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REVIEW OF CYTOLOGY VOLUME10
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INTERNATIONAL
REVIEW OF CYTOLOGY VOLUME10
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 10
Prepared Under the Auspices of The International Society for Cell Biology
ACADEMIC PRESS, New York and London 1960
Copyright 0, 1960,
by
ACADEMIC PRESS INC. All Rights Reserved NO PART OF THIS BOOK MAY BE REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.
ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK3, N. Y . United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 17 OLDQUEEN STREET, LONDON S.W. 1
Library of Cortgress Catalog Card Nurnbcr 52-5203
PRINTED I N THE UNITED STATES OF AMERICA
Contributors to Volume 10 F. DOLJANSKI, Departwent of Experimental Medicine and Cancer Research, Hebrew University, Jerusalem, Israel FREDERICK H. KASTEN,Department of Biology, Agricultural and Mechanical College of Texas, College Station, Texas
A. GEDEON MATOLTSY, Department of Dermatology, University of Miami, School of Medicine, Miami, Florida YOSHIMINAGATANI, Biological Institute, Faculty of Literature and Science, Yamaguchi University, Yamaguchi, Japan ARCHANASHARMA,Cytogenetics Laboratory, Department of Botany, Calcutta University, Calcutta, Indict ARUNKUMARSHARMA, Cytogenetics Laboratory, Department of Botany, Calcutta University, Calcutta, India SAULWISCHNITZER, Department of Anatomy, N e w York Medical College, Flower and Fifth Avenue Hospitals, N e w York, N e w York LEWISWOLPERT, Zoology Department, Kings College, London, England
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CONTENTS
...............................................
v
CONTENTSOF PREVIOUS VOLUMES..........................................
xi
TO VOLUME 10 CONTRIBUTORS
The Chemistry of Schiff’s Reagent
FREDERICK H . KASTEX
. . . .
I I1 I11 IV V. VI VII VIII IX . X XI XI1
.
. .
. . .
Introduction ...................................................... Historical Background ............................................ Methods of Preparing Reagent .................................... Factors Affecting Sensitivity of Reagent .......................... Specificity of Reagent ............................................ Chemical Nature of Schiff’s Reagent .............................. Reaction with Aldehydes .......................................... Development of Schiff -Type Reagents ............................. Kinetics of the Schiff-Aldehyde Reaction .......................... Applications of the Reagent to Cytochemistry ...................... Absorption Curve Analyses of Schiff-Polyaldehyde Binding in Situ Conclusions ...................................................... Addendum ....................................................... References .......................................................
1 2
4 11 22
34 37
46
..
56 65 81 89 91 93
Spontaneous and Chemically Induced Chromosome Breaks ARUNKUMARSHARMA A N D ARCHANA
.
I I1. I11 IV V VI VII . VIII IX X.
. . . .
. .
SHARMA
Introduction ...................................................... Spontaneous Breakage ............................................ Technical Limitations in the Study of Chromosome Breaks ........ Action of Alkaloids. Pigments. and Coumarin Derivatives Action of Vegetable Oils. Fats. and Essences ...................... Action of Drugs and Bacterial Products .......................... Action of Hormones and Other Growth-Promoting Substances Action of Mustards. Related Compounds. and Phenols .............. Other Compounds ................................................ Conclusion ....................................................... References .......................................................
.......... ....
101 102 107 110 112 112 113 116 119 122 123
T h e Ultrastructure of the Nucleus and Nucleocytoplaemic Relations
SAUL WISCHNITZER I. I1 I11 IV . V. VI VII VIII.
. .
. .
Introduction ...................................................... Nomenclature .................................................... Nuclear Envelope ................................................ Nucleolus ........................................................ Chromosomes .................................................... Nucleoplasm ...................................................... Discussion ....................................................... Conclusion ........................................................ References ........................................................ Addendum .......................................................
137 138 139 142 143 148 148 158 158 162
The Mechanics and Mechanism of Cleavage
LEWISWOLPERT
. . . . . . .
Introduction ...................................................... Geometry of Cleavage ............................................ Theories of Cleavage ............................................ Mechanical Properties of the Cell Surface ........................ Discussion of Surface Force Theories ............................ Astral Relaxation Theory ........................................ Application of Astral Relaxation Theory to Other Cells and Forms of Cleavage .................................................... VIII. Biochemistry of Cleavage .......................................... I X. Summary and Conclusions ........................................ References ....................................................... I I1 I11 IV V VI VII
164 165 169 175 183 186 195 205 211 213
The Growth of the Liver with Special Reference to Mammals
F. DOLJANSKI I. Introduction ...................................................... I1. Development of the Liver during Embryonic and Postnatal Growth of the Organ ...................................................... I11. Liver Growth Response to Various Conditions in the Body .......... I V Liver Regeneration after Partial Hepatectomy ...................... V Concluding Remarks and Summary ................................ References ........................................................
. .
217 218 234 236 236 239
Cytological Studies on the Affinity of the Carcinogenic Azo Dyes for Cytoplasmic Componentn YOSHIMINAGATANI
.
Introduction ...................................................... Methods for Demonstrating the Cellular Affinity of Carcinogenic Azo Dyes for Cytoplasmic Components .......................... I11 Affinity of the Carcinogens for Cytoplasmic Components of Amphibian Erythrocytes .................................................... IV Mitochondria of Mammalian Differentiated Erythrocytes .......... V Affinity of Carcinogens for Amphibian Somatic Cells ............... V I Conclusion ........................................................ References ........................................................
I I1
243
. .
. . .
244 246 255 257 305 306
Epidermal Cells in Culture A . GEDEON MATOLTSY
I. I1. I11. I V. V.
Introduction ...................................................... Properties of Epidermal Cells hi V i m ............................ Properties of Embryonic Epidermal Cells in Vitro .................. Properties of Postnatal Epidermal Cells in Vitro .................. Remarks on Keratinization ........................................ References ........................................................
315 317 323 331
344 348
AUTHORINDEX...........................................................
353
SUBJECT INDEX..........................................................
368
..................................
377
CUMULATIVE SUBJECT INDEX. VOLUMES 1-9
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Contents of Previous Volumes Aspects of Bacteria as Cells and as Volume 1 Organisms - STUART MUDDAND Some Historical Features in Cell EDWARD D. DELAMATER Biology -ARTHURHUGHES Nuclear Reproduction- C. LEONARD Ion Secretion in Plants- J. F. SUTCLIFFE Hus~ms Enzymic Capacities and Their Rela- Multienzyme Sequences in Soluble Extracts -HENRYR. MAHLER tion to Cell Nutrition in Animals The Nature and Specificity of the GEORGEW. KIDDER Feulgen Nucleal Reaction--. A. The Application of Freezing and DryLESSLER ing Techniques in Cytology-L. G. Quantitative Histochemistry of PhosE. BELL phatases -WILLIAML. DOYLE Enzymatic Processes in Cell Membrane Penetration -TH. ROSENBERCAlkaline Phosphataee of the Nucleus M. CH~VREMONT A N D H. FIRKET A K D W. WILBRANDT Gustatory and Olfactory Epithelia Bacterial Cytology - K. A. BISSET A. F. BARADI AND G. H. BOURNE Protoplast Surface Enzymes and Absorption of Sugar-R. BROWN Growth and Differentiation of ExReproduction of Bacteriophage -A. planted T i m e s - P. J. GAILLARD D. HERSHEY Electron Microscopy of Tissue SecThe Folding and Unfolding of Protions-A. J. DALTON tein Molecules as a Basis of Os- A Redox Pump for the Biological motic Work-R. J. GOLDACRE Performance of Osmotic Work, and Nucleo-Cytoplasmic Relations in AmIts Relation to the Kinetics of Free phibian Development -G. FAKK- Ion Diffusion Across Membranes HAUSER E. J. CONWAY Structural Agents in Mitosis--. M. A Critical Survey of Current ApSWANK proaches in Quantitative Histo- and Factors Which Control the Staining Cytochemistry DAVIDGLICK of Tissue Sections with Acid and Nucleo-Cytoplasmic Relationships in Basic Dyes -MARCUSSINGER the Development of Acetabularia. The Behavior of Spermatozoa in the Report of the Conference of Tiwue Neighborhood of Eggs -LORD Culture Workers Held at CoopersROTHSCHILD town, New York- J. HXMMERLING The Cytology of Mammalian Epider- AUTHOR INDEX-SUBJECT INDEX. mis and Sebaceous Glands -WILLIAM MONTAGNA Volume 3 The Electron-Microscopic InvestigaThe Nutrition of Animal Celh tion of Tissue Sections-L. H. CHARITYWAYMOUTH BRETSCHNEIDER Caryometric Studies of Tissue CulThe Histochemistry of Esterases - G. t u r e ~ OTTU BUCHER GOMORI The Properties of Urethan ConaidAUTHOR INDEX-SUBJECT INDEX. ered in Relation to Its Action on Volume 2 Mitosis -IVOR CORNMAN Quantitative Aspects of Nuclear Nu- Composition and Structure of Giant Chromosomes -MAX ALFERT cleoproteins - HEWSONSWIFT Ascorbic Acid and Its Intracellular How Many Chromosomes in MamLocalization, with Special Refermalian Somatic Cells?-R. A. ence to Plants- J. CHAYEN BEATTY
-
-
xi
xii
CONTENTS O F PREVIOUS V O L U M E S
The Significance of Enzyme Studies Volume 5 on Isolated Cell Nuclei - ALEXAN- Histochemistry with Labeled AntiDER L. DOUNCE ALBERT H. COONS The Use of Differential Centrifuga- The Chemical Composition of the tion in the Study of Tissue EnBacterial Cell Wall-C. S. CUMzymes-CHR. DE DUVEAND J. MINS BERTHET Theories of Enzyme Adaptation in Enzymatic Aspects of Embryonic Microorganisms -J. MANDELSTAM Differentiation -TRYGGVE The Cytochondria of Cardiac and GUSTAFSON Skeletal Muscle- JOHN W. HARAzo Dye Methods in Enzyme HistoMAN chemistry -A. G. EVERSON PEARSEThe Mitochondria of the Neuron Microscopic Studies in Living MamWARREN ANDREW mals with Transparent Chamber The Results of Cytophotometry in Methods -ROY G. WILLIAMS the Study of the Deoxyribonucleic The Mast Cell - G. ASBOE-HANSEN Acid (DNA) Content of the NuElastic Tissue -EDWARD W. DEMPSEY cleus--. VENDRELY AND C. VENAND ALBERTI. LANSING DRELY The Composition of the Nerve Cell Protoplaamic Contractility in Relation Studied with New Methods- SVEN- to Gel Structure: TemperatureOLOFBRATKARDAND HOLCER HYDEN Pressure Experiments on CytokineAUTHOR INDEX-SUBJECT INDEX. ds and Amoeboid Movement DOUGLAS MARSLAND Volume 4 Intracellular pH - PETER C. CALDWELL Cytochemical Micrurgy - M. J. KOPAC The Activity of Enzymes in MetaboAmoebocytea -L. E. WAGGE lism and Transport in the Red Cell Problems of Fixation in Cytology, T. A. J. PRANKERD Histology, and Histochemistry Uptake and Transfer of MacromoleM. WOLMAN cules by Cells with Special ReferBacterial Cytology -ALFRED MARence to Growth and Development SHAK A. M. SCHECATMAN Histochemistry of Bacteria -R. Cell Secretion; A Study of Pancreas VENDRELY and Salivary Glands - L. C. U. Recent Studies on Plant MitochonJUNQUEIRA AND G. C. HIRSCA dtia-DAVID P. HACKETT The Acrosome Reaction- JEAN C. The Structure of Chloroplasts -K. DAN MUHLETH ALER Cytology of Spermatogenesis Histochemistry of Nucleic Acids -N. VISHWA NATH B. KURNICK The Ultrastructure of Cells as ReStructure and Chemistry of Nucleoli vealed by the Electron Microscope W. S. VINCENT FRITIOFS. SJOSTRAND O n Goblet Cells, Especially of the Intestine of Some Mammalian Species AUTHOR INDEX-SUBJECT INDEX. HARALD MOE Volume 6 Localization of Cholinesterases at The Antigen System of Paramecium Neuromuscular Junctions -R. aurelia-G. H. BEALE COUTEAUX Evidence f o r a Redox Pump in the The Chromosome Cytology of the Ascites Tumors of Rats, with SpeJ. Active Transport of Cations--. cial Reference to the Concept of the CONWAY Stemline Cell - SAJIRO MAKINO AUTHOR INDEX-SUBJECT INDEX.
CONTENTS OF PREVIOUS VOLUMES
...
s111
The Structure of the Golgi Apparatus Hypothalamo-neurohypophysial NeuARTHURW. POLLISTER AND PRISCILLA rosecretion- J. C. SLOPER F. POLLISTER Cell Contact - PAULWEISS An Analysis of the Process of Fertili- The Ergastoplasm: Its History, U1zation and Activation of the Egg trastructure, and Biochemistry A. MONROY FRAN~OISE HACUENAU The Role of the Electron Microscope Anatomy of Kidney Tubules in Virus Research-ROBLEY C. JOHANNES RHODIN WILLIAMS Structure and Innervation of the InThe Histochemistry of Polysacchaner Ear Sensory Epithelia - HANS rides - ARTHURJ. HALE ENCSTROM AND JAN WERSALL The Dynamic Cytology of the Thy- The Isolation of Living Cells from roid Gland- J. GROSS Animal Tissues--. M. J. RINALRecent Histochemical Results of DIN1 Studies on Embryos of Some Birds AUTHOR INDEX-SUBJECT INDEX. and Mammals - ELIOBORCHESE Carbohydrate Metabolism and Em- Volume 8 bryonic Determination -R. J. The Structure of Cytoplasm O’CONNOR CHARLESOBERLINC Enzymatic and Metabolic Studies on Wall Organization in Plant Cells Isolated Nuclei - G. SIEBERT AND R. D. PRESTON R. M. S. SMELLIE Recent Approaches to the Cytochemi- Submicroscopic Morphology of the Synapse - EDUARDO DE ROBERTIS cal Study of Mammalian Tissues GEORGEH. HOGEBOOM, EDWARD L. The Cell Surface of Paramecium AND E. L. POWERS KUFF,AND WALTER C. SCHNEIDER C. F. EHRET The Kinetics of the Penetration of The Mammalian Reticulocyte- LEAH MIRIAM LOWENSTEIN Nonelectrolytes into the Mammalian The Physiology of Chromatophores BOWYER Erythrocyte - FREDA MILTON FINCERMAN AUTHOR INDEX-SUBJECT INDEX. CUMULATIVE SUBJECT INDEX The Fibrous Components of Connec(VOLUMES 1-5). tive Tissue with Special Reference to the Elastic F i b e r - D A ~ D A. Volume 7 HALL Some Biological Aspects of Experi- Experimental Heterotopic Osdfication- J. B. BRIDGES mental Radiology: A Historical ReA Survey of Metabolic Studies on Isoview--. G. SPEAR lated Mammalian Nuclei-D. B. The Effect of Carcinogens, HorRWDY N mones, and Vitamins on Organ Cultures -ILSE LASNITZKI Trace Elements in Cellular Function BERTL. VALLEEAND FREDERIC L. Recent Advances in the Study of HWH Kinetochore -A. LIMA-DE-FARIA Autoradiographic Studies with S35- Osmotic Properties of Living Cells D. A. T. DICK Sulfate -D. D. DZIEWIATKOWSKI The Structure of Mammalian Sperma- Sodium and Potassium Movements in Nerve, Muscle, and Red Cells-I. tozoon -DON W. FAWCETT M. GLYNN The Lymphocyte-0. A. TROWELL The Structure and Innervation of Pmocytosis - H. HOLTER Lamellibranch Muscle -J. BOWDEN AUTHOR INDEX-SUBJECT INDEX.
xiv
CONTENTS OF PREVIOUS VOLUMES
Volume 9 The Influence of Cultural Conditions on Bacterial Cytology- J. F. WILKINSON AND J. P. DUGUID Organizational Patterns Within Chromosomes- BERWINDP. KAUFMANN, HELENGAY, AND MARGARETR. McDONALD Enzymic Processes in Cells- JAY BOYDBEST The Adhesion of Cells-Lm~Am WEISS
Physiological and Pathological Changes in Mitochondrial Morphology - CH. ROUILLER The Study of Drug Effects at the Cytological Level -G. B. WILSON Histochemistry of Lipids in Oogenesis -VISHWANATH Cyto-Embryology of Echinoderm and Amphibia -KATSUMADAN The Cytochemistry of Non-Enzyme R. COWDEN Proteins-RONALD AUTHOR INDEX-SUBJECT
INDEX.
The Chemistry of Schiff's Reagent FREDERICK H. KASTEN Department of Biology. dgricirlhral arid Mechanical College of Texas. College Station. Texas Page I . Introduction ....................................................... 1 I1. Historical Background ........................................... 2 I11. Methods of Preparing Reagent ..................................... 4 A . Sources of Sulfur Dioxide ..................................... 4 B. Use of Strong Acids in Reagent ............................... 5 C. Stability of Reagent .......................................... 10 I V. Factors Affecting Sensitivity of Reagent ........................... 11 A,. Basic Fuchsin and Impurities ................................. 11 B. Treatment of Reagent with Charcoal ......................... 15 C. Influence of Acids and Salts. ................................. 16 D. S&r Dioxide Concentration ................................. 19 E. Influence of Heat ............................................ 21 V . Specificity of Reagent ............................................ 22 V I . Chemical Nature of Schiffs Reagent ............................. 34 VII . Reaction with Aldehydes ......................................... 37 A . Condensation Theory ......................................... 37 B . Abnormal Schiff Reactions with Certain Aromatic A.ldehydes ... 38 C. Alkyl-Sulfonic Acid Theory .................................. 39 D. Wieland and Scheuing Theory ................................ 42 VIII . Development of Schiff-Type Reagents ............................. 46 IX: Kinetics of the Schiff-Aldehyde Reaction ........................... 56. 56 A . Studies in Vitro ............................................. B. Studies of Stained Tissues ................................... 62 X . Applications of the Reagent to Cytochemistry ....................... 65 A . Use in Feulgen Reaction ..................................... 65 B. Use in Plasma1 Reaction ..................................... 71 C. Use in the Bauer and Casella Oxidizing Techniques ............ 73 D . Use in the Periodic Acid-Schiff Reaction ...................... 74 E. Use in Performic Acid- and Peracetic Acid-Schiff Techniques . . 76 F. Use in Detection of Proteins ................................. 77 G. Use in Direct Staining of Lipids ............................... 78 H. Recent Use in Multiple-Staining Reactions .................... 78 X I . Absorption Curve Analyses of Schiff -Polyaldehyde Binding in Sittr 81 XI1. Conclusions ...................................................... 89 Addendum ........................................................ 91 Acknowledgments ................................................. 93 93 References ........................................................
I . Introduction Schiff's reagent has played a major role in the development of cytochemical techniques used in the study of deoxyribonucleic acid (DNA) and polysaccharides. Other methods of detecting lipid aldehydes and pro1
2
FREDERICK €I. KASTEN
teins also utilize this reagent. The widespread use of Schiff’s reagent in cytochemistry has heightened the need for further information concerning reaction mechanisms. A more complete understanding of the basic principles underlying any cytochemical reaction is of paramount importance, especially when quantitative applications are made. The complexities of the Feulgen reaction are not adequately understood even though vigorous studies were carried out about 35 years ago. Past review articles and recent histochemistry texts have summarized the Feulgen reaction and the application of the technique to biological problems (Swift, 1953 ; Lessler, 1953; Swift 1955 ; Kurnick 1955 ; Leuchtenberger, 1958 ; Casselman, 1959 ; Walker and Richards, 1959 ; Pearse, 1960). The histochemistry of polysaccharides was recently reviewed (Hale, 1957). This article will attempt to provide a fuller account of Schiff’s reagent from the standpoint of historical backgrdbnd and theories of reactions with aldehydes. Other major topics will include applications in cytochemistry, development of Schiff-type reagents, and absorption curve analyses of stained cells. Cytochemical techniques which make use of Schiff’s reagent will not be considered in detail here since the emphasis will be on those phases of the reaction that directly involve the reagent and polyaldehyde moieties. A complete review of the Feulgen reaction is in preparation (Kasten, 1960~).
11. Historical Background Before his death in 1915 at the age of 81, Hugo Schiff had written about 250 scientific papers which had appeared in French, German, and Italian chemical journals. Born in Frankfurt am Main, he obtained a Ph.D. in chemistry at the age of 24 from the University of Turin. H e was associated during his scientific career with the Institute of Higher Studies at Florence, Italy. Here he had the position of Professor of Chemistry and became Director of the Institute. In addition to articles published in scientific journals, he wrote three books and contributed to the Italian Encyclopedia of Chemistry. Schiff’s contributions to organic chemistry embraced several areas. Any detailed discussion of his work would go beyond the boundaries of this article. However, it is significant that he carried out extensive research involving reactions of aromatic amines with aldehydes; known today as Schiff-base reactions (Schiff, 1865a, b). One of the amines he studied was the triphenylmethane basic dye, rosaniline, also known as fuchsin. The name fuchsin was given to the dye because of its fuchsia or rose color, but magenta was a name also used, in honor of the victory of Napoleon I11 in the Battle of Magenta in 1859. In 1 8 6 1 8 6 7 Schi< reported that a
THE CHEMISTRY OF SCHIFF'S REAGENT
3
red-violet color returned to the dye solution, decolored by SOZ, upon addition of a few drops of aldehyde. His interpretation of the mechanism of formation of the re-formed dye was that there was a direct combination of dye and aldehyde, regardless of the presence or absence of HzS03 in the reacting solution. This interpretation is now known to be incorrect, although there is still considerable debate about the true mechanism of reaction between the SO2-treated dye solution and aldehydes. In subsequent discussion, the term Schiff's reagent or fuchsin sulfurous acid (FSA) refers to the colorless derivative produced by the action of sulfur dioxide in water on basic fuchsin or any of its component dye -.Yl'E&ies including pararosaniline, rosaniline, and new fuchsin. The terms leucofuchsin or leuco dye have also been used, but can be misleading, since other leuco derivatives may be obtained from basic fuchsin. Treatment with a r-ng agent forms a leuco dye which is easily oxidized to basic fuchsin. The ca%il base of fuchsin is obtained by treating the dye with ammonium hydroxide or other alkali. The leuco compound formed from ammonium hydroxide reacts with aldehydes to form a colored product (Wang, 1932) but this reaction is unrelated to the Schiff-aldehyde reaction. The attachment of sulfite ester, -O-SOzH, to the methane carbon of fuchsin produces a colorless compound that is nonreactive with aldehydes (Wieland and Scheuing, 1921). The early popularity of Schiff's reagent was due to a large extent to some publications by Schmidt (1880, 1881). Schiff had little to say about the reagent. Many other studies involving Schiff's reagent were reported, especially in the chemical literature. Of special importance to biologists and biochemists was the use of this reagent in cytology to detect deoxyribonucleic acid in nuclei (Feulgen and Rossenbeck, 1924). Feulgen and Voit (1924) also found a substance in the cytoplasm of cells which differed from nucleic acid but gave a positive Schiff reaction. This reaction became known as the plasma1 reaction. I n 1933, Bauer employed Schiff's reagent in conjunction with chromic acid to detect glycogen in tissue sections. Some years later the periodic acid-Schiff ( P A S ) technique was developed (McManus, 1946; Lillie, 1947a, b ; Hotchkiss, 1948) for the histochemical visualization of 1,2 glycols or aminohydroxyl compounds in tissues. For all practical purposes, polysaccharides are the usual compounds stained. Unsaturated fatty acids and keratin have been studied using the performic acid-Schiff ( P F A S ) and peracetic acid-Schiff (PAAS) reactions (Pearse, 1951). An ultraviolet Schiff method, claimed to specifically demonstrate double bonds, was published (Belt and Hayes, 1956). In recent years Schiff's reagent has been applied to the cytochemical detection of proteins after treating sections with oxidative deaminating agents such as ninhydrin, alloxan, or chloramine T (Yasuma and Ichikawa, 1953 ; Burstone, 1955).
4
FREDERICK H. KASTEN
111. Methods of Preparing Reagent
SOURCES OF SULFUR DIOXIDE The original reagent reported by Schiff was prepared by saturating an aqueous solution of fuchsin with sulfur dioxide. The literature is now replete with dozens of modifications. It is surprising that users of Schiff’s reagent have been so willing to work with solutions that contain added ingredients, since the only essential components are fuchsin and sulfurous acid. Many of these modifications are shown in Table I, listed according to sulfur dioxide source and date of. publication. Unfortunately, many of thein involve minor variations of previously published formulas. Some of the “recipes” were designed for special characteristics such as extra sensitivity, reduced sensitivity, easy preparation, etc. For strictly chemical studies, SO2 gas has -used by many workers. Although the method has not been easy to standardize, some have bubbled sufficient gas into the dye solution to add the desired number of grams of SOz,or have titrated it with iodine to obtain the desired concentration. The usual procedure has been to add SOz sufficient to saturate the solution and remove excess gas by a vacuum (Josephson, 1923). This was one of the methods used by Feulgen and Rossenbeck (1924) in their pioneering work on cytochemical detection of DNA. It has been claimed (Tobie, 1942) that Schiff’s reagent, prepared with a definite amount of SOz, 0.2 gm./100 ml. (0.03 M),is supersensitive. In Tobie’s study, the reagent gave pink colors with aldose sugars, whereas a conventional reagent gave a negative reaction. Rafalko also reported (1946) that small and diffuse chromatin elements could be detected in the Feulgen reaction by using a fuchsin solution saturated with SOz. He could not stain these same elements using the DeTomasi ( 1936) reagent, which contains sodium metabisulfite. Ris and hlirsky (1949) later showed that Rafalko’s reagent did not give as intense a Feulgen reaction in mouse liver as did the metabisulfite reagent of Coleman ( 1938). However, the comparison may not be justified, since the Coleman reagent employs carbon to remove interfering impurities. Of particular interest is some nice work reported recently by Elftman ( 1959a), who adjusted the sensitivity of his Schiff’s reagent by titrating with Lugol’s iodine solution. In this way, he obtained maximal histochemical staining of PAS-reactive material in rat pituitary. H e employed a 0.2 M solution of HzS03 in the Schiffs reagent; this was sensitive enough for mod histochemical purposes. H e found that the most sensitive histochemical reagent staining rat pituitary had a 0.02 M SO2 concentration. This agreed very well with the concentration used by Tobie (1!342), A.
THE CHEMISTRY OF SCHIFF’S REAGENT
5
which was 0.03 M. It would be interesting to use Schiff reagents of different sensitivities to determine whether maximal staining of different aldehyde elements can be achieved at the same SO2 concentration. Sodium bisulfite, NaHS03, was used as long ago as 1890 (Mohler) in combination with sulfuric acid and fuchsin solution to provide H2S03. A reagent containing these components, with HC1 in place of H2SOa, was used in cytochemistry by Feulgen and Rossenbeck (1924) and by other workers in the field. Various other sulfur compounds have been used in place of sodium bisulfite. A sensitive reagent containing sodium hydro-, NazS204, was introduced by Prud’homme (1904). A reagent with this compound and a very low amount of fuchsin was reported by Wertheim (1922) to have very desirable properties. The reagent does not require the use of carbon, boiled water, or hydrochloric acid in preparation, is cololless, will not stain the container or the hands, may be kept in clear glass bottles in the laboratory without refrigeration, and can be prepared very quickly. Except for a recent report confirming these advantages and the application to histo- and cytochemistry (Kasten and Burton, 1959), the reagent has not been used in this field. Sodium sulfite, Na2S03, was used as a source of SO2 in the reagent by a few chemists, but has been little used in histochemical applications (note Mowry, 1958). The sulfur compound that seems to be commonly used is potassium metabisulfite, K2S206. It was first used by DeTomasi ‘ (1936), who pointed out that sodium bisulfite is hard to keep anhydrous. Because sodium metabisulfite, Na2Sz06, is unstable, DeTomasi prepared a reagent with potassium metabisulfite. The reagent was popularized by Stowell ( 1945). Thionyl chloride, SOC12, was employed in another modification of Schiff’s reagent by Barger and DeLamater (1948). This compound reacts with water to produce HCl and S 0 2 . It is a convenient way to make a Schiffs reagent since one only adds a few drops of thionyl chloride to the dye solution. It is evident from Table I that there is wide variation in the amounts of the various sulfur compounds used in different Schiff reagents. The optimal concentrations for producing a sensitive solution have been studied (Atkinson, 1952 ; Longley, 1952) and will be discussed in another section. B. USEOF STRONG ACIDSIN REAGENT Almost all sulfur compounds used in Schiff reagents require some acid to generate the important component SO2. The exception is sodium hydrosulfite, which gives good results without acid (Wertheim, 1922 ; Alexander et al., 1950; Kasten and Burton, 1959). Sulfuric acid has been used in a few cases, but most techniques call for 10 to 20 meq. hydrochloric acid in
TABLE I COMPOSITION OF SCHIFF'S REAGENTS IN 100 ML. Source of SO, and references
Sulfur compound (gm.)
Basic fuchsin km.1
Schiff, 1866 Chautard, 1886
-
?
-
0.05
Francois, 1897
-
0.012
OF
SOLUTION
Acid
-
-
1.2 ml. H,SO,
Bitto, 1897 Tolman and Trescott, 1906 Chace, 1906
-
0.25
-
0.5
0.05 0.05
-
1.6
Woodman and Lyford, 1908
2.0
0.05
__
Mulliken, 1914 Josephson, 1923
-
Feulgen and Rossenbeck, 1924 Cracker, 1925 Porter, et al., 1927 Widstrom, 1928
-
0.5
-
0.06 0.1 0.35
-
Schibsted, 1932
0.085
1.o
-
Tobie, 1938
0.5
0.05
-
-
o. 1 0.025
-
t-
-
Comments
To detect acetone in urine T o detect aldehyde in ether (H,SO, reduces sensitivity)
-
Aldehyde in whiskey Citral in lemon extracts Benzaldehyde in almond extracts Excess SO, removed by vacuum Excess SO, removed by vacuum
-
-
Excess SO, removed by vacuum 50% ethanol soh. used for fatty aldehydes 0.1 gm. carbon
*I
i
m
E
G w
c
2
TABLE I (continued) Source of SO., and references-
Sulfur compound km.>
Basic fuchsin km.)
Schreiner and Fuson, 1940 Tobie, 1942
Acid
Comments
1
SO, (continued) half-saturated at room temp. 0.2
0.05 0.1
Rafalko, 1946
0.5
Moree, 1917
0.1
Adams and Johnson, 1949 Cason and Rapoport, 1950 Wild, 1953 Itikawa and Ogura, 1954
0.05 0.05 0.05 0.5
11.3
-
1.5 ml. of 5.6% H2S0,
Rosin, 1955 Feigl, 1956 Vogel, 1956 Linstead and Weedon. 1956 Staple, 1957
-
0.5 0.025 0.1 0.1 0.1 0.5
Na2S20.4 Prud’hornme, 1904 Wertheim, 1922
0.87 2.0
0.013 0.002
Alexander et al., 1950
0.5
0.5
-
0.2 gm. carbon; supersensitive For small and diffuse chromatin elements For sensitive staining of chromatin
-
E i! 0
e
z
-
0
For acetaldehyde Stop at red-violet with SO,; decolors later
v)
-
-
70% ethanol is solvent
1 ml. H2S0, -
Sensitive reagent To make more sensitive, boil 1 min.
-
e
w
c, q W”
m
-
w
Source of SO, and references
Sulfur compound (gm.)
Basic fuchsin
(gm.1
Acid
Comments
1.5 ml. concentr. HCl 1 ml. concentr. HCl 2 ml. concentra. HCl 4 ml. concentr. HCl
-
Na,SO, Fincke, 1914 Elvove, 1917 Scott, 1945 Mowry, 1958 SOCl, Barger and DeLamater, 1948
2.5 1.0 1.o 2.5
0.1 0.1 0.1 1.o
Modified cold Schiff; store at room temp.
H
m tl
m
0.25 ml.
0.25
-
0.45 0.95 0.91 0.98 0.98 1.9 0.5 0.98 0.55 0.3
0.45 0.48 0.45 0.49 0.49 1.0 0.5 0.49 0.29 0.3
10 ml. 1 N HCl 10 ml. 1 N HCI 10 ml. 1 N HCl 10 ml. N HCl 10 ml. 2 N HCI 100 ml. 0.15 N HCl 100 ml. 0.15 N HCl 10 ml. 2 N HCl 1.25 ml. concentr. HCI 10 ml. N H,SO,
-
K2S206
(Na,S,O, sometimes used) DeTomasi, 1936 Coleman, 1938 Stowell, 1945 Hotchkiss, 1948 Ritter and Oleson, 1950 Lillie, 1951a Longley, 1952 Glegg et al., 1952 Mowry et al., 1952 Hormann ct 01.. 1958
~
b
Adds carbon
-
Cold Schiff
-
70% ethanol is solvent Na,SO, 10 H,O as buffer with H,SO,, pH 1.7
-
E
w3: P e E
rn
TABLE I (coittinzced) Source of SO, and references NaHS03 Mohler, 1890
Sulfur compound (gm.)
Basic fuchsin km.)
8.6
0.013
1
Acid
Comments +I
2.0 0.45
0.1
1.5 ml. H,SO,
Bitto, 1897 Feulgen and Rossenbeck. 1924 Margolena, 1932 Bauer, 1933 Carey et al., 1933 Snell and Snell, 1937
0.5 1.7 2 ml. satur. 1.o
0.45 0.83 0.1 0.1
20 ml. 5 N HCI 1.4 ml. concentr. HCl 1 ml. concentr. HCl 1 ml. concentr. HCI
Benseley, 1939 Blaedel and Blacet, 1941
0.83 1.0
0.83
0.1
20 ml. 1 N HCl 17 ml. 6 N HCl
Lillie, 1947a McManus, 1948 Atkinson, 1952 Wild, 1953
1.7 0.45 1.04 0.5
0.83 0.45 0.39 0.125
Kramm ct al., 1955 Vogel, 1956
0.62 1.o
0.29 0.1
0.45
2 ml. concentr. HCl 20 ml. 1 N HCI
k kz
To detect aldehydes in ethyl alcohol
c)
-
Y
v)
+I
20 20 10 1 ml.
ml. 1 N HCl ml. 1 N HCl ml. 2 N HCl concentr. H,SO,
27 ml. 6 N H,SO, 1 ml. HCl
-
2 0
w
-
v)
For methanol by oxidiz. to formaldehyde
4 q-
Add little H2S0, to reduce sensitiv. -
-
For methanol by oxidiz. to formaldehyde
-
-
v)
56
PI
>
c)
M
z
+I
10
FREDERICK H. KASTEN
100 ml. of reagent. Unreacted acid in the reagent undoubtedly reduces the sensitivity (Biddle, 1913). This accounts in part for the fact that the extra-sensitive Schiff reagents are those produced by bubbling in SO2 (Tobie, 1942 ; Rafalko, 1946 ; Elftman, 1959a) without introducing mineral acid. Although Wertheim’s reagent contains only 2 mg. of fuchsin in 100 ml. of 2% hydrosulfite, it seems to be an adequate histochemical reagent (Kasten and Burton, 1959). One probable reason is that it does not contain any strong acid. Blaedel and Blacet (1941) took advantage of the effect of strong acids to reduce the sensitivity of their reagent. They added a little sulfuric acid to a solution already containing 100 meq. of hydrochloric acid. Since many Schiff reagents are prepared with pararosaniline hydrochloride, it is possible that a small amount of hydrochloric acid is released. OF REC. STABILITY The longevity of Schiff’s reagents varies from several hours to 6 months or longer, depending on the ingredients and the method of storing. The useful life of Schiff’s reagents has been reported as 6 hours (Alyea and Backstrom, 1929), 2 days (Chace, 1906), 10 days (Woodman and Lyford, 1908), 6 weeks (Elvove, 1917), several months (Mulliken, 1914), 5 months (Tobie, 1938), 6 months (Lhotka and Davenport, 1949), and 13 months (Tobie, 1942). In the writer’s laboratory, large quantities of the Barger and DeLamater variety of Schiff’s reagent are stored in stoppered dark bottles in the refrigerator. The reagent gives good staining results for at least six months. Some of the factors involved in preservation were considered by Elftman (1959a). They include the evaporation of SO2 and the chemical change of sulfite to sulfate. Loss of SO2 raises the pH, while decrease in sulfite due to oxidation lowers it. The presence of a pink or red color is usually a sign of dissociation of sulfurous acid from the dye; the reagent deteriorates and loses its specificity. Various methods have been employed to prevent loss of S02. Two of the obvious methods are storing in a stoppered bottle (Chautard, 1886; Mulliken, 1914 ; Feulgen and Rossenbeck, 1924 ; Finholt and Thorvick, 1943) and maintaining the solution at a low temperature (Ely and Ross, 1949; Elftman, 1959a). The solubility of SO2 increases with a decrease in temperature as one might expect. Evaporation is also minimized by covering the surface with xylene (Alexander et al., 1950) or mineral oil (Elftman, 1959a). Oxidation of sulfite to sulfate is lessened by using inhibitors such as isopropanol and secondary butanol ( Alyea and Backstrom, 1929), using boiled water in the preparation of the reagent, storing the reagent in completely filled dark bottles (Finholt and Thorvick, 1943; Ely and Ross, 1949), adding antioxidizing agents, such as 0.1%
THE CHEMISTRY OF SCHIFF’S REAGENT
11
pyrogallol (Middleton and Hymas, 1931) and 0.5% hydroquinone (Elftman, 1959a), or using very acid solutions (Reinders and Vles, 1925). Storing in the refrigerator (Ely and Ross, 1949 ; Elftman, 1959a) is simple and probably is the most effective method. Some dye occasionally precipitates from Schiff’s reagent during storage. According to Longley (1952), this results in part from the use of hot solutions during preparation and in part from the use of 1.0% dye solutions. He did not get any precipitation from a 0.5% “cold Schiff” stored in the refrigerator for 3 months. Unfortunately, it does not seem possible to prepare a solid form of Schiff’s reagent that is stable. The solid may be precipitated (Wieland and Scheuing, 1921), but it quickly turns pink. Apparently, SO2 groups are attached loosely to the amine groups and are lost easily. In solution the reagent will retain its staining efficiency for at leasXmo?ths, if kept in well-filled and tightly corked bottles at 0-5OC (Lhotka and Davenport, 1949). It was proposed recently (Beutner, 1954) that a standardized form of Schiff’s reagent be made available commercially. This would involve mixing basic fuchsin, charcoal, and sulfite and adding it to a dilute acid solution. It was suggested by Beutner that formaldehyde could be used on samples of the solution to standardize it before sealing and storing it. The method suffers from the disadvantage that charcoal is used during the sulfite treatment rather than afterwards (Longley, 1952). A simpler approach here might be to use a variation ’of the reagent described by Wertheim (1922). This reagent has sodium hydrosulfite and fuchsin but no charcoal or acid. A stable reagent of controlled sensitivity was reported, which gave reproducible analytical results for 23 days (Kramm and Kolb, 1955).
