in zones of bone and cartilaginous resorption. Acid phosphatase is localized on the surface of cartilaginous erosion in chondroclasts and very probably in areas of vessels in active erosion, but in the bone the major activity corresponds to osteoclasts. When this distribution is present (chondroclasts, vessels in erosion, and osteoclasts) and when endochondral growth is very active and the zone of ossification very high, the sections, if seen with topographic magnification, show two bands with maximum enzymic reaction. These bands correspond successively to the cartilaginous resorption layer and the metaphyseal trabeculae erosion layer. If the incubation time is lengthened, a positive reaction of osteo-

HISTOCHEMISTRY OF OSSIFICATION

295

blasts and of all the neighboring mesenchymal cells can be seen, as this permits the detection of less active cells ; under similar conditions, all the cells which form the perichondrium can be seen. A more prolonged incubation shows a slight response of the cartilaginous cells, especially in the layer where these elements are in proliferation, but on the other hand when “hypertrophic” cartilage becomes calcified, there would appear to be a marked decrease in enzymic activity. The behavior is similar in direct ossification, and here again osteoclasts have a maximum enzyme content. Recently, Burstone (1959a) has observed enzymic activity in newly formed bone and cartilaginous ground substance.

3. $-Glucuronidase In the last few years, techniques have been developed which permit histochemical demonstration of 0-glucuronidase in various tissues ; these techniques are based on the hydrolysis of some compound of glucuronic acid which produces a precipitable stain (Burton and Pearse, 1952; Seligman et al., 1954 ; Fishman and Baker, 1956 ; Pearse and Macpherson, 1958). The enzyme can be easily detected in bone tissue in formation (De Bernard et al., 1955; Monesi, 1958; Monesi and Bettini, 1958; Cabrini and Schajowicz, 1958). Material should be fixed in formalin or formalin chloral hydrate ( Fishman and Baker, 1956) ; other fixations and also sections not fixed, when obtained with the cryostat system, have an incomplete or irregular reaction. In general, decalcification leads to enzymic loss which is in proportion to the time and p H used. A decalcification lasting 2 or 3 days with a p H 5 can be carried out without excessive loss of enzyme. The presence of calcium salts does not appear to spoil the reaction, because P-glucuronidase acts on a wide p H range, and therefore the elimination of calcium, prior to decalcification, does not appear to be necessary, as is the case with acid phosphatase. Short incubation periods with Fishman’s method easily show large quantities of P-glucuronidase in the cytoplasm of active osteoblast cells, and there is also a marked response of the osteoclasts. With longer incubation there is response in other cellular elements ; cartilaginous tissue reaction is always very weak (Cabrini and Schajowicz, 1958). However, Monesi ( 1958) finds zones of great activity in the hypertrophic cartilaginous layer and in proliferation. Membranous and endochondral ossification behave in a similar manner. The chondroclasts also react intensely in endochondral ossification, but the behavior of the vessels in erosion is not so clear.

296

ROMULO L. CABRINI

4. Succinic Dehydroyenase During these last few -years, techniques sufficiently precise to enable the demonstration of succinic dehydrogenase have been developed, based on the reduction of tetrazolium salts for the formation of crystallized and stained formazanes. Tetrazolium chloride and tetrazolium blue were the first substances to be used but were not very sensitive, and very thick sections were necessary. However, modifications now exist such as the introduction of cyanide, which blocks cytochrome oxidase and traps oxalacetic acid (Rosa and Velardo, 1954), or the use of other salts, nitro blue tetrazolium chloride (nitro-BT) (Nachlas et d.,1957) and 2-p-iodophenyl-3nitrophenyl-5-phenyl ( I N T ) (Pearson and Defendi, 1954) : all these methods are of greater sensitivity. Follis and Melanotte (1956) studied the behavior of dehydrogenase in cartilage and found enzymic activity in the chondrocytes. With the use of nondecalcified sections, especially those prepared with the cryostat or similar methods, it has been possible to study the distribution of this enzyme in zones of ossification. An enzymic reaction of great intensity is observed in osteoclasts and chondroclasts ( Schajowicz and Cabrini, 1960) ; the enzyme being found to react exclusively in the cytoplasm. When longer incubations are carried out, especially when using nitroBT and I N T , there is a weak response from osteoblasts and cartilaginous cells. In any case, the reaction is very much weaker than that obtained in giant cells. Tonna (1958a,b) has described an enzymic reaction of the periostium, though he does not give details of the reacting cells. The technique for histochemical demonstration of succinic dehydrogenase is, in general, easy to use, but the enzyme is very labile and is quickly lost, rendering experimental study difficult. At the moment, there is no method which permits previous decalcification of the material.