IV.
Factors Affecting Sensitivity of Reagent
A. BASICFUCHSIN A N D IMPURITIES Rosaniline was used by Schiff (1866) in his original formulation of the aldehyde reagent. It is a triphenylmethane dye containing three amine groups (Conn, 1953). It is commonly called fuchsin or magenta. It is often found associated with the closely related dyes pararosaniline or parafuchsin and, possibly, with magenta 11. The mixture is known as basic fuchsin (C. I. 42510) ; it contains pararosaniline in the greatest concentration, some rosaniline, and small amounts of magenta I1 (Conn, 1953). The chemical structures of these dyes are shown on page 12. In addition to these dyes, colorless Schiffs reagents may be obtained with new fuchsin, C. I. 42520, and Doebner’s violet, both of which are triphenylmethane derivatives. According to Prud’homme (1904),
12
FREDERICK H. KASTEN
7-
H2N=
c)@
NH*
Pararoeaniline
o=c
CHa
f
H*N=
\
4
CHa
Magenta I1
diazofuchsin is also a colorless aldehyde reagent, after reaction with sulfurous acid. The chemical constitution of this dye is uncertain. Malachite green decolors with sodium sulfite and reacts selectively with aldehydes, whose solutions have been neutralized, to give a green color (Frehden and Fiirst, 1939). The mechanism of reaction is not the same as that shown by fuchsin sulfurous acid, since malachite green has no primary aniine groups (Conn, 1953). The malachite green reagent does not work in the Feulgen test for DNA (Kasten, unpublished observations). The sulfonated derivative of basic fuchsin, acid fuchsin (C. I. 42685), is a potentially reactive aldehyde reagent (Lefevre, 1896), but in tissue sections the sulfonic groups bind other tissue components as well (Kasten, 19564. A large spectrum of Schiff -type reagents was recently described (Kasten, 1958a and 1959) although most of these do not decolor appreciably with HtS03. Rosaniline may be supplied as the hydrochloride or acetate salt. The spectrophotometric characteristics are unaffected by the nature of the salt (Peterson et d.,1934). In 47.5% ethanol the visible absorption maxima are 547 mp for pararosaniline salt and 549-550 mp for rosaniline salt (Emery and Stotz, 1953). By taking absorption measurements at two selected wavelengths, the two dyes may be distingushed from one another in 47.5% ethanol (Peterson et al., 1934 ; Emery and Stotz, 1953). Figure 1 shows absorption curves of these dyes in water. The visible absorption peaks are almost identical in water. Table I1 summarizes the ultraviolet and visible absorption peaks of various batches of basic fuchsin. For quanti-
THE CHEMISTRY OF SCHIFF'S REAGENT
13
tative analyses, basic fuchsin is titrated with titanium chloride (Peterson et al., 1934). It is possible to purchase pararosaniline or rosaniline and either of these rather than basic fuchsin may be desired for quantitative application of Schiffs reagent. However, these batches, as well as those supplied under the name basic fuchsin, are frequently contaminated with yellowish brown impurities to varying degrees. Such contaminants interfere with the aldehyde reaction in vitro (Crocker, 1925) and in the Feulgen staining reaction (DeTomasi, 1936). As a result of this variable, commercial fuchsins vary
WAVELENGTH IN Mp FIG.1. Absorption curves of different batches of basic fuchsin and related dyes. Key to curves: , basic fuchsin (Harleco, Cert. No. LF-21); ---, basic fuchsin ( Difco, Cert. No. DF-8) ; x-x-x-x, pararosaniline HCI (Matheson, Coleman and Bell, Batch 490614); - - - - - , rosaniline HCI (National Aniline, Batch No. 8073).
in their sensitivity. I n some cases, better samples provide twice as much sensitivity as the poorer samples (Crocker, 1925). It has been shown in test tube studies (Carey et al., 1933) that some fuchsin solutions cannot be bleached with sulfurous acid and that these amber-colored solutions are less sensitive than colorless ones. In an extended study of 80 samples of basic fuchsin DeTomasi (1936) found that only a few commercial samples could be decolored to a light yellow and give good Feulgen staining. H e also found that fuchsins from the same source varied from time to time. O n the other hand, when special care was taken to obtain specially purified batches of pararosaniline, good staining results were obtained. Accordingly, it was thought that the best approach was to start with the best grade of pararosaniline base and con-
14
FREDERICK H. KASTEN
vert it carefully into the salt, preferably the acetate, since it was more soluble than the hydrochloride (Scanlan and Melin, 1937). As an alternative, in the case of unsatisfactory batches of fuchsin special purification methods with SOz were given. The problem of purification of pararosaniline was raised recently (Yarbo et al., 1954), and new methods of achieving this were described. TABLE I1 SPECTROPHOTOMETRIC DATA FROM DIFFERENTBATCHESOF BASIC FUCHSIN AND RELATED DYES" Dye Basic fuchsin Basic fuchsin Basic fuchsin Basic fuchsin Basic fuchsin Basic fuchsin Basic fuchsin Fuchsin Pararosaniline Rosaniline hydrochloride Rosaniline hydrochloride Rosaniline hydrochloride
Certif. No. or batch no.
U.V. peak
Visible peak
Difco Difco National Aniline National Aniline National An i1ine Schaar & Co. Harleco Ciba Coleman & Bell Coleman & Bell National Aniline
DF-8 DF-4
284 284
535 536
NF56
284
536
NF68
283
536
NF64
-
284 281 283 285
535 536 536 536
490614
285
534
-
284
536
8073
283
536
Schaar & Co.
-
285
536
Company
x 6 .
LF21
Data obtained from Beckman DK-1 recording spectrophotometer. Dyes prepared in aqueous solutions at concentrations of 10 mg./l. and used in a 1-cm. cuvette. Absorption peaks are given in mp.
Tobie (1941, 1942) states that commercial fuchsins deteriorate with age, forming a brown-yellow water-soluble material and a black waterinsoluble material. One batch, certified by a company, gave good results when first purchased, but after several years produced considerable impurities. However, these could be reduced markedly by carbon treatment and adjustment of pH. Aqueous solutions have been generally used to prepare Schiff's reagent. Basic fuchsin is about 7% soluble in ethanol at room temperature (Conn, 1953). This property has been useful in preparing alcoholic Schiffs
THE CHEMISTRY OF
SCHIFF’SREAGENT
15
reagents. A 50% ethanol solution was used as a solvent for the reagent by Schibsted (1932) to detect fatty aldehydes; a 70% ethanol reagent was used by Staple (1957). An alcoholic Schiff’s reagent was essential to demonstrate the presence of the water-soluble polysaccharide dextran in mouse kidney and liver (Mowry et al., 1952). A few Schiff’s-type reagents, such as typogen brown and Magdala red, are insoluble in water and may be prepared in alcohol solution (Kasten, 1959). They are likely to be very useful in demonstrating water-soluble polyaldehydes in tissues. It is apparent from Table I that there is wide variation in concentration of basic fuchsin in the various modifications of Schiff’s reagent. The range is from 2 mg. (Wertheim, 1922) to 1 gm. (Schibsted, 1932 ; Lillie, 1951a) in 100 ml. of solution, In general, chemists employ low concentrations of dye, about O.l%, whereas histochemists use 0.5 or even 1.0% solutions. With formddehyde, more dye is produced from a Schiff‘s reagent containing 0.3% fuchsin than from a reagent having 0.1% fuchsin (Kramm and Kolb, 1955). According to Longley ( 1952), solutions containing more than 0.5% basic fuchsin will in time precipitate large amounts of fuchsin sulfurous acid. Longley concluded that reliable Schiff staining is likely to be obtained from reagents containing at least 0.125% basic fuchsin. However, he had used a metabisulfite reagent containing hydrochloric acid; the results might have differed in an SOz-Schiff’s reagent lacking mineral acid. Some progress has been made by the Biological Stain Commission (Conn, 1943) in standardizing basic fuchsin for use as a Schiff’s reagent in histochemistry. Five different tests are made on each batch of dye submitted. One of these is a Feulgen histochemical test. As a result, the user may be assured that certified batches will give good results. Dye manufacturers now produce better batches of fuchsin so that the general situation is greatly improved over that of 25 years ago. It is hoped that European dye manufacturers will work out a similar system so that their users also will have the benefit of reliable fuchsins for histochemical studies. OF REAGENT WITH CHARCOAL B. TREATMENT The procedures employed by Scanlan and Melin (1937) to rid fuchsin samples of interfering yellow-brown impurities prompted Coleman ( 1938) to devise a simpler method of achieving the same end. H e used decolorizing carbon to remove objectionable impurities that were not bleached by sulfite treatment. Since the carbon treatment caused some SO2 to be lost, Coleman increased the concentration of metabisulfite in the solution. The The added decolorizing action of carbon allowed poor batches of fuchsin to be used in the Feulgen test, Decolorizing carbon was used independently
16
FREDERICK H. KASTEN
by Mann and Saunders (1936) and Tobie (1938) for the same purpose, and this procedure has generally been incorporated into all Schiff modifications devised since then. Some batches of decolorizing carbon or activated charcoal may lose their desirable decolorizing qualities upon storage (Longley, 1952). Most users extract the yellow-brown impurities with charcoal after the dye solution has been reduced with H2S03.Barger and DeLamater (1948) mentioned that the charcoal could precede the treatment with a reducing agent. Longley (1952) showed that charcoal had a greater affinity for untreated fuchsin than for fuchsin which had been reduced with H2S03. Consequently, the heavier yellow impurities were more likely to be removed by charcoal after the sulfite treatment. It has been recommended that the amount of charcoal be less than 50 mg./100 ml. of Schiff’s reagent (Hediger ct al., 1940). Longley (1952) used 300 nig./100 ml. of reagent, and Segal (1951) recommended 2e&fig./lOO ml. Too much carbon abstracts some of the decolored dye and reduces the sensitivity of the reagent. The contact time between charcoal and reagent is also important. These two factors were considered by Kraniin and Kolb (1955), and their recommendation was that 200 nig. of charcoal should be mixed with 100 ml. of Schiff’s reagent for 45 seconds. The mixture should then be filtered through a Biichner funnel by vacuum filtration for 2 or 3 minutes. This procedure was devised to help provide a stable reagent of controlled sensitivity for the detection of formaldehyde in cellulose acetate formal. Fuller’s earth also has been used to remove the impurities (Scott, 1945), but this was not recoiniiiended by Longley (1952). C.
INFLUENCEOF ACIDSAND SALTS
Over 60 years ago it was pointed out that the sensitivity of the reagent could be reduced by adding sulfuric acid (Francois, 1897). The influence of acids on the aldehyde reaction was studied in detail by Biddle (1913), who demonstrated that the reaction with formaldehyde was inhibited in in the presence of strong mineral acids. As the concentration of hydrochloric acid was increased, the sensitivity of the reagent decreased. On the other hand, with such organic acids as acetic acid or propionic acid the reaction was accelerated. As the concentration of organic acid was increased, the speed of reaction was likewise increased. Organic acids with a weaker dissociation constant had a greater effect in speeding up the reaction. These observation are summarized in Figs. 2 and 3. The effects mentioned above seem related to the hydrogen ion concentration and the tendency to form quinones (Biddle, 1913), but further studies are needed taestablish the mode of action. Some work has been reported on the hydrogen ion concentration of
THE CHEMISTRY OF SCHIFF'S REAGENT
17
different Schiff's reagents. According to Longley (1952), pH values vary from 1.24 for the thionyl chloride reagent (Barger and DeLamater, 1948) to 4.80 for a sodium hydrosulfite reagent (Alexander et al., 1950). One SO,-treated fuchsin solution had a pH of 1.45 but according to 24 22
10
N TARTARIC "ID> ACIO-
4
2 0
0
4
8
12
I6
20
24
28
32
INCREASE I N COLOR INTENSITY
FIG.2. Effect of different organic acids and hydrochloric acid on development of color in formalin-treated Schiffs reagent. Data redrawn from Biddle (1913). I"
0.1 N ACETIC ACID-
0 . 2 N ACETIC ACID
I
10-
2 N ACETIC ACID
INCREASE IN COLOR INTENSITY
FIG.3. Effect of different concentrations of acetic acid on development of color in formalin-treated Schiff's reagent. Data redrawn from Biddle (1913).
18
FREDERICK H. KASTEN
another report (Itikawa and Ogura, 19-54), this kind of a reagent had a p H of 1.8-2.4. The in vitro sensitivity of the reagents was reduced upon acidification with concentrated hydrochloric acid, but there was no decline in histochemical effectiveness (Longley, 1952). I n the p H range of 1.2-6.9, an SO2-treated fuchsin solution gave optimal Feulgen staining at p H 3.0 and 4.3; optimal staining in the P A S reaction was at p H 2.4; pH optima in the Bauer and Casella reactions were at 3.0-4.3; Lillie’s performic acid reaction gave good staining at p H 6.9 (Itikawa and Ogura, 1954). These studies were carried out on sections of horse liver. It seems desirable to extend such tests to other polyaldehydes, especially in regions of low concentrations of reacting moieties. In addition, it would be of interest to study the histochemical effects after adding mineral acids and organic acids to an SO2-treated Schiff’s reagent. Some data from stained cells were recently reported, in which dye CMknt per nucleus was found to increase progressively as p H varied from 0.8 to 3.6 (Swift, 1955). Basic fuchsin is an indicator dye, changing from purple to red in the p H range of 1.2-3.0, and from red to colorless in the range of 11.6-14.0. It is not surprising that variation in the acid concentration of Schiff‘s reagent produces a difference in the spectrum of stained nuclei (Barka, 1959). It was shown that in the Schiff reaction with hydrolyzed DNA in vitro, pH is an important factor for color development (Caspersson, 1932). The color reaction proceeds in two‘ parts; the first part is color producing and results from aldehyde addition, while the second part is a degradative reaction tending to reduce color formation. Each part is influenced by pH, and the optimal p H for color intensity in this system is 2.3. The system is complicated by the fact that the same solution contains hydrochloric acid, DNA, all the products of DNA hydrolysis, buffer solution, and Schiff’s reagent. Mulliken (1914) pointed out that alkali salts of weak acids cause the reagent to become red. It was shown that p H influences this effect (Josephson, 1923). At a pH of less than 3, acetate and phosphate buffers do not produce a coloration. Mulliken (1914) added a slight amount of sodium acetate to resensitize an old reagent. False positive reactions with certain proteins do not occur in very acid solutions (Josephson, 1923) and may be accounted for by a failure to neutralize the acid groups of the reagent. I n spite of the fact that some investigators have used buffered Schiff’s reagent without noting a red color (Widstrom, 1928 ; Caspersson, 1932), it seems desirable to avoid this possible complication.
THE CHEMISTRY OF SCHIFF’S REAGENT
D.
19
SULFUR DIOXIDE CONCENTRATION
Sulfur dioxide is an essential component in the aldehyde reaction. It is well known that an excessive amount of SO2 decreases the sensitivity of the color reaction in vitro (Mulliken, 1914; Wieland and Scheuing, 1921 ; Schibsted, 1932 ; Rumpf, 1932 ; Tobie, 1942 ; Atkinson, 1952 ; Hormann et al., 1958). A minimum of SO2 is essential to produce the bis-N-sulfinic acid of fuchsin leucosulfonic acid. A deficiency of SO2 causes incomplete color development and variation in color intensity (Caspersson, 1932). Excess SO2 above this minimum may react directly with aldehyde to form aldehyde sulfinic acid, a colorless compound (Wieland and Scheuing, 1921). In the presence of a low concentration of aldehyde and a high concentration of SO2 the aldehyde sulfinic acid reaction markedly impairs the sensitivity of the Schiff-aldehyde reaction. Ely and Rose41949) reported that an increase in metabisulfite content increased the color intensity in hydrolyzed DNA preparations. These results are in conflict with those of other workers (Wieland and Scheuing, 1921 ; Caspersson, 1932 ; Atkinson, 1952 ; Hormann et al., 1958). According to Lison (1936), the reagent of Wermel (1927) is extrasensitive. It is a sodium bisulfite reagent (Feulgen and Rosenbeck, 1924) that has a little added acetaldehyde. The violet color produced is discharged by adding more hydrochloric acid and sodium sulfite. The rationale behind this treatment is that one of the reactive amine sulfite sites supposedly is blocked by acetaldehyde, leaving one other site free for aldehyde reaction and color development. Whether or not this reagent truly behaves this way has not been determined. The ideal proportion of fuchsin : SO2: aldehyde is hard to determine because of the complexity of the reaction and the fact that the reaction differs with different aldehydes (Shoesmith et al., 1927;Hormann et al., 1958). Table I11 shows the color intensities obtained when the proportion of SO2: acetaldehyde is varied. Large concentrations of both SO2 and aldehyde gave the best results. In other studies with formaldehyde, it was shown that the optimal molar ratio of fuchsin: sodium bisulfite was 1 : 10 (Atkinson, 1952). These studies were carried out with a single concentration of formaldehyde, 0.02%. Other sensitive Schiff’s reagents were reported to contain 0.03 moles( Tobie, 1942) and 0.02 moles of SO2 (Elftman, 1959a). I n another study, the optimal concentrations of SO2 for determination of formaldehyde was 0.028-0.048 moles (Kramm and Kolb, 1955). Figure 4 shows that this concentration is optimal for different aldehyde concentrations. An alcoholic Schiff’s reagent that was claimed to be very sensitive to fatty aldehydes had 0.45 mole S02:l mole fuchsin in 1% dye and 50% ethanol (Schibsted, 1932). It was stated that in analytic
20
FREDERICK H. KASTEN
tests involving nine different forms of Schiff's reagent, the reagent of Longley ( 1952) was most sensitive (Giral et al., 1955). TABLE 111 EFFECT OF VARYING THE RATIO OF SO,: ACETALDEHYDE ON COLOR INTENSITY~
SO, (moles)
Acetaldehyde (moles)
Per cent of max. color intensity
1 1 1 2 2 2
0.6 1 4 2
2 6 9 24
3
37 37
5
3
3
4 5 10 10 10 10 10 10
4 5 2
51 61 66 40
.**
3
63
4 5
80 90 100 100
6 10
0 Data from Wieland and Scheuing (1921). Concentration of fuchsin leucosulfonic acid was constant at 0.78 gm./l.
A thorough study of the effect of SO? concentration on dye development was recently reported (Hormann et al., 1958). With formaldehyde, the maximal amount of dye formed is obtained at a ratio SO, : CHaO of 1 : 1. When more or less SO:! is present, the color intensity drops. The same is true with acetaldehyde, although the ratio is slighter greater than 1: 1.
'
"
d'
eo
40 60 80 TRANWTTAW€ IN PER ENT
loo
FIG.4. Loci showing variation of Schiff's reagent sensitivity with reagent sulfur dioxide content for fixed formaldehyde levels. A, 0.250 mg. H C H O ; B, 0.125 mg. H C H O ; C, 0.050 mg. of HCHO. Ordinates D and E enclose zone of optimum reagent response. Data from Kramm and Kolb (1955).
THE CHEMISTRY OF
SCHIFF’SREAGENT
21
The number of moles of SOz and of aldehyde is as important as the ratios used (Wieland and Scheuing, 1921) . Since more aldehyde sulfinic acid is produced with acetaldehyde than with formaldehyde, the color is unstable with acetaldehyde and stable with formaldehyde (Wieland and Scheuing, 1921 ; Hormann c t al., 1958). Some data from Feulgen-stained nuclei were recently reported by Swift (1955). He found that at a p H of 2.1, an increase in SO, concentration of the reagent, from 0.3 to 10.0%, resulted in a progressive decrease in dye content per nucleus. For histochemical purposes a Schiff’s reagent of maximal sensitivity is not always desired. Such a reagent has a low concentration of SO, and is easily oxidized. For routine studies it seems desirable to achieve specificity at some sacrifice of sensitivity. For a few quantitative purposes and for detection of low amounts of polyaldehydes in tissues the supersensitive reagents may be desired. Staining reactions in tissues are often undiminished in intensity when reagents with excess sulfite are used, especially at fuchsin concentrations of 0.5% (Longley, 1952). Reagents produced by bubbling in SO2 are difficult to standardize at low concentrations of SOn. It is customary in some laboratories to recharge the reagent with SO, before each use to achieve uniform staining conditions.
E. INFLUENCE OF HEAT Boiling off all the SO2 from Schiff’s reagent causes the solution to turn red and remain colored. Other phenomena result if Schiff’s solution is heated for a short time to remove only part of the SOz. The solution becomes colored and stays colored while hot but, when cooled, it gradually becomes colorless again. This process may be repeated several times with the same solution (Kastle, 1909). Since these interesting changes also occur in an oxygen-free atmosphere (Kastle, 1909; Damianovitch, 1910) it is likely that fuchsin leucosulfonic acid either dissociates under the influence of heat or, less likely, is hydrolyzed by water (Kastle, 1909; Shoesmith et al., 1927). Both of these suggested reactions are shown on page 22; it should be kept in mind that the second and third steps of the hydrolytic reaction scheme are unlikely to occur. Wertheim (1922) claimed that his Schiff’s reagent was more sensitive after having been boiled for 1 minute, and he noted the color changes described above. The phenomena were also studied by Shoesmith et d. (1927). Increased temperature causes an increase in the amount of dye re-formed with formaldehyde (Atkinson, 1952). Since dissociation may occur simultaneously with the aldehyde reaction, heat should be avoided. An inverse relationship p a s reported with hydrolyzed DNA as the reacting aldehyde (Ely a d Ross, 1949). The aldehyde reaction takes place
22
FREDERICK H. KASTEN
Further heating# Cooling
x+ Complete loss of
+
+
H,N=
S9.b boiling
Suggeuted chemical changes during dissociation of Srhiff's reagent by heating
OH
+X-
N--f%&H
NHz
o-18'"
Heated (SO1 lost)
Cooled
(Sor regained)
5-
NHz Colorless
Schiff's reagent
HzN=
+x-
HzO
&SOa
c
NH2
Pararosaniliie Hydrolytic dissociation of Schiff'e reagent at low temperatures in the test tube as well as in the Feulgen reaction in tissue sections. The latter is illustrated in Fig. 5. The Feulgen absorption curve is unaffected by increased temperature of the dye complex (Kasten, 1958b). V. Specificity of Reagent The first studies aimed at verifying the aldehyde specificity of Schiff's reagent were reported by Schmidt (1880, 1881). H e discussed some work by Car0 in which a variety of aldehydes had been shown to give the color reaction. Closely related nonaldehyde compounds were not reactive. For example, chloral, CCl,CHO, gives a violet reaction, .whereas the close relative chloral hydrate, CClsC( OH) 2, is nonreactive. This example by
T H E CHEMISTRY OF SCHIFF'S REAGENT
23
Schmidt is not ideal since chloral and chloral hydrate are indistinguishable in water solution. Schmidt used the reagent to help identify the aldehyde nature of some reaction products he was studying. H e was enthusiastic about the aldehyde specificity of Schiff's reagent and considered it a very useful reagent for this purpose. Production of color with aldehydes is not immediate nor does it occur at a uniform rate with all aldehydes, Some Schiff's reagents are more sensitive than others. This is probably one of the reasons why some compounds have been reported as both Schiff-positive
1 A
1
400 I410
415 42s 4W 445 455 I 4(LsI 480 l 4W r l 510 l l 530 l I 545 I I 565 I I 5SO I I El5 I I $45 I I 6RI I I TO5 I I
WAVELENGTH IN Mp FIG.5. Transmission curves from three frog liver nuclei obtained with the automatic double-beam ratio-recording microspectrophotometer of Pollister and Ornstein (1955). Feulgen reaction occurs rapidly (2 minutes) at 6" C., as shown by peak at point B. Remainder of DNA-polyaldehyde was stained by chrysoidine Y-SO, (45 minutes) with peak at point A . Data from Kasten (1960b).
and Schiff-negative. For example, Tobie (1942) was able to detect aldehydes in some aldose sugars by using a supersensitive reagent, but these same compounds were Schiff-negative with a conventional reagent. Some aromatic hydroxyaldehydes give a very slow reaction (Lorenz, 1881) compared with the rapid reaction given by aliphatic aldehydes. Some of the differences are probably due to the poor solubility of aromatic aldehydes in Schiff's reagent. Yellow precipitates often result with o-hydroxyaroniatic aldehydes, in contrast to the soluble red-violet-to-blue dyes produced by other aldehydes (Shoesmith et al., 1927). Some aromatic aldehydes are not colored by Schiff's reagent despite the fact that closely related aldehydes do give a positive reaction (Pauly and von Buttlar, 1911 ) .
24
FREDERICK H. KASTEN
The Schiff test has been used to detect aldehydes in various substances. For example, it has been applied to alcohol (Mohler, 1890, 1891 ; Paul, 1896), ether (Franqois, 1897; Phelps and Rowe, 1926; Middleton and Hymas, 1931 ; Cary et nl., 1933; Hediger et al., 1940), whiskey (Tolman and Trescot, 1906; Tobie, 1938, 1941), citral adulterants in lemon extracts (Chace, 1906), almond extracts for benzaldehyde determination (Woodman and Lyford, 1908), milk, red wines, and fruit juices for formaldehyde (Fincke, 1914), methanol (Elvove, 1917), certain organic products (Hoffpauir et al., 1943), oils and butter fat for fatty aldehydes (Schibsted, 1932), and the colorimetric determination of lactic acid and glycerol by conversion to acetaldehyde and acrolein respectively ( Snell and Snell, 1937). Table IV lists some of the compounds reported to give a positive Schiff reaction. The nonaldehydic compounds which also give a positive reaction include certain ketones (Schmidt, 1881 ; Lorenz, 1881 ; Bitto, 1897; Otte and von Pechmann, 1889; Muller and von Pechmann, 1889). Acetone is one of the ketones frequently mentioned as giving a violet color with Schiff’s reagent. In fact, this reaction has been used as the basis for detecting acetone in the urine of diabetics (Chautard, 1886). Although acetone reacts more slowly than do aliphatic aldehydes, it forms a color as fast as many aromatic aldehydes. Lower aliphatic ketones give a strong color reaction, whereas many aromatic ketones do not react. Some aromatic diketones give a positive Schiff reaction (Muller and von Pechmann, 1889). Acetals may react slowly as the acid present in Schiff’s reagent catalyzes their hydrolysis to aldehydes (Porter ct al., 1927). Certain unsaturated compounds give a color reaction with Schiff’s reagent. According to Lison (1932), oleic acid, completely free of aldehyde, gives a positive reaction. This was verified by Chu (1950), who suggested that aldehydes may be produced from unsaturated fatty acids by oxidation during the Schiff test. Isosafrol also gives a positive reaction. Paradoxically, the aldehyde derivative of isosafrol, piperonal, is Schiff-negative (Lison, 1932). Various aldehydes may be produced from non-aldehydic compounds by suitable techniques. This forms the basis for the histochemical applications of Schiff’s reagent. Deoxyribonucleic acid is hydrolyzed by hydrochloric acid to form aldehyde residues (Feulgen and Rossenbeck, 1924). Acetal phosphatides, known also as plasmalogens, react with mercuric chloride to form higher aldehydes or plasmals (Feulgen and Voit, 1924). Carbohydrates and a few amino acids are oxidized by periodic acid (Malaprade, 1928) and other oxidants, giving aldehyde groups. Proteins may be detected by using oxidative deaminating reagents, such as ninhydrin
THE CHEMISTRY OF SCHIFF’S REAGENT
25
TABLE IV in Vitro COMPOUNDS THATGIVE A COLORREACTIONWITH SCKIFF’SREAGENT Group Aldose sugars D-Galactose D-Glucose D-Mannose L-Arabinose L-Xylose Rhamnose 2-Deoxy-~-glucose 2-Deoxy -n-galactose
Reference Tobie, 1942 Tobie, 1942 Tobie, 1942 Tobie, 1942 Tobie, 1942 Tobie, 1942 Overend, 1950 Overend, 1950 Overend, 1950 Overend, 1950
2-Deoxy-~-ribose Glucofuranose, 5 : 6-monocarbonate 2-Deoxy-n-g~ucofuranose,5 :6Overend, Monocarbonate Overend, 3 : 4-Dimethyl-2-deoxy-~-ribopyranose Overend, 3 :5-Dimethy1-2-deoxy-n-ribofuran0~e 3 : 4 : 6-Trimethyl-2-deoxy-~-gala~topyrmose Overend, 3 : 5 : 6-Trimethyl-2-deoxy-~-galactofura1iose Overend, Aliphatic aldehydes Acetaldehyde Acrolein Aldol Butylchloral Butylaldehyde Chloral Crotonaldehyde Decylaldehyde Dodecylaldehyde Formaldehyde Glycolaldehyde Glyoxal Heptaldehyde Isobutylaldehyde Isovaleraldehyde Methylethyl acrolein Nonyl aldehyde Oenanthaldehyde Paraldehyde Propionaldehyde Tiglicaldehyde Amino oxides N-Oxide of piperidine N-Oxide of pipecoline ~~
1950 1950 1950 1950 1950
Schmidt, 1881 ; Bitto, 1897; Francois, 1916; Crocker, 1925 ; Feigl, 1956; Chu, 1950 Schmidt, 1881 ; Francois, 1916 ; Shoesmith, et al., 1927 Bitto, 1897 Schmidt, 1881; Bitto, 1897 Chu, 1950 Schmidt, 1881; Chu, 1950 Bitto, 1897; Francois, 1916 Chu, 1950 Crocker, 1925 Shoesmith ct al., 1927 Shoesmith et al., 1927 Crocker, 1925; Chu, 1950 Bitto, 1897; Shoesmith et al., 1927 Schmidt, 1881 Bitto, 1897 Chu, 1950 Schmidt, 1881 Schmidt, 1881; Bitto, 1897 Schmidt, 1881 Bitto, 1897 Lison, 1932 Lison, 1932
26
FREDERICK H. KASTEN
TABLE IV (confinued) Group Amino oxides (continued) N-Oxide of lupetidine N-Oxide of cicutine N-Oxide of copellidine Aromatic aldehydes Anisaldehyde Benzaldehyde
5-Bromo-2-naphthaldehyde 2-Carboxy-3 : 4-methylene dioxybenzal Cinnamic aldehyde Cuminaldehyde 2,3-Dihydroxybenzaldehyde 2,4-Dihydroxybenzaldehyde 2,s-Dihydroxybenzaldehyde Furfural 3-Hydroxybenzaldehyde 4-H ydroxy benzaldehyde
2-Hydroxy-5-chlorobenzaldehyde Isopropylbenzaldehyde m-Fluorobenzaldehyde m-Bromobenzaldehyde 3 : 4-Methylenedioxybenzaldehyde m-Methoxybenzaldehyde m-Nitrobenzaldehyde a-Naphthaldehyde 2-Nitro-Cmethoxybenzaldehyde 3-Nitro-4-methoxybenzaldehyde o-Chlorobenzaldehyde o-Fluorobenzaldehyde o-Methoxybenzaldehyde o-Nitrobenzaldehyde 3-hydroxy-4-methoxyvanillin p-Bromobenzaldehyde Phenylacetaldehyde Piperonal
Reference Lison, 1932 Lison, 1932 Lison, 1932 Bitto, 1897; Pauly and Buttlar, 1911; Crocker, 1925 ; Feigl, 1956 Schmidt, 1881; Wolffenstein, 1892; Bitto, 1897; Prud’homme, 1904; Woodman and Lyford, 1908; Crocker, 1925 ; Shoesmith, et al., 1927; Alyea and Backstrom, 1929; Lison, 1932; Chu, 1950; Feigl, 1956 Shoesmith et al., 1927 Shoesmith et al., 1927 Schmidt, 1881; Bitto, 1897; Crocker, 1925; Feigl, 1956 Schmidt, 1881; Bitto, 1897 Pauly and Buttlar, 1911 Chu, 1950 Pauly and Buttlar, 1911 Schmidt, 1881; Bitto, 1897; Crocker, 1925 ; Chu, 1950; Feigl, 1956 Pauly and Buttlar, 1911; Shoesmith et al., 1927 Feigl, 1956 Chu, 1950 Shoesmith ef al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927; Chu, 1950; Feigl, 1956 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al., 1927; Feigl, 1956 Pauly and Buttlar, 1911 Shoesmith e t al., 1927 Chu, 1950 Pauly and Buttlar, 1911; Crocker, 1925
THE CHEMISTRY OF SCHIFF’S REAGENT
27
TABLE IV (continued) Group Aromatic aldehydes (contitwed) p-Dimethylaminobenzaldehyde p-Methylbenzaldehyde p-Methoxybenzaldehyde p-Nitrobenzaldehyde Protocatechualdehyde Salicylaldehyde Tolylaldehyde Vanillin Diketones Diacetyl Sec. a#-diketoheptane a-B-Diketohexane Sec. u,p-diketohexane a,fl-Diketohexylene a,p-Diketopentane Ethy lphenyldiketone Methy lbenzyldiketone Methylphenyldiketone Ketones Acetone Acetophenon Ethylbutylketone Methylhexylketone Methyl-N-amylketone Methylpropylketone Organic bases Pyrimidine N-hydroxypyridine Piperidine Pyridine Polysaccharides Oxycellulose Unsaturated compounds Cinnamic acid Isoeugenol Isosafrol Lecithin Linoleic acid Linolenic acid Oleic acid
Reference Chu, 1950 Shoesmith et al., 1927 Shoesmith et al., 1927 Shoesmith et al, 1927 Lison, 1932 Pauly and Buttlar, 1911; Lison, 1932; Crocker, 1925; Chu, 1950; Feigl, 1956 Chu, 1950 Bitto, 1897; Pauly and Buttlar, 1911; Crocker, 1925; Chu, 1950 Otte and Pechmann, 1889 Otte and Pechmann, 1889 Otte and Pechmann, 1889 Otte and Pechmann, 1889 Otte and Pechmann, 1889 Otte and Pechmann, 1889 Muller and Pechmann, 1889 Muller and Pechmann, 1889 Muller and Pechmann, 1889 Lorenz, 1881; Schmidt, Chautard, 1886; Bitto, Lison, 1932 Lorenz, 1881 Chu, 1950 Bitto, 1897 Chu, 1950 Bitto, 1897 Lison, 1932 Zinner, 1957 Semmens, 1940 Semmens, 1940 Flint and Tollens, 1892 Lison, 1932 Lison, 1932 Lison, 1932 Chu, 1950 Chu, 1950 Chu, 1950 Lison, 1932; Chu, 1950
1881; 1897;
28
FREDERICK H. KASTEN
TABLE V SUBSTANCES THATMAY REACTWITH SCHIFP’S REAGENT IN HISTOCHEMICAL REACTIONS Histochemical reactions
Substances
Feulgen
Deoxyribonucleic acid
Plasma1
Acetal phosphatides (plasmalogens). Widely distributed in animal tissues. Choline plasmalogen : ram sperm, beef heart Ethanolamine plasmalogen : brain
Periodic acid-Schiff
Acid mucopolysaccharides Certain heparins Epithelial mucins Uterine gland much Respiratory tract mucin Corneal mucopolysaccharide Glycolipids Gangliosides Cerebrosides Phrenosin Kerasin Inositol phosphatides Miscellaneous substances Adrenaline Ascorbic acid Muco- and glycoproteins Blood group A substance Chorionic gonadotrophin Fractions of serum albumin and serum globulin Collagen Reticulin Gastric mucin Lens capsule polysaccharide Pituitary gonadotropins Pituitary TSH Salivary gland mucin Seroid much Thyroid colloid Neutral mucopolysaccharides Gastric mucins Bacterial capsule polysaccharides Chitin
THE CHEMISTRY OF SCHIFF’S REAGENT
Histochemical reactions
29
TABLE V (contittucd) Substances Polysaccharides Cellulose Dextrans (water-soluble) Galactogen Glycogens Starches Unsaturated lipids and phospholipids Ceroid Cephalin Cardiolipin Lipofuscins Sphingomyelin
Bauer’s chromic acid-Schiff technique
Some a-glycols Certain carbohydrates
Casella’s permanganate-Schiff nique
Ethylene groups a-Glycols Certain carbohydrates
tech-
Performic acid- and peracetic acidSchiff reactions
Disulfide groups Unsaturated lipids
Ninhydrin- and Alloxan-Schiff actions
a-Amino acid proteins
Ultraviolet-Schiff reaction
re-
Unsaturated lipids
and alloxan, followed by Schiff’s reagent (Yasuma and Ichikawa, 1953). Table V lists compounds or moieties that may be detected histochemically from a color reaction with Schiffs reagent. Bromine has been reported to give a color reaction with Schiff’s reagent (Guareschi, 1913 ; Wunsche, 1919 ; Fujimoto, 1956). The mechanism of reaction probably differs from that of the aldehyde reaction, because Hoffniann’s violet (C.I. 42530), another triphenylmethane dye, gives the bromine reactictn (Guareschi, 1913) but not the aldehyde reaction. The bromine reaction is probably at the central carbon. Alcohols, free of aldehydes, also have been reported to give a positive Schiff reaction (Schmidt 1881; Lorenz, 1881), but these results were not substantiated by Chautard (1886). It would be impossible to produce a colorless alcoholic Schiff’s reagent (Schibsted 1932 ; Mowry et nl., 1952), if alcohol itself reacts with the decolorized dye. Table VI lists some compounds which fail to give a color reaction with Schiffs reagent. Various oxidizing agents, such as chlorates, peroxides, and atmospheric
30
FREDERICK H. KASTEN
TABLE VI COMPOUNDS THATFAILTO GIVEA COLORREACTION WITH SCHIFF'S REAGENT in
Vitro References
Group Acids Acrylic acid Crotonic acid Formic acid Fumaric acid Maleic acid
Lison, 1932 Lison, 1932 Schmidt, 1881 Lison, 1932 Lison, 1932
Alcohols Chloral hydrate Glycol Heptyl alcohol Hexyl alcohol Isobutyl alcohol Octyl alcohol Pinacol Trimethylcarbinol
Schmidt, Schmidt, Schmidt, Schmidt, Schmidt, Schmidt, Schmidt, Schmidt,
'
1881 1881 1881 1881 1881 1881 1881 1881
Aliphatic aldehydes Glyoxal Thioacetaldehyde Sulfaldehydes
Bitto, 1897 Shoesmith et al., 1927 Bitto, 1897
Aromatic aldehydes 2,4-Dihydroxybenzaldehytle 3,4Dihydroxybenzaldehyde Ethyl vanillin 4-Hydroxybenzaldehyde
Crocker, 1925; Feigl, 1956 Crocker, 1925 Feigl, 1956 Crocker, 1925; Pauly and Buttlar,
1911 2-Methoxy-1-naphthaldehyde p-Aminobenzaldehyde p-Dimethylaminobenzaldehyde P-Homosalicylaldehyde Piperonal Vanillin
Shoesmith et al., 1927 Feigl, 1956 Lison, 1932; Feigl, 1956 Bitto, 1897 Lison, 1932 Feigl, 1956
Glucosides Esculin Salicin
Tobie, 1942 Tobie, 1942
Ketone acids Acetoacetic ester Levulinic acid Pyroracemic acid
Bitto, 1897 Bitto, 1897 Bitto, 1897
T H E CHEMISTRY OF SCHIFF’S REAGENT
31
TABLE V I (continued) Group Ketones Acetone Acetophenon Benzilidenacetone Benzophenon Diethylketone Methylnaphthyl ketone Monobromoacetophenon Monochloracetone Naphthylphenylketone Phenyl thienylketone Salicylresorcylketone
References Carey et al., 1933; Chu, 1950 Schmidt, 1881; Bitto, 1897; Lison, 1932 Bitto, 1897 Schmidt, 1881; Bitto, 1897; Lison, 1932 Bitto, 1897 Chu, 1950 Bitto, 1897 Bitto, 1897 Bitto, 1897 Bitto, 1897 Bitto, 1897
Organic bases Antipyrine Pyrrol
Lison, 1932 Lison, 1932
Phenols Cinnamylguaiacol Guaiacol Hydroquinone Orcin Phenol Pyrocatechol Pyrogallol Quinone Resorcin
Lison, 1932 Schmidt, 1881 Schmidt, 1881 Schmidt, 1881 Schmidt, 1881 Schmidt, 1881 Schmidt, 1881 Schmidt, 1881 Schmidt, 1881
Polysaccharides Inulin Potato starch
Tobie, 1942 Josephson, 1923
Sugar alcohols D-Mannitol D-sorbitot Dulcitol Meso-inositol hydrate
Tobie, Tobie, Tobie, Tobie,
Sugars Glucose Invert sugar Lactose
1942 1942 1942 1942
Schmidt, 1881 ; Bitto, 1897 ; Josephson, 1923 ; Overend, 1950 Bitto, 1897 Schmidt, 1881
32
FREDERICK H. KASTEN
TABLE VI (continuedl ~~
GrouD Sugars Levulose Raffinose Sucrose Trehalose D-Galactose o-Ribose 2 : 3 : 4 : 6-TetramethylD-glucopyranose Unsaturated compounds Eugenol Safrol
References Schmidt, 1881 ; Bitto, 1897 ; Tobie, 1942 Tobie, 1942 Bitto, 1897; Tobie, 1942 Tobie, 1942 Overend, 1950 Overend, 1950 Overend, 1950 Lison, 1932 Lison. 1932
oxygen, may regenerate the original dye (Kastle, 1909). The color formed is red and is easily distinguished from the violet reaction products given in the true Schiff reaction. Regeneration of basic fuchsin may occur also by heating (Kastle, 1909) or by treating the reagent with other substances that remove SO*, such as mild alkalies and amines( Noller, 1957). According to Valdiquie (quoted by Lison, 1932), pyrimidine reacts with Schiff’s reagent to produce a color different from that of fuchsin. If this is true, the reaction may be due to the unsaturated bonds and not to the basic properties of pyrimidines. Piperidine and pyridine restore the original color to the reagent (Semmens, 1940) because of their basic properties (Barber and Price, 1940). Basic groups of amino acids remove SO2 from Schiff’s reagent to re-form the original fuchsin (Bhattacharya, 1954a). Salts of weak acids, such as acetates, borates, and phosphates, give a red color with Schiff’s reagent (Josephson, 1923). By using Schiff’s reagents of different sensitivities, unknown concentratiohs of ammonium acetate are said to be detectable by their color reactions (Bhattacharya and Ghose, 1954a). In general, it seems desirable that buffered Schiff’s reagents be avoided, to minimize the possibility of false reactions. The reagent has been proposed as an acid-base indicator (Bhattacharya and Ghose, 1954b), since it gives a sharp rise in color at the equivalence point. It has been suggested that, if treatment with Schiff’s reagent produces a dye whose color differs from that of the original fuchsin dye, it should be called a true reaction regardless of the nature of the reacting compound (Lison, 1932). True reactions would be produced by aldehydes, certain ketones, and some unsaturated compounds. On the other hand, when the original dye is regenerated, a “pseudoreaction” is responsible. Substances
THE CHEMISTRY OF SCHIFF’S REAGENT
33
causing such regeneration include oxidizing agents, salts of weak acids, milk alkalies, and amines. Application of heat also causes regencration of the red dye because of dissociation of sulfurous acid from the reagent. It is likely that the true reaction actually comprises reactions that differ according to the compound and the conditions involved. The final dye product(s) in each case differs from the original fuchsin. The term true Schiff reaction should be considered as referring to the formation of a new dye product (s), without implying that there is a single constant mechanism or that aldehydes are the only groups capable of reacting. A spectrophotometric analysis of the colored product should help to determine whether certain compounds give a true Schiff reaction. A brief survey of organic chemistry textbooks in regard to the specificity of Schiff’s reagent is illuminating. Although some aldehydes do not react with Schiff’s reagent and some ketones give a definite reaction, emphatic statements are made which often disregard these facts. For example, several authors state that “ketones do not react” (Moore and Hall, 1933; Kamm, 1937; Cheronis, 1943; Richter, 1947; Conant and Blatt, 1948; Adams and Johnson, 1949; Cason and Rapoport, 1950; Fieser and Fieser, 1950; McElvain, 1953; Vogel, 1956; Muldoon and Blake, 1957; Noller, 1957). Probably the most accurate statements that may be made at present are the following : 1. If a strong red-violet-to-violet or blue color develops almost immediately upon addition of a few drops of Schiff’s reagent to an aqueous solution of a substance, it is probably a simple aliphatic aldehyde and less likely a ketone. 2. If a violet or pink color develops slowly, no conclusion may be drawn, since many compounds behave this way. 3. Schiff’s reagent is not specific for aldehydes, especially in test tube studies. Fortunately, the above facts have been recognized by a few authors and are found in some texts (McGookin, 1955; Linstead and Weedon, 1956; Cheronis and Entricken, 1957). Although Schiffs reagent is not specific for aldehydes in analytic tests, the histochemical applications are much more reliable. I n the spectrum of tissue reactions in which this reagent is employed, the reactive groups of stained compounds generally have been proved to be aldehydes. In a few cases ketones have been thought to react, but the base-acid test of Oster and Mulinos (1944) distinguishes ketones from aldehydes. The use of suitable control sections also helps to detect pseudoreactions resulting from oxidation of Schiff’s reagent or from other causes.
34
FREDERICK H. KASTEN
VI. Chemical Nature of Schiff’s Reagent Hugo Schiff had been working with reactions between amines and aldehydes at the time he discovered that fuchsin solution was decolored with SO2 (Schiff, 1866, 1867a, b, c). H e may have been influenced by a knowledge of these reactions in interpreting the structure of the colorless reagent. Although he referred to the decolored solution as rosaniline sulfite, he did not consider a sulfur derivative to be an integral part of the colorless moiety. We now know that this view is incorrect. His interpretation of the reaction mechanism is shown : CHI
CHs
yy/ + ““Q%
O N H 2
HsN-
2&0 sor
+X-
NH2
NHz
F’uchsin
+ His04
Schiff’s reagent according to Schiff (1866)
Further attempts were made to study the reaction products of rosaniline and H2S03. Hantzch and Osswald (1900) obtained a white crystalline precipitate from a supersaturated solution of rosaniline that had had SOz bubbled into it. The precipitate apparently was unstable because it turned faintly red on drying. However, they believed it to be the solid fuchsin sulfurous acid since it could be redissolved in water and the solution gave a color reaction with aldehydes upon addition of SOZ. They considered the reagent to have the structure shown:
Fucbsin leucosulfonic acid: Schiff’s retqent according to Hantzch and Osswald (1900)
Since other triphenylmethane dye solutions also could be decolored with SO2, their structures also were considered to have a sulfite ester group
attached to their central carbon atom and a benzoid ring in place of the original quinone. o N Z i H 6 ) ,
O.”:o SOsH
N(CzHdr
Brilliant green leucosulfonic acid (Hantach and Osswald, 1900)
35
THE CHEMISTRY OF SCHIFF’S REAGENT
Further studies were made by Durrschnabel and Weil (NOS), using the free bases of the dyes and not their salts. They isolated several reaction products of the type obtained by Hantzch and Osswald (1900), one of which was a colorless leucosulfonic acid. Since derivatives of the free bases differed in constitution and properties from the H2SOs derivatives of the dye salts, the results were not comparable with those obtained by Hantzch and Osswald (1900). Some of the interpretations of Durrschnabel and Weil (1905) regarding the constitution of their reaction products of pararosaniline and H2S03 were disputed by Wieland and Scheuing (1921), who made a thorough study of the problem. Apparently, Durrschnabel and Weil thought they had isolated sulfinic acid derivatives (R-S02H) when actually they had isolated sulfite ester compounds (R-0-S02H) . Wieland and Scheuing also showed that the fuchsin sulfonic acid of Hantzch and Osswald (1900) did not give a color reaction with aldehydes. T o give a color reaction, this compound had to have sulfur dioxide added. Wieland and Scheuing recognized that in the decolorizing of fuchsin, one mole of sulfurous acid entered as the sulfite ester group binding to the central carbon atom. In contrast to free sulfurous acid, this group could no longer be oxidized with iodine. They found that sulfur dioxide then added to the amino group to form the N-sulfinic acid. In another step, ii excess SO2 were present, a second mole of SO2 might add to the other amino group. This sulfinated compound could not be isolated because dissociation occurred so easily that the sulfinic acid groups were removed. Wieland and Scheuing worked out the scheme of reactions for the forniation of Schiffs reagent as shown.
Pararosaniline (red solution)
PararoFaniline leucosulfonic acid (colorlw) sol lt
NHa N,N-Sulfinic acid of pararosaniline leucosulfonic acid (colorless)
N-Sulfinic acid of ararosaniline leucosulfonic mi8 (colorless): Schiff’s mgent Formation of Schiff’s reagent according to Wieland and Scheuing (1921)
36
FREDERICK H . KASTEN
Many of the discussions of this important work have not mentioned that Wieland and Scheuing (1921) also prepared the same kind of derivatives from Doebner’s violet that they prepared from pararosaniline. The two dyes are similar in that they are triphenylmethanes. Of greater importance here is the fact that each contains at least one amino group. This is discussed more fully in the section concerning Schiff-type reagents. Wieland and Scheuing showed that the N-sulfinic acid of Doebner’s violet may be produced with SO, and that this compound is also a reagent for aldehydes. Doebner’s violet, which decolors with SO2 as do pararosaniline and fuchsin, gives a deep blue-violet color with aldehydes.
+
O N E L
X-
““O-”:O Doebner’s violet
Regarding the formation of amino sulfinic acids (sulfaminic acids), Rumpf (1935) could find no evidence of the existence of these derivatives in his study of the Schiff aldehyde reaction. Recently, the problem was investigated again in vitro (Hormann et al., 1958). The results regarding the nature of the new dye formed after addition of aldehyde are at variance with those of Wieland and Scheuing (1921). This problem will be considered in the next section. There seems to be no question that the first step in the formation of Scliiff’s reagent is the introduction of sulfite ester at the central carbon atom. The loss of the quinoid structure causes the molecule to decolor. This molecule is commonly called fuchsin leucosulfonic acid. I t is unlikely that the linkage to the central carbon is a true sulfonic acid. It is probably
I
a sulfite ester linkage (-C-O-S02H)
I
; the term sulfonic acid, is often
used when sulfite ester is meant. Certainly, the concentration of SO2 in the reacting solution has a strong bearing on the subsequent chemical steps. Wieland and Scheuing indicate that one or both amino groups add sulfur dioxide, depending on the SO2 concentration. Very likely, both species of sulfinic acid derivatives may exist together under conditions of equilibrium. These interpretations have been accepted for the most part by chemists and histochemists. However, Hormann et ul. (1958) now suggest that a Mannich-type reaction occurs (see Section VII, C.).
37
T H E CHEMISTRY OF SCHIFF’S REAGENT
VII. Reaction with Aldehydes One of the early ideas about the reaction was that the original fuchsin is regenerated. It was shown by Prud’homme (1904) and by many others since that the new dye is more violet than fuchsin. The new dye also differs from fuchsin in its chemical properties (Franqois, 1916). A new dye is also formed in the Feulgen histochemical reaction (Stowell and Albers, 1943 ; Kasten, 195813). The idea of fuchsin regeneration has been generally discarded but is unfortunately still mentioned in a few textbooks.
A. CONDENSATION THEORY According to this theory, first suggested by Schiff (1866, 1867a, b, c), a union is formed between reduced rosaniline and aldehyde with elimination of water. The same compound is formed that is obtained in the absence of SOz. In other words, a Schiff-base product results that lacks sulfur in the molecule.
2RCHO +
?-
HEN=
o=c
2HI0
N4HR
Aldehyde dye product ScW-aldehyde reaction according to LSchiiT (1866; 1867a; b; c)
Urbaine (18%) also emphasized the fact that a condensation product was formed in the reaction, which was not decolored by sulfurous acid. This view was speculative and was not supported by chemical analyses. Schiff’s idea was modified somewhat by Hantzch and Osswald (1900), who studied the problem further. They obtained the leucosulfonic acids X-
X-
2RzHo
+
+ HzSOr
H&=o+
NH2
NHz
Perarosaniline leucosulfonic acid
Parrrrossniline
X-
+
+ 2HtO
HzN=
Aldehyde dye product ScW-aldehyde reaction tiecording to Hantech and Osswald (1900)
38
FREDERICK H. KASTEN
of fuchsin and related dyes and recognized that fuchsin leucosulfonic acid was a component in the reagent. They considered this colorless compound to be split by aldehydes to reform fuchsin, which underwent a condensation reaction with aldehydes. We now know the condensation theory to be incorrect, since derivatives of sulfurous acid are present in the reaction products (Franqois, 1916 ; Wieland and Scheuing, 1921).
SCHIFFREACTIONS WITH CERTAIN AROMATIC ALDEHYDES B. ABNORMAL It is known that some aromatic aldehydes give yellow precipitates with Schiff’s reagent in place of the usual violet soluble dye. This phenomenon was studied extensively and was found generally with o-hydroxy aromatic aldehydes (Shoesmith et al., 1927). The reactions involved in the production of these yellow insoluble products differed from the usual reactions obtained with other aldehydes. Apparently, an addition reaction was involved in which sulfurous acid acted as a catalyst. Similar results were obtained when hydrochloric acid was substituted for sulfurous acid.
Pararosaniline leucosulfonic acid
Yellow precipitate Schfl-aldehyde reaction with certain aromatic aldehydes
In the Schiff -aldehyde reaction with aromatic aldehydes given here (see the formula) the sulfinated Schiff’s reagent is not shown. According to Shoesmith et al. (1927), the sulfonic acid derivative reacts when low concentrations of SO, are present. Under conditions of high concentration of SO,, the Wieland and Scheuing (1921) scheme of reactions occurs. Accordingly, at certain intermediate concentrations of SO2 some red-violet dye is produced, as well as yellow precipitate. It is not clear why these
THE CHEMISTRY OF SCHIFF’S REAGENT
39
particular aldehydes behave this way, especially since certain exceptional o-hydroxy aromatic aldehydes do not give this reaction. C. ALKYL-SULFONIC ACIDTHEORY This theory has been offered in various forms, first by Damianovitch (1910) and later by Bucherer (1921), Rumpf (1935), and Hormann et al. (1958). The common idea presented in the work of these investigators is that the final aldehyde-dye reaction product has the structure R
I
(-NH-CH-SO,H)
2
where R-CH is the alkyl component of the aldehyde. It is seen that the aromatic amine is attached to an alkyl sulfonic acid. In the theory of Wieland and Scheuing (1921), the structure attached to the dye is R -NH-SO*-CH
/
1
OH
Since these views have not been dealt with to any extent in the recent histochemical literature, it seems desirable to present the work and a fuller description of the reaction sequences. The work of Damianovitch (1910) and Bucherer (1921) contains some errors. For example, Damianovitch assumed that the first step in the Schiff -aldehyde reaction was the formation of aldehyde bisulfite addition product which he thought reacted with the aromatic amino group to give the structure R
I
-NH-CH-SOSH
It was later shown by Wieland and Scheuing (1921) that the aldehyde bisulfite addition product does not give the dye upon the reaction with Schiff’s reagent. Bucherer ( 1921) correctly stated that fuchsin leucosulfonic acid is the first product formed from fuchsin and sulfurous acid. HOWever, he then postulated that sulfite ester is lost from the central carbon and is replaced by OH to give a carbinol compound. There is no evidence to support the idea of carbinol compounds’ being present as intermediate products. Although his formulation is unacceptable, it is given here in its entirety, because the final dye product contains aldehyde bisulfite. In excess aldehyde and sulfurous acid, the reaction shown below with one amino group is repeated for other amino groups on the molecule.
FREDERICK H. KASTEN
CHOH t
so,
0 7 N-
HsN=
-401H
O=q-JH*
Schiff reaction with formaldehyde according to Bucherer (1921)
Rumpf (1935) took issue with some of the conclusions of Wieland and Scheuing ( 1921) , chiefly because he could find no evidence of the existence of amino sulfinic acids and had concluded that they do not play a role in the Schiff reaction. The basis for this statement, however, rests on work with simpler aromatic aniines than fuchsin. The sweeping statements of Rumpf seem to this reviewer unjustified when the facts are considered. On the other hand, Rumpf presented the view that strong acid complexes result from combining aromatic primary or secondary aniines with formaldehyde and sulfurous acid. Some of these complexes were found to be aminoalkylsulfonic acids&*NH-CH2-SOsH ) . Rumpf took the view that the same kind of reac(lon also occurs between Schiff's reagent and formaldehyde and probably all aldehydes. Similar views of the nature of the reaction were presented by Horniann et a1 (1958). Another point raised by both Rumpf (1935) and Horniann et al (1958) concerned the interpretation of color changes in the Schiff -aldehyde reaction. They explained these color changes on the basis of ionization phenomena, and disputed the quinoid theory. Their idea has been considered by others, especially in relation to the theory of color (Steiglitz, 1903). Simply stated, color in the triphenylmethane derivatives depends on the tendency of the negative group on the central carbon to ionize (Porter et al.. 1927).
41
THE CHEMISTRY OF SCHIFF'S REAGENT
Halogens tend to ionize more than do -OH or -SOSH groups. Ionization is affected by solvents and by substituents in the phenyl groups. It was proposed that -NH-CHR-SOSH groups are formed when formaldehyde is treated with fuchsin in the presence of SO2 in a Mannichtype reaction (Runipf, 1935 ; Hormann et al., 1958). They thought that
/
/
CHs
O'E--$-SO,H
H ' 0SOiH 7 C < N-C--SOaH oHI CH: \I
+ HI% Aldehyde dye product Interpretation of formation of SchiiT's reagent and reaction wit.hformaldehyde according to H6rmann el d. (1958)
42
FREDERICK H. KASTEN
when two of the three amino groups are substituted, SO2 dissociates from the central carbon as S03H-, leaving a carbonium ion. This ion is in inesomeric equilibrium with the quinone structure. Although the ideas expressed above regarding color production are of theoretical interest, the end result in the above scheme and in the Wieland and Scheuing scheme is a colored quinone structure. What does seem to be significant is the view that alkylsulfonic acids are attached to amino groups. There are also new data obtained on the kinetics of the reaction, a subject which is discussed in another section. The scheme of reactions shown requires that two amino groups be substituted to obtain a color product with formaldehyde (Hormann et al., 1958). The fact that more than one violet product can be demonstrated by spectrophotometry (Bhattacharya, 1954b ; Elftman, 1959a) and by paper chromatography (Kasten, unpublished observations) does not support this view.
D. WIELANDA N D SCHEUING THEORY Extensive analytic studies have been reported by Wieland and Scheuing ( 1921), who recognized the complex nature of the aldehyde reaction with Schiff’s reagent and made valuable contributions. Fortunately, their studies were carried out with acetaldehyde, which is a more typical aldehyde than formaldehyde. The chemical sequences involved in the production of the aldehyde-dye compound involve a number of equilibrium reactions, some of which lead to colorless products. There are basically three different series of reactions that may be in progress during the Schiff-aldehyde test. Each reaction series is related to the others, but has been drawn separately here, to simplify the presentation. Although most of the reactions shown are taken from the work of \Vieland and Scheuing, some other intermediate steps have been added to complete the picture. Som . recent work by Bhattacharya (1954b), Elftman ( 1959a), and Kasten fihpublished) provides evidence of the production of more than one dye as a reaction product. This has been incorporated into the scheme. Other reactions not pictured possibly occur, depending on variations in pH, presence of mineral acids, and different concentrations of reactants. According to the reactions illustrated, a complex mechanism is involved. Sufficient SOz must be present to allow the N-sulfinic acid derivative of pararosaniline leucosulfonic acid to be formed. Pararosaniline leucosulfonic acid itself will not &?act with aldehyde to give a color reaction. If a great excess of SO2 is present, however, it may react directly with aldehyde to
T H E CHEMISTRY OF SCHIFF’S REAGENT
43
give aldehyde bisulfite product (reaction b) . This compound is colorless and will reduce the over-all color intensity to the extent that aldehyde bound here is not free to react with Schiff’s reagent. In addition, aldehyde sulfinic acid may detach from the pararosaniline moiety. In this case, color is also reduced. This accounts for the fact that many Schiff-aldehyde dye products are unstable and lose color after standing several hours. Formaldehyde is the exception here. In the Feulgen cytochemical reaction, the color product is attached to an insoluble aldehyde moiety (DNA) and does not fade (Stowell, 1945 ; Ris and Mirsky, 1949). Aldehyde sulfinic acid presumably also may be lost from various intermediate products shown in the scheme of reactions, but is illustrated as being detached from one compound, the unstable colorless product. According to Wieland and Scheuing, aldehyde sulfinic acid does not give a color reaction in the presence of parafuchsin leucosulfonic acid. The aldehyde and sulfinic acid must be freed from combination to give the color reaction. When aldehyde sulfinic acid is formed in the two cases described above, the sensitivity of the aldehyde reaction is reduced. Although excess SO, diminishes sensitivity from this sandpoint, it also has an opposite effect in favoring the formation of N-sulfinic acid groups. An excess of aldehyde favors complete reaction with Schiff’s reagent, even when aldehyde sulfinic acid also is being formed. The genuine dye of the aldehyde reaction is water-soluble and, according to Wieland and Scheuing ( 1921), consists of fuchsin leucosulfonic acid, in which both amino groups carry aldehyde sulfinic acid groups. In such a complex series of reactions, it is possible that a mixture of aldehyde compounds is formed, in which one or both amino groups are involved. It was mentioned earlier that by paper chromatography it has been possible to separate at least two different violet dye products as well as the original fuchsin (Kasten, unpublished observations). In this experiment involving formaldehyde and Schiff’s reagent, fuchsin was detected only at the beginning of the reaction. It is possible that some of the aldehyde Fndenses directly with fuchsin in a Schiff-base reaction but it is more likely that the two violet dyes represent the fully reacted and the partly reacted sulfinic aldehyde dye products. By spectrophotometric analysis, Bhattacharya (1954b) also detected two violet dyes formed in the acetaldehyde-Schiff reaction. By using S36-labeled Schiff‘s reagent, it has been possible to detect as many as six compounds with formaldehyde, depending on the proportion of aldehyde to Schiff’s reagent and on the concentration of SOz in the reagent (Barka, 1959; Barka and Ornstein, 1959). The further use of spectrophotometry, chromatography, electrophoresis, and a radioactive Schiff’s reagent may elucidate these complex series of reactions.
44
FREDERICK H. KASTEN
The addition of aldehydes to the colorless aniinosulfinic acid compounds renders the molecules unstable. As a result, sulfurous acid is lost from the compounds to re-form colored quinoid structures. The aldehyde-dye compounds formed differ from the original fuchsin. Since the color is more violet or blue than the original red of fuchsin, the absorption curve is shifted accordingly. The fact that more than one dye is produced as a reaction product may lead to difficulties when attempts are made to quantitate aldehyde reactions by measuring color intensities. In most cases where this has been studied in vitro, especially with aliphatic aldehydes, it has been recognized that there is no simple relationship between color intensity and aldehyde concentration (Mohler, 1891). On the other hand, aldehydes vary in their reaction mechanisms with Schiff’s reagent, as was demonstrated by Horniann et al. (1958). They found that with formal-
Aldehyde bisulfite addition product (colorless)
+XNH, Pararosmiline leucosulfonic mid
Schiffs reagent RCHO
lt
RCHO Jt
45
THE CHEMISTRY OF SCHIFF'S REAGENT
U
I
OH Unstable colorless product
Single aldehyde dye product of N,N-~ulfinic acid derivative of pararoeaniline
+X-
0 . H .
HaNo7c:0 SOaH
NHI
Pararosaniline leucosulfonic acid
+2
[I1 R-,-SOaH
I
OH Double aldehyde dye product of N,N-sulfinic acid derivative of pararosaniline
Mechanism of Schiff-aldehyde reaction based partly on work of Wieland and Scheuing (1921); other reactions included to account for more than one colored product
dehyde, the amount of dye formed is proportional to the square of the aldehyde concentration. In the case of the polyaldehyde of starch which has been oxidized with periodate, there is a linear relationship. Similar findings have been reported for DNA polyaldehyde in the Feulgen reaction in vitro (Widstrom, 1928 ; Caspersson, 1932 ; Lessler, 1951) and in stained nuclei (Swift, 1955). Further discussion of these cytochemical aspects is found in another section. The chemical analyses carried out by Wieland and Scheuing (1921) indicated that aminosulfinic groups on the decolored dye molecule are the reactive sites for acetaldehyde in the Schiff reaction.
46
FREDERICK H. KASTEN
VIII. Development of Schiff-Type Reagents The term Schiff-type reagents is used for dyes other than basic fuchsin that react specifically with aldehydes in the presence of sulfurous acid (Kasten, 1958a, 1959). These reagents resemble Schiff’s reagent in their specific aldehyde reactivity, but are not necessarily colorless like the true Schiff’s reagent. Their reaction mechanism with aldehydes is probably the same basically as that which occurs between Schiff’s reagent and aldehydes, since they all require an amino group (Kasten, 1959). Over 60 years ago a stormy controversy took place between Cazeneuve (1896, 1897) and Lefevre (1896, 1897) on the question of whether acid fuchsin, or fuchsin S as it was known then, behaved as an aldehyde reagent in sulfurous acid solution. Lefevre emphasized the similarities between basic fuchsin and its sulfonated derivative, acid fuchsin, in the Schiffaldehyde reaction. On the o t h e r m n d , Cazeneuve was struck with the difficulty in decoloring acid fuchsin with sulfurous acid and the relative insensitivity of this reagent compared with that of the basic fuchsin reagent. Recent spectrophotometric and cytochemical studies carried out with the acid fuchsin-SO2 reagent (Kasten, 1956a) demonstrate that the dye certainly decolors, although not always easily, and that formaldehyde produces a new violet dye with this reagent just as with the conventional reagent. Some absorption curves illustrating these changes are shown in Fig. 6. The acid fuchsin reagent also stains DNA in cell nuclei in the Feulgen reaction. A dye known as diazofuchsin was shown to decolor with sulfurous acid and to behave as an aldehyde reagent (Prud’homme, 1904). A variety of aromatic aldehydes gave a blue color reaction with this reagent (Noelting, 1904). The dye is not mentioned in the Colour Index (1958) and its chemical constitution is uncertain. I n the extensive study of Wieland and Scheuing (1921) a second Schifftype reagent is mentioned, Doe ner’s violet. This dye is a triphenylmethane derivative and decolors in sul ’bus acid. The reagent is shown to behave in the same way as Schiff’s reagent, giving a blue-violet color with aldehydes. There seems to have been no use made of this reagent in studies undertaken since 1921. The chemical structure of this dye was shown in Section VI. I n 1948 Ostergren reported briefly that several other dyes could replace basic fuchsin in Schiffs reagent in the Feulgen cytochemical reaction. He found that treatment of brilliant cresyl blue, indoin blue, neutral red, safranine 0, and thionine with sulfurous acid produced a reagent that gave specific nuclear staining following the usual Feulgen hydrolysis of tissues.
2
THE CHEMISTRY OF SCHIFF’S REAGENT
47
Ostergren predicted that toluidine blue 0 also should substitute for basic fuchsin, and this has been confirmed (Kasten, 1958a). Azure A also was added to this list of potentially reactive dyes (Atwood and Ornstein, 1949). Thionine-SOz was used for staining microorganisms (DeLamater, 1951). The specificity of this reagent for nuclear structures in bacteria was questioned (Bisset, 1953). Further studies are indicated to sustain such criticism, although it seems likely that the technique is a valid one. A double-staining technique for Chlamydomonas was devised which involves the use of azure A-S02 (DeLamater et d., 1955). It is claimed that free aldehydes in. the cell membrane and DNA are stained. 100
J
so
-
so
-
X Emox. 40
-
wAVELENOTH IN Mu.
FIG.6. Absorption curves of solutions of acid fuchsin (-)
and formaldehyde-reacted acid fuchsin-SOz (-----). The curve is shifted to a higher wavelength, just as with the conventional Schiff’s reagent. Data from Kasten (19%).
Unaware initially of the reports cited above, Kasten (1956a, 1958a, 1959) made an extensive survey of dyes to find new Schiff-type reagents, to e m o n s t r a t e by standard cytochemical method? the aldehyde specificity of such reagents, to utilize these colorimetric reagents in multiple-staining techniques, and to use such reagents to study cytochemical polyaldehyde reaction mechanisms. Several dozen new Schiff-type reagents were found in a survey of over 400 different dyes. Table VII gives a list of important Schiff-type reagents and some of their properties. The specificity of these diverse colored reagents was proved by a variety of tests summarized in Table VIII. The different Schiff-type reagents are not associated with any one dye class. The more than 40-dye reagents available for cytochemical studies
P 00
TABLE VII OF DYESTHATARE OF IMPORTANCE AS SCHIFF-TYPE CHARACTERISTICS REAGENTS Color of
Dye Acridine brown Acridine yellow Acriflavine Auramine 0 Azure A Bismarck brown R Bismarck brown Y Brilliant cresyl blue Chrysoidine R Chrysoidine Y Chrysophosphine 2G Coriphosphine 0 Cresyl violet Flavophosphine N Neutral red Neutral violet Phenosafranin
C.I. no.
Dye class
-
Acridine Acridine Acridine Diphenyl-methane Thiazine Disazo Disazo Oxazine Monoazo Monoazo Acridine Acridine Oxazine Acridine Azine Azine Azine
46025 46OOo
41000 52005 21010 21000 51010 11320 11270 46040 46020
-
46065 50040 50030 50200
?
Primary amine groups
1,2 2 2 1 1 2b 2b 2 1 1 2 1 1
2 1 1
2
Spectrophotometric data (mp)
stained aldehyde moiety
U.V. peaka
Visible peak"
Broa' ' Yellow Yellow Yellow Blue Brown Brown Blue Orange Orange Yellow Yellow Violet Yellow Brown-red Red Red
267 264 261 249 284 283 276 236 278 276 265 265 268 266 275 276 275
458 442 445 430 625
447 435-455 615 450 440 432 453 580 443 515 558 515
Cyto-
Feulgen chemical stained fluorescence cells detectable
455 465
-
595 465 455 605 460 -
-
+ + + -
-
-
+ ++ -
500 540
-
450 570 450
-
a Spectrophotometric data obtained at concentrations of 0.05 to 0.20 mg. dye/l. water in a 1-cm. cuvette in Beckman DKI recording spectrophotometer. h Bismarck brown has four primary amine groups. Commercial preparations are considered to be dihydrochloride, which leaves two amine groups per molecule unattached to hydrochlorides. c Although many batches of pyronin are potentially reactive Schiffs-type reagents, only one batch was found whose reactive dye component is also fluorescent in cytochemical studies ; this one was pyronin B from Chroma-Gesellschaft.
3:
TABLE VII (contirzued)
Color of
Dye
C.I. no.
Dye class
Phosphine 5G Phosphine GN Proflavine Pyronin B Rheonin AL Rhodamine 3 G 0 Safranin 0 Spirit blue Thionin Toluidine blue 0
45210 50240 42775 52000 52040
Acridine Acridine Acridine Xanthene Acridine Xanthene Azine Triaryl-methane Thiazine Thiazine
Typogen brown
11285
Monoazo
46035 46045 46OOo 45010 46075
Primary amine groups 2 2
2
0
1
1 2 40
2 1 1
stained aldehyde moiety Yellow Yellow Yellow Red Yellow Red Red Blue Blue Blue to blue-green Brown
Spectrophotometric data (mp)
U.V. peak5
245 276 285 283 278
Visible peak5 575 441 441 548 440 550 514 576 598 636
242
449
267 262 260 256 266
cyto-
Feulgen chemical stained fluorescence cells detectable
550 530
++ + + + -
615 605
-
-
-
450 470
-
-
+e
50
FREDERICK H. KASTEN
TABLE VIII EVIDENCE FOR SPECIFICITY OF SCHIFF-TYPE REAGENTS FOR TISSUEPOLYALDEHYDES 1. 2. 3. 4. 5.
6.
7. 8. 9. 10.
Specific staining of structures known to contain potential aldehyde groups. Nonspecific staining after omission of essential aldehyde-producing step. Sulfur dioxide required in dye solutions for positive Feulgen and PAS reactions. Identical optimal hydrolysis times for all reagents in Feulgen technique. Reaction with aldehyde-blocking reagents (hydroxylamine thiosemicarbazide, phenylhydrazine) Reactivity and blockade with formaldehyde. Extraction procedures (trichloroacetic acid, perchloric acid). Use of enzymes to check staining specificity (DNAase, diastase, RNAase, pepsin). Positive film tests. Dye reagents all have at least one amino group as a reactive site.
.
have their color peaks throughout the visible spectrum. Absorption curves of some of these dyes are shown in Figs. 7 and 8. Absorption curves from stained nuclei (Feulgen) and other structures (PAS) are shown in Figs. 9 and 10. Shifts in the absorption p a k may be detected in different polysaccharides stained in the PAS reaction. This is shown in Fig. 11 with phenosafranin-SOz as the reactive dye reagent. Some Schiff-type reagents may prove to be better suited for quantative measurements of FeulgenDNA than the conventional Schiff’s reagent. Studies are in progress to analyze the quantitative possibilities of the new reagents (Kasten and Aviles ; unpublished). Some cytophotometric data are shown in Figs. 12 and 13. The spread in DNA values seems to be greater with the reagents shown than with Schiffs reagent, although phenosafranin gives a good distribution. Many of the Schiff-type reagents are highly fluorescent (Kasten rt al., 1959) ; fluorescence emission spectra of some of the untreated dye solutions are shown in Fig. 14. Tissue polyaldehydes stained with many of the fluorescent reagents are easily visible in the fluorescence microscope. Auramine O-SOz emits a particularly intense green-yellow fluorescence from stained nuclei (Feulgen), even when an AH4 100-watt low-pressure mercury lamp is used as a s o w of ultra violet light. Various polysaccharide moieties often emit different fluorescent colors when stained with the same fluorescent reagent. This may facilitate detection of particular polysaccharides in tissue sections, In addition, detection of low concentrations of polyaldehydes is made possible by the fluorescence approach. It has . been calculated that 100 molecules of acriflavine may be detected in a square micron using an Osram HBO 500 mercury arc (Ornstein et aZ., 1957). It may be possible to detect DNA in the mature oocytes of sea urchins by this technique without modifying the Feulgen reaction (Burgos, 1955). Where abnormally high concentrations of DNA are present, as in tumor cell nuclei, intense fluorescence results after Feulgen staining.