5. Cytochrome Oxidase Techniques for the demonstration of cytochrome oxidase have existed for many years (G-Nadi reaction) but, unfortunately, they lack the sensitivity and accuracy necessary for making valuable histochemical studies. Tonna ( 1958a,b), using the classical G-Nadi reaction, described enzymic activity in the periostium, but without being able to determine with any accuracy the type of cell which reacts. Using the same reaction, Follis (1952) believes he saw a positive reaction of the osteoblasts. The introduction of new substrates (Burstone, 1960) has permitted

HISTOCHEMISTRY OF OSSIFICATION

297

better studies on the behavior of cytochrome oxidase in bone tissue. With the use of these new techniques, an intense activity centered on the osteoclasts is observed, i.e., it coincides with the zones of greater activity of succinic dehydrogenase (Burstone, 196Oc). 6. Pkosphorylase It has been possible to detect phosphorylase with a procedure based on the formation of polysaccharides, starting with glucose-l-phosphate. These polysaccharides must be histochemically stained later by different procedures (Cobb, 1953 ; Takeuchi, 1956). Although good results are obtained with the use of material showing a high enzyme content, certain defects are still to be found in these techniques, as, for example, the solubility of the formed polysaccharide and the difficulty of differentiating it from that which already exists. Histocheinical study of this enzyme in ossification zones has shown areas of localized activity, especially in the cartilage, in hypertrophic chondrocytes in the process of ossification and also in osteoblasts (Cobb, 1953).

7. Estcrascs M'ith the use of new substrates, Burstone (1957) detected esterases in bone tissue. These techniques respond to specific inhibitions. Unlike other enzymes, esterases appear in the bone with an exclusively extracellular localization, the largest quantities being found in cartilaginous zones in a state of calcification and also in the recently formed bone matrix. This enzyme has not been detected in osteoblastic and osteoclastic cells. It is apparent that this enzyme settles in those sectors where the mucoproteic matrix starts calcification.

V. Histochemistry of Bone Formation The correlation of all these histochemical findings may permit the understanding of functional processes. It must be pointed out that although histochemical results are reliable with reference to the existence or nonexistence of a given substance, and within certain limits even the proportions in which it is present, it cannot be asserted that all the substance has been detected, as in many cases a large quantity is lost during histochemical processing. This fact is especially important in the study of enzymes, where in many cases one part is closely related to the cell (desmoenzyme) while another part is soluble in the media, this latter portion being quite important in some cases (Hannibal and Nachlas, 1959).

298

ROMULO L. CABRINI

A. CARTILAGINOUS TISSUE Few substances appeaf in appreciable ' quantities in the cells during cartilage formation. Glycogen appears in moderate quantities and is also to be found in resting zones. The zone of cartilaginous proliferation seems to have a slight increase in acid phosphatase, succinic dehydrogenase, and $-glucuronidase content.

-

-----A@-

p.0.

m@& \+

*-

/--

Basophilio

Glycogen

DNA

>--\/

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

HISTOCHEMISTRY O F OSSIFICATION

299

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.