THE CHEMISTRY OF SCHIFF’S REAGENT
51
Photomicrographs are shown in Fig. 15 A and B of biological material stained with fluorescent Schiff -type reagents in the Feulgen and PAS
WAVELENGTH IN
&
FIG.7. Absorption curves of solutions of potential Schiff-type reagents before treatment with SO,. Concentrations are 10 mg. per liter. Data obtained with Beckman DK-1 recording spectrophotometer. Solid line curve : acriflavine HCl ; dotted line curve: azure A.
WAVELENGTH IN
MJL
FIG.8. Absorption curves of a few potential Schiff-type reagents. Data obtained as in Fig. 7. Key: , cresyl violet; - - - -, phenosafranin ; ----, chrysoidine R.
--
52
FREDERICK H. KASTEN
A
FIG.9. Transmission curves of nuclei stained in Feulgen reaction by thionineSO,. Data obtained with automatic recording microspectrophotometer. Curve A : onion root tip nucleus ; curve B : frog liver nucleus.
FIG.10. Tracings illustrating the precision of the double-beam ratio-recording microspectrophotometer. Time for each curve is about 9 minutes. Neutral redSchiff stain (Feulgen), frog liver.
THE CHEMISTRY OF SCHIFF’S REAGENT
53
A few potentially reactive dyes such as spirit blue, Magdala red, and typogen brown are insoluble in water and must be used in organic solvents. They may find special use in the staining of water-soluble polysaccharides, such as inulin and dextran. I t is uncertain at present whether their use for this purpose has any advantage over the alcoholic Schiff’s reagent of Mowry et al. (1952). It was demonstrated by Van Duijn (1956) and Himes and Moriber (1956) that the Feulgen and PAS staining techniques could be carried out on the same tissue section using two different aldehyde
- ’ 0
580 6CM
WAVELENGTH
IN
Mp
FIG. 11. Absorption curves from mouse tissues stained with phenosafranin-SOz in Feulgen and PAS reactions. Curve from goblet cells of duodenum differs significantly from the other curves. (Unpublished data of N. Aviles.) Key to curves: , PAS-goblet cells of duodenum (4 curves) ; ---, PAS-cartilage ground substance of trachea (5 curves) ; - - - -, Feulgen-liver (4 curves).
reagents. Thionine-SO2 (Feulgen) followed by Schiff’s reagent (PAS) #as used by Van Duijn (1956). This combination gives blue nuclei and a red PAS pattern. In an independent study a triple-staining technique was developed involving the above two reactions with azure A-SO2 in place of thionine-SO2 and the naphthol yellow S technique for basic proteins (Himes and Moriber, 1956). Striking staining combinations resulted in several tissues. Both azure A and thionine are thiazine dyes and give intense blue stains to tissue polyaldehydes. However, they suffer the disadvantage of staining certain acidic tissue constituents by virtue of their metachromatic properties, while DNA is stained blue in the Feulgen reaction. For quantitative DNA measurements the absorption
54
FREDERICK H. KASTEN
peak of the blue dye is not as well separated from the violet Schiff complex as one might desire. This may not cause trouble if there is definitely no overlap of stained structures and the measured wavelength selected is distant from the peak of the other dye. Other combinations of Schiff-type reagents may usefully circumvent problems indicated above. For example, many of the yellow acridine dye reagents provide good contrast with red, violet, or blue reagents. The
j I
PHENOSAFRANIN- SOe
,
.
//\
,
, -* , .
,
0
ii
BISMARCK BROWN Y-SO*
ACRIFLAVINE
Isr
SCHIkf’S
-
S%
REAQENT
20 40 60 80 100 RELATIVE AMOUNT OF DNA (FEULOEN)
FIG.12. Cytophotometric data from sections of mouse kidney stained with Schiff’s reagent and different Schiff-type reagents in the Feulgen reaction. Measurements were taken near the absorption peaks of each DNA-dye complex. Sections stained with Bismarck brown Y-SO, h y ? a great spread of D N A values. (Unpublished data of N. Aviles.)
Feulgen reaction should be carried out before the PAS reaction. Glycogen is removed by acid hydrolysis (van Duijn, 1956; Kasten, 1959). However, with some dye combinations the second dye reagent replaces the first one, so that structures stained in the Feulgen and PAS techniques all appear in one color. This phenomenon has provided information about reaction kinetics and is discussed further in another section. Table VIII lists the dye combinations that are known to give good color contrast in doublealdehyde staining reactions. Some Schiff-type reagents have added advantages over the conventional
THE CHEMISTRY OF SCHIFF’S REAGENT
55
. Schiff’s reagent in phase contrast microscopy and in autoradiography. We
recently found that certain brown reagents, such as Bismarck brown R-SOz and Bismarck brown Y-SO2, stain nuclei intensely in the Feulgen reaction. These preparations allow clearer observation of nuclear and chromosome phenomena by phase contrast microscopy than the violet stained preparations using Schiff’s reagent. Various treatments involved in processing
I
PHENOSAFRANIN- SO2
BISMARCK
I0l
BROWN Y-SOz
-
SCHIFF’S REAOENT (PARAROSANILINE S q )
RELATIVE
AMOUNT
OF
DNA (FEULOEN)
FIG.13. Cytophotometric data from sections of adult mouse liver stained as in Fig. 12. Differences are seen in distributions of polyploid classes. (Unpublished data of N. -4viles.)
autoradiographic preparations do not extract dye from nuclei stained with Schiff-type reagents. Tissues stained with the brown reagents seem easier to study in autoradiography than the conventional reagent. The possibility of using specific Schiff-type reagents in triple-aldehyde reactions should not be overlooked. The ninhydrin-Schiff and alloxanSchiff techniques for proteins (Yasuma and Ichikawa, 1953) may lend themselves to combination with the Feulgen and PAS techniques. The performic acid-Schiff and plasma1 reactions also might be looked into. Since diverse colored aldehyde-specific reagents are available, the limitations restricting multialdehyde staining sequences involve other basic considerations :
56
FREDERICK H. KASTEN
1. The treating of a tissue section for staining one aldehyde constituent should not influence the qualitative distribution of the other aldehyde constituents to be stained. 2. There should be no quantitative loss of aldehyde constituents by a previous treatment or loss of aldehyde-dye moiety by treatment later. 3. The reactive aldehyde groups should not be altered or made unavailable for staining by Schiff -type reagents.
41)
000
590
140
560
600
160
6ZO
640
WAVELENGTH IN hlp FIG.14. Fluorescence spectra of aqueous solutions of two dyes that may be used
, Coriphosphine 0, actias fluorescent Schiff-type reagents. Key to curves: vated h = 450 mp; ---, Coriphosphine 0, activated h = 270 m p ; ------, Phosphine GPN, activated 1 = 445 mp. ~
4. If two different aldehytle constituents have the same cytological distribution, their stained moieties will not be visibly distinct from one another and an intermediate color will be seen. Absorption curve analyses may be used to detect the relative proportions of the two aldehyde-dye components.
IX. Kinetics of the Schiff-Aldehyde Reaction A.
STUDIES in Vitro
Most of the early work with Schiff's reagent was concerned with the specificity of the reaction, qualitative use of the reagent, and chemical analyses of reaction products. However, as long ago as 1890 Mohler
T H E CHEMISTRY OF SCHIFF’S REAGENT
57
pointed out that the reagent could not be used quantitatively, since the intensity of color produced was not proportional to the concentration of aldehyde. The first detailed studies of this problem appear to have been those reported by Biddle ( 1913). While studying the reaction with formaldehyde, he observed that the velocity of reaction seemed proportional to the square of time. This indicated that a complex reaction was involved which was not a first-order reaction. Various workers have mentioned that formaldehyde behaves differently, in its color-forming properties with Schiff’s reagent, from other aldehydes. For example, the aldehyde test for formaldehyde is made extrasensitive by heating the reagent (Crocker, 1925). The same treatment reduces the sensitivity to most other aldehydes. In the usual Schiff reaction, color intensity reaches a maximum within a few hours and remains stable with formaldehyde but is lost with acetaldehyde (Rumpf, 1935 ; Hormann et al., 1958), butyraldehyde, isobutyraldehyde and propionaldehyde (Rumpf, 1935). It therefore does not seem necessary to expect reaction mechanisms and kinetics to be identical among different aldehydes. In addition to formaldehyde (Rumpf, 1935 ; Hormann et al., 1958), acetaldehyde and butyraldehyde (Rumpf, 1935) give at least two successive reactions with Schiff’s reagent, since the amount of dye formed is proportional to the square of the aldehyde concentration. On the other hand, with polyaldehydes produced from periodate-oxidized starch, there is a linear relation between aldehyde concentration and dye intensity&&Iormann et al., 195s). In the case of DNA in protein-free solutions, a linear relation was found to hold, when 0.5 to 2.0 mg. of hydrolyzed DNA and Schiffs reagent (Widstrom, 1928) were used. According to Caspersson (1932), the upper limit measurable is 10 mg. of DNA. The experimental conditions required for measuring DNA ilz vitro are very rigid, because such variables as pH, time of reaction, SO, concentration, presence of proteins, and time of hydrolysis exert marked effects on final color development (Widstrom, 1928 ; Caspersson, 1932 ; Shibatani, 1953a, b) . Furthermore, the intensity of color developed is a reflection of the number of aldehyde groups exposed by hydrolysis, and not a true measure of total DNA ; i.e., only a part of the nucleic acid participates in the color development. A standard DNA sample with known phosphorous content always must be used for a basis of comparison. The Feulgen reaction also has been studied by using strong acid extracts of DNA and fuchsin from Feulgen-stained cell nuclei (Shibatani, 1954), by extracting fuchsin from Feulgen-stained nucleoprotein samples (Hiraoka, 1957), and by analyzing material from Feulgen-stained bacteria (Dondero et al., 1954; MacEntee, 1959). These studies are difficult to evaluate,
58 FREDERICK H. KASTEN
w
0
FIG.15A. Photomicrographs from fluorescence microscope irradiated with AH4-100-watt mercury lamp. ( a ) Smear of bull sperm stained with auramine 0-SO, in Fedgen reaction. ( b ) Human cervical smear stained with auramine 0-SO, in
.B
Feulgen reaction. (c) Section from human omentum of patient with cancer ; fluorescence from nuclei stained with acriflavine-S02 in Feulgen reaction; some autofluorescence appears from unstained cytoplasm. ( d ) High-power magnification of portion of field from c, showing cancer cells.
FREDERICK H. KASTEN
FIG.15B. Photomicrographs from fluorescence microscope irradiated with AH4-100-watt mercury lamp. (u) Section of pig submaxillary gland stained with auramine 0-SO,in PAS reaction. ( b ) Human cervical smear stained with flavophosphine N-SO, in PAS reaction; PAS-positive droplets intensely fluorescent compared with squamous epithelial cells. (c) Squad3
THE CHEMISTRY OF SCHIFF'S REAGENT
61
preparation of salivary gland chromosomes ot ~ r o s o p h i kmcrcmogoster ~ stained with auramine 0-SO, in Feulgen reaction (unpublished work of C. Klingman and F. Kasten). ( d ) Fluorescent nuclei and chromosomes from Ehrlich-LettrC mouse ascites tumor cells stained with auramine 0-SO, in Feulgen reaction.
62
FREDERICK H . KASTEN
partly because of special techniques required to produce colored material from decolored extracts. For example, adjustments in pH were carried out (Shibatani, 1954; Hiraoka, 1957), formaldehyde was added to the extract (Dondero et al., 1954), and bisulfite was added after hydrolysis (Dondero et al., 1954 ; MacEntee, 1959). These special procedures depart from the traditional Feulgen technique and add new complications to a reaction that is still not understood. According to the Wieland and Scheuing (1921) theory of the Schiffaldehyde reaction, two aldehyde groups react with one molecule of Schiff’s reagent. Just as with aliphatic aldehydes, except formaldehyde, the development of color in vitro with DNA-polyaldehyde rises to a maximum and then falls. According to Caspersson (1932), the first part of the curve is caused by a rapid reaction with excess Schiff’s reagent. The second part of the curve results from destruction of dye by degradative reactions. Such a reaction could be the formation of aldehyde sulfinic acid from the aldehyde dye product. Wieland and Scheuing (1921) showed that this compound may appear during the reaction. Since it does not become colored, the over-all effect would be to reduce the color intensity. The compound remaining after loss of aldehyde sulfinic acid would be a leucosulfonic acid derivative of fuchsin. Probably, the initial production of color is the net effect of a series of equilibrium reactions. Excess aldehyde bvors this initial color reaction. As the reaction proceeds, the concentration& reacting aldehyde is reduced, and aldehyde sulfinic acid is liberated. One must also consider the possibility that prolonged incubation in Schiff’s reagent induces further hydrolysis of DNA. If this occurs, the molecular integrity may be destroyed, with a concomitant decrease in color intensity.
B. STUDIES OF STAINED TISSUES The two principal cytocheq&$ reactions involving Schiff’s reagent are the Feulgen reaction and the periodic acid-Schiff (PAS) reaction. Very little work has been done with either of these reactions in regard to rate of color production and factors influencing formation of colored reaction products. This is especially surprising in the case of the Feulgen reaction because of the large number of quantitative studies carried out during the past 15 years. Swift (1955) measured DNA in tetraploid mouse liver nuclei after they had been stained for various lengths of time. He found that the initial reaction was extremely *id. The amount of dye formed remained at a maximal level for a long timqbefore any decrease occurred. Since a Schiff’s
T H E CHEMISTRY OF SCHIFF’S REAGENT
63
reagent was used that contained hydrochloric acid, it is likely that the dye-aldehyde moiety was hydrolyzed slightly between 24 and 48 hours of treatment, causing some loss of dye. Hydrolysis in Schiff’s reagent is minimized by using a reagent lacking HC1 (Kasten, unpublished). It would be of interest to carry out kinetics studies on a variety of biological material. The initial reaction velocity is rapid at room temperature and at low temperatures. Staining for 2 minutes at 6’ C. produces appreciable colored product. This is illustrated in Fig. 5. It would be desirable to make kinetics studies on cells that are stained at different temperatures. Quantitation of the Feulgen reaction has been carried out using photometric methods. Valid relative data are obtained that are highly reproducible (Pollister, 1952 ; Leuchtenberger, 1954 ; Richards et al., 1956). Further stoichiometric information is needed, however, concerning the reaction. Steps in this direction were taken by Walker and Richards (1957), who obtained measurements from stained objects at 260 mp and 560 mp. Their results led to no conclusive findings of interest here, but the approach is an interesting one and may be expected to furnish needed information. Ordinarily, tissues stained by Schiff’s reagent in the Feulgen reaction retain all the chemically-bound dye during subsequent washing, dehydration, and clearing processes. However, it is possible to displace some or all of the dye from reactive sites and bind a new dye at these sites (Kasten, 1%0b). These changes are easily visible if the two dye reagents are of contrasting colors. For example, a section of mouse duodenum was stained in the Feulgen reaction using a standard Schiff’s reagent. After normal washing in SO2 water, water, and 70% ethanol, the same section was restained in acriflavine-SO2 for 30 minutes. This was followed by routine washing and dehydration. However, after the section was mounted it was found to have a brown-yellow color instead of the expected violet color in nuclei. Acriflavine-SO2 ordinarily stains hydrolyzed nuclei yellow in a regular Feulgen reaction. To ensure proper interpretation of these anonialous results, suitable control sections were used. It was found that all wactive aldehyde groups were stained by dye reagent 1 before dye reagent 2 had been introduced. Moreover, no new reactive aldehyde groups were produced during incubation in dye reagent 2. The most reasonable interpretation is that some of the Schiff chromophore is displaced from the aldehyde and replaced by the acriflavine reagent. When the order of staining is reversed in the above experiment, so that reagent 1 is acriflavine-SO2 and reagent 2 is Schiff’s reagent, the nuclear color remains yellow. No replacement occurs in the reverse sequence. A competition for the aldehyde sites exists between the two reagents, but the latter are not equally matched. The equilibrium constants apparently
64
FREDERICK H. KASTEN
differ sufficiently to allow the remarkable results mentioned above. An illustration of these schemes is given for single and multiple reactions.
DYE B-%
DYE B-SOd71
POLYALDEHYDE
4
I
L k y ? L W E C POLYALDEHYD - ~ DNA
Representation of separate reactions between DNA-polyaldehyde and various Schiff type reagents. Each reaction involves a different equilibrium constant.
-
piq POLYALDEHYD
-'*
,-,
Representation of replacement phenomena in sequential staining reactions.
Further data and discussion of this important problem have been given elsewhere (Kasten, 1960b). The significance of these findings must await complete analyses. It is apparent that the insoluble Feulgen-DNA dye complex is capable of further reaction at the aldehyde site, so that the previously bound dye is released. The DNA dye product is not inert in the proper environment. L.,,.
T H E CHEMISTRY OF
SCHIFF’SREAGENT
65
X. Applications of the Reagent to Cytochemistry The application of Schiff’s reagent in the cytological detection of chemical moieties was carried out by Feulgen and his collaborators with great thoroughness. Milovidov (1949) lists about 35 of their publications, beginning with some early chemical studies of Feulgen (1912). The scientific career of Feulgen was intimately concerned with cytochemical applications of Schiff’s reagent. His development of the nucleal reaction, which is generally called the Feulgen reaction, provided an important tool in cytological studies of deoxyribonucleic acid (DNA). His study of lipid aldehydes with the plasmal reaction was likewise important in this field. Since Schiff‘s reagent is so useful in the Feulgen and plasmal reactions, it has also been used by other investigators to study aldehyde moieties. A. USE IN FEULGEN REACTION The reaction was described by Feulgen and Rossenbeck (1924) and involved two vital steps ; the first was treatment of sections fixed in sublimateacetic acid with 1 N HC1 at 60’ C. for 4 minutes ;the second was exposure of the hydrolyzed section to Schiff’s reagent for 1-142 hours for animal material and for 3 hours for plant material. All nuclei were selectively stained by this procedure, and the newly formed dye resisted extraction by water and alcohol. Sections were placed in one or more rinses of sulfurous acid solution after staining in Schiff’s reagent. The rationale for this step was to prevent any Schiff’s reagent in the tissue from being oxidized to give a spu6ous color reaction. The acid hydrolysis procedure was shown to be essential since unhydrolyzed sections did not stain. By various tests it was established that the, stained moiety was DNA. For example, Feulgen and Rossenbeck wrote on writing paper and on glass slides with a pen dipped in a 1 or 10% solution of deoxyribonucleate. They exposed the paper and slide to hydrochloric acid and then to Schiff’s reagent under the same conditions as tissue sections. Specific staining resulted on these materials but not on the controls which were not hydrolyzed. Various other tests were carried out by these workers to confirm the cytochemical specificity of the nucleal reaction. The method became very popular among cytologists for specific staining of nuclei and of chromosomes in dividing cells. An extensive list of references was compiled by Milovidov (1949, 1954) in his Physik und Chemie des Zellkernes. Cytophotometric measurements of Feulgen-DNA in cell nuclei provided new approaches at the intracellular level to the quantitative analyses of DNA changes. The extent of these studies is indicated by review articles in this field (Swift, 1953, 1955 ; Swift and Rasch, 1956 ; Leuchtenberger, 1958 ; Pollister and Ornstein, 1959).
66
FREDERICK H . KASTEN
The theory of the reaction, according to Feulgen and Rossenbeck ( 1924), involves first the splitting of purines from DNA by mild acid hydrolysis. This frees aldehyde groups, which then react with Schiff’s reagent to give a violet color. The production of aldehydes in the initial step seems a well founded fact. Aldehyde-blocking reagents such as hydroxylaniine and semicarbazide (Lessler, 1951) inhibit the color reaction after sections are hydrolyzed. When the aldehyde moiety was reduced to an alcohol, the color reaction was also blocked (Lhotka and Davenport, 1951). It was shown by in vitro experiments that 2-deoxy-~-ribosewill react in its aldehyde form with Schiff’s reagent (Overend, 1950). The intensity of color increases when the pyranose form of the sugar is inhibited. Acid hydrolysis ruptures the glycosidic bonds between deoxyribose and purines of DNA. Consequently, deoxyribofuranose components are converted to aldehydes (Overend and Stacey, 1949; Brown and Lythgoe, 1950). Theoretically, an aldehyde linkage may be present on each nucleotide after hydrolysis. With a molecular weight of 8 million for DNA, there are as many as 24,000aldehyde sites per DNA molecule. Since all sites may not be freely accessible, the figure represents an upper limit. Furthermore, all glycosidic bonds may not be cleaved under a given set of conditions to give aldehyde residues. The sugars remain attached to the main DNA chain through phosphate linkages. The molecular weight and structural configuration of the hydrolyzed DNA molecule may be altered, but the evidence supports the idea that DNA for the most part remains insoluble. With excessive hydrolysis pyrimidines are liberated, a h a soluble aldehyde, y-hydroxylevulaldehyde, may appear (Stacey et al., 1946 ; Overend, 1950). There are a few reports that by optimal hydrolysis time for Feulgen staining considerable DNA is lost (Shibatani and Fukuda, 1953 ; Woods, 1957). Further studies have not supported these views (Savage and Plaut, 1958; Taylor, 1958). According to Lee and Peacocke (1952), very mild acid or alkali treatment of DNA (0.025 N foGJO seconds at room temperature) hydrolyzes the C1 linkage between de6xyribose and phosphate in a small number of labile linkages. Some straight chain sugars result with exposed aldehydes, which couple with Schiff‘s reagent, to give a color reaction. This is not the usual hydrolysis effect encountered, and has not been observed in histological sections. Other evidence, that the site of color in the Feulgen reaction is DNA, is based on experiments with deoxyribonuclease (Catcheside and Holmes, 1947) and with a Schiff-type reagent (Kasten, 1 9 5 6 ) . The possibility of other substances, contributing to the nuclear color reaction has been studied. Acid proteins were considered potential binders of Schiff’s reagent (Stedman, 195Q., but this was disproved by using an acid dye
THE CHEMISTRY OF SCHIFF’S REAGENT
67
analogue of basic fuchsin (Kasten, 1956a). It has been claimed that hydrolysis causes a false localization of DNA in plant cells (Chayen and Norris, 1953). The Feulgen reaction in Acanthanzoeba and Tetrahynzena is inhibited by uranyl or ferric ions (Cerroni and Neff, 1959). The second step of the reaction involves exposure of hydrolyzed tissues to Schiff’s reagent. The chemical basis for this reaction has been considered to agree with the Wieland and Scheuing (1921) theory (Swift, 1955; Walker and Richards, 1959; Lessler, 1953 ; Leuchtenberger, 1958). This theory was based on chemical studies of the reaction between acetaldehyde and Schiffs reagent. Since the theory was discussed in detail earlier, it seems necessary to make only a few pertinent points. Formaldehyde differs from acetaldehyde in the reaction with Schiff’s reagent. For example, the color with formaldehyde is stable and even increases with time (Hormann et al., 1958), while the color with acetaldehyde fades (Rumpf, 1935). The test with formaldehyde is more sensitive than that with acetaldehyde (Feigl, 1956). There are marked differences in the limits of sensitivity with various aldehydes (Crocker, 1925). The aliphatic aldehydes differ from the polyaldehydes of complex compounds in the Schiff reaction. In the case of formaldehyde, the amount of dye formed is proportional to the square of the aldehyde concentration (Hormann et al., 1958), while the color with the polyaldehyde of hydrolyzed DNA is directly proportional to the DNA concentration up to 1.0% (Caspersson, 1932). Other polymeric polyaldehydes also given a linear color response with concentration, viz., periodate-oxidized hyaluronic acid (Jorpes et al., 1948), glycogen (Hooghwinkel and Smits, 1957), and starch (Hormann et al., 1958). Although the Wieland and Scheuing theory explains many phenomena observed in Schiff-aldehyde reactions, the fact remains that the theory is based on chemical analyses involving a relatively simple aliphatic aldehyde. Acetaldehyde is more representative of aliphatic aldehydes than is formaldehyde, but is still not an adequate substitute for a complex polyaldehyde such as hydrolyzed DNA. The theory of Rumpf (1935) and Horman et al. (1958) regarding binding of alkyl sulfonic acids by fuchsin leucosulfonic acid has not been sufficiently examined. Some kinetic studies were carried out with the polyaldehyde of hydrolyzed DNA (Caspersson, 1932 ;Widstrom, 1928; Lee and Peaco&e, 1952). It was shown that the reaction is complex and involves at least two separate reactions (Caspersson, 1932). There have been no chemical studies using DNA-polyaldehyde, such as were carried out by Wieland and Scheuing. The complexity of the aldehyde moiety may preclude such studies. I t is not known to what extent DNA-polyaldehyde-sulfinic acid is formed, although it was assumed that this colorless complex accounted
68
FREDERICK H . KASTEN
for the degradative process in vitro (Caspersson, 1932). It is not known how many different color products are formed in the Feulgen reaction. It has been claimed that in the aliphatic aldehyde reaction two moles of aldehyde must bind to one mole of reagent before the new dye is formed (Wieland and Scheuing, 1921 ; Hormann et al., 1958). It was mentioned earlier that more than one violet product is detectable by spectrophotometry ( Bhattacharya, 1954b ; Elftman, 1959a) and paper chromatography (Kasten, unpublished). This indicates that the monosubstituted reagent also can give a colored compound, or that the aldehyde reacts directly with amine groups in a Schiff-base type of reaction (DeLamater, 1948; Arzac, 1950). It has been claimed that a trisubstituted product is favored in the reaction between Schiff’s reagent and polyaldehydes (Hormann et al., 1958). I t is difficult to see why this should occur in aqueous solutions since there is a proton on one of the three amine nitrogens. This ainine group is the salt-forming group and may behave differently from the other two amines. If pararosaniline base is used to prepare Schiff’s reagent in a nonaqueous solvent, the three amino groups are identical to one another. In this case it is likely that trisubstituted derivatives could be formed in the reaction between Schiff’s reagent and polyaldehyde moieties. Possibly the trisubstituted product results from the replacement of a second hydrogen on the amine, but there was no evidence given for this. It has been postulated that the Feulgen reaction is facilitated by the fact that the sulfur-to-sulfur distance in Schiff’s reagent is about the same as that found between alternate nuclebtides (Lessler, 1951). I t was assumed that purines are on alternate nucleotides. According to the doublehelix structure of DNA (Watson and Crick, 1953), a pair of nucleotides are hydrogen-bonded to one another by their bases, adenine to thymine or guanine to cytosine. This means there is a purine on each nucleotide plane and not on alternate nucleotides. Another criticism concerns the fact that Schiff-type reagents work in the Feulgen reaction, apparently in the same basic mechanism asethe conventional Schiff‘s reagent (Kasten, 1958a ; 1959). The number o r amino groups attached to each dye varies in each case as does the distance between sulfur atoms of polyamino reagents. Some reagents have only one reactive amino group for sulfur attachment. It therefore seems unlikely that stereochemical phenomena are involved in the manner suggested (Lessler, 1951), or that aldehydes on alternate nucleotides are the only ones that’react. Other cytochemical studies of the reaction concern the nature of the changes occurring during hydrolysis and the factors affecting them (Bauer, 1932; Di Stefano, 1948; Ris and Mirsky, 1949; Ely and Ross, 1949; Woods, 1957 ; Savage and Plaut, 1958 ; Taylor, 1958). Hydrolyzed DNA
69
THE CHEMISTRY OF SCHIFF'S REAGENT
4
u ar-P-
Su a - P 4 u
f Pyine
f Purine
1 1
h i Pyrimidine
I
Pdmidine
ar--P Pyrimidine
I !
I
I
I
I
I
Phihe
I
Rigar-P4uga-P+ugarJ,
1N HCl 80°C.
--Sugar--P--1Suga-P-
\H Pyrimidine
--
8 Pyrimidine H '
/ ! I
I
1
Su ar-
f
Pyrimidine+Purines O H
Sugar-----P-Sugar---P----Sugar Representhion of hydrolyzed DNA
II
/
8ahi'a
1reagent 4
ar----P+u rA Pyrimidine OH SO,, H
u ar-
P----Su
OHGbH H-N
+
I
I
0 \//Hcl \\
0
0 4 N-H
Pyrimidine H*N+X- Pyrimidine
I
x6
HO&-&O-H
1
OH kOaH
----Sugar---P4ugar---P--SugaAldehyde dye product
\I /
Mechanism of Feulgen reaction
Pareparations also have been studied in vitvo (Widstrom, 1928 ; Caspersson, 1932 ; Hillary, 1939 ; Lessler, 1951 ; Shibatani, 1953a, b). Other acids have been proposed as substitutes for hydrochloric acid in the hydrolysis procedure (Di Stefano, 1952; Hashim, 1953; Itikawa and Ogura, 1954; Bloch and Godman, 1955). Bromine in carbon tetrachloride was found to replace hydrochloric acid (Barka, 1956), but since bromine itself reacts with Schiff's reagent (Guareschi, 1913), obvious complications may arise. The composition of the sulfite rinse after staining affects the color intensity (Shibatani and Naora, 1952). Recently an analysis was carried out to determine the effects of diff etent sequences of washing Feulgen-stained
70
FREDERICK H. KASTEN
cells, especially the effects of the sulfite rinse and alcohol dehydration (Srinivasachar and Patau, 1959). In the conventional washing, dehydration, and mounting procedures there was no selective loss of dye from stained nuclei. However, when cells are put through this procedure once and are then demounted and brought back to SOz water, there is a differential loss of dye material from cells, even from cells in differing mitotic stages. Fixation is another factor that affects the Feulgen reaction by its influence of the hydrolysis curve (Bauer, 1932) and on the intensity of the reaction in different areas of a tissue section (Swift, 1953). A more complete discussion of the topics mentioned briefly in this section and aspects of the reaction unrelated to Schiff’s reagent may be found elsewhere (Swift, 1955 ; Casselman, 1959 ; Pearse, 1960). Although more knowledge about the basic chemistry of the Feulgen reaction must come from studies in vitro, it is not necessarily true that the results may be applied freely to the histochemical reaction. The Feulgen reaction exhibits certain noteworthy differences, depending on whether it is carried out on DNA preparations in vitro or on the fixed DNA moiety in intact cells in sections and smears. For many years proteins were considered to interfere with the Feulgen reaction. This was indicated by Widstrom (1928) and Caspersson (1932), referring to in vitro studies. Later it was found that in vitro the Feulgen absorption curve shifts with variation in protein concentration (Shibatani, 1950 ; 195313). The implication of these facts for quantitative cytochemistry necessitated critical studies of Feulgen-stained cells. It was clearly shown that the Feulgen absorption curve from stained nuclei is not affected by changes in nuclear protein content (Kasten, 1956b). It was also shown that in mouse kidney nuclei the amount of Feulgen DNA per nucleus is unaffected by removal of nuclear protein (Kasten, 1958b). In cells of varying protein content Feulgen-DNA remains constant (Swift, 1955). The color yield from Feulgen-stained cells was also unaffected by the ratios of protein to DNA (Shibatani and Naora, 1953; Sliibatani, 1954). Beer’s law is followed in stained cells in concentrations%f as much as 30% (Swift, 1955), which is 300 times higher than the optimal concentration found by Lessler (1951) in gelatin-DNA drops, and 30 times higher than that found in proteinfree solutions (Caspersson, 1932). It is apparent that in vitro studies of the Feulgen reaction cannot be relied on always to provide accurate information about the condition of stained cells. The cellular environment in fixed material is complex both chemically and physically. The intranuclear nucleoprotein complex is precipitated, and in eitEer solid or colloidal form. There is little that can be said at present about the molecular configuration of DNA in this dehy-
THE CHEMISTRY OF SCHIFF’S REAGENT
71
drated and stained state. It is unlikely that it is the extended double-helix state of the Watson-Crick model (Watson and Crick, 1953). Chemical reactions nevertheless occur at aldehyde sites of hydrolyzed DNA. The use of Schiff-type reagents to study the stability of the dye complex was mentioned earlier. Feulgen-stained nuclei are not decolored by exposure to light (Stowell, 1946 ; Ris and Mirsky, 1949), nor do their absorption spectra vary under diverse conditions, with minor exceptions (Kasten, 1956b, 1958b). There is some evidence that Feulgen-DNA differences exist between species (Lessler, 1956) and in an ascites tumor cell population (Kasten, 196Oa). With more vigorous studies and the development of new techniques perhaps the chemistry of the Feulgen reaction may be better understood at the microscopic level. Although it was correctly stated almost 20 years ago (Baker, 1942) that “Feulgen’s test has been much used but less understood,” considerable progress has been made since. The problem of the proportion of DNA bound by the Feulgen dye is an important one and needs further study (Patau and Swift, 1953). It cannot be stated with certainty that cells containing identical ‘amounts of DNA per nucleus will bind identical amounts of dye in the Feulgen reaction. Within a certain range of error the statement is probably correct according to agreement between cytophotometric data and biochemical analyses (Ris and Mirsky, 1949 ; Leuchtenberger, 1954). It is desired however, to obtain more precise information ; possibly a single amino Schiff-type reagent with one reactive site will provide a better quantitative tool than the conventional Schiff’s reagent, which has at least two reactive sites. The fact that our knowledge of the reaction is incomplete should be no deterrent in the quantitative measurements of bound dye. Evidence favoring quantitation of FeulgenDKA is summarized by Swift (1955). With proper precautions and objective ways to reduce potential errors, reliable data are obtained from Feulgen-stained cells in the same way that they are obtained from colorimetric reactions in vitro, whose chemical mechanisms are incompletely under.stood. i.. B. USEIN PLASMAL REACTION The plasma1 reaction was worked out by Feulgen and Voit (1924). They found that lipid aldehydes or plasmals could be liberated from a lipid precursor, plasmalogen, by mild treatment with mercuric chloride or dilute hydrochloric acid. Later studies by Feulgen and Behrens (1928, 1938) and Feulgen and Bersin (1939) provided evidence of the chemical nature of plasmalogen. It was shown to be an acetal phosphatide. The structure shown is a glycerophosphatide in which a fatty aldehyde is joined by an acetal linkage to two of the hydroxyl groups of glycerol. According to
72
FREDERICK H. KASTEN
Feulgen, mercuric chloride acts to split the acetal linkage to release palmital, stearal, and other higher aldehydes. These then react with Schiff’s reagent to produce a coloration, usually cytoplasmic. H
-(CHl)l&Hs
/
’
HI
$4
0-
1 Palmitic aldehyde group
NHI+ } Cholamine phosphate group
a-Palmitalplasmalogen
H
I
I I
H-(2-4 H-C--0
H I H-&OH
H ‘L(CH2)nCH8
/
H-
0
H H
I
I
I
I
HIO
1
I
0-
I
--OH
0
II
H H
“FixH-c+P--O-c--C--NH3+I --t
H-~O--lj--o--C-C-NH8+ H
A
H+
H H
I
I
I I I
H 0- H H Glywrophosphrtte of cholamine
0
+
\\C-(CHl)nCHa /
H
Phmal (stearic or palmitic) aldehyde J
x-
Schiff’s went
d’
H
I
N-ffO-C-(CHp)nCH OH I H
I
N-SO--C-(CH~)nCH~ OH Aldehyde dye product Mechanism of plasma1 reaction
a
THE CHEMISTRY OF
SCHIFF’SREAGENT
73
The technique employed by Feulgen and his collaborators of visualizing plasmalogens involved the use of unfixed sections and control sections unexposed to mercuric chloride. Other workers used formalin-fixed sections (Gerard, 1935), but this preliminary treatment has been considered to oxidize tissue lipids (Gomori, 1942) and fatty acids (Verne, 1929), thus producing Schiff-positive groups (Hayes, 1949). The preliminary oxidizing action has been referred to as a pseudoplasmal reaction (Cain, 1949). The subject is very controversial, primarily because results are strongly influenced by the techniques used. Other workers who studied the histochemistry of the plasmal reaction include Lison (1936) and Danielli (1949). The subject has been thoroughly reviewed by Pearse (1960). Although the plasmal reaction plays a relatively unimportant role in present-day histochemistry, the presence of acetal phosphatides may complicate the interpretations of other aldehyde reactions. For example, a special type of false positive staining in the Feulgen reaction which involved a lipoprotein complex has been reported (Wolman, 1954). C. USEIN
THE
BAUERAND CASELLAOXIDIZING TECHNIQUES
Chromic acid was employed by Bauer (1933) to oxidize glycogeii in tissue sections. This was followed by treatment with Schiffs reagent for a demonstration of the exposed polyaldehyde residues. Other carbohydrate components, such as thyroid colloid, also were visualized by this technique (Dempsey and Wislocki, 1947). Similar results were obtained by Casella (1942), who used potassium permanganate as the oxidizing agent. Both techniques were studied extensively by Lillie (1951b) and compared with the periodic acid-Schiff technique. The Bauer and Casella techniques did not give as intense staining of tissue components as did periodic acid. Apparently, oxidation of carbohydrates with chromic acid, permanganate, and periodic acid occurs in the same way, but the first two agents further oxidize the aldehyde residues. According to Deane et 01. (1946), there is a correlation between the optical density of glycogen in .-diver sections stained by the Bauer method and the chemical determination of glycogen. This correlation probably was a chance occurrence, since the microscopic data seem to be transmission values rather than optical densities. The irregular distribution of stained material also would be expected to create errors in measured values. The attempt at quantitation was a noteworthy one, considering the lack of knowledge at that time about potential errors in cytophotometry. The Bauer and Casella techniques are of only historical interest, since the periodic acid-Schiff technique has superseded them.
74
FREDERICK H. KASTEN
D. USEIN THE PERIODIC ACID-SCHIFF REACTION Periodic acid exerts an oxidative action on 1:2-glycols that cleaves carbon-carbon bonds (Malaprade, 1928). This action also results with compounds containing 1 : 2-hydroxyaldehydes, 1 : 2-ketols, 1 : 2 ketonealhydes, 1 : 2 diketones, and 1 : 2-amino alcohols (Jackson, 1944). Dialdehydes are produced after the periodic oxidation of 1 : 2-glycol moieties or corresponding amino derivatives. When used under proper conditions, this oxidizing agent does not further oxidize the resulting aldehydes. Periodic acid was first used on tissue sections by McManus (1946), in connection with Schiff's reagent, to demonstrate mucins. The histochemical significance of this technique was recognized in the independent studies of Lillie (1947a, b), Hotchkiss (1948), and McManus (1948). A numCHzOH
CHzOH
CHzOH
Part of a glycogen molecule
5 HI04 CHiOH
CHiOH
CHiOH
0 Schifi's
1reagent CHIOH
CHIOH
CHIOH
Aldehyde dye product
H,I!iX-
+
Mechanism of periodic acid-Schiff reactiou
THE CHEMISTRY OF SCHIFF’S REAGENT
75
ber of carbohydrate and carbohydrate-protein substances in tissues give a positive PAS reaction. These are listed in Table V. The most recent reviews of this reaction are given by Hale (1957)’ Casselman (1959), and Pearse (1960). The reaction involves two separate steps. The first is the production of dialdehydes by periodic oxidation of responsive 1 :2-glycol or related . moieties, and the second is the reaction of such aldehydes with Schiff’s reagent. It may seem logical from a stereochemical standpoint that adjacent aldehydes on a glucose residue of a polysaccharide molecule react with adjacent sulfinic groups on a single molecule of Schiff’s reagent. Although there is no evidence to support this assumption, the reaction is shown this way for convenience. The PAS technique has been modified with regard to oxidizing agent (Crippa, 1951), necessity for a reducing rinse after oxidation, solvent (Mowry et d.,1952), time of reaction, and temperature. According to recent studies (Davies, 1952; Glegg et al., 1952; Braden, 1955; Hooghwinkel and Smits, 1957; Leblond et al., 1957), the acid mucopolysaccharides, chondroitin sulfate, and hyaluronic acid are now considered to be PAS-negative. By varying the conditions of the PAS reaction for staining polysaccharides on paper, it was possible to eliminate glycogen from the reaction and to stain only galactogen (Aufsess, 1959). According to Wolman (1950, 1956), unsaturated lipids may react in the PAS reaction. The PAS reaction was applied to the staining of bacteria in milk, using,Schiff -type reagents (Moats, 1959). Studies of human kidney sections were recently reported (Ornstein et d.,1957), in which acriflavine-SO2 was used in the PAS reaction to detect low concentrations of stained material by fluorescence microscopy. Similar studies are in progress in which a variety of fluorescent Schiff-type reagents (Kasten et al., 1959) are used to detect PAS-positive substances in vertebrate tissues (Kasten and Calder ; unpublished). The histochemical reaction has been used in a semiquantitative way by ~ n n n yworkers. This involved visual estimations of dye content or concentration in microscope preparations. An extension of the semiquantitative approach involved the cytophotometric measurements of PAS-stained material in Arvelius male germ cells (Leuchtenberger and Schrader, 1950). The use of the reaction in this quantitative way has not been adequately justified. Test tube studies of starch (Hormann et al., 1958), glycogen (Hooghwinkel and Smits, 1957), and hyaluronic acid (Jorpes et al., 194.8) indicate a linear relation between concentration of periodateoxidized polysaccharides and intensity of color reaction with Schiff’s reagent. However, numerous other tests, especially at the cytological level,
76
FREDERICK H. KASTEN
need to be carried out before cytophotometry of the PAS reaction becomes an acceptable procedure. According to Pollister and Ornstein (1955) and our own work (Kasten and Aviles; unpublished), the PAS-dye complex in stained tissues varies according to the polysaccharide moiety. This type of metachromasia can only complicate attempts at quantitation. Possibly, the difficulties may still be overcome; there is an obvious need for such studies. A unique approach involving a radioactive Schiff’s reagent was carried out by Barka and Ornstein (1959). These workers found it possible to determine the relative amount of Ss6-labeled Schiff’s reagent bound to periodic acid-oxidized tissue sections. Radioactivity was measured in tissue sections with a gas-flow counter. The quantitation was complicated by the fact that some labeled Schiff’s reagent was also bound without periodic acid oxidation. The manner of producing the radioactive Schiffs reagent did not preclude the possibility that other SWabeled compounds also were present in the reacting solution. With proper control of specific activity of the reagent it seems possible that quantitation may also be carried out using autoradiographic techniques.
E. USEI N PERFORMIC ACID-AND PERACETIC ACID-SCHIFFTECHNIQUES Performic acid and peracetic acid have been used as selective oxidizing agents in histochemistry. They were used on lipids, ceroid (Lillie, 1954)’ and hair (Pearse, 1951). The test for lipids seemed to depend on the oxidation of ethylene bonds to aldehydes (Lillie, 1952), which then reacted with Schiff’s reagent. The reaction was prevented by bromination before oxidation (Lillie, 1952). Lillie’s interpretation of the action of per-acids on unsaturated lipids and ceroid was not supported by the study of Findlay (1955) involving pure substances in vitro. The disagreement apparently has not been resolved, although older studies (Verne, 1929 ; Cain, 1949) have indicated that aldehydes are the reactive groups present in oxidized lipids. It is of interest that Lison (1932) claimed that unsaturated lipids could react dir‘ealy with Schiff’s reagent. However, the per-acid reaction required oxidative treatment before exposure to Schiff’s reagent. The reactions shown were adopted from Lillie (1952) but other reactions are possible. The action of performic acid on hair cortex followed by Schiff’s reagent produced a violet color (Pearse, 1951). This reaction was considered to result from the selective oxidation of cystine composing keratin. A number of intermediate products were considered to be potential reactants with Schiff’s reagent ; these included derivatives containing sulfonic acid, sulfinic acid, and aldehyde. The sulfinic acid and aldehyde derivatives
77
THE CHEMISTRY OF SCHIFF’S REAGENT
0 R-HC=CH-R Unsaturated lipid
+ 2HCOtH
/ \
+ R-HC
Performic acid
\o’
and by rearrangement
.--)
H
2 R - L
Schll’s
CH-R or
R - H O
+ O=CH-R H
H
H
bH H
X-
reagent 4
I
N-SOz-GR I
bH Aldehyde dye product Mechanism of performic arid-Schiff reaction according to Lillie (1952)
were shown to give a Schiff-positive reaction, but Lillie and Bangle (1954) showed that an unsaturated lipid moiety probably was involved.
F. USEIN DETECTION OF PROTEINS Schiff’s reagent was employed in the ninhydrin-Schiff and alloxan-Schiff reactions of Yasuma and Ichikawa (1953). These histochemical reactions were based on the fact that ninhydrin splits a-amino acids into three compounds-aldehyde residue, carbon dioxide, and ammonia-during the first stage of the reaction. The stable aldehyde residue was then exposed to Schiff’s reagent to give a red or purple color at protein sites. This color reaction is different from that obtained in the ninhydrin reaction, as employed by biochemists. In this case the reaction produces a violet color due to a condensation of ninhydrin, ammonia, and the reduction product of ninhydrin. The histochemical test employs ninhydrin as an oxidative deaminating reagent which does not enter into the final color product, The specificity of the chemical reactions for proteins was confirmed by tests with enzymes, by comparison of results with other protein reactions, and by spot tests. Alloxan, which also is an oxidative deaminating compound, gave results identical with those of ninhydrin (Yasuma and Ichikawa, 1953). Isatin was expected to act similarly. The ninhydrin-Schiff and alloxan-Schiff reactions were studied by Burstone (1955), who confirmed the claims of Yasuma and Ichikawa (1953). a-Amino acids can also be oxidatively deaminated by compounds which release active chlorine. It was found that chloramineT may be used in the same way as ninhydrin or alloxan in a Schiff reaction for proteins (Burstone, 1955). Other compounds, such as
78
FREDERICK H. KASTEN
R-CH-COOH
I
NH2 a-amino acid
+
a72<0
+
ninhvdrin
R--CCOOH
co
AH
R-C-COOH AH
+ HnO
---t
yo + NH,
R--CH
+ Con
H
0 Schfls x2R-C-H// reagent --t H,N=o=C’
+
H
(-JLOA-R AH H
‘ a S o L bH Aldehyde dye product Mechanism of ninhydrin-Sch8 reaction according to Yasuma and Ichikan a (1953)
sodium hypochlorite and calcium hypochlorite, do not produce stable aldehydes from tissue proteins (Burstone, 1955).
G. USEIN DIRECT STAINING OF LIPIDS
It was mentioned earlier that unsaturated lipids may be stained directly by Schiff’s reagent after formalin fixation (Lison, 1932; Chu, 1950). This has been referred to as a pseudoplasmal reaction, because it may complicate the plasmal reaction and has been confused with it. Since oxidation of lipids may occur in formalin or during treatment with Schiff’s reagent, the possibility exists that a reaction similar to the plasmal or PAS may occur. Unsaturated fatty acids may contain peroxides that regenerate Schiff’s reagent. For this reason, Lison (1953) suggested that the reaction at the -C=Cgroup be r e m m i n e d . Recently a report appeared describing an ultraviolet-Schiff reaction for unsaturated lipids (Belt and Hayes, 1956). The color reaction is unstable. This is another case in which oxidation at the double bond may give rise to aldehydes.
H. RECENT USEIN MULTIPLE-STAINING REACTIONS Substances normally stained separately in the PAS and Feulgen reactions were stained simultaneously in Schiff’s reagent after exposure to 1.0%sodium bismuthate in 40% phosphoric acid for 40 minutes at room temperature (Hashim et al., 1953) or in 0.5%periodic acid in 50% phos-
79
THE CHEMISTRY OF SCHIFF'S REAGENT
phoric acid for 10 minutes at room temperature (Afifi and Acra, 1955). Both groups of polyaldehyde substances were stained essentially the same color in these concurrent techniques. They were stained different colors when a Schiff-type reagent was used as well as the conventional Schiff's reagent in sequential reactions (Himes and Moriber, 1956 ; van Duijn, 1956). The technique employed by Himes and Moriber (1956) also included a third staining reaction for basic proteins with naphthol yellow s. Other useful combinations of Schiff-type reagents are listed in Table IX. TABLE IX PAIRSOF ALDEHYDE-SPECIFIC REAGENTS" THATWEREFOUND TO GIVE POLYCHROMATIC STAINING IN THE FEIJLGEN-PAS REACTIONS (DOUBLE-ALDEHYDE) Colors of polyaldehyde moieties Dye reagent 1 (Feulgen) Acridine yellow-SO,
Acriflavine-SO,
Azure .4-S0, Basic fuchsin-SO, (Schiff's reagent) Bismarck brown R-SO, Bismarck brown Y-SO, Chrysoidine R-SO,
Chrysoidine Y-SO, Coriphosphine 0-SO, Cresyl violet-SO, Flavophosphine N-SO,
Dye reagent 2 (PAS) Basic fuchsin-SO, Pyronin B-SO, Rhodamine 3GO-S02 Safranin 0-SO, Spirit blue-SO, Azure A-SO, Brilliant cresyl blue-SO, Neutral violet-SO, Pyronin B-SO, Thionine-SO, Acridine yellow-SO, Pyronin B-SO, Chrysoidine R-SO, Chrysoidine Y-SO, Pyronin B-SO, Spirit blue-SO, Toluidine blue 0-SO, Basic fuchsin-SO, Basic fuchsin-SO, Brilliant cresyl blue-SO, Cresyl violet-SO, Basic fuchsin-SO, Azure A-SO, Safranin 0-SO, Chrysoidine R-SO, Basic fuchsin-SO, Neutral violet-SO,
Nuclei Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Blue Blue Violet Violet Brown Brown Brown Brown Orange Orange Orange Orange Yellow Yellow Violet Yellow Yellow
PAS + Structures Violet Violet Red Red Blue Blue Blue Red Violet Blue Yellow Violet Orange Orange Violet Blue Blue Violet Violet Blue Violet Violet Blue Red Orange Violet Red
80
FREDERICK H . KASTEN
TABLE IX (continued) Colors of polyaldehyde moieties Dye reagent 1 (Feulgen)
Neutral violet-SO, Phenosafranin-SO, Phosphine GN-SO, Phosphine GN-SO, Proflavine-SO, Safranin 0-SO, Spirit blue-SO, Thionine-SO,
Dye reagent 2 (PAS)
Nuclei
PAS + Structures
Phenosafranin- SO, Pyronin B-SO, Rhodamine 3GO-S02 Spirit blue-SO, Chrysoidine R-SO, Chrysoidine R-SO, Flavophosphine N-SO, Thionine-SO, Toluidine blue 040, Basic fuchsin-SO, Rhodamine 3GO-S02 Basic fuchsin-SO, Cresyl violet-SO, Safranin 0-SO, Safranin 0-SO, Phenosafranin-SO,
Yellow Yellow Yellow Yellow Red Red Red Red Red Yellow Yellow Yellow Red Blue Blue Blue
Red Violet Red Blue Orange Orange Yellow Blue Blue Violet Red Violet Violet Red Red Red
0 Dye reagents are prepared as 0.5% solutions, except for acridine dyes which are prepared as 0.1% solutions for P A S reaction, filtered, and then treated with SO, gas for 1 to 2 minutes. Tissue sections are stained in Feulgen reaction for 45 minutes, washed in SO, water, in water, stained in PAS reaction by second dye reagent for 20 minutes, washed in SO, water, debdrated in ethanol, and mounted from xylol. Spirit blue reagent is prepared in 70% ethanol. Violet color in pyronin B-SO,-stained structures results from nonfluorescent impurities in many batches of pyronin B. Dye pairs having a fluorescent reagent may also be used in fluorescence microscopy, viz., phenosafranin-SO, (Feulgen) and flavophosphine N-SO, (PAS) stained sections have brown nuclei and orange-to-yellow PAS-fluorescent structures when viewed in a fluorescence microscope.
The Feulgen and PAS reactions were used separately with Muller’s colloidal ferric oxide technique &r acidic polysaccharides (Mowry, 1958) and with alcian blue (Mowry and Morard, 1957). A combined PAStrichrome stain was reported (Lazarus, 1958), which was claimed to be more specific than Pearse’s trichrome-PAS (Pearse, 1953). The PAS reaction was combined with an alkaline phosphatase technique for demonstrating these two groups on the same tissue section (Moffat, 1958). It was recently reported that cytological details in the kidney were discerned particularly well by combining a luxol-fast blue stain with the PAS reaction (Shanklin and Nassar, 1959). The application of Schiff’s reagent and Schiff-type reagents in multiple aldehyde reactions was discussed earlier in Section VIII.
THE CHEMISTRY OF SCHIFF’S REAGENT
Sl
XI. Absorption Curve Analyses of Schiff -Polyaldehyde Binding in Situ The application of photometric techniques to stained cells brought about great progress in quantitative cytochemistry. Numerous reviews have appeared and are listed by Swift (1955). As better techniques have been developed and potential errors more fully appreciated, the cytophotometric technique (Moses, 1952) has been applied widely to biological and biochemical problems. Cytophotometry is used for intracellular studies of nucleoproteins, and provides a useful tool for analyzing the dynamics of dye-binding by polymeric chemical moieties in cytochemical reactions. Measurements of dye at several wavelengths are made easily by using a monochromator between the light source and the microscope. Several commercial instruments are available and provide light of adequate purity when strong tungsten light sources are used. Technical details regarding the proper use of such instruments and other essential components are discussed in recent reviews (Swift and Rasch, 1956; Naora, 1958). The accuracy of absorption curves from stained cells will be affected by such obvious factors as the degree of purity of light used to irradiate the specimen, the accuracy of the wavelength setting of the monochromator, the distribution of stained material in the measured field, and the density of stained material. Many of the factors involved in absorption curve analyses were studied by Kasten (1956b; 1957; 1958b). Changes in absorption curves with impure light depend on the intensity of stain. In a densely stained nucleus, the extinction value decreases and the curve shape tends to become broader. In lightly stained nuclei these changes are less marked or are absent. In critical studies regarding the nature of stained dye complexes, it is certainly desirable to use the narrowest slit width possible that will still allow sufficient light in the system for detectable and accurate measurements. A full band width of 15 mp or less seems desirable for such absorption curve analyses. It is relatively simple to check the wavelength setting of a monochromator if a mercury lamp is available. The emission lines at a few wavelengths, such as 491.6 and 546 mp, should correspond with the wavelength setting of the monochromator when narrow slits are used (Mellon, 1950). Even with a new instrument, the writer has found the wavelength setting to be as much as 5 mp in error. Accurate absorption curves are best obtained from stained cells with homogeneous dye distributions. With theoretical approaches, Ornstein (1952) and Patau (1952) showed the errors caused by neglecting this factor. In practice, these errors are made with material that is difficult to measure, such as fragmented nuclei, nuclei with abnormal
82
FREDERICK H. KASTEN
shapes, and chromosomes in mitosis. The effect on the absorption curve is to broaden it and to reduce the extinction values, especially near the absorption peak. Clumps of chromatin in cells of ordinary paraffin sections may appear to be poor material for photometric curve analyses, but the clumps themselves may be distributed at random. If the measured region is representative of this population of clumps, the data obtained will give an accurate picture of spectral light absorption by the DNA-dye moiety. In practice, this seems to be true with formalin-fixed material. The problem was analyzed using Feulgen-stained nuclei of mouse liver cells (Kasten, 1958b). In one case, nuclei were isolated in cold sucrose before fixing in formalin, to obtain a very homogeneous distribution of chromatin. In the other case, curves were obtained from nuclei in paraffin sections of liver tissue. Resulting curves were almost identical with one another, although the clumping was obvious in paraffin sections. However, the measured amount of dye per nucleus was influenced by the clumping and varied with the core region measured. The Feulgen-DNA absorption curve both in vitro and in stained cells has been studied by various workers. In one case, all the reacting fluids involved in color production were present, such as Schiffs reagent, acid hydrolysis solution, DNA, buffer (if used), newly formed dye, and all intermediate reacting compounds. In the other case, all reactants were washed out of the tissue except the precipitated DNA and its attached dye moiety( s) . In a pioneering study in this field, Stowell and Albers (1943) obtained absorption curves from cells stained by various techniques including the Feulgen technique. They placed slides with stained and unstained tissues in front of the exit slit of a monochromator. Unfortunately, even with a densely nucleated organ such as thymus gland the nonhomogeneity of dye material can lead to broad absorption curves. The same effect is often seen when using strips of stained filter paper. These stained strips may be made translucent by adding a little oil. When the strips are placed in front of the exit slit of a spectrophotometer, absorption curves are broader than those obtained by using the dye solution in a cuvette. The broad Feulgen absorption curves reported by Stowell and Albers (1943) on rabbit thymus gland undoubtedly were in error. They obtained only two curves under conditions that were far from ideal in respect to homogeneity of absorbing material in the measured field. The absorption maximum was at 550-570 mp. Errors in position of the Feulgen-DNA curve appeared in the work of Yoshida (1958) and Naora (1955, 1958). Absorption peaks at 540 mp were reported from three stained nuclei of ox retina
THE CHEMISTRY OF SCHIFF’S REAGENT
83
by Yoshida (1958). A single curve from tissue of unnamed origin was shown by Naora to have the absorption maximum at 540-550 mp. The curve plotted by Naora is extremely broad, having approximately 70% of the peak extinction at 500 mp. Curves such as those shown by Naora and Yoshida would be expected from red-stained nuclei and not from the typical red-violet-to-violet stains seen in most Feulgen preparations. Presumably, instrumental errors, aberrant dye batches, or abnormalities arising from staining manipulations are responsible. Shibatani and Naora ( 1953) mentioned that the peak could be shifted by staining in various glyciiie buffer mixtures, sodium metabisulfite, and Schiff’s reagent for 3 to 4 hours, instead of by using Schiff’s reagent alone for a shorter time. Unfortunately, these studies were based only on measurements at two wavelengths (546 and 580 mp), and the significance of these changes in terms of the entire DNA dye C U N e was not brought out. The broad absorption curve of the Feulgen-DNA complex mentioned above was resolved by Moses (1951) into two distinct maxima, a primary one at 585 mp and another one at about 550 mp. These studies were carried out on sections of anthers from Trillium erectum. Since this information was reported only in an abstract, it is not clear whether the two peaks were present to the same extent. The question is important, because its .answer could indicate whether more than one dye moiety were present. If the peak at 550 mp was a minor one, it would preferably be called a secondary shoulder. In this case, it could represent the normal pattern seen in curves of triphenylmethane dye derivatives. The Feulgen-DNA absorption curve not only is shifted to longer wavelengths than the curve obtained from cells stained with basic fuchsin, but is of a different nature, especially around the secondary shoulder. These differences are shown in Fig. 16. The altered dye produced by adding formaldehyde to basic fuchsin also has an absorption curve that is shifted to longer wavelengths, but in this case the curve retains the same shape as the original curve of basic fuchsin. Figure 17 shows curves from nuclei stained with these two solutions. Some absorption curves from Feulgen-stained cells were reported by Swift (1955) and Swift and Rasch (1956). Extensive studies of this dye complex in situ were carried out by Kasten (1956b; 1957; 1958b). More than 250 individual absorption curves from various cell types were obtained. The studies clearly showed the Feulgen-DNA absorption peak to be at 570 my for most batches of basic fuchsin used in this Schiff’s reagent. With some dye batches an identical absorption curve was obtained, but shifted to a higher wavelength, so that the absorption maximum could lie as high as 580 or even 585 mp. A secondary shoulder was seen at 530-550
84
FREDERICK H. KASTEN
mp. However, this portion of the curve showed some variation. For example, the secondary shoulder was absent or barely perceptible in stained cells previously isolated by homogenization methods (Kasten, 1956b). In addition, the acid hydrolysis procedure affected this portion of the curve when tissues had been fixed previously in formalin (Kasten, 1956b) or acetic alcohol (Swift, 1953 ; Kasten, 1956b). Cells fixed in Zenker’s fluid gave the same absorption curve, regardless of their previous exposure to loo.
m.
60.
i Y
Y40.
20
.
- m w o S m S m e o o a m YvLLowoTn IN nyl
FIG. 16. Absorption curves of nuclei stained by basic fuchsin and in the Feulgen reaction. Curve from Feulgen-stained cells is shifted to a higher wavelength and is broader than that from fuchsin-stained nuclei. Data from Kasten, 195813. Key to curves : * - , basic fuchsin (8 curves) ; * - - - - ., Feulgen (56 curves) ; . . . ., basic fuchsin shifted 15 mp
-
+.
.
acid hydrolysis. In the case of. the other fixatives mentioned above, the main peak appeared early in hydrolysis and was followed later by the secondary peak. This phenomenon suggests that a newly available component is produced in late hydrolysis. Possibly, the component is removed or made unavailable by Zenker’s fluid or sucrose homogenization, since the secondary peak is absent here. Another explanation is that the secondary peak arises from unusual staining mechanisms. For example, it is known that amino groups of basic fuchsin can react with DNA after acid hydrolysis in a Schiff-base reaction to give red-stained nuclei (Arzac, 1950; Kasten, unpublished). A small proportion of molecules reacting
THE CHEMISTRY OF SCHIFF'S REAGENT
85
this way in the Schiff test could alter the absorption curve. Further studies of this problem may provide valuable information about reaction mechanisms. Absorption curves from Feulgen-stained nuclei are shown in Fig. 18. Transmission curves that were obtained with an automatic ratio-recording microspectrophotometer are illustrated in Fig. 19. Some details of this instrument were described by Pollister and Ornstein ( 1959). Various biological factors that may alter the nature of the Feulgen absorption curve have been studied. The DNA-dye complex is remarkably 100
80
60
2i
40
w
I# PO
FIG.17. Absorption curves of nuclei stained by basic fuchsin ( - - - - - - ) and by basic fuchsin plus formalin () solutions. Cells stained with the formalintreated dye solution have their absorption curves shifted (10 m p + ) to a higher wavelength but curve shape (. . .. .) is unaltered.
.
stable under a number of conditions. The absorption curve is the same after removal of nuclear protein by enzyme digestion or by histone extraction, different methods of fixation, and isolation of liver cells in cold sucrose solution or citric acid (Kasten, 1956b). The color complex is also the same in different cell types in a tissue, different organs, and some different species (Kasten, 1958b). Absorption curves from nuclei of different cancers are identical with those from normal cells (Kasten, 1957). Other technical factors found to have no appreciable effect on Feulgeii curve shape included type of microscope objective (achromatic orapochromatic) , temperature of DNA-dye complex (20 or 40" C.), hydrogen-ion concentration (pH 1.6 and 7.0), and concentration of DNA-dye:
86
FREDERICK H. KASTEN
complex. Slight differences in curve shape were found between mouse spermatozoa and primary spermatocytes ( Kasten, 195813). Transmission curves from onion root tips were slightly broader than those from rat and frog liver cells. More studies involving different plant and animal species need to be carried out to confirm these observations. There is a report that the color complex differs among different stained moieties in the PAS reaction (Pollister and Ornstein, 1955). This may be of general significance for the identification of PAS-positive substances.
FIG.18. The complete Feulgen absorption curve in the visible range. Group 1 (x-x, sections of paraffin embedded tissues) was stained with a different batch sections of paraffin of SchifT's reagent from groups 2 and 3. Groups 2 (0-0, embedded tissue) and 3 (0-0, isolated in sucrose or in citric acid) were stained with the same batch of Schiff's reagent. Data from Kasten (1958b).
Studies along these lines are being carried out in our laboratory by N. Aviles, especially with certain Schiff-type reagents. It was shown in Fig. 11 that phenosafranin-S02 produces different absorption spectra in Feulgen- and PAS-stained structures. Fluorescence spectra also differ among certain structures stained by fluorescent Schiff -type reagents in the PAS reaction. Absorption curve analyses at the cytological level are also useful in studying reaction kinetics. In an earlier section it was indicated that the dye products in the Feulgen and PAS reactions are not chemically inert.
THE CHEMISTRY OF SCHIFF’S REAGENT
87
The dye may be completely or partly displaced and then replaced by a different aldehyde-dye reagent. In some cases there is no displacement. Chrysoidine Y-S02 stains nuclei orange in the Feulgen reaction ; trans-
0
WAVELENGTH IN b$t (B) FIG.19. A. Transmission curve of onion root tip nucleus stained by Schiffs reagent in Feulgen reaction. Data obtained with automatic recording microspectrophotometer. B. Transmission curves of frog liver nuclei stained by Schiff’s reagent in Feulgen reaction. Data obtained with automatic recording microspectrophotometer.
88
FREDERICK H. KASTEN
mission curves from these nuclei have a broad peak with a maximum at about 470 mp (Fig. 20). Feulgen-stained rat liver cells retain the violet chromophore even after treatment with chrysoidine Y-SO2. The absorp-
WAVELENGTH
IN MJA
FIG.20. Transmission curves of rat liver cells stained by chrysoidine Y-SO2 in Feulgen reaction. Considerable nonspecific absorption is seen between 400 and 500 mp in unstained cytoplasm.
WAVELENGTH IN MJI FIG.21. Transmission curves illustrating failure of chrysoidine Y - S O2 (.45 min-
Utes in cold) to displace Schiff’s reagent after Feulgen reaction (45 minutes in cold) in rat liver cells.
THE CHEMISTRY OF SCHIFF’S REAGENT
89
tion curve has the peak at 570 mp with no sign of a peak at 470 my (Fig. 21). Chrysoidine R is very similar to chrysoidine Y and is partly replaced by brilliant cresyl blue-S02 from stained nuclei. The absorption curves are shown in Fig. 22. If all of the DNA-polyaldehyde is not bound by the violet chromophore because of a short staining time, then the remaining aldehyde groups are free to react with chrysoidine-SOz. The absorption curve in this case has both peaks (Fig. 23). Similar phenomena of this nature are amenable to absorption curve analyses.
XII. Conclusions Schiffs reagent has been used to detect aldehydes for over 90 years. The specificity for aldehydes in vitro has been questioned since positive reactions also are given by some ketones, fatty acids, and bromine. There is a possibility that other substances in fatty acids may be responsible for the color reaction. Some aromatic aldehydes do not give a positive reaction. The histochemical applications of the reagent have been shown to be reliable in terms of aldehyde specificity. The original dye may be regenerated by oxidizing agents and by substances that remove sulfur dioxide. A number of factors have been shown to affect the reagent and reactivity. These include pH, temperature, concentration of sulfur dioxide, presence of mineral and organic acids, presence of salts and dye contaminants, and method of storing reagent. Test tube results generally are more strongly influenced by changes in these factors than are the histochemical results. However, further histochemical studies of some of these factors are needed. Schiff’s reagent is considered to be the N-sulfinic acid derivative of fuchsin leucosulfonic acid. According to Wieland and Scheuing (1921), this compound reacts with an additional molecule of sulfur dioxide and two moles of aldehyde to form an unstable compound, which becomes colored upon loss of sulfurous acid and production of a quinoid structure. It is obvious that the sequence of reactions is even more complicated than Wieland and Scheuing indicated. The modern interpretation of the reaction is that a Mannich-type reaction is involved (Hormann et al., 1958). This reaction is more predictable than the reactions shown by Wieland and Scheuing. Further chemical studies are needed to help settle this important problem. At the present level of knowledge it is concluded from an analysis of the literature that the mechanism may vary, depending on the aldehyde and the conditions of reaction. Further chemical studies with polyaldehydes are needed to provide information of possible use in understanding reactions in stained tissues. Schiff -type reagents provide new approaches to the study of reaction kinetics at the histochemical level.
90
FREDERICK H. KASTEN
WAVELENGTH IN Mp FIG.22. Transmissioa curves illustrating partial replacement of chrysoidine RSO, (16 hours) from Feulgen-stained rat liver nuclei by brilliant ctesyl blue-SO, (1 hour). Point A marks peak of chrysoidine R-DNA complex. Point B marks peak of brilliant cresyl blue-DNA complex.
0
z
0 i7j
$
2
E 8
FIG.23. Transmission curves from stained frog liver nuclei illustrating the rapid reaction of Schiffs reagent with DNA to form a peak at about 580 mp (2 minutes in cold). Unreacted DNA-polyaldehyde may then react with a second reagent, chrysoidine R-SO, (45 minutes).
THE CHEMISTRY OF SCHIFF’S REAGENT
91
The Feulgen-dye moiety is susceptible of analysis by studies of absorption curves. Our understanding of the mechanism of aldehyde staining with Schiff’s reagent has not kept pace with the histochemical applications of the reagent. The Feulgen reaction for DNA is the only reaction involving Schiff’s reagent that has been shown to be usable for quantitation by cytophotometry. All histochemical reactions involving Schiff’s reagent require suitable controls for proper interpretation of stained structures. The field of histo- and cytochemistry had its start with the application of Schiff‘s reagent in the Feulgen reaction about 35 years ago. Since that time, Schiff’s reagent has been recognized as the most valuable histochemical reagent for detection of aldehydes. Advances may be expected in the use of the reagent and Schiff-type reagents for the staining of chemical moieties in new histochemical reactions and in multiple aldehyde reactions. It is hoped that progress will be made also in our knowledge of reaction mechanisms. ADDENDUM Since this paper was set in type, several other pertinent publications have come to the attention of the reviewer. Gayon (1928, Compt. rend. acad. sci., 105, 1182) used Schiff’s reagent to detect aldehydes in commercial alcohols. A number of workers measured methanol in ethanol by oxidizing methanol in the presence of HzS04 to formaldehyde followed by treatment with Schiff’s reagent. This test has been frequently called Denigb’ method (Scudder, 1905, J . Anz. Chem. SOC.,27, 892; Denigb, 1910, Compt. rend. mad. sci., 150, 529; Denigh, 1910, Compt. rend. acad. sci., 150, 832; Simmonds, 1912, The Analyst, 37, 16; Schaffer, 1912, U.S. Naval Med. Bull., 6, 392; Gettler, 1920, J . Biol. Chem., 42, 311 ; Chapin, 1921, Ind. Eng. Chem., 13, 543; Georgia and Morales, 1926, Ind. Eng. Chem., 18, 304). It was emphasized by Rosenthaler (1923, Der Nachweis organischer Verbindungen, 2nd ed., p. 120) that not all aldehydes react with Schiff’s reagent. H e mentioned that no reaction occurs with phenolaldehyde, glyoxal, and sulfaldehyde whereas acetone and a few other ketones do react. In cytochemical studies, the silver-Feulgen method was used to demonstrate chromatin by electron microscopy (Bretschneider, 1949, Proc. Kon. Acad. v. Wetensch., 62, 301 ; Bradfield, 1954, Nature, 173, 184; Bradfield, 1956, Proceedings of the Third International Conference on Electron Microscopy, p. 321 ; Jurand, Deutsch, and Dunn, 1958, J . Royal Micr. SOC., 78, 46). In this technique, Schiff‘s reagent is replaced by ammoniacal silver hydroxide. Aldehyde residues in DNA formed by hydrolysis reduce the silver compound to metallic silver which is deposited in areas where reduction took place. It appears brown in the light microscope and as
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small granules in electron micrographs. The technique was used many years ago by Feulgen and Voit (1924) to demonstrate that this procedure blocks aldehyde groups and prevents the Schiff reaction. In electron microscopy, observations were made of DNA in bull sperm, bacteria, and various animal cells. Unfortunately, cytoplasmic silver grains may also be deposited which is suggestive that other reducing groups are responsible here. The silver-Feulgen reagent is not a Schiff-type reagent since the mechanism of staining aldehydes differs in the two cases. According to Adler and Zelle (1957, J . Bacteriol., 73, 526), it is valid to make counts of Feulgen stained bodies in B . mycoides. Schiff’s reagent, azure A-SO2, and thionin-SOz are useful for this purpose. Renewed criticism of the Feulgen reaction has come from Chayen (1959, Exptl. Cell Res. Suppl. 6, 115; 1960, Exptl. Cell Res., 20, 150) who does not consider the reaction to be valid for studying the intracellular distribution of DNA. It is difficult to reconcile his views with the wealth of supporting evidence, such as the autoradiographic distribution of labeled precursors in DNA. Chayen’s studies on plant root tips may have only minor significance. It seems desirable to the reviewer that the studies need to be refuted or confirmed by other workers. An interesting paper by Barka and Ornstein appeared (1960, J . Histochem. and Cytochent., 8,208) which dealt with the reaction between Schiffs reagent and formaldehyde. They were able to demonstrate 3 to 7 compounds by paper chromatography. Absorption curves also were obtained from eluted spots. The peaks ranged from 535 to 574 mp. The formation of these compounds was influenced by the SO2 content of the Schiff reagent and the molar ratio of aldehyde to Schiff reagent. In the reviewer’s laboratory, it was recently demonstrated that Schiff ’s reagents of various recipes may themselves give more than one dye without exposure to aldehyde. When formaldehyde is added, at least 3 to 5 new dyes are formed. The basic fuchsin used in these Schiff’s reagents had been purified and contained only one dye. Further studies by Cerroni and Goldstein (1960, Exptl. Cell. Res., 20, 258) on the effect of iron on Tetrahymena cells demonstrated that FeulgenDNA content per nucleus is reduced by 3 2 4 % although no differences in DNA were detected by non-Feulgen biochemical analyses. Glycogen and mucins were demonstrated histochemically with Schiffs reagent after oxidation with chromyl chloride in an anhydrous solvent (Mende and Chambers, 1957, J . Histochem. and Cytochem., 6, 606). On prolonged exposure, a typical Feulgen nuclear reaction was obtained. The classical concept of natural plasmalogens has been challenged recently. There is conclusive evidence by Rapport and Franzl (1957,
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J. Neurochem., 1, 303) that a vinyl ether type linkage occurs between the fatty aldehyde and one of the glyceride carbons rather than an acetal linkage. A very important paper was published which deals with a spectrophotometric study in vitro of the Schiff-formaldehyde reaction (Naunian, West, Tron, and Gaeke, 1960, Anal. Chem., 32, 1307). By use of simple amines and by comparison with known ultraviolet and visible spectra, the final dye reaction product was shown to be an alkyl-sulfonic acid derivative. It would have been desirable if infrared spectral studies had also been made to support this conclusion. The results are in agreement with those of Rumpf (1935) and Hormann et al. (1958). This latest study emphasizes the need for a re-evaluation of our concepts of Schiff-polyaldehyde reactions. New critical experiments are needed to determine whether the Wieland and Scheuing theory has any validity in histochemistry and should be abandoned in favor of the alkyl-sulfonic acid theory.
ACKNOWLEDGMENTS This work was aided by Grant C-3420 (C2) from the National Cancer Institute of the National Institutes of Health, U.S.P.H.S., Contract No. AT- (40-1)-2432 from the U.S. Atomic Energy Commission, and a grant from the Biological Stain Commission. Workers in this laboratory who contributed materially to the article include Vivian Burton and Norman Aviles. Dr. A. F. Isbell and Dr. H. Weidmann were liberal with their time in reading the manuscript and making useful suggestions. My wife has given generously of her time in reading and criticizing the manuscript.