304

ROMULO L. CABRINI

REFERENCES Akamine, R. N., Engel, M. B , and Sarnat, B. G. (1954) J. Bone and Joint Swg. %A, 1166. Albaum, H. G., Hirschfeld, A., and Sobel, A. E. (1952) Proc. SOC.Exptl. Biol. Med. 79, 682. Atkinson, W. B. (1952) Science 116, 303. Baratieri, A. (1955) Rev. ital. stomatol. 10, 3. Bevelander, G., and Johnson, P. L. (1950a) Anat. Record 108, 1. Bevelander, G., and Johnson, P. L. (1950b) Trans. 3rd Josiah Mary. Jr. Conf. Metabolic Interrelations 3, 25. Bhaskar, S. N., Mohammed, C. I., and Weinmann, J. P. (1956) J . Bonr and Joint Surg. S A , 1335. Birge, E. A., and Imhoff, C. E. (1952) Am. J. Clin. Pathol. 22, 192. Borghese, E. (1952) Boll. soc. ital. biol. sper. 28, 801. Bourne, G. H. (1942) J . Physiol. (London) 101, 327. Bourne, G. H. (1943a) J . Physiol. (London) 102, 319. Bourne, G. H. (1943b) Quart. J. Exptl. Physiol. 32, 1. Bourne, G. H. (1948) J . Anat. 82, 81. Bourne, G. H., ed. (1956a) “Biochemistry and Physiology of Bone.” Academic Press, New York. Bourne, G. H., ed. (1956b) I n “Biochemistry and Physiology of Bone,” p. 251. Academic Press, New York. Brachet, J. (1947) “Embryologie chimique.” Masson, Paris. Burstone, M. S. (1957) A.M.A. Arch. Pathol. 63, 164. Burstone, M. S. (1958a) J. Drntal Research 37, 5. Burstone, M. S. (1958b) J . Natl. Cancrr Inst. 21, 523. Burstone, M. S. (1959a) J . Historhein. and Cytochem. 7, 147. Burstone, M. S. (1959b) J . Histochem. and Cytochem. 7, 39. Burstone, M. S. (1960a) J . Historhem. and Cytochem. 8, 63. Burstone, M. S. (1960b) J. Histochem. and Cytochem. 8, 112. Burstone, M. S. ( 1 W c ) J . Histochrin. and Cytochrm. 8, 225. Burton, J. F., and Pearse, A. G. E. (1952) Brit. J . Exptl. Pathol. 33, 87. Cabrini, R. L., and Schajowicz, F. (1958) 21st Intern. Physiol. Coirgr. Birrnos Aires (Abstr. and Commzm.) p. 49. Cappellin, M. (1948) Rass. biol. umuna 3, 35. Cappellin, M. (1949) Chir. org. movbnento 33, 410. Carranza, F. A., Jr., and Cabrini, R. L. (1955) Rev. odontol. Buenos Aires 43, 206. Caspersson, T. (1941 ) Natiirzisscnschaften 29, 33. Caspersson, T. (1950) “Cell Growth and Cell Function.” Norton, New York. Cessi, C. (1954) Btill. soc. atal. biol. sper. 30, 629. Cobb, J. D. (1953) A.M.A. Arch. Pafhol. 66, 496. De Bernard, G. L., Lorenzi, C. L., and Castellani, A. (1955) Bull. soc. ital. biol. sper. 31, 1303. Dotti, I. B., Papero, G. P., and Clarke, B. (1951) Am. J. Clin. Pathol. 21, 475. Fishman, W. H., and Baker, J. R. (1956) J. Histochem. and Cytochem. 4, 570, Fishman, W. H.,-Anlyan, A. J., and Gordon, E. (1947) Cancrr Research 7, 808. Follis, R. H., Jr. (1949a) Bull. Johns Hopkins Hosp. 85, 360. Follis, R. H., Jr. (194913) Federation Proc. 8, 355. Follis, R. H., Jr. (1950a) Federation Proc. 9, 330. Follis, R. H., Jr. (1950b) Bd1. Johns Hopkins Hosp. 87, 181.