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Wermel, E. (1927) 2. Zellforsch. u. mikroskop. Anat. 6, 400. Wertheim, E. (1922) J . Ant. Chem. SOC.44, 1834. Widstrom, G. (1928) Biochem. Z . 199, 298. Wieland, H., and Scheuing, G. (1921) Ber. deut. Chem. Ges. M, 2527. Wild, F. (1953) “Estimation of Organic Compounds,” Cambridge Univ. Press, London. Wolffenstein, R. (1892) Ber. deut. Chem. Ges. 25, 2777. Wolman, M. (1950) Proc. SOC.exptl. Biol. N . Y . 75, 583. Wolman, M. (1954) J . Pathol. Bacteriol. 68, 159. Wolman, M. (1956) Stain Technol. 81, 241. Woodman, A. G., and Lyford, E. F. (1908) J . Am. Chem. SOC.90, 1607. Woods, P. S. (1957) J . Biofihys. Biochem. Cytol. 3, 71. Wunsche, F. (1919) Arch. exgtl. Pathol. Pharmacol. Naunyn-Schmiedeberg’s 84, 328. Yarbo, D. L., Miller, B., and Anderson, C. E. (1954) Stain Technol. !29, 299. Yasuma, A., Ichikawa, T. (1953) J. Lab. Clin. Med. 41,296. Yoshida, M. (1958) Japan. J . Physiol. 8, 57. Zinner, G. (1957) 2. A d . Chem. 156, 412.
Spontaneous and Chemically Induced Chromosome Breaks ARUN KUMAR SHARMA A N D ARCHANA SHARMA Cytogcnetics Laboratory, Departinent of Botany, Calcutta University, Calcutta, India
Page 101 I. Introduction ..................................................... 102 11. Spontaneous Breakage ............................................ 111. Technical Limitations in the Study of Chromosome Breaks ........ 107 A. Temperature Induced Breaks .................................. 107 108 B. Breaks Induced by Water .................................... 109 C. Breakage by Dyes ............................................ IV. Action of Alkaloids, Pigments, and Coumarin Derivatives .......... 110 112 V. Action of Vegetable Oils, Fats, and Essences ...................... 112 VI. Action of Drugs and Bacterial Products .......................... VII. Action of Hormones and Other Growth-Promoting Substances ...... 113 VIII. Action of Mustards, Related Compounds, and Phenols .............. 116 119 IX. Other Compounds ................................................ X. Conclusion ........................................................ 122 123 References ........................................................
I. Introduction The problem of the mechanism of chromosome breakage, both spontaneous and induced, is an old one, and its interpretation is still controversial. As the manifestation of the visible cytological effect is far removed from the initial step, deductions have to be made on the basis of indirect observations. Since the first induction of chromosomal mutations by chemicals (Auerbach and Robson, 1946) and the demonstration of definite chromosome breakage by Oehlkers (1943), such a vast multitude of chemicals have been shown to possess the chromosome-breaking property that the problem has become increasingly complex. A large number of both organic and inorganic compounds are at present on record which show radiomimetic activity. The range is very wide, extending from water to the highly complex nucleic acid salts. Most of them produce similar physical effects, such as stickiness, pycnosis, etc., along with the most important radiomimetic expression-chromosome breakage. A generally held view is that a disbalance in the metabolic setup of the chromosome, especially with relation to the surrounding medium, is responsible for chromosome breakage. Most workers have mainly dealt with the radiomimetic property of the chemicals only, disregarding its physico-chemical basis (Hadorn and Niggli, 1946; Lettrk, 1946; Meyer, 1948; D’Amato, 1949a, b, 1950b, 1952a, b, 1954b; D’Amato 101
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and Avanzi, 1949a ; Lopane, 1950 ; Battaglia, 1950b ; Keck and HoffmanOstenhof, 1951 ; Hoffman-Ostenhof and Keck, 1951 ; MascrC and Deysson, 1951; Linnert, 1951; Boyland, 1952; Hair, 1952; Fahmy and Bird, 1952; Sharma and Mukherji, 1955; etc.), while others, who have attempted to probe into the details of their mode of action, have made varying inferences (Gavauden, 1939, 1947 ; Oehlkers, 1947, 1949, 1952 ; Dustin, 1947, 1949a, b ;Allsop and Catcheside, 1948; Catcheside, 1948; Deysson, 1948; Koller, 1949; Darlington, 1950 ; Bird, 1952 ; Gray, 1952 ; Loveless, 1952 ; Butler, 1954; D’Amato, 1954c; Revell, 1955). This difficulty in interpreting the data is chiefly due to the fact that, while dealing with the chemical treatments and their cytological expression, it is impossible to check other indirect metabolic disbalances caused by chemical treatment, which obviously exert indirect influence on chromosome metabolism. The cytological expression varies because of this indirect influence. This is shown by the observation of nonlocalized breaks by the majority of authors and of localized breaks by others (Levan and Lotfy, 1949 ; McLeish, 1952). However, all workers including Moutschen-Dahmen (1958a, b) agree that by comparison with radiation breaks, chemically induced breaks are more localized. Certain workers, when comparing radiation and chemically induced breaks, emphasize the inefficiency of chemicals in causing direct breaks on chromosomes (Lea, 1946). However, later data from different studies clearly show that radiation breaks too are not direct (Koller, 1949) but are caused indirectly, e.g., through chemical interference in the cytoplasm (Darlington and Koller, 1945, 1947). The disruption of hydrogen bonds is regarded by some as principally responsible for chromosome breakage (Ambrose and Gopal Ayenger, 1952; Butler, 1954, etc.), whereas others hold that the mode of action is mainly through an effect on sulfhydryl groups (Dustin, 1947, 1949a, b; Auerbach, 1952). Another controversial point is the cell stage which i s most susceptible to chemical treatment. Susceptibility also varies in different tissues, and it has been established that, e.g., in plants, meristematic tissues, being more labile, are more susceptible than others. 11. Spontaneous Breakage
Spontaneous breakages occur frequently in plants ( McClintock, 1939, 1941, 1942a, b, 1943, 1944; Darlington and Upcott, 1941 ; Olenov, 1949; D’Amato, 1952d). Records show the presence of a large number of species in the monocotyledons than in the dicotyledons. As early as 1940, Giles studied natural mutations in species of Tradescantia. Nichols (1941) recorded the same phenomenon in Allium cepa, and since then spontaneous mutation has been observed in a large number of species, such as
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Nothoscordum (D’Amato, 1948b), Bromus hybrids (Sparrow and Sparrow, 1950), Trillium (Walters, 1950, 1951), Antirrhinum, Vicia (Kihlman and Levan, 1951 ; Sharma and Bhattacharyya, N. K., 1956c) ; Hyacinthus (La Cour, 1952), Trituum (Camara, 1941 ; Smith, 1947), Pisum (D’Amato, 1951b, 1952a), and Scilla (Rees, 1952), as well as others (Beadle, 1937; Chakravorti, 1948 ; HHkansson, 1950; Gisquet et ad., 1951; Tjio and (Sstergren, 1954; Sharma and Bal, 1959a). A majority of chromosome breaks generally occur in root tips, except in a few plants such as Antirrhinum, Paeonia (Marquardt, 1952b), and Tradescantia, where they occur in meiosis and in pollen grain division as well. Recently, the mechanism of breakage in chromosomes of endosperm nuclei (La Cour and Rutishauser, 1953 ; Brock, 1954 ; Rutishauser, 1956; Rutishauser and La Cour, 1956a,b) has been studied. With the accumulation of data on natural chromosome breakage, the causal factor underlying it is also being investigated. After the outstanding discovery by Muller of induced mutation through X-rays (1928, 1940), the general tendency was to attribute spontaneous mutability to natural radiation (D’Amato, 1952d). Later, Giles ( 1940, 1947) clearly explained that natural radiation cannot account for the high frequency of mutation rate, especially in the pollen of Tradescantia. Evidently, therefore, some other factor induces spontaneous mutation. Another theory was the concept of thermal agitation as noted by Timofeeff-Ressovsky and Zimmer (1939). This idea was also not very explicit and there was not enough evidence to warrant its universal application. A new line of approach, initiated by Navashin (1938) and his school, emphasized that the internal factors, rather than the external ones, cause spontaneous chromosome breakage. According to them, a difference in the metabolic setup in immature tissues is responsible for spontaneous mutation. Since then several workers, namely, Pet0 (1933), Nichols (19429, Walters (1950, 1951), D’Amato (1952a), and Gunthardt et al. (1953), have noted that aging of seeds has a profound influence on natural mutability. Nichols also noted a proportionate increase in spontaneous mutation rate with seed aging. Some workers are of the opinion that the common cytological phenomena such as hybridity or duplication of chromosomes may account for natural breaks (D’Amato, 1952d). This explanation cannot be applied to true diploid species, such as Tradescantia, Vicia, Lathyrus. Although a high frequency of breakage occurs in hybrids and polyploids, the mechanism there appears to be entirely different from spontaneous chromosome changes. Chromosome breakage in hybrids and polyploids is generally conditioned by the internal failure of correlation between the different components of the genome rather than by any cytoplasmic or nutritional factor.
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It has been suggested that with seed aging (D’Amato, 1952d) there is an accumulation of certain metabolites within the cell, especially due to the decomposition of a number of reserve products. The injurious effects of these decomposition products are responsible for chromosome breakage. This theory appears to be reasonable in view of the fact that a number of organic and inorganic compounds have recently been shown to be mutagenic in nature. The loss of viability subsequent to seed aging has also been attributed to the accumulation of decomposition products exerting a toxic effect. Levan and Lotfy (1950) noted spontaneous fragmentation in seedlings of V i c k faba following presoaking in water and suggested that soaking in water favors fragmentation. They claim that the partially anaerobic conditions during the 24 hours in which the dormant vital processes are awakened may involve a change in the metabolism, with the formation of a high concentration of metabolites, finally leading to chromosome breakage. On the other hand, in Vicia sativa, although Sharma and Bhattacharyya, N. K. (19564 noted the formation of chromosome fragments with aging, they did not find any correlation with the soaking in water previous to germination. Different workers have subjected plants to treatment with extracts of aged seeds. Marquardt (1949a, b) demonstrated that a water extract of aged Oenothera seeds caused marked fragmentation of chromosomes in Paeonia. A nearly similar effect was noted with putrescin-a decomposition product of diaminoornithine. Keck and Hoff man-Ostenhof ( 1952) obtained breaks with a water extract of aged seeds of Phuseolus vulgaris on Allium cepa. They also ( 1951) studied.the property of water extracts of A . cepa in inducing chromosome aberrations in Crocus. Similar observations have been reported by other workers too (Mota, 1952; Scarascia, 1954; Scarascia and Scarascia-Venezian, 1954). A rare case has been noted in soybean extracts in which fragmentation was recorded but was found to have no relation with age. It is likely that in this case, products in aged seeds do not decompose so readily as in other seeds. The minimum of decomposition needed for fragmentation in such cases persists for a long time after reaching that level. It is evident that the mutagenic substances produced by these plants may have an automutagenic property too. In this connection, Marquardt noted that the water extract of aged Oenothera seeds caused aberrations in Oenothera hybrids. Gisquet and his associates (1951) obtained abnormal seedlings, less viability, and chromosomal irregularities in plants germinated from old seeds. Water extracts of Allium cepa have been found to cause chromosomal irregularities in the same plant. Resendk (1951) re-
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corded an effect of old scales of onion bulbs on their roots. Hoffman-Ostenhof and Keck (1951) studied the action of diallyl disulfide, a constituent of Allium sativum. From the above-mentioned results, as inferred by several workers (Nichols, 1941 ; Marquardt, 1949a, b ; D'Amato, 1952d, etc.) , it is evident that the susceptibility to spontaneous mutation is conditioned by the decomposition of metabolic products. The acceleration of mutation after presoaking in water with Vich faba (Levan and Lotfy, 1950) and the absence of this effect in Vicia sativa (Sharma and Bhattacharyya, N. K., 1956c) show that it is also controlled by a specific physiological setup, which is different in different species. D'Amato (1952d) has pointed out that excessive production of metabolites or the production of new compounds or a change in the physiological condition may not be favored by the tissue. The injurious effects of these are manifested by chromosome fragmentation. In this connection, the observations of Takenaka (1950), indicating disturbances in Colchicum autumnale at 25"-35"C., and of Levan and Steinegger ( 1947), showing autotoxicosis of Colchicum under certain physiological conditions, have been mentioned. D'Amato ( 1952d) has emphasized that the production of spontaneous mutations is conditioned by special metabolic or physiological conditions. His observations, as well as those of others, indicating that chromosomal aberrations are also present in the polyploid nuclei of the elongation zone of the onion root, have further shown the importance of the basic physiological state in the origin of spontaneous mutations. It appears that the tissue of every organ of an individual is well organized and works in complete harmony, on a limited and strictly controlled metabolic level, having a specific physiological organization. No doubt mutability is conditioned chiefly by the internal factors, but the external medium also plays a role in it. Any change in the external medium, such as temperature (Laczynska, 1947), oxygen level (Conger and Fairchild, 1952), and even deficiency of calcium and magnesium, as pointed out by Steffensen (1953, 1955), has serious repercussions on the internal medium, thus causing chromosome breakage in a majority of cases. The embryonic stage, in which most of the mutations are noted, is very susceptible to external effects, as meristematic tissues are physiologically rather unstable (Dolcher, 1949). I n the mature organs, which are comparatively better equipped to resist the injurious effects of the change, the same effects have been noted but not in such a marked degree. The spontaneous breaks, like some of the chemical ones, result mostly in localized breaks, as compared to the random breaks induced by X-rays. Localized spontaneous breaks have been recorded by Levan and Lotfy
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(1950) and Sharma and Bal (1959a) in Vicia fubu and Allium sutivum respectively. Heterochromatic segments of chromosomes are considered to be more labile than the euchromatic ones. Both maleic hydrazide and diethyl ether cause chromosome breakage in the heterochromatic segments. Similar localized breaks have been noted by Moutschen-Dahmen (1958a, b) in Hordeum vulgare and ViCiu fuba following treatments with dimethane sulfonyloxybutane and 8-ethoxycaffeine. Therefore, an analogy has been drawn between spontaneous and chemical breaks. On the basis of this analogy as well, it is claimed that spontaneous breaks may arise through the accumulation of chemical substances within the cell. It is yet to be seen how far this accumulation influences chromosome breakage in a direct or indirect way. It appears that both direct and indirect actions are involved in the process. The preceding statement implies that the accumulated substances may directly interfere with nucleoprotein synthesis of certain segments of chromosomes, causing them to break at certain loci; also the synthesis of certain precursors of chromosome metabolism may be affected. Regarding localization, Levan and Lotfy’s observations (1950) as well as those of Sharma and Bal (1959a) indicate breaks at the secondary constriction regions. Undoubtedly, this is an indication of the labile nature of these segments. These are connected with the ribonucleoprotein metabolism of the nucleus, especially with regard to the organization of the nucleolus. In view of their specialized function, these segments are expected to be differentiated in their chemical setup from the rest of the chromosome. This difference possibly involves substances concerned with precursors of nucleolar organization. Although it is uncertain whether the nucleolar substance is actually synthesized in these regions or whether the regions represent loci of nucleolar formation, it is evident that certain chemical peculiarities must exist there which permit nucleolar formation in them. The best interpretation, under the existing circumstances, is that they contain certain precursors which help at least in the final stage of nucleolar formation. Assuming the greater susceptibility of the secondary constriction segments in comparison with other regions of chromosomes in Vicia faba and Allium sativum, it is implied that the spontaneous breaks are mainly concerned with the ribonucleoprotein metabolism of the nucleus. This also supports the idea that the heterochromatic segments are, in general, more labile. Other works (Vanderlyn, 1949, Brachet, 1957) show that heterochromatic segments are directly concerned with the RNA-protein turnover of the nucleus, though they may not be directly concerned with the production of the nucleolus, like the secondary constriction regions.
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111. Technical Limitations in the Study of Chromosome Breaks
A. TEMPERATURE INDUCED BREAKS The most important factor, other than chemicals, capable of inducing chromosome fragmentation, is temperature ( Peto, 1933; Schkarnikov and Navashin, 1934 ; Sax, 1937 ; Laczynska, 1947 ; Marquardt, 1952a; Nakahara and Komoto, 1957). Changes in temperature, especially an increase, are very effective in inducing chromosomes to break at different loci. High temperature not only induces breaks when applied alone, but also accelerates X-ray breaks and as such may add to technical errors. The exact steps involved in the temperature breaks are still obscure. A number of alternatives can be suggested to account for this process. The existence of a specific end-result of temperature variation in different groups of plants indicates that finally a common step in metabolism becomes affected through a number of initial different steps, which may vary in different species. The initial steps are held to be different because the reserve products and nutritional setup of the different species are not the same. Alternatively, it may be suggested that a common metabolic path, which occurs universally, is susceptible. The latter suggestion seems unlikely in view of the fact that a change in temperature is likely to affect all the metabolic paths, a number of which would have an indirect effect on chromosome fragmentation. Whatever may be the metabolic path influenced, the way through which the effect operates appears to be obvious. The increase in temperature may cause an unusual acceleration of some of the enzymic steps within the cells. This will lead to the excessive production of certain metabolites, which may have a harmful effect, either on the chromosomes themselves or on their precursors. The temperature change may on the other hand retard some of the enzymic activities, causing the accumulation of unutilized metabolites. The latter may exert a harmful influence on the nucleoprotein metabolism of the cell. It is well known that .temperature shift may often cause the colchicine effect. If, in Levan’s words (1949), the C-mitotic effect is supposed to be a manifestation of narcosis, then the induction of fragmentation-a manifestation of subnarcotic influenceby less drastic temperature treatment is explainable. Whatever may be the final step leading to chromosome breakage, it is a fact that both spontaneous and temperature breaks are due to certain disturbances in the metabolic setup. Any such disturbance can cause radiomimetic effects in the end. The results of Poussel (1945), showing the induction of chromosome fragmentation by a change in the osmotic pressure of the cell, or even by decapitation as pointed out by
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D’Amato (194&), support this suggestion. Obviously, this change in osmotic balance affects the intake of different metabolites within the cell, which may in turn have an ultimate effect on chromosome breaks.
B. BREAKSINDUCEDBY WATER At present, it is difficult to assess clearly the relative potency of different mutagenic agents. Excluding cases of spontaneous breaks and mutation through plant extracts, discussed earlier, a large number of chemical agents having the property of inducing chromosome fragmentation can be listed. These agents include a number of special drugs, antibiotics, plant products, plant hormones, vitamins, and growth regulators, in addition to a number of organic compounds mainly of the mustard group, phenols, heterocyclic bases, some inorganic salts, metals, and gases, and even the halogens. The deficiency of certain necessary elements in the plant tissue, such as magnesium, calcium, etc., may cause breakage. Continuous keeping in water may even cause fragmentation. The chemistry of fragmentation, induced through these diverse groups of chemicals, is very likely different in different treatments, but the final step leading to a break in the chromosome thread may be the same in all. The first case to be discussed is the radiomimetic effect of water. Mere water treatment in Allium cepa causes a viscosity change in the plasma (Sharma and Sen, 1954a) and ultimately a chromosome break. Even distilled water shows this effect, not only when distilled in a metallic distillator, but also when distilled in a glass retort. Only with redistilled water, twice distilled in glass, is this effect not found. These results indicate that even a minute trace of any chemical left in water which has been distilled once in a metallic or glass container is quite enough to cause chromosomal changes. Evidently, a subtle, metabolic equilibrium exists within the cell, a disbalance of which may even result in chromosome breaks. This disbalance can be caused by transferring the plant to any medium different from its norma] environment. Obviously, the metabolic equilibrium is maintained by an extremely complex but well organized enzymic setup under strictly controlled conditions. Mutation through water alone emphasizes the fact that spontaneous mutation need not be due to any special mechanism: it may just arise out of a change in water balance having a far-reaching repercussion within the cell. Therefore, in interpreting chromosome breaks induced through chemicals dissolved in water, extreme caution must be used.
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C. BREAKAGE BY DYES Before dealing with fragmentation through chemicals, it is desirable to deal with certain aspects of the technique used for detecting breaks. Usually, after pretreating the tissue in the mutagenic chemical, the tissue is fixed in a fixing fluid for a certain period, followed by staining with certain dyes. It has recently been shown that some of these dyes can cause chromosome breaks. D’Amato ( 1951a) reports that with brilliant cresyl blue, chromosomes often break. Battaglia ( 1948, 1950a) observed chromosome fragmentation following methyl blue and toluidine blue, but negative results were obtained following treatment with basic fuchsin, crystal violet, aniline blue, erythrocine, uranine, safranine, quinoline blue, orseillin BB, hematoxylin. Becker (1932, 1933), too, noted the effect of methyl blue and the azine series. The effect of neutral red was noted by Patterson as early as 1942. A high frequency of chromosome fragmentation has been reported to occur following orcein staining (Sharma and Roy, 1955). With other dyes, such as carmine, no such effect was noted. The most interesting point in orcein breakage is that the tissue must be pretreated with some fluids to permit manifestation of the breaks. Another prerequisite is that orcein must be added when hot. All control experiments for checking whether the break is due to any other step in the process show positively that only orcein in a hot state is capable of inducing fragmentation. It has been suggested that pretreatment causes certain changes in some of the important components of the chromosome. That component is, in all probability, the nucleic acid. It is likely that, to some extent, depolymerization of nucleic acid takes place following pretreatment. The sticky nature of the chromosomes also supports this fact. It is not known as yet whether any other chemical change is involved in the process. In all likelihood, especially in view of the breaks observed later, the DNA-protein linkage is made labile. Therefore, at the time of orcein treatment, the nucleic acid molecule is depolymerized and lies in a labile state, susceptible to separation from protein by any slightly severe treatment. Evidently, the change undergone by DNA molecules is not uniform throughout the chromosome length, and certain segments retain more nucleic acid than the others. This behavior is manifested in the formation of eroded segments of chromosomes, often noted in such preparations. When orcein is applied in a hot state for a short time, the nucleic acids come out from the segments, where they are present in a labile condition. Finally, if orcein treatment is prolonged, the protein thread may be attacked and breaks may occur. These observations clearly show that heated orcein solution is responsible for the breaks and imply the necessity of a cautious
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application of this technique in detecting mutagenic properties of chemicals. In the study of chemical mutagens, three technical errors, involving respectively the temperature, the solvent of the chemical, and the dye used for staining the preparations, must be checked. If water and orcein are the two ingredients used, caution is necessary in interpreting the results. Margin should be left in assessing the data for the possibility of error due to their inherent effects.
IV. Action of Alkaloids, Pigments, and Coumarin Derivatives The alkaloids form one of the most important groups of radiomimetic chemicals. The most used and most discussed alkaloid of this group is colchicine. Since the discovery of its polyploidizing effect on plants by Blakeslee and Avery (1937), it has been extensively used on different groups of plants. The parent plants of this chemical are Colchicum autumnale and Cokhicum luteum, though recently, other species of the same genus have been shown to yield the same chemical (Eigsti and Dustin, 1957). It is not necessary to go into the details of colchicine action, as it has been thoroughly discussed by a number of authors (Duhamet, 1939, 1945 ; Levan, 1940 ; Karpachenko, 1940; Hawkes, 1942 ; Steinegger and Levan, 1947a, b ; Eigsti and Dustin, 1957 ; D’Amato, 1948a ; Bauch, 1949a ; Stalfelt, 1949). Tjio (1951) recorded that the addition of colchicine to other subnarcotic agents reduced their radiomimetic effects. Levan (1949) suggested that C-mitosis possibly excluded the radiomimetic effects. Therefore, the report of fragmentation of chromosomes through colchicine is interesting. Other alkaloids in which the radiomimetic action has been worked out include putrescin (Marquardt, 1949a), podophyllotoxin (Cova, 1953 ; Lona, 1953 ; Sullivan and Wechsler, 1947), protoanemonine (Ericksson and Rosen, 1949), veratrine (Sharma and Sarkar, 1956), theobromine, theophylline, caffeine (Kihlman and Levan, 1949 ; Kihlman, 195Oa, b), berberine (Rossi, 1955a, b ; Sharma, 1959), and other derivatives (Gosselin, 1940). The chromosome breakage noted in these cases may be due to either physical or chemical effects or both. The extent of the effects of nonspecific physical or specific chemical reactions is not known. Fragmentation may often result from a nonspecific manifestation of stickiness, which may ultimately cause difficulty in chromosome division and breakage at certain loci. The actual cause of stickiness, whether it is due to mere physical changes or a chemical reaction, is not known. In the case of podophyllotoxin (Cortini, 1955), the effect varies according to the way in which the alkaloid is used, the plants from which it is
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extracted, and the group to which it belongs. This suggests that the effect is not merely a generalized physical one but that also some subtle chemical change is involved. With certain alkaloid salts such as berberine sulfate (Rossi, 1955a), the effects become distinct after a certain period of recovery in water. This may indicate that in addition to causing radiomimetic changes, the general toxic effect of the chemical does not allow the cells containing fragments t o divide further. On removal of this toxic effect by recovery in water, these cells regain their vitality and divide, manifesting radiomimetic effects. The effect of alkaloids on differentiated tissue has been also tested. Definite chromosome breaks occur in the differentiated and meristematic tissue of Allium cepa following treatment in extracts of the bark of AZstonia scholaris as well as of Holarrhoenu antidysentrica (Sharma and Bal, 1959b ; Sharma, 1959). This fact indicates that the subnarcotic response of chromosomes to some specific chemicals is not limited only to meristematic regions, but is found in adult tissues as well. Of the large number of plant alkaloids, only a few have been tested for this effect, and an immense scope of research exists in this direction. Lately, research in this laboratory (Sharma and Majumdar, 1959a, b) has revealed that plant pigments too are capable of inducing fragmentation in Allium chromosomes. Until now pigments extracted from members of Chlorophyceae, Cyanophyceae, and Xanthophyceae, as well as chlorophyll of higher plants, have yielded positive results. Although with direct treatment fragments are noted, they increase in frequency when the tissue has recovered in KnopS medium. Investigations are now in progress in which pigment mixtures extracted from plants are separated, and the action of each of them is being studied. In addition to products mentioned above, a number of coumarin derivatives (Cornman, 1947 ; Ostergren, 1948 ; Ostergren and Wakonig, 1954 ; Giacometti, 1954 ; Quercioli, 1954, 1955a, b ; D’Amato and Avanzi, 1954 ; D’Amato, 1955 ; Sharma and Bal, 1956; Sharma and Chaudhuri, unpublished) have also been tried on root cells of Allium cepa. Coumarin, in fact, is the orthocoumaric acid lactone and is responsible for the well-known soft scent of new-mown hay. Derivatives of coumarin, such as aesculin, aesculetin, daphnetin, etc., involve the addition of hydroxyl groups in different positions of the coumarin ring. Coumarin and aesculetin especially show a high frequency of fragments. Experiments have been started in this laboratory (Sharma and Chaudhuri, unpublished) now to find out the degree of viscosity change caused by these substances and its correlation with their molecular structure and fragmenting property. It may be
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noted in this connection that salicin too shows the same fragmenting effect on Allium chromosomes (Sharma and Bal, 1956). V. Action of Vegetable Oils, Fats, pnd Essences
Recently, fragments have been noted in (he germinating roots of Triticum after soaking the seeds in a number of vegetkble oils and fats (Swaminathan and Natarajan, 1956). The breakage occurs at the time of chroniosome reduplication and is spread over a period of time. This fact suggests that the action is mainly through a disruption in the nucleic acid cycle of the cell. The nonsynchronized nucleic acid metabolism at different gene sites, due to this disruption, makes the chromonema thread liable to breakage at the time of reduplication. D’Amato and Avanzi (1949b) have carried out tests on Allium with a number of essential oils, namely, eugenol, lavender, eucalyptol, fennel, Oleum cudinum, turpentine, etc., and have shown that toxicity including radiomimetic effect is more pronounced in distilled water than in tap water solution. The same behavior has been noted with pervitine chloride (D’Amato, 1948a). It appears that since tap water alone can cause fragmentation, radiomimetic chemicals and tap water prove antagonistic to each other, thus causing no breakage.
VI. Action of Drugs and Bacterial Products A number of drugs have also been investigated, including the DDT insecticides by Vaarama ( 1947), sulfa compounds by Fuller ( 1947), Bauch (1949a), and Dustin (1947), BAL and arrhenal by D’Amato (1949d), and antibiotics by Wilson (1950), Wilson and Bowen (1951), Levan and Tjio (1951), and LettrC (1948). Of the sulfa compounds, sulfadiazine, guanidine, merazine, pyridine, and thiazole have been tested. Of the antibiotics, penicillin, aureomycin, streptomycin, terramycin, chloromycetin, actidione, etc., have been investigated. The radiomimetic property of some of these, especially penicillin, is remarkable. It is not possible to state their mode of action with certainty. It is clear, however, that their medical application is not without any genetic significance. The same problem arises in the application of DDT as an insecticide. Kaplan (1948) and Montezuma de Carvalho (1955) noted radiomimetic effects following use of a number of bacterial products on roots of Viciu fubu. It has been suggested that possibly these products cause the decomposition of proteins, which in turn is responsible for chromosome breaks. The idea of mutagenicity of soil has been presented, and it has been emphasized that spontaneous mutation may arise out of external and not internal influences. Without disregarding the influence of external factors on spon-
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taneous mutations, it may be pointed out that at the present state of our knowledge, as shown in the preceding part of the discussion, the influence of internal factors cannot be ignored.
VII. Action of Hormones and Other Growth-Promoting Substances The hormones are another important group of plant products possessing the property of inducing chromosome fragmentation. Their capacity to accelerate growth in plants is well known. In very low concentrations, some of them, like indolylacetic acid, indolylbutyric acid, etc., can induce roots to develop in cuttings. Fragmentation is caused by hormones when applied in concentrations much higher than that needed for production of growth. At a concentration below the subnarcotic level, some hormones even cause clarification of karyotypes ( Sharma and Mookerjea, 1954a). This clarification is thought to involve a viscosity change in the cytoplasm after an upset of the nucleic acid balance of the cell. A further upset in the balance following treatment in higher concentration results finally in chromosome breakage. Fragmentation of chromosomes by hormone treatment shows the necessity of their cautious use in inducing growth in plant cells. Levan and Lotfy (1949) noted the Occurrence of chromosome fragments in adult cells following naphthalene acetic acid treatment. Berger and Witkus (1948) also noted this effect on plant tissue. They suggest that the fragments originate when the chromosome thread is single, after the last meristematic telophase. The adult cells divide endomitotically. This suggestion implies that NAA acts probably as a detector of structural changes, inherent in the chromosomes of old cortical cells. The effects of a number of other hormones on root-tip cells have also been studied. Sharma and Mookerjea (1954a) noted that in meristematic cells, five different hormones, namely, 0-naphthoxyacetic acid, phenylacetic acid, indolylpropionic acid, indoleacetic acid, and indolylbutyric acid are capable of causing chromosome breakage. These results, however, do not show a correlation between the solubility and the concentration required for optimum effect. It has been suggested that all these hormones show a number of common effects on chromosomes and cytoplasm. These effects may be attributed to their similarity in chemical properties. An invariable aftereffect of hormone treatment is the appearance of erosion in a number of chromosome segments prior to breakage. This finding obviously indicates that certain chromosome sites become devoid of the full quota of nucleic acid and appear as thinner areas compared to the rest of the chromosome segments. These areas, because of this change in their constitution, become labile and ultimately break.
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Huskins and others (Huskins and Steinitz, 1948; Therman, 1951) noted that hormones, such as indolylacetic acid, can induce division in adult nuclei. It is now well established that adult cells of the differentiated region are not in a resting condition, but in an endomitotic state. The chromonema of these nuclei do not separate further and do not enter into metaphase. The later stages of division can be induced in them by hormone treatment. As they divide, fragments appear due to nonsynchronized separation. Systematic attempts (Sharma and Mookerjea, 1954b) to induce division in adult nuclei through different groups of substances, including nucleic acid and its constituents, show that the apparently nondividing state of the adult nuclei is due to their deficiency in the full quota of nucleic acid. This deficiency is caused by insufficient synthesis of the sugar component of nucleic acid, and it can be counteracted by growing the roots in different solutions of vitamins, hormones, etc. This implies that these compounds help indirectly in the nucleoprotein metabolism of the nucleus and that nucleic acid is essential for gene reduplication and separation. Earlier, it has been shown that (Sharma and Sen, 1954b) fragmentation may be due to disbalance in the nucleoprotein metabolism of the nucleus. The addition of nucleic acid also causes fragmentation, as was shown by Woll (1953). The implication follows that hormones can both help in the synthesis of NA and retard its metabolism. As these two effects are quite opposite, possibly they are influenced by additional factors. These supplementary factors, possibly modify the effect of hormones into an injurious one on the plant cells. Another growth-promoting substance with radiomimetic properties, other than organic acids, is maleic hydrazide (Bertossi, 1950; Greunlach and Atchison, 1950; Darlington and McLeish, 1951 ; McLeish, 1952). As it is a structural isomer of uracil, a heterocyclic bace, their effects have been compared. McLeish (1952) noted heavy fragmentation of chromosomes in root-tip cells of Vicia faba with maleic hydrazide treatment. But an allied species, Vicia satirua, did not show any effect (Sharma and De, 1954). V . faba is characterized by mainly long chromosomes, while those of V. sativa are comparatively much smaller. It is not yet known whether the differential response of the two species is due to their size. A worthwhile problem is to find out if this difference in response occurs with X-ray treatments too. If it is due merely to size, then the behavior of the two species may apparently signify that the number of fragments will decrease with a decrease in chromosome size. But even then, the complete absence of induced fragments in V. sativa cannot be explained. This differential effect can perhaps be explained by comparing the
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general morphology of the chromosomes of the two species. In V . faba, the chromosomes possess prominent heterochromatic segments other than the secondary constriction regions, while in V . sativa, the only prominent heterochromatic segments are the secondary constriction regions. The abundance of heterochromatic segments in chromosomes of V . faba may be responsible for their fragmentation with maleic hydrazide. Heterochromatic segments are evidently more susceptible to maleic hydrazide than the euchroniatic ones. Auerbach (1949a) referred to Koller and Caspersson, who had suggested that carcinogens act preferentially on heterochromatin. Auerbach, however, noted negative results in her material. In this connection, the genomic differences of the two species may be mentioned. They may have a genic difference with regard to susceptibility to maleic hydrazide, but in that case the data so far gathered cannot be generalized. McLeish’s (1952) observations on maleic hydrazide are similar to results obtained by Loveless and Revell (1949) with di(2 :3-epoxypropyl) ether. With both substances, breakage seems to be localized in the heterochromatic segments. It may be stated here that heterochromatin located at the secondary constriction regions has been assumed to be similar to that of other heterochromatic segments of chromosomes. How far the two can be considered to be identical is as yet a debatable point. McLeish has shown certain fundamental differences between the actions of diethyl ether and maleic hydrazide. The former follows the law of mass action and induces breakage mainly in short chromosomes, whereas the latter does not follow the law and affects mainly the secondary constriction regions. The present authors think that this fact also implies fundamental differences between the heterochromatin present in secondary constriction regions and that of other segments, which is brought out by chemical treatment. McLeish assumes that probably different systems of chromosome synthesis become affected by these chemicals. The systems are blocked in different stages of completion by the two reagents. If this contention is correct, then it must be admitted that the modes of operation of the different chromosome-fragmenting chemicals are different. Revell (1952) has suggested the reason for the higher susceptibility of the heterochromatic segments. He states that the action of chemicals is never at random. Possibly they inhibit chromosome synthesis directly or indirectly. The substrate required by the chromosome locus for metabolism may be affected, or the chemical may interfere with chemical processes in relation to this locus. H e also suggests that possibly some of the labile sites along the chromosome length, which normally break at the meiotic crossing-over, are more susceptible. Breakage is conditioned,
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according to him, by chromosome pairing. This, however, is not homologous since it may involve any two heterochromatic segments, a process facilitated by their sticky nature. This reason underlies the so-called specificity of the heterochromatic regions. With regard to the data of Revell, it must be pointed out that heterochromatic pairing is not universal for all biological objects. In a number of plants, this is not visible, although in species like Cicer arietinum, its occurrence has been recorded (Thomas and Revell, 1946). The question arises as to whether inadequacy of technique is responsible for the nonvisibility of heterochromatic pairing in a majority of plants. Otherwise, some other suggestion for heterochromatic specificity must be made. It appears that the chemical nature of these segments is responsible for their high susceptibility. It has been definitely proved that their chemistry is different from that of their euchromatic counterparts, since these segments are at the same time less condensed and so more liable to breakage. Several growth-promoting substances, the vitamins, such as ascorbic acid, succinic acid, nicotinic acid, and thiamine, as well as calcium pentothenate, etc., have been tried on differentiated cells of roots of Allium cepa, and their fragmenting property has been demonstrated (Sharma and Datta, 1956; Sharma and Bhattacharyya, B., 1956). Similarly the effect of vitamin K has been noted by Nybom and Knutsson (1947). It has been shown that in adult cells the former four substances as well as calcium pentothenate can induce division. During the process of separation of polytene threads, fragments arise due to differential tension on different chromosome segments which are induced to proceed to metaphase artificially. A number of growth regulators, such as 4-chloro-2-methylacetic acid, 2,4-dichlorophenoxyaceticacid' and 2,4,5-trichlorophenoxyacetic acid, etc., have been tried on somatic cells and a similar capacity for chromosome fragmentation noted (Carlton, 1943 ; Doxey and Rhoades, 1949 ; Nygren, 1949). As these substances control to a large extent the growth processes of plants and accelerate them, if applied in proper dosage, it is likely that their effect becomes injurious when the threshold concentration, responsible for their beneficial activity, is surpassed.
VIII. Action of Mustards, Related Compounds, and Phenols The most important group of chemicals proved to be mutagenic is the mustard group (Auerbach, 1947, 1949a, b, 195Oa, b, 1952; Auerbach and Robson, 1944, 1947a, b ; Auerbach et al., 1947; Gustafsson and Mackey, 1948; Novick and Sparrow, 1949; Koller, 1952). A number of vesicant compounds, namely, all$ isothiocyanate, chloracetone, etc., have been
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tried and their capacity for inducing chromosome aberrations worked out. Some authors (Dustin, 1947) believe that this group of compounds reacts mainly with the sulfhydryl groups, though objections to this idea have also been raised, suggesting that esterification of acid groups is responsible for radiomimetic action (Loveless and Revell, 1949). Auerbach ( 1949a) noted that Lewisite, in spite of its high affinity for sulfhydryl groups, is not mutagenic. She has also objected to the possibility that the mustard compounds act on phosphokinases, mainly on the basis of the experimental fact that mutations are very difficult to obtain if eggs are treated in the ovary. Several authors (Goldacre, et d., 1949; Loveless and Revell, 1949) have suggested that two reactive groups in the mustard molecule are responsible for its radiomimetic action. Chromosome breaks and bridges have been attributed to interchromatid cross linkage by single polyalkylating molecules. However, in addition to polyfunctional molecules whose action they studied, compounds having monofunctional groups have been shown to induce radiomimetic effects by Biesele et al. (1950). Although the latter authors do not deny the possibility of polyfunctional alkylating agents effecting chromosome breaks, they suggest another alternative suggestion. They consider that as 3-membered heterocyclic radicals are the common feature for all nitrogen mustards tested, it is better to consider that the radiomimetic activity is related to this common chemical property. According to Auerbach, some of these poisons act in the same way as radiations, namely, through localized contacts with certain governing centers of cell activity, mainly by direct transfer of energy to the chromosomes. This statement can be verified through dosage experiments with chemical mutagens, a scheme limited by the difficulty of penetration of these chemicals. She also infers that some chemical mutagens influence the metabolic rate and “hazards of protein synthesis may lead to error in gene reduplication.” The metabolic setup can be changed by a shifting of viscosity, hydrogen ion concentration, permeability of the cell membrane, etc. In order to work out the metabolic path involved in nuclear changes following mustard gas treatment, experiments based on the use of antagonistic chemicals are necessary. In mustard gas treatment, the condition of application and the stage of cell cycle to be treated must be taken into account. Several workers have noted specificity of effect with mustards. Ford obtained exclusively median breaks in short chromosomes of Vick faba root cells after applying nitrogen mustard (194%, b). In strong contrast, X-ray breaks are more or less homogeneous throughout the chromosome.
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Auerbach (1952) observed that mustards cause both gene mutation and rearrangement, but the proportion is entirely different from that obtained by high energy or ultraviolet radiation. This specificity of action and dependence on the cell cycle, as well as other metabolic factors, indicate that the action of mustards is possibly different from that of X-rays. Revell’s (1952) comments on the stage of the cell cycle most susceptible to breakage are noteworthy. According to him, treatment in the early resting stage produces the highest proportion of breaks, which gradually decreases with the progress of cell division. This can be explained by the fact that the effect of a chemical is mainly through a disbalance in the metabolism, which is maximum in the so-called resting stage. Evidence so far accumulated indicates that a change in the metabolic rate is the initial reaction involved. However, the possibility of both direct and indirect action, as emphasized by Auerbach, cannot be overruled. The phenol group of chemicals has been noted by Levan and others (Levan and Tjio, 1948a, b ; Parmentier and Dustin, 1948, etc., Therman 1949; Sharma and Bhattacharyya, N. K., 1956a) to be capable of inducing fragmentation. Levan has obtained a marked number of chromosomal aberrations by testing a number of phenols, including pyrogallol, guaiacol, hydroquinone, resorcinol, etc. He observed subnarcotic action following treatment with a number of them. Due to the frequent occurrence of pseudochiasmata in his preparations, it appears that most of the breaks originate through irregular and delayed division of certain chromosome segments. It is well known that the occurrence of stickiness and pseudochiasmata is due to the failure of certain regions to reduplicate while the adjoining segments duplicate a s usual. This causes a tension at the joining point, and finally results in breaks at these loci. Levan is of the opinion that fragmentation by phenol treatment indicates that certain chromosome loci are specifically attacked. He is as yet uncertain as to whether pseudochiasmata should be included under this set of reactions or not. It appears to the present authors, however, that the locus of pseudochiasma formation is the locus of chromosome breakage as well, for the reason mentioned above. Of all the phenols so far tested, a very high frequency of fragments has been noted with pyrogallol, hydroquinone, p-phenylenediamine, pyrocatechol, p-aminophenol, hydroxyhydroquinone, and benzoquinone. They are all readily oxidized and have their -OH group in the ortho or para position. The rest of the phenols, namely, resorcinol, phloroglucinol, etc., are comparatively less active. Levan suggests that oxidation is in some way concerned with their activity. They may directly affect the oxidation-
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reduction system of the cell, or their oxidation products may be responsible for their activity. The implication of this fact is far-reaching. If the addition of oxygen or removal of hydrogen may be initially to some extent responsible for the breakage, then certainly the phenol-induced breaks have some parallel with the X-ray breaks. In addition to the resemblance in the structural aberrations of chromosomes induced by both (Levan and Tjio, 1948a, b ; Hohl, 1948; Ehrenberg ct al., 1949; FabergC, 1950; and Lea, 1946), there is likely to be some fundamental similarity'in the mode of reaction involved. The correlation between radiosensitivity and oxygen concentration has been dealt with by many authors (Nilan, 1956). A decrease in oxygen concentration or irradiation of the tissue in an oxygen-free atmosphere shows a great reduction of X-ray breaks. A considerable decrease has been noted if the germinating seeds are irradiated in a vacuum and an increase has been observed in an atmosphere containing increased oxygen. It has been suggested that increase in oxygen may have an indirect action on radiation effects. It may help in accelerating the production of OH or HzOz radicals which, according to some authors (Goodspeed and Uber, 1939; Hohl, 1948; Giles and Riley, 1949; Rees, Giles and Beatty, 1952), have a profound effect on chromosome breakage. Levan's results with phenols, indicating that oxidation has an effect on chromosome breakage, suggest a similarity between the mechanism of radiation and chemically induced breaks. In order to work out exactly how the metabolism is deranged after treatment with a specific chemical, it is desirable to perform experiments involving the use of protective chemicals as has been done in the case of X-rays (Patt et al., 1949; Michaelson, 1952; Riley, 1954, 1957; Wolff, 1954 ; Plaine, 1955 ; Read and Kihlman, 1956). Experiments are now being carried out in this laboratory involving the induction of chemical breakage following treatment with different groups of chemicals in root tissue and subsequent growth of these tissues under different nutrient media for recovery. I t is expected that recovery from the injury in the particular medium will indicate the derangement of a metabolic step and the consequent deficiency caused by the fragmenting chemical, which will be compensated for later by supplying that specific chemical in the nutrient medium. IX. Other Compounds Demerec (1947a, b, 1948, 1949) has listed a number of hydrocarbons and azo compounds which can induce chromosome aberrations. Among the hydrocarbons, a definite effect has been induced following treatment with 1,2,5,6-dibenzanthracene; 3,4-benzpyrene ; 1,2-benzanthracene ; and
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among the azo compounds, with p-hydroxyazobenzene, p-dimethylaminoazobenzene, 2-aminoazotoluene, etc. Hydrocarbons have also been tested by Patton and Nebel (1940). The effects of some of the heterocyclic bases, namely, adenine, guanine, uracil, etc., have been studied (Kihlman, 1950b; Sharma and De, 1956) and their property for inducing fragments observed. These bases are the natural constituents of the nucleic acid of plant chromosomes. It appears anomalous that these substances can at the same time induce chromosome breakage, which is a manifestation of an injury to cell activity. This fact shows that an excess of a metabolite within the cell can cause mutation, whether it is essential for vital activity or not. It may be emphasized that spontaneous chromosome breaks may be caused, not only through a severe external shock, but rather through any sufficiently serious disorder in the internal setup. The same explanation applies to the hormones and growthpromoting substances, discussed earlier. No doubt, different chemical steps, as pointed out by various authors, are responsible for the ultimate break, but in all cases it cannot be denied that an initial disorder in metabolism is responsible for the breakage. This disorder may be manifested in the form of its influence on various steps of chromosome metabolism. Of the chemicals tested by Kihlman and Levan (1949) and Kihlman (1955a, b), the importance of a number of methylxanthines, caffeine, theophylline, theobromine, etc., deserves mention. They, especially 8-ethoxycaffeine, can induce fragmentation of chromosomes in root tips of Allium cepa. Since these chemicals have been shown to have a narcotic effect, they can be expected to show radiomimetic activity at the subnarcotic level. An important problem is the extent of mutagenic action of these chemicals on their parent plants. For, example, colchicine cannot cause polyploidy in Colchicum (Mehra and Khoshoo, 1948). The parallel effect of these chemicals on their parent plant species should be investigated. In such cases, a natural immunity may be developed against the toxic action of these chemicals by the plants concerned. The fragmenting properties of a number of other purine derivatives have been studied by Kihlman (1949, 1952). An increase in toxic effect has been noted when methyl groups are substituted by other alkyl groups. The ally1 group is less toxic than the propyl one. As the derivatives are very sparingly soluble in water, difficulties were encountered in testing their effects. In general, it has been recorded that anaphasic chromosomal changes, that is, fragmentation, translocation, etc., are induced by %ethers of caffeine and especially tetramethyluric acid rather than by caffeine itself. Trimethyluric acid, dimethylhypoxanthine and tetramethyl4,Sdimethoxydihydrouric acid are practically inactive. By comparing the
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effect of caffeine with 1-ethyltheobromine and also 1, 3, 7-triethylxanthine, theophylline with 1, 3-diethylxanthine, and finally tetramethyluric acid with 9-ethyltrimethyluric acid, it has been noted that there is a reduction in effect if an N-bound methyl group is substituted by an ethyl group. When several methyl groups are substituted by ethyl groups, the action of the purines practically stops. The length of the carbon chain in the oxygenbound alkyl groups in 8-ethers of caffeine does not control the effect to a marked degree. With progressive increase in N-methylation and more electronegative substituents in positions 6 and 8, there is an acceleration of the effect. A correlation between the solubility and degree of chromosomal changes can also be worked out, as specially pointed out by Kihlman (1949, 1952), referring to the works of Weil-Malherbe (1946). A number of other chemicals have been found to be mutagenic as well. These include the naphthalene derivatives (Avanzi, 1950) ; benzpyrene (Casabona, 1948) ; benzene vapor (Berger et al., 1944) ; morphine ( Malheiros-GardC, 1950) ; ethylene glycol (D’Amato, 1948a) ; parascorbic acid (Cornman, 1947) ; acridine derivatives (Massart et d., 1947 ; D’Amato, 1950c, 1951c, 1952b, 1952c, 1954a ; D’Amato and Avanzi, 1954) ; rhamnose (Sharma and Jash, 1961) ; @-naphthoquinoline (Tarabusi, 1951) ; a number of sugars (Sharma and Mookerjea, 1954b) ; m nitroxylene and p-dichlorobenzene ( Simonet, 1940 ; Sharma and N. K. Bhattacharyya, 1956b) ; propiolactone and acetyl pyridine chloride (Smith and Srb, 1951 ; Smith and Lotfy, 1954) ; quinoline (Simonet and Igolen, 1944; Mello-Sampayo, 1950) ; gammexane (D’Amato, 1949c, e, 195Oa ; Sharma and Chaudhuri, 1959) ; chloroform (Steinegger and Levan, (1947a) ; urethane and cyclohexylcarbamate (Battaglia, 1949; Cornman, 1954; Nambiar, 1955) ; diethylmalonylurea or veronal (Deysson and Rollen, 1951) ; hydrogen peroxide (Rondoni and Bassi, 1951) ; pentavalent arsenic (Mangenot, 1940, 1941) ; chloranil (Yakar, 1952) ; azotoriprite (Battaglia, 1948) ; trypaflavin (LettrC, 1941; Bauch, 1948, 1949b) ; acriflavin (Ephrussi, 1949) ; phenylmercuric hydroxide, phenylmercuric nitrate, and mercury compounds (Macfarlane, 1950, 1951, 1953, 1954 ; Macfarlane et al., 1951) ; 2,4-dichlorophenoxyacetate (D’Amato, 1948d) ; guanidine nitrate (Shaw, 1952) ; azine series (Becker, 1932) ; phenanthrene derivatives (Steinegger and Levan, 1948) ; a number of alkalis (LorenzoAndreu, 1951) ; chromic acid and potassium dichromate (D’Amato, 1949a, e) ; versene (Hyde, 1956 ; Sarkar, 1957) ; acenaphthene (Kostoff, 1938 ; Navashin, 1938; Levan, 1940 ; D’Amato, 1949b) ; ethylenediaminetetraacetic acid (McDonald and Kaufmann, 1957) ; gallic acid (Sharma and De, 1954) ; 0-isopropyl N-phenyl carbamate (Ennis, 1948) ; halogen derivatives (Simonet and Guinochet, 1939 ; Tarabusi, 1951) ; phosphates
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(Galinsky, 1948) ; ethyl urethane (Deufel, 1951, 1952) ; oxygen (Conger and Fairchild, 1952) ; phosphomanganate (Doring and Stubbe, 1938) a number of phosphates (Galinsky, 1948) ; and sodium p-aminosalicylate (Levan, 1945; Mascr6 and Deysson, 1950, 1951) ; the salicylic series (Mcletti, 1953) ; and xyloquinone (Ganguly and Datta, 1956). In general, von Rosen (1954, 1957) classified the radiomimetic substances under the halogen series, strong and weak metals, alkalis, acids, acid salts, and organic compounds. All the halogens are found to be radiomimetic. Of the metals, T1, Cd, Cu, Os, Hg, Ag, Ti, Au, Cr, Co, Ni, Pd, La, and Ce show strong action, and weaker activity is seen with Zu, Li, Al, Ca, Mn, Fe, Se, Rb, Sr, Zr, Sn, Sb, Cs, W, and Th. Of the alkali solutions, the action of ammonium sulfide and sodium hydroxide is less than ammonia and ammonium sulfide. The latter mixture shows lesser action than sodium hydroxide and potassium hydroxide, and finally these two show again lesser effect than a mixture of ammonium hydroxide and ammonium sulfide. Of the acids, in order of action, can be arranged sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, and acetic acid. Among the acid salts, sodium dihydrogen phosphate is stronger than sodium hydrogen phosphate and sodium phosphate. Under the organic compounds listed by von Rosen, according to the intensity of action, come the mustards, quinoline, mustard oil, acetic acid, acetone, sulfhydryl groups, gallic and ascorbic acid, ethyl alcohol, acetaldehyde, caffeine, pyrogallol, and urea. The above discussion shows that in addition to the chemicals listed by von Rosen, a number of others as well are radiomimetic in action. All of them mainly fall under either inorganic salts or under aliphatic, allocyclic, aromatic, and heterocyclic compounds.
X. Conclusion This resumk of spontaneous and chemically induced fragmentation reveals clearly the complexity of the problem involved. Fragmentation occurs following the action of a variety of chemicals, having widely differing properties. It is difficult to assume that all these varied chemicals can act identically within the cell. The mode of action cannot be deduced from a recording of the end results. Some of these chemicals may affect to some degree the sulfhydryl groups of proteins, as suggested by some workers. Others, on the contrary, may act only through their influence on hydrogen bonds of nucleic acid. Still others may cause a disturbance in the oxidation-reduction system within the nucleus. I t has been discussed in this paper that all these theories have been derived through the use of certain chemicals and the evidence produced in support of each theory is
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well warranted. On the other hand, nothing could be inferred about the mode of action of a large number of chemicals shown to possess radiomimetic property. At the same time, none of the theories presented by different workers is universally applicable. Nucleic acid is a very complex macromolecule. Leaving aside its polymerized nature in the chromosome, each nucleotide is composed of several less complex substances-the bases, the sugar, and the acid. The metabolism of each of these components is very complicated in nature. It is also further influenced by a number of other intra- and extranuclear factors. Therefore, that the synthesis of nucleic acid is complex can easily be conceived. The importance of nucleic acid in protein synthesis is unquestionable. It is no wonder that different chemicals, the actions of which vary widely, can cause chromosome breaks. In our opinion, their action can be explained by the fact that hazards in protein synthesis, which are responsible for breaks, can easily be brought about by causing a disruption in the metabolism of nucleic acids, which in all possibility normally act either as a screen (Frey-Wyssling, 1953) or trapping agent favoring protein synthesis in the genes as visualized by Danielli (1950). This disruption can be induced by influencing any of the requisite metabolic steps and maybe by affecting the hydrogen bond of nucleic acid through alkylation of -NH2 groups and possibly esterification of acid groups as well, through direct or indirect means. It is likely that with certain compounds at least, an indirect action on -SH groups of proteins too is exerted in addition, on which thorough investigation is desired. The data of various authors have revealed different facets of the same problem, all ultimately implying the derangement of this vital metabolism of the cell. The only inference that we can draw regarding the mode of action of chemically induced breaks is that the chemicals act through different means, all of which finally upset the nucleic acid metabolism. The latter being absolutely responsible for gene reduplication, this upset brings about the hazards in protein reduplication, resulting in chromosome breaks. This suggestion, though broad in its implications, embodies a reconciliation of the varied data so far presented.
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Auerbach, C. (1949a) Discovery of the mutagenic action of mustard gas. Proc. 7th Intern. Congr. Genet., Stockholm, 1949, Hereditas, Suppl., 128-147. Auerbach, C. (1919b) Chemical mutagenesis. Biol. Revs. Cambridge Phil. SOC. 24, 355-391. Auerbach, C. (1950a) Differences between effects of chemical and physical mutagens. Pubbl. staz. zool. napoli 22 Suppl., 1-23. Auerbach, C. (195Ob) Some recent results with chemical mutagens. Hereditas 37, 1-14. Auerbach, C. (1952) Sensitivity of Drosophila germ cells to mutagens. Symposium on chromosome breakage. Heredity 6 Suppl., 247-257. Auerbach, C., and Robson, J. M. (1944) Mutation chemically induced. Production of mutations by ally1 isothiocyanite. Nature 154, 81. Auerbach, C., and Robson, J. M. (1946) The chemical production of mutations. Nature 167, 302. Auerbach, C., and Robson, J. M. (1947a) The production of mutations by chemical substances. Proc. Roy. SOC.Edinburgh B62, 271-283. Auerbach, C., and Robson, J. M. (1947b) Tests of chemical substances for mutagenic action. Proc. Roy. SOC.Edinburgh B62, 284-291. Auerbach, C., Robson, J. M., and Carr, J. G. (1947) The chemical production of mutations. Science 106, 243-268. Avanzi, M. G. (1950) Frequenza e tipi di aberrazioni chromosomiche indotte da alcuni derivati dell’ a-naftalene. Caryologia 3, 165-180. Avanzi, S. ( 1954) Osservazioni sull’attivita citologica della 8-aminochinolina e di due derivati naftolici. Caryologia 6, 128-133. Battaglia, E. (1948) Sull’azione mutagenica dell azotoiprite (Ny) Reddic. Accad. Naz. Lincei, C1. Sc. Fis. Ser. (8a), 4, 771-776. Battaglia, E. (1949) Sull’azione citologica dell’etil-carbamato (uretano) e del ciclo-esil-carbamato. Caryologia 1, 229. Battaglia, E. (1950a) Osservazioni sull-azione citologica di alcune sostanze coloranti. Caryologiu 2, 223-228. Battaglia, E. (1950b) Nuove sostanze inducenti fragmmentazione cromosomica. Pubbl. staz. 2001. napoli !22 Suppl., 125-157. Bauch, R. (1947) Trypaflavin als typhus der chromosomengifte. Naturwissenschaften 34, 356-357. Bauch, R. (1948) Irreversible chromens chadigungen durch Trypaflavin. Planta 36,536-554. Bauch, R. (1949a) Sulfonamide und Colchicin. Pharmuzie 4, 1-7. Bauch, R. (1949b) Selective speicherung von trypaflavin durch die nukleoprotide der chromosomen. Biol. Zentr. 68, 113-118. Beadle, G.W. (1937) Chromosome aberration and gene mutation in sticky chromosome plants of Zea mays. Cytologia (Tokyo) 2,43-56. Becker, M. W. A. (1932) Influence des colorants de la serie des azinas sur la marche de la cinese, somatique dans les racines chez 1’Allium cepa. Rev. g h . Botan. 44, 24-29. Becker, M. W. A. (1933) Vitalbeobachtungen iiber den einfluss von Methylenblau. Beitrag. zur Pathologie der mitose. Cytologb (Tokyo) 4, 135-157. Berger, C. A., and Witkus, E. R. (1948) Cytological effects of alphanaphthalene acetic acid. J . Heredity 89, 117-120. Berger, C. A., Witkus, E. R., and Sullivan, B. J. (1944) The cytological effect of benzene vapor. Bull. Torrey Bofan. Club 11, 620-623.
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The Ultrastructure of the Nucleus and Nucleocytoplasmic Relations’ SAUL WISCHNITZER Department of Anatomy, New York Medical College, Flower and Fifth Avenue Hospitals, New York, New York Introduction ..................................................... Nomenclature .................................................... Nuclear Envelope ................................................ Nucleolus ........................................................ Chromosomes .................................................... A. Interphase Chromosomes ..................................... B. Mitotic Chromosomes ........................................ C. Meiotic Chromosomes ........................................ D. Giant Chromosomes .......................................... VI. Nucleoplasm ..................................................... VII. Discussion ....................................................... A. Nuclear Constituents ........................................ B. Nucleocytoplasmic Relations .................................. VIII. Conclusion ....................................................... Acknowledgment ................................................. References ....................................................... Addendum .......................................................
I. 11. 111. IV. V.
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“The morphologist, on the one hand, strives to elucidate the structure of protoplasm down to its finest details; the biochemist, on the other, with his apparently ruder yet still more searching methods, seeks to determine the chemical functions of the same protoplasm ; broadly speaking, they are only dealing with two different sides of the same thing.” Hofmeister, 1901.
I. Introduction In 1934, two years after inventing the first electron microscope, Ruska succeeded in achieving high resolutions with .such an instrument. By 1940 technical advances had continued to the point where such microscopes had resolving powers of 25A. and commercial production was initiated. More than an additional decade elapsed before this instrument could be intensively applied in biological research. This was made possible by such technical improvements as the introduction of accurately symmetrical magnetic objectives and the use of high voltage accelerating potentials, together with advances in preparation and sectioning of the material. Thus by 1953 the methodology had reached a point where the procedures employed were as routine as those used in classical histology (see review 1 This work was supported in part by a U. S. Public Health Research Grant RG-5803 (Cl) . 137
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by Selby, 1953a). Since the early 1950’s most of the research in electron inicroscopy has been devoted to an elucidation of the fine structure of the cytoscme and its organoids, and enormous progress has been made (Porter, 1956; Jackson and Randall, 1958; Palay, 1958). So far as the nucleus is concerned the results have been decidedly limited. The direct aim of such studies obviously has been the attempt to uncover the basic structure of the nuclear constituents, especially the chromosomes, which lies beyond the level of resolution of light microscopy. The ultimate aim of such studies, however, extends beyond the boundaries of morphology. The nucleus, by virtue of the fundamental role which it plays in the biological processes associated with heredity and morphogenesis ( Mazia, 1952), is considered to be the most significant component of the cell. Knowledge as to the exact mechanisms by which the nucleus exerts its influence on cellular metabolism is as yet extremely limited (Mather, 1958) . All available evidence suggests that nucleocytoplasmic exchange takes place to a large extent on a macromolecular level (Anderson, 1953), which is at the lower limits of resolution of the electron microscope (Hall, 1956). Research on this problem is, however, limited by the fact that the electron microscope directly presents only a morphologically static view of the cell. Nevertheless, it is still felt that, on the basis of an understanding of the fine structure of the nuclear elements, some insights into nuclear physiology can be deduced. This review will therefore be exclusively concerned with a presentation of the fine structure of the nucleus, especially of the nuclear envelope and nucleolus, the elements which have been thus far most satisfactorily and therefore most extensively studied. As a result of the advances made by means of electron microscopy on the elucidation of nuclear morphology, a number of possible modes of functional nuclear expression have been uncovered, and the aforementioned raison d’ztre for this cytological approach to an understanding of nuclear physiology is beginning to be realized. 11. Nomenclature Since a number of new descriptive cytological terms have been introduced by electron microscopists, these will therefore now be defined. The morphological boundary between the nucleus and cytoplasm will be called the nuclear envelope since it is composed of two nuclear membranes. The region between the membranes will be referred to as the intermembranous zone. The nuclear envelope when viewed in perpendicular section is interrupted by discontinuities. These discontinuities correspond to the holes, pores, or fenestrae referred to by other authors. In tangential sections of the nuclear envelope, these are seen as annuli,
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which others have also called pores, rings, or nodes. The cylindrically disposed material associated with each of the discontinuities will be called the annular complex. The nucleoli may be made up of two components, an amorphous matrix, within which is embedded the nucleolonema (Estable and Sotelo, 1951), an organized coiled structure. The chromonema, that is, the chromosomal axis as seen with the light microscope, becomes resolved by electron microscopy into one or many microfibrils. 111. Nuclear Envelope In 1950, Callan and Tomlin, working with isolated oocyte nuclei from salamanders, were the first to demonstrate by electron microscopy (using the replica technique) that the nuclear envelope was composed of two membranes. They found that the outer one was about 300A. thick and that it had circular discontinuities about 400A. in diameter. The inner membrane was reported to be uninterrupted and to be only 150A. thick. In the next two years Bovey (1952), studying nuclei primarily from locust and cockroach oocytes, and Bairati and Lehmann (1952) and Harris and James (1952), working with Amoeba proteus, in general confirmed the original electron microscope observations of Callan and Tomlin. The major difference, aside from dimensional ones noted in Amoeba, was the reversal of the position of the two layers. Later studies by Bahr and Beermann (1954) of sectioned salivary gland and mid-gut cells of Chironomus reported the presence of annuli in tangential sections of the nuclear envelope, while in perpendicular sections, discontinuities at identical points in both membranes were observed. Annuli were originally noticed by Callan and Tomlin as well as by Bovey and later by Gall (1954a), but they considered these structures to be artifacts arising during the preparation of the material. Subsequently, Gall (see Callan, 1955) studied sectioned oocytes from fish, echinoderms, and annelids and concluded that annuli exist in vivo. Watson (1955), in a survey of a number of different tissues, convincingly established the intimate relationship between the annuli and discontinuities. H e concluded as have a number of others (Dawson et d., 1955 : Haguenau ahd Bernhard, 1955 ; Pappas, 1956) that both structures are views of the same formation seen in different orientations. On the basis of the aforementioned and subsequent sudies, it can be concluded that the basic structure or skeleton of the nuclear envelope of virtually all cells, both animal and plant (De, 1957 ; Hassenkamp, 1957), normal and malignant (De Groodt et al., 195S), is bilamellar, and that it is interrupted by circular discontinuities produced by the union of both
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membranes (Fig. 1). The exact dimensions of the individual membranes, the perinuclear space, and the discontinuities vary among different cell types. Generally, however, each of the membranes is usually about 100A. thick with the intermembranous zone being 150A. Thus, the entire nuclear envelope has a diameter of about 350A. The discontinuities are usually about loo0 A. in diameter.
FIG.1. The nuclear envelope. Schematic representation of the nuclear envelope “skeleton” which is characteristic of virtually all cells. It consists of a bilamellar envelope interrupted by circular discontinuities at the places where both the membranes unite.
This concept of the morphology of the nuclear envelope is, however, complicated by a number of additional observations. Thus, it has been found in some cells that a band extends across the discontinuity (see references in Wischnitzer, 1958), and that a central granule is present in some annuli (Pollister et al., 1954; Watson, 1955; Gay, 1956a; Swift, 1956; Wischnitzer, 1958). Watson (1955) has suggested that the band may serve as a mechanism for the .formation of discontinuities. More recently, however, Barnes and Davis (1959) have shown that the bands are section artifacts in the sense that they simply represent the margins of the annular openings at the level of the nuclear envelope. The small granules, on the other hand, have been considered to represent material passing through the annuli from the nucleus to the cytosome or to be structural components of the nuclear envelope (Pollister et al., 1954). These interpretations of the aforementioned observations await further experimental confirmation. Also complicating the situation are the observations made on sectioned oocytes from both vertebrates (Wischnitzer, 1958 ; Swift, 1958) and invertebrates (Afzelius, 1955 ; AndrC and Rouiller, 1956), which have shown their nuclear envelopes to have what, until very recently, was thought to be a more complex organization than had been demonstrated in somatic cells. The electron micrographs of oocytes have shown dense, rectangular or partly elliptical masses which are intimately associated
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with the discontinuities (Fig. 2). These structures were interpreted (Afzelius, 1955 ; Wischnitzer, 1958) as representing vertical or oblique sections respectively of a tube (Fig. 3 ) , a concept which is supported by the observation of annuli on levels above and below the nuclear envelope (Wischnitzer, 1958; Swift, 1958). There is also evidence to suggest that the tubes themselves have a more complex structure than simple cylinders. Thus high resolution micrographs have shown that the annuli are in reality composed of subannuli (Gall, 1956 ; Rebhun, 1956; Wischnitzer, 1958; Swift, 1958).
FIG.2. Results of two types of sections through the nuclear envelope of oocytes. Schematic representation of the results of two types of sections through the nuclear envelope of oocytes. Sections tangential to the surface of the envelope will result in rows of annuli. The micrograph on the right demonstrates such structures, some of which have a central granule (arrows). In actuality the micrograph represents a section which is oblique rather than perfectly tangential; thus only one row of annuli is seen. In sections perpendicular to the nuclear envelope, individual membranes and discontinuities containing dense masses of various shapes (see Fig. 3) will be produced. The micrograph demonstrating such a section is on the left. All these observations can be interpreted as suggesting that the nuclear envelope of oocytes is highly specialized just as it is in the amoeba (Pappas, 1956). On the other hand, Dawson et al. (1955) described the nuclear envelope in spinal ganglion cells as containing a tubelike organization within the discontinuities. Further evidence emphasizing the structural similarity between the nuclear envelope of oocytes and somatic cells has very recently appeared. Thus Watson (1959) showed that granular material is cylindrically arranged around the discontinuities in the nuclear envelope of various somatic cell types.
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FIG.3. Sections through a cylindrical composite associated with a discontinuity in the bilamellar nuclear envelope. Schematic representation of a minute portion of the nuclear envelope of an oocyte in which cylindrically disposed material is located within and around a discontinuity. The illustration demonstrates the results of vertical sectioning of such a structure at three different points. A. A rectangular mass results from a section through the wall parallel to the long axis of the cylinder. B. In a section through the lumen of the cylinder a rectangular mass is produced, whose margins are denser because of their increased thickness as compared to the grooved center. C. A partly elliptical section is produced by an oblique cut. Figures similar to sections A and C can be seen in the electron micrograph accompanying Fig. 2 on the left side.
IV. Nucleolus The extensive studies of this organelle by electron microscopy have been carried out by Bernhard et al. (1955) on both normal and malignant cells. They have shown that in their preparations of vertebrate cells, the nucleoli contain a coiled structure, the nucleolonema. The latter is composed both of round to oval granules 100-20A. in diameter and filaments of varying lengths but about 1OOA. in diameter. Yasuzumi et al. (1958) considers the dense granules to be gyres of helically coiled filaments which make up the nucleolonema. This organized portion appears to be embedded in an amorphous or diffused matrix which itself is made up of minute, less dense particles. Horstmann and Knoop (1957) have reported that the granules making up the nucleolonema are organized into rows both along and across the longitudinal axis, giving this structure a “striated” appearance. In many cases the nucleolonema cannot be distinguished (Wischnitzer,
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1958; Serra, 1958; Bolognari, 1959). This may be due to the masking effect produced by the very dense and evenly granulated amorphous portion of the nucleolus. But even in such cases, Horstmann and Knoop (1957) claim to be able to discern organized rows of granules. All this has suggested to Bernhard (1958) that while the presence of a nucleolonema is the best criterion for identification of the nucleolus, this organoid, depending on the functional state of the cell, can also appear as a homogeneous or amorphous body. The appearance of the nucleolus, however, is relatively constant within the same tissue. In some types of cells, e.g., protozoa (Cohen 1957) and some plant cells (Lafontaine, 1958), very dense spherical or irregularly shaped granules have been observed within the nucleoli. The significance of these bodies is as yet unknown. The presence of a nucleolar membrane could not be confirmed in any study thus far made by means of electron microscopy. Yasuzumi et al. (1958) have observed the presence in some cells of a nucleolus-associated body made up of helically coiled, tightly packed filaments 15-30A. thick. These investigators suggest that this structure may correspond to the Feulgen positive body which many earlier studies, using the light microscope, have found to be connected with the nucleolus.
V. Chromosomes In general it can be stated that insofar as the elucidation of the fine structure of chromosomes by electron microscopy is concerned, this new cytological technique has failed to confirm the presence of any highly organized structures. The latter was implied by light microscope observations, and was presumed to exist on the basis of cytogenetic evidence. The observations which have been made will be discussed under the headings of interphase, mitotic, meiotic, and giant chromosomes. CHROMOSOMES A. INTERPHASE The live interphase nucleus usually appears optically homogeneous by light microscopy, suggesting that the chromosomes have lost their structural integrity. However, experiments using the ultracentrifuge (see references in Brachet, 1957) have shown that fine filaments having a characteristic chromosomal appearance can be isolated from disrupted interphase nuclei. A number of studies of such isolated “chromosomes” by means of shadow casting techniques have suggested that they may be in the form of a definite spiral structure consisting of a filamentous central core embedded in a matrix (Hovanitz, 1947; Van Winkle et al., 1953). A more
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complex picture of such chromosomes as consisting of at least 32 microfilaments was also presented (Yasuzumi, 1955). It however has been pointed out by Denues and Senseney (1952) that in using such procedures it is possible to mistake casual contaminants for isolated chromosomes. In addition, it is not known how the isolation procedure affects the structure of the chomosomes themselves. Thus, strong doubt is cast on the reliability of this approach. Ris (1955) has noted that initial chromosome studies of interphase nuclei from the rat and lily, obtained by squash techniques, have also shown chromosomes to be made up of masses of microfibrils each ZOOA. thick. In studies of sectioned material of interphase nuclei of somatic cells, the chromosomal material appears only as amorphous masses irregularly distributed within the nucleoplasm or along the nuclear envelope. The masses themselves can be resolved into granules 100-200A. in diameter and minute filaments less than 1OOA. in diameter. Bernhard (1958) suggests that the absence of coarser structures directly comparable to the isolated filaments is due to the fact that they probably are highly coiled and thus in cross section appear as granules or short threads.
B. MITOTIC CHROMOSOMES Progress in studies of the fine structure of such chromosomes has been impeded by their thickness, which makes them opaque to the electron beam. Thus, initial studies reported the mitotic chromosomes as being structurally homogeneous and were unable to uncover any internal structure (Beams et al., 1950a,b; Rosza and Wyckoff, 1950, 1951; Sedar and Wilson, 1951). As techniques improved so has there been an increase in the ability to resolve structural, detail. Thus, Selby (1953b) found density variations in metaphase chromosomes. Kurosumi (1957), working with sea urchin eggs, also noted that such chromosomes had periodic striations. In cross section, these were shown to consist of an electron-dense cortex and a less opaque internal matrix. The cortex was reported to be composed of tightly arranged strands 300A. in diameter. It was suggested that these strands may be the chromonemata responsible for the cross-striated appearance of the chromosome, around which they are spirally wound. These observations appear to have a general similarity to those reported somewhat earlier by Kaufmann and De (1956), who worked with the staminate hairs of Tradescantia. They studied thin sections of early prophase chromosomes and interpreted the results as indicating that such chromosomes consist of several (up to 64) orders of helically arranged chromonemata. These are arranged as pairs, which in turn form a hier-
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archy of pairs all the way up to half-chromatids. The ultimate unit resolved was found to have a diameter of about 125A.
C. MEIOTICCHROMOSOMES There now exist a considerable number of electron microscope studies which strongly suggest that a fibrillar core is present along the axis of meiotic prophase chromosomes of animal cells (see literature summary in Moses, 1958). Fawcett (1956) was able to resolve this complex into a pair of dense fibrils, each about 500A. in diameter, which are equidistant from a thinner (120A.) central linear density (Fig. 4). In the most
FIG.4. Nucleus of a spermatocyte. Schematic representation of a nucleus of a spermatocyte in prophase of the first meiotic division as seen by means of the electron microscope. The chromosomes ( c h r ) are dense masses, within which there appear three parallel linear densities ( I d ) . At other points ( x ) these structures are seen in &oss section.
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recent study Moses (1958) showed that the complex occurs exclusively during the synaptic period of meiosis. He also feels that each of the lateral linear elements can be considered as representing the “core” of the individual meiotic chromosomes which is surrounded by finely granular material. The central density was considered to be the result of the pairing phenomenon. De Robertis (1956), who studied the prophase and metaphase chromosomes in the spermatocytes of the grasshopper, has been the only one not to find any evidence of the presence of a central core in such material. H e reported that the bulk of each of the chromosomes appears to be made up of very fine and irregularly coiled filaments ranging from 50 to 100A. in diameter. Ris (1955) studied the prophase meiotic chromosomes from the pachytene stages of a number of plants and animals by shadow casting methods. H e reported that they are composed of bundles of microfibrils 500400A. thick. Very recently Bopp-Hassenkamp ( 1959), studying sectioned material of plant pachytene chromosomes, found that the microfibrils were about 125A. thick and that these in turn were made up of “subfibrils” which had a diameter of about 25A.
D. ,GIANTCHROMOSOMES Most of the early studies of the salivary gland chromosomes confirmed the difficulties involved in elucidating their fine structure (Palay and Claude, 1949; Pease and Baker, 1949; Schultz et al., 1949; Herskowitz, 1952; Borysko, 1953). Yet Yasuzumi et d. (1951) reported that the banded regions consisted of twisted and tightly packed fibers which gave the appearance of granules, while in the interband region the 500A. thick fibers ran mainly parallel to the long’axis of the chromosomes. Bahr and Beermann (1954) later showed that the basic fibrillar unit was 50-1OOA. thick and that the interband region was made up of granules 300A. in diameter. These observations were essentially confirmed in the most recent study of these chromosomes (Lowman, 1956). A somewhat earlier study by Gay (1955) reported that the fibers were about 250A. in diameter and that a mature chromosome from Drosophila contains from 1000 to 2000 such fibers. With reference to the fine structure of the second type of giant chromosomes, namely, the lateral loop or lampbrush chromosomes, electron microscopy has not resolved the conflicting viewpoints as to their structure which developed from earlier light microscope studies (Duryee, 1941 ; Dodson, 1948 ; Gall, 1954b ; Wischnitzer, 1957). In the first extensive study of these chromosomes by electron microscopy, Guyknot and Danon (1953) came out in support of the chromomeric
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FIG.5. Current concepts of morphology of the lampbrush or lateral loop chromosomes. ch, chromomere; cm, chromonema; Zl, lateral loop; mf,microfibrils. The chromomere hypothesis (A) is based on light and phase contrast microscope observations. It postulates that each chromosome has a chromonema along which are located the Feulgen positive chromomeres. The lateral loops are considered to be outgrowths from these chromomeres. Gall (19558) has advanced the interpretation (B) that there are two greatly elongated chromatids, each a few hundred angstroms in diameter, making up each chromosome. The chromomeres are considered to be regions where the chromatids are tightly coiled. The chromatids are thought to serve as the axis of the loops, which are paired and arise by the partial unwinding of the chromomeres. The bulk of each loop is made up of material of varying consistency which accumulates around the loop axis. Lafontaine and Ris (1958) have presented an interpretation (C) in which the two fine strands serve to make up the chromomeres and the loops. The former also are considered to represent regions where the chromatids are highly coiled, but need not necessarily be associated with a loop. Each strand itself is considered to represent a bundle of microfibrils (*). Each loop, which according to this concept need not be paired, is made up solely of the coiled strand, and its diameter depends on the degree of uncoi!i?g of the individual microfibrils as shown at (**).
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interpretation (Fig. 5A). This concept postulates that each chromosome is composed of one or at the most two fine chromonemata along which are located true Feulgen positive chromomeres. The lateral loops are considered to be outgrowths from these chromomeres. Gall (1955), changing his earlier opinion ( 1954b), conceived (Fig. 5B) each of the chromosomes as being made up of two greatly elongated chromatids, a few hundred angstroms in diameter, with the chromomeres being regions where the chromatids are tightly coiled. The chromatids are thought to serve as the axis of the loops, which arise by the partial unwinding of the chromomeres. Granules or dense masses of material envelop the loop axis. Gall was able to demonstrate the existence of a loop axis by electron microscopy as well as the presence of an interchromomeric axis which was a few hundred angstroms thick. Ris ( 1956), revising his original interpretation ( 1945), has presented a concept (Fig. 5C) in which the loops are made up of a helically coiled bundle of submicroscopic fibers. Each fiber is thought to be 500A. thick and may itself be further subdivided, The chromomeres are considered to have the same fine structure as loops. Electron microscopic evidence in support of this concept was recently presented (Lafontaine and Ris, 1958). Gall (1958) has shown the similarity between both types of giant chromosome, structurally, in terms of their basic composition in very fine (1W2WA.) fibers, and functionally, in terms of the similarity of the lateral loops with the Balbiani rings as expression of chromosomal activity.
VI. Nucleoplasm
a.
Considerable progress has been made in obtaining a better understanding of the basic structure of cytoplasm (Sjostrand, 1956 ; Porter, 1957). However, as far as the nucleoplasm is concerned, it is devoid of any highly organized structural elements comparable to the ergastoplasm of the cytosome. As generally seen with the electron microscope, the interphase nucleus simply contains small, randomly distributed granular patches between large “chromosomal” masses. Recently Watson ( 1959) described the nucleoplasm as consisting of masses of dense material separated by regions of lower density, the latter being interpreted as channels which are associated with the discontinuities (Fig. 8A).
VII. Discussion From the preceding review of our knowledge of the ultrastructure of the nucleus, it is apparent that progress in this area has not been comparable to that reported for the cytosomal constituents. The study of the
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nucleus by electron microscopy appears to be hindered mainly by the fact that there are no striking differences in contrast between most of its internal constituents, which also lack any membranes enclosing them. Moreover, there is some evidence suggesting that osmium fixation may not be particularly suited for the preservation of chromosomal material (Ris, 1955). Thus, the formulation of a three-dimensional picture of the nucleus by the use of thin sections becomes a very formidable task.
A. NUCLEAR CONSTITUENTS When we compare the observations made by electron microscopy to those obtained earlier by the conventional microscope systems, we find that as far as the nucleus is concerned, this new instrument has: (a) confirmed the presence of structures whose existence was already suggested by the older techniques, (b) extended our knowledge of the ultrastructure of the known nuclear constituents, and (c) revealed some fundamentally new structures. Thus, the concept of the morphological boundary between the nucleus and cytoplasm being bilamellar in composition was originally suggested by Flemming (1882) and supported by later studies using various cytological techniques (see references in Baud, 1953). Electron microscopy has confirmed these interpretations and has shown that in virtually all cells the nuclear envelope has a common skeletal structure. Moreover, by means of this technique it has been possible to demonstrate that the envelope is interrupted by discontinuities with which are intimately associated cylindrical configurations. It also has been shown that the nuclear envelope, in places, is continuous with the endoplasmic reticulum. The latter observation has led to the conception of the nuclear envelope as being a cytoplasmic constituent, namely, a specialized portion of the endoplasmic reticulum. This interpretation is supported by the recent studies on the origin of the nuclear envelope by Barer et al. (1959) and Yasuzumi (1959). Hertwig in 1929 was able to demonstrate threadlike structures within the nucleolus. In 1951 this observation was supported by light microscopy (Estable and Sotelo, 1951) and electron microscopy (Borysko and Bang, 1951). Also by means of the latter technique, some advances have been made in elucidating the fine structure of both components of the nucleolus. On the basis of the limited observations .made thus far on the nucleolus by means of electron microscopy, it is apparent that we are dealing with a plastic organoid whose variable morphology suggests that it is dependent on the physiological state of the cell as a whole (Estable and Sotelo, 1955; Stich, 1959).
.
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Functionally, the nucleolus has for more than half a century remained an enigma. The experimental results which have appeared in the last few years strongly indicate that this long-standing cytological problem is becoming resolved. The early hypotheses of nucleolar function were derived from morphological studies (see review by Vincent, 1955). The cytochemical studies by Caspersson and Brachet nearly twenty years ago demonstrating the presence of R N A in the nucleolus strengthened the hypothesis that this structure was linked to protein synthesis (Caspersson, 1950). In the last few years, advances in our understanding of nucleolar function come from experiments using autoradiographic techniques and studies on starfish oocyte nucleoli isolated by differential centrifugation in sucrose solutions. Such methods have demonstrated that there is a rapid rate of incorporation of nucleic acids and protein precursors in the nucleoli (Ficq, 1955 ;Vincent, 1955) and that the nucleoli contain a high concentration of nucleoside phosphorylase and DPN-synthesizing enzyme (Baltus, 1954). These and other studies led Brachet (1957) to the conclusion that functionally the nucleolus is characterized by its metabolically very labile RNA, ability to incorporate amino acids into proteins, and by its accumulation of enzymes associated with nucleotide metabolism. Electron microscopy is one of the tools being used to obtain an understanding of chromosome organization on a macromolecular level with the aim of making it possible to integrate morphological concepts with existing biochemical data. From the classic cytological studies (see references in Denues, 1958),there has emerged a generalized picture of a chromosome as consisting of two or more helically coiled chromonemata on which are located the beadlike chromomeres (Fig. 6). Such strands are thought to be embedded in a matrix which may be enclosed by a limiting membrane, the pellicle. Prior to the advent of electron microscopy, evidence obtained by various cytological means strongly suggested that the chromosome was a composite of a number of subdivisions (see review in Swanson, 1957). On the basis of studies carried out by electron microscopy, both Kaufmann and McDonald (1956)and Ris (1956)have concluded that the chromonema is made up of a number of microfibrils which are 150-25012.in diameter. These are considered to be the basic morphological units of the chromosome. On the other hand, the studies of the fine structure of the lampbrush chromosomes and the work on meiotic chromosomes by Moses (1958) and others, which was discussed earlier, has implied that each chromosome contains but a single microfibril along its axis. It is apparent that additional data are needed before acceptance of the concept that a
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basic morphological thread of constant diameter exists as the elementary component of all chromosomes. The evidence available simply suggests that many chromosomes have a fibrillar organization whose fundamental unit is 100-500A. in diameter. The elucidation of a basic morphological unit existing on a macromolecular level would fit into the hierarchal concept of the structure of the salivary gland chromosome as formulated by Ambrose (1956). It would also allow formulation of a hypothesis of the molecular organization of nucleic acids and proteins in morphological terms (Lawman, 1956;
FIG.6. The classic chromosome. The generally accepted concept of the morphology and internal structure of a metaphase or anaphase chromosome as developed from light microscope studies. ch, chromonema; m, matrix; fl, pellicle; ch, chromomeres ; sc, secondary constriction; k, kinetochore.
Ris, 1956). I t however requires the introduction of a new concept of chromosome reproduction on the basis of the replication of the elementary fibrils (Ris, 1956). Results obtained from studies of chromosome duplication after their incorporation of tritiated thymidine indicate that in Vicia faba, biochemically speaking and on a microscopic level, each chromosome behaves as though it were composed of two units (Taylor et al., 1957). The most plausible way of visualizing the organization of a small, compact chromosome or chromosomal unit, in the light of its high DNA concentration, is by assuming that in terms of its finer structure it is a composite of microfibrils. Taylor et al. have presented a model of a ribbon-shaped structure made up of two multistranded units folded together. Such a
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model may offer a basis for the satisfactory correlation of the aforementioned light microscopic and audioradiographic evidence with some of the results obtained by electron microscopy. As to the nature of the chromomeres, here electron microscopy has not yet been helpful in clarifying the conflicting interpretations which resulted from earlier light microscope studies. Thus from observations of the fine structure of the banded regions of salivary gland chromosomes, the chromomeres have been interpreted as being made up of tightly packed fibrils (Yasuzumi et al., 1951) and as distinct granules (Lowman, 1956). Clarification of the fine structure of the chromomeres of the lateral loop chromosome is especially necessary, since in the interpretations presented by both Gall (1956) and Lafontaine and Ris (1958), the chromomeres are considered to be regions where the chromonemata are tightly coiled. Electron microscopy has produced no evidence to support the concept of the existence of a limiting membrane around the chromosome. Also the granular material enclosing the linear densities of meiotic prophase chromosomes (Moses, 1958), is the only indication as to what can be interpreted as a chromosomal matrix.
B. NUCLEOCYTOPLASM IC RELATIONS From the limited observations made by means of the electron microscope, it is obvious that our current understanding of the fine structure of the nucleus is quite restricted in scope. As a result, any discussion of the subject of nucleocytoplasmic exchange (see also review by Prescott, 1960) can, at this time, only be preliminary in nature. The problem can be resolved into two parts : (a) pathways that may possibly be used in the interchange of material, and (b) the nature of the material being transferred on the macromolecular level. 1. Pathways for Exchange Electron microscopy has uncovered a number of possible sites and mechanisms by means of which, theoretically at least, material should be able to be transferred in both directions. The nuclear envelope has been confirmed by electron microscopy to be a morphological barrier. It is obvious that such a structure, either actively or passively, will influence the nature of the exchange of material that takes place. It is therefore of particular interest that by means of the new technique locations in the envelope which would appear to permit contact between the nucleus and cytoplasm have been demonstrated. Thus, the circular discontinuities seen in numerous cell types can be interpreted as being “pores” whichmay permit the exchange of material up to and including particles of macromolecular dimensions (Fig. 7A). However,
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all available evidence points to the fact that the discontinuities are complex structures and cannot be considered to be simple openings in the nuclear envelope (Kautz and DeMarsh, 1955 ; Gall, 1959). Thus, tubelike struc-
A
B
C
D
E FIG.7. Diagrammatic representation of the possible pathways for nucleocytoplasmic interchange as uncovered by electron microscopy. (The cytoplasm is located above the nuclear envelope and the nucleoplasm below it.) A. Extruded nuclear material arranged cylindrically around the cytoplasmic surface of several discontinuities. (Watson, 1959). B. Cylindrically arranged material on both sides of the discontinuities (Afzelius, 1955 ; AndrC and Rouiller, 1957; Wischnitzer, 1958.) C. Structural continuity between the endoplasmic reticulum and the nuclear envelope (Watson, 1955). D. Outpocketing of the nuclear envelope which may become pinched off and the contents of which may be liberated into the cytoplasm (Gay, 1956b). E. Delamination of membranous sheets from the nuclear envelope which become arranged in the cytoplasm as the “annulate lamellae” (Swift, 1956 ; Merriam, 1959).
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tures have been observed, in both somatic and germinal material, to be intimately associated with the annular openings (Fig. 7B). At the present time it is uncertain whether the cylindrically disposed material is an integral part of the structure of the nuclear envelope or represents matter which is being passed across the nucleocytoplasmic boundary (Swift, 1958). A better understanding of the events taking place at such possible sites of exchange must therefore await clarification of the ultrastructure of the annular complex. Calculations by Watson (1955) have shown that from 5 to 15% of the nuclear surface is exposed in the form of the annular openings. Such potential sites of exchange are further increased in the cases where the surface area of the nuclear envelope is enlarged by the formation of outpocketings which have been observed in both normal (De Robertis, 1956; Moses, 1956; Gay, 1956a, b ; Wischnitzer, 1958; Karasaki, 1959) and malignant (Schultz, 1957 ; Wessel and Bernhard, 1957) nuclei. Watson (1955) appears to have uncovered a second possible site for the transfer of material into the cytosome. He reported that in some cells the endoplasmic reticulum was continuous with the nuclear envelope (Figs. 7C and 8). This passageway is an indirect one as compared with the discontinuities, and may be specialized as far as the constituents which it serves to transport. The other mechanisms for exchange uncovered by the use of electron microscopy appear to be restricted to the transfer of nuclear material. Moreover, these mechanisms may operate only in certain cell types or under special physiological conditions. Thus Gay (1956b) showed that in salivary gland cells of Drosophila, outpocketings of the nuclear envelope are intimately associated with specific regions of the chromosomes. She suggested that such blebs may become detached and thereby material of chromosomal origin may be directly transferred into the cytoplasm (Fig. 7D). Another method for the transfer of nuclear material that has been found (Fig. 7E) involves the replication of portions of the nuclear envelope (Merriam, 1959) which usually become organized as a distinct cytoplasmic organelle. It usually consists of a parallel array of basophilic membranous sheets which are collectively called the “annulate lamellae” (Swift, 1956). Such membrane systems may serve as the means whereby synthesizing mechanisms are transferred to the cytoplasm especially at a time of high cellular anabolic activity. 2. Nature of Materials Exchnged The classical studies at the turn of the century by the pioneers in cytological research presented a number of hypotheses as to the nature of
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FIG.8. The cell as seen with the electron microscope. Schematic representation of the structural components of a hypothetical cell as seen by means of the electron microscope. The cytoplasm contains the ergastoplasm (er) which is made of its two elements, the endoplasmic reticulum and its attached small particles or microsomes. Near the nucleus is seen the Golgi apparatus (9), which is made up of closely packed and flattened cisternae and many small vesicles. Mitochondria ( m t ) , seen in cross and longitudinal section, are distributed throughout the cytosome. The nucleus is surrounded by its nuclear envelope (we) which is made up of two membranes (nm) enclosing the intermembranous ZOhe (p). At the places where the membranes unite, discontinuities (d) are present whose margins are sometimes evident as a band (b). The discontinuities are associated with tubelike structures ( t ) , and sometimes they are found to contain a central granule ( c ) . The connection between the nuclear envelope and ergastoplasm is also illustrated. Within the nucleus, the nucleolus (n) is seen as being made up of two elements: a coiled nucleolonema (nl) and a finely granular matrix (m) . Nucleolar material extends toward the discontinuities, which may be one of the mechanisms for nucleocytoplasmic exchange. The chromosomes (chr) each contain a linear density as their axis.
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the materials involved in nucleocytoplasmic interchange (see Wilson, 1925). The instruments and techniques available at that time were not sufficiently refined to allow concrete progress to be made in this area. With the development of electron microscopy some of the earlier restrictions, such as limited resolution, have been overcome. I n spite of the technical progress made, little direct evidence for exchange has been presented. Pollister et al. ( 1954), as was mentioned earlier, reported filamentous structures passing through the discontinuities of the nuclear envelope in amphibian oocytes. Similarly Anderson and Beams (1956) reported that in nurse cells from the ovary of the insect Rhodnius, granules which appear to be similar to those making up the nucleolus extend from aggregates within the nucleus through the “pores” of the nuclear envelope into the adjacent cytoplasm. It has also been found in somatic cells (Horstmann and Knoop, 1957) as well as in oocytes (Wischnitzer, 1958; AndrC, 1958) that strands of granular material appear to extend from the nucleoli to the nuclear envelope. Such evidence suggests that the granular components of the nucleoli, which are on the order of magnitude of RNA-containing particles (Gall, 1956), may serve as one of the means for the transmission of nuclear material, on the macromolecular level, into the cytoplasm. It is also possible that the material which makes up the tubelike structures associated with the discontinuities represents material passing out of the nucleus (Swift, 1958; Watson, 1959). The fact that the evidence for the particulate exchange of material is so limited suggests that transfer of RNA and other substances of macromolecular dimensions, to a large extent, takes place in an agranular form (Swift, 1958). It may at this point be worthwhile examining how some of the morphological observations described above fit into our existing knowledge of nucleocytoplasmic exchange from the physiological point of view. It has been demonstrated that the nucleus exclusively contains an enzyme needed for the synthesis of disphosphopyridine nucleotide (Hogeboom and Schneider, 1952), a compound which after being transferred has a decisive influence on the metabolic processes of the cytoplasm. Electron microscopy, however, cannot be used in studies of the transfer of such nonspecific mechanisms of nuclear control since they take place on a molecular level. On the other hand, the nucleus contains specific mechanisms which have a decisive effect on the nature and activity of the cell. Thus, the theory that nuclear R N A may be a precursor of cytoplasmic R N A has been debated for quite some time. The reason for this is that the evidence obtained from biochemical and cytological experiments appears to be con-
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tradictory and the problem could not be resolved. Reviewing the evidence as it stood up to 1957, Brachet (1957) concluded that it appears that two mechanisms for RNA synthesis coexist in the cell, one intranuclear (followed probably by transport to the cytoplasm) and the other intracytosomal. In the last two years autoradiographic studies have yielded additional evidence to support the hypothesis that the R N A of the chromosomes is a primary site of synthesis of nuclear RNA, which is rapidly transported to nucleoli where it remains for longer periods (Goldstein and Micou, 1959a). Additional evidence has been obtained that nuclear RNA in some form passes into the cytoplasm where it contributes to the formation of cytoplasmic RNA (Zalokar, 1959; Woods and Taylor, 1959 ; Goldstein and Micou, 1959b), and the latter may be involved in the cytoplasmic synthesis of special proteins involved in the processes of differentiation (Mirsky and Allfrey, 1958; Ficq, 1959). As a result we now visualize a potential narrowing of the gap between biochemistry and cytology to the point where a morphological basis can be postulated for biochemical events. Thus, a working hypothesis can be presented whereby the chromosome expresses its genetic influence by the elaboration of RNA, which as nucleolar ribonucleoprotein granules or in nonparticulate form passes through the existing potential sites of exchange into the cytoplasm where it participates in the morphogenetic activities of the cytosome. Current physiological concepts are of a dual nuclear function : long-term so far as heredity and reproduction are concerned, and short-term as affecting the mechanism involved in cellular differentiations. The transmission of nuclear influence into the cytoplasm may well not be a phenomenon occurring exclusively on the macromolecular level. The evidence suggesting the transmission of such components as nucleoli or large nucleolar fragments into the cytoplasm is extensive (Gates, 1942; Altmann, 1955 ; Raven, 1958). It is probable that these are specialized mechanisms called into action only at times of great metabolic activity. From what has been said it is apparent, therefore, that our knowledge of nucleocytoplasmic interchange on a macromolecular level as has thus far been determined by electron microscopy is very limited. What is needed is a concerted effort using several different approaches, such as by means of serial sections and the study of nuclei in different physiological states. The correlation of electron microscope and light microscope cytochemical studies using alternating thin and thick sections (Moses, 1956) has proven to be of some value. Further exploitation of such material as oocytes and protozoans should be fruitful. Ultimately the development
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of cytochemical (Dettmer and Schwartz, 1954; Sheldon et al., 1955 ; Seligman, 1956; Barrnett and Palade, 1958; Nelson, 1959; Gersh, 1959) and autoradiographic (Liquier-Milward, 1956; O’Brien and George, 1959) techniques to be used specifically in conjunction with electron microscopy, should allow a more intensive study of the problem of nucleocytoplasmic interchange to be made. It is probable that much progress along these lines will be forthcoming in the not too distant future.
VIII. Conclusion Electron microscopy, for technical and other reasons, has not succeeded in uncovering the ultrastructural organization of the nuclear elements to an extent comparable to that which has been reported for the cytoplasmic constituents. The nuclear envelope of almost all cell types has been found to have a bilamellar organization which is interrupted by discontinuities. The structural organization around these discontinuities is complex and remains to be clarified. The nucleoli have, in many cases, been found to be made up of two components, a coiled nucleolonema embedded in a granular matrix. The fine structure of chromosomes remains to be definitively established, but all indications point to a fibrillar organization. Paralleling the limited progress made in uncovering the fine structure of the nuclear constituents, there has been a corresponding difficulty in elucidating the mechanisms and the nature of the material involved in nucleocytoplasmic interchange on the macromolecular level. The current state of our knowledge in this area has been presented and discussed, and suggestions for additional studies which may aid in resolving this problem have been offered. ACKNOWLEDGMENT The author is grateful to Dr. M. S. Jacobs of New York Medical College for his critical reading of this manuscript.
REFERENCES~ Afzelius, B. A. (1955) Exptl. Cell Research 8, 147. Altmann, H. W. (1955) Klin. Wochschr. 39, 306. Ambrose, E. J. (1956) Progr. in Biophys. and Biophys. Chem. 6, 25. Anderson, E., and Beams, H. W. (1956) J . Biophys. Biochem. Cytol. 2, 439. Anderson, N. G. (1953) Science 117, 517. AndrC, J. (1958) Bull. Microscop. Appl. 8, 93. AndrC, J., and Rouiller, Ch. (1957) in “Proceedings of the Stockholm Conference on Electron Microscopy” (F. S. Sjostrand and J. Rhodin, eds.), p. 162. Academic Press, New York. 2
The survey of literature pertaining to this review was concluded in March 1960.
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Gall, J. G. (1956) J . Biophys. Biochem. Cytol. 2, Suppl. 393. Gall, J. G. (1958) in “A Symposium on the Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), p. 103. Johns Hopkins Press, Baltimore, Maryland. Gall, J. G. (1959) J. Biophys. Biochem. Cytol. 6, 115. Gates, R. R. (1942) Botan. Rev. 8, 337. Gay, H. (1955) Thesis, University of Pennsylvania Doc. Diss. Series. Publ. No. 11407, Univ. Micro Films, Ann Arbor, Michigan. Gay, H. (1956a) Cold Spring Harbor Symposia Quunt. Biol. 21, 257. Gay, H. (1956b) I. Biophys. Biochem. Cytol. 2, Suppl. 407. Gersh, I. (1959) in “A Symposium on Molecular Biology” (R. Zirkle, ed.), p. 230. Univ. Chicago Press, Chicago, Illinois. Goldstein, L., and Micou, J. (1959a) J. Biophys. Biochem. Cytol. 6, 301. Goldstein, L., and Micou, J. (1959b) J . Biophys. Biochem. Cytol. 6, 1. Guyhot, E., and Danon, M. (1953) Rev. szcisse zool. 60, 1. Haguenau, F., and Bernhard, W. (1955) Bull. Cancer 42, 537. Hall, C. E. (1956) Proc. Natl. Acad. Sci. U.S. 42, 801. Harris, P., and James, T. W. (1952) Experientia 8, 384. Hassenkamp, G. (1957) Natunksenschajten 44, 334. Herskowitz, I. H. (1952) I. Heredity 43,227. Hertwig, G. (1929) in “Handbuch der Mikroskopischen Anatomie des Menschen,” (W. v. Molendorff, ed.), Vol. 1, Part 1. Springer, Berlin. Hofmeister, F. (1901) “Die Chemische Organization der Zelle.” F. Vieweg und Sohn, Braunschweig. Hogeboom, G. H., and Schneider, W. C. (1952) 1. Biol. Chem. 197, 611. Horstmann, E., and Knoop, A. (1957) 2. Zellforsch. u. mikroskop. Anat. 46, 100. Hovanitz, W. (1947) Genetics 32, 500. Jackson, S. F., and Randall, J. T. (1958) Proc. Roy. SOC.Bl48, 290. Karasaki, S. (1959) Embryologia 4, 273. Kaufmann, B. P., and De, D. N. (1956) J. Biophys. Biochem. Cytol. 2, Suppl. 419. Kaufmann, B. P., and McDonald, M. R. . (1956) Cold Spring Harbor Symposia Quunt. Biol. 21, 233. Kautz, J., and De Marsh, Q. B. ‘ (1955) Exptl. Cell Research 8, 394. Kurosumi, K. (1957) Protoplasma 49, 116. Lafontaine, J. G. (1958) I. Biophys. Biochem. Cytol. 4, 229. Lafontaine, J. G., and Ris, H. (1958) J. Biophys. Biochem. Cytol. 4, 99. Liquier-Milward, J. (1956) Nature 117, 619. Lowman, F. G. (1956) Chromosoma 8, 30. Mather, K. (1958) Proc. Roy. SOC.B148, 362. Mazia, D. (1952) in “Modern Trends in Physiology and Biochemistry” (E. S. Guzman-Barrbn, ed.), p. 77. Academic Press, New York. Merriam, R. W. (1959) J. Biophys. Biochem. Cytol. 6, 117. Moses, M. J. (1956) J. Biophys. Biochem. Cytol. 2, 215. Moses, M. J. (1958) J. Biophys. Biochem. Cytol. 4, 633. Mirsky, A. E., and Allfrey, V. (1958) in “A Symposium on the Chemical Basis of Development” (W. D. McElroy and B. Glass, eds.), p. 94. Johns Hopkins Press, Baltimore, Maryland. Nelson, L. (1959) Exptl. Cell Research 16, 403. Q’Brien, R. T., and George, L.A., 11. (1959) Nature 285, 1461.
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Yasuzumi, G. (1955) Biochim. et Biophys. Acta 16, 322. Yasuzumi, G. (1959) Z . Zellforsch. u. mikroskop. Anat. SO, 110. Yasuzumi, G., Odate, Z., and Ota, Y. (1951) Cytologia, 16, 233. Yasuzumi, G., Sawada, T., Sugihara, R., Kiriyama, M., and Sugioka, M. (1958) 2. Zellforsch. u. mikroskop. Anat. 48, 10. Zalokar, M. (1959) Nature la, 1330. ADDENDUM Since this article was written, a number of pertinent papers dealing with the subject of the ultrastructure of the nucleus have appeared. Of particular significance are those which can be found in the following two books : Bargmann, W. (ed.) (1960) “Fourth International Conference on Electron Microscopy. Proceedings Volume I1 : Biological-Medical Part.” Springer, Berlin. Mitchell, F. R. S. (ed.) (1%0)
“The Cell Nucleus.” Academic Press, New York.
The Mechanics and Mechanism of Cleavage LEWIS WOLPERT Zoology Department. Kings College. London. England Page 164 165 165 166 169 169 170 171 171 175 175 176 179
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I Introduction ...................................................... I1. Geometry of Cleavage ............................................. A . General Relationships ........................................ B . Sea Urchin Egg ............................................. I11 Theories of Cleavage ............................................. A . Theories Involving the Mitotic Apparatus .................... B. Diffusion-Drag Theory ...................................... C Growth Theories ............................................ D. Surface Force Theories ...................................... I V Mechanical Properties of the Cell Surface ......................... A . Cell Membrane .............................................. B. Methods of Measurement .................................... C. Mechanical Analogies ........................................ D Changes in the Mechanical Properties of the Surface during Cleavage .................................................... 180 E. Structural Changes .......................................... 183 V Discussion of Surface Force Theories ............................. 183 A . Expanding Membrane Theory ................................ 184 B. Cortical Gel Contraction Theory ............................. 185 C. Surface Tension Theories .................................... 185 V I . Astral Relaxation Theory 186 A . Mechanics ................................................... 186 B . Discussion of Mechanics ..................................... 187 C. Polar Differentiation ........................................ 190 D Mitosis and the Coordination of Cleavage ...................... 191 E. Energy Requirements ........................................ 194 VII. Application of Astral Relaxation Theory to Other Cells and Forms of Cleavage ..................................................... 195 A . Polar Body Formation ...................................... 1% B. Polar Lobe Formation ....................................... 197 C. Eccentric Cleavage .......................................... 197 D. Tissue Cells ................................................. 199 E Unequal Cleavage of a Neuroblast ........................... 200 F. Amoeba ..................................................... 202 G. Amphibian Egg .............................................. 203 VIII. Biochemistry of Cleavage 205 A . The Muscle Protein Analogy .................................. 205 B. Structure of the Cell Membrane .............................. 207 C. Action of Chemical Agents ................................... 208 D. “Dynamic” Properties of the Cell Membrane .................. 210 IX. Summary and Conclusions ......................................... 211 Acknowledgment ................................................. 213 References ....................................................... 213
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I, Introduction It is the purpose of this paper to review current theories of cleavage (see also Swann and Mitchison, 1958), to show that these are not satisfactory, and to present a theory of cleavage that can account for the cleavage of a wide variety of cells. The cleavage of the animal cell may, from one point of view, be regarded as a problem in mechanics. This is because the cleavage of the cell in general involves considerable changes in form of the cell, and these changes in form must be due to forces acting on the cell. The change in form of any body can only arise from the action of forces on it, whether these be of external or internal origin. The consideration of these forces will form the basis of this paper, and the ability of a particular theory to account for cleavage in terms of forces will be one of the major criteria by which it will be judged. The other major criterion by which theories will be assessed is their ability to account for the organization of cleavage, especially the relation between cleavage and mitosis. It will be seen that cleavage is closely related to mitosis both with regard to timing and the position of the cleavage plane. Most theories of cleavage have been developed with reference to the first cleavage of the sea urchin egg, since it is on this egg that the major portion of experimental work on cleavage has been carried out. Initially this paper will deal almost exclusively with the sea urchin egg, and only later will other cells be considered. The approach adopted here is as follows. The geometry of cleavage (Section 11) will be considered first, as it is essential to know just what changes in form are to be explained. This is particularly true of changes in the configuration of the surface during cleavage. Theories of cleavage (Section 111) may be classified according to the type and origin of the forces which they postulate. It will be shown that theories involving the mitotic apparatus as the active agent, the theory based on diffusion forces, and those based on “growth” cannot account for the cleavage of the sea urchin egg. This leaves only that group of theories which places the forces at the cell surface. The assessment of surface force theories requires a somewhat detailed consideration of the mechanical properties of the cell surface (Section IV ) and of the changes in these properties during cleavage. It is only after this aspect has been reviewed that surface force theories will be discussed (Section V). It will be shown that present theories are unsatisfactory, and the new theory will be given in detail (Section V I ) . The possibility of applying the new theory to other cell types and
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other forms of cleavage will then be considered together with special theories t b t have been developed with reference to them (Section VII). Finally, the biochemistry of cleavage will be dealt with (Section VIII). The separation of biochemical consideration from the mechanics and organization of cleavage until so late a stage is done here in the belief that the failure to separate them in the past has led to some confusion. Thus, mechanical aspects of theories have been objected to on grounds more relevant to physico-chemical considerations, and there have been unjustified “extrapolations” from biochemical data to mechanical principles.
11. Geometry of Cleavage A. GENERALRELATIONSHIPS It is essential to know the precise nature of the changes in form of the cell if one is to propose forces to account for these changes in form. However, apart from the movements associated with the cleavage of any particular cell, there are certain simple geometrical relationships that arise from the division into two of any body. A somewhat idealized case is the division into two of a sphere without change in volume-a case that probably approximates quite closely the cleavage of a large variety of cells. Such a division involves a minimum increase of surface area of 28% if the division results in two similar spheres, and an increase of 50% if the division remains in two similar hemispheres. These increases in surface area can be brought about in two main ways-by stretching the existing surface or by forming a new surface (see Fig. 1). Division by stretching
FIG.1. The two main ways by which the surface area of the cell can be increased at cleavage: 1, by stretching the original surface; 2, by building a new cell wall.
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of the existing surface to form two similar spheres involves a considerable rearrangement of the geometry of the body, and though there are an infinite number of ways by which this transformation may be brought about, two essential requirements are that the circumference AB must shrink to nothing and the length of the arc ACB must increase by 60%. By contrast, the building up of a new surface to cause division into two hemispheres (Fig. 1) need involve neither a change in form of the cell nor surface movements. Although division can be a combination of both types, the distinction between them is fundamental to an investigation of the mechanics of the process. For, while it is reasonable to look for forces that could give rise to the former type in terms of deformation of the body, in the second type deformations are not involved. Rather it is a problem of the mechanism by which a new surface is built up, and this will involve properties and forces of a quite different kind. In general, cleavage of animal cells involves considerable changes in form, but it does not necessarily follow that new surface is provided by stretching. This is considered with respect to the sea urchin egg in the next section, and the amphibian egg in Section VII, G. Wall building or cell-plate formation is typical of higher plants and is not considered here. B.
SEAURCHINEGG
The cleavage of this egg corresponds closely with the division of a sphere into two similar spheres and occurs without change in volume (Hiramoto, 1958). Investigations into the changes in the configuration of the surface during cleavage has largely been carried out by Dan and his co-workers (Dan et ul., 1937; Dan and Ono, 1954) by observing the movements of particles attached to the surface. Their resuks are in general confirmed by the detailed studies of Hiramoto (1958). The main oonclusions to be drawn from such studies are : (1) The new surface is formed by stretching of the old (Dan et ul., 1937). (From Fig. 2B it can be shown that the 27% increase in area is provided by the stretching of the existing surface.) Also, the equator shrinks to nothing and the length of the arc ACB (Fig. 1) increases by 60%. These are the requirements for the formation of new surface by stretching. (2) Along the line of the greatest optical section there is an initial linear expansion in the polar region and contraction in the furrow region ; this is followed by a wave of expansion spreading from the pole to the furrow as cleavage proceeds (Fig. 2A).
MECHANICS AND MECHANISM OF CLEAVAGE
1G7
P
I
.
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50 30 15 STAGE OF CLEAVAGE
70
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FIG.2A
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50 3 0 1 5 0 STAGE OF CLEAVAGE
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FIG.2B
,
70 50 3 0 1 1 0 STAGE OF CLEAVAGE
KEYTO FIG.2
FIG.2C FIG.2A. Linear changes of the surface along the line of the greatest optical section during the cleavage of the sea urchin egg. 0, length between C, and C,; 0, between C, and C,; x, between C, and C,; 0, between C, and C,. The stages of cleavage refer to the ratio of the diameter of the furrow at C, to the initial diameter. After Hiramoto (1958). FIG.2B. Changes in area of the surface of the sea urchin egg during cleavage. The areas are those defined by the lengths of surface in Fig. 2A. The broken line gives the total area of the egg. After Hiramoto (1958). FIG.2C. Linear changes of the surface in the direction perpendicular to the axis of the sea urchin egg during cleavage. Constructed after Hiramoto (1958).
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(3) Changes in area show a pattern similar to the linear changes, except that the furrow region remains shrunken throughout the major portion of cleavage (Fig. 2B). (4) Annuli considered at right angles to the egg axis show contraction in the furrow region, and expansion in the polar region (Fig. 2C). Changes in this direction have been largely ignored previously, and Fig. 2C has been constructed from Hiramoto’s data. Between the region of expansion and contraction there is a transition zone in which no change in diameter occurs. The existence of rings whose diameter remains constant was first pointed out by Ishizaka (1958), who has called them stationary rings. Ishizaka’s method consists in superimposing successive contours of a cleaving egg, the stationary rings being defined by the intersection of such contours (Fig. 3).
FIG