HISTOCHEMISTRY OF OSSIFICATION

305

Follis, R. H., Jr. (1952) Trans. 4th Josiah Macy, Jr. Conf. Metabolic Interrelations 4, 11. Follis, R. H., Jr., and Melanotte, P. L. (1956) Proc. SOC.Exptl. Biol. Med. 93, 382. Freiman, D. G. (1951) Am. J . Clin. Pathol. 24, 227. Gendre, H. (1938) Bull. histol. appl. et tech. microscop. 37, 502. Gerzelli, G., and Bottino, D. (1957) Acta Histochem. 3, 243. Glock, G. E. (1940) J . Physiol. (London) 98, 1. Gomori, G. (1939) Proc. SOC.Exptl. Biol. Med. 42, 23. Gomori, G. (1941) A.M.A. Arch. Pathol. !J2, 189. Gomori, G. (1943) Am. J. Pathol. 19, 197. Gomori, G. (1950) Stain Technol. 26, 81. Greep, R. O., Fischer, G. J., and Morse, A. (1947) Science 106, 666. Greep, R. O., Fischer, G. J., and Morse, A. (1948) J. Am. Dental Assoc. 36, 427. Gutman, A. B. (1951) Bull. Hosp. Joint Disrnses 12, 78. Gutman, A. B., and Yii,T. F. (1950) Trans. 2nd Josiah Macy, Jr. Conf. Metabolic Interrelotions 2, 167. Hahn, F. L., and Reygadas, F. (1951) Science 114, 462. Hale, C. W. (1946) Nature 107, 802. Ham, A. W. (1953) “Histology.” Lippincott, Philadelphia, Pennsylvania. Hannibal, M. J., and Nachlas, M. M. (1959) J . Biophys. Biochem. Cytol. 6, 279. Harris, S. H. A. (1932) Nature 130, 996. Heller-Steinberg, M. (1951) Am. J . Anat. 89, 347. Henrichsen, E. (1956) Exptl. Crll Research 11, 115. Hilleman, H. H., and Lez, C. H. (19533 Staiit Techizol. 28, 285. Hirschman, A. (1954) Aimt. Record 118, 312. Horowitz, N. H. (1942) J . Dent. Research 21, 519. Hotchkiss, R. D. (1948) Arch. Riochciw. 16, 131. Ichikawa, M. (1954) Osaka Daigokii Igaku Znssi 6, 243. Jackson, S. F., and Smith, R. H. (1955) Symposia SOC.Exptl. Biol. 9, 89. Kabat, E. A., and Furth, J. (1941) Am. J . Pathol. 17, 303. Lillie, R. D., and Greco, J. (1947) Stain Technol. 22, 67. Lillie, R. D., Laskey, A., Greco, J., Burtner, J., and Jones, P. (1951) Am. J . Cliiz. Pathol. 21, 711. Lison, L. (1933) Acad. roy. Belg. classe sci. dlriir. 19, 1332. Lison, L. (1935a) Arch. biol. (Lidge) 46, 599. Lison, L. (1935b) Compt. rend. so(. biol. 118, 82. Lison, L. (1954) Stain Techizol. 29, 131. Lorber, M. (1955) Anat. Record 11, 129. Lorch, I. J. (1946) Nature 158, 269. Lorch, 1. J. (1947) Quart. J . Microscop. Sci. 88,326. McManus, J. F. A. (1946) Natrcrc 168, 202. Majno, G., and Rouiller, C. (1951) Arch. pathol. Anat. t i . Physiol. Virchozc’s 321, 1. Malinsky, J. (1959) dcta Histochem. 7, 107. Mancini, R. E. (1948) Anat. Rernrd 107, 149. Monesi, B. (1958) 42nd Congr. Ital. Ort. e Traiiin, p. 219. Monesi, B., and Bettini, G. (1958) Arch. Pzitti 10, 326. Morse, A.,and Greep, R. 0. (1947) ‘4nnt. Record 99, 379. Morse, A., and Greep, R. 0. (1948) J . Dcntal Research 27, 725. Morse, A., and Greep, R. 0. (1949) Anat. Record 103, 77.

306

ROMULO L. CABRINI

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

Page 307 308 310 314 320 322 322 324 329 333 333

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.

307

308

C. M. POMERAT

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.

CINEMATOGRAPHY, INDISPENSABLE TOOL FOR CYTOLOGY

309

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.

310

C. M . POMERAT

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.

C I N E M A T C G R A P H Y , I N D I S P E N S A B L E TOOL FOR CYTOLOGY

31 I

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.)

314

C. M . POMERAT

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.

CIXEAIATCGRAPHY, IKDISPENSABLE TOOL FOR CYTOLOGY

315

316

C. M. POMERAT

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.

CINEMATOGRAPHY, INDISPENSABLE TOOL FOR CYTOLOGY

317

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.)

318

C. M . POMERAT

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.

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

319

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.

320

C. M . POMERAT

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

CINEMATOGRAPHY, INDISPENSABLE TOOL FOR CYTOLOGY

321

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.

322

C. M. POMERAT

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.)

CINEMATOGRAPHY, INDISPENSABLE TOOL FOR CYTOLOGY

323

324

C.

M . POMERAT

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,

CINEMATOGRAPHY, INDISPENSABLE TOOL FOR CYTOLOGY

325

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.

326

C. M . POMERAT

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.

CIKEMATOGRAPHY, INDISPENSABLE TOOL FOR CYTOLOGY

327

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.)

328

C. M. POMERAT

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.

CINEMATOGRAPHY, INDISPENSABLE

TOOI, FOR CYTOLOGY

329

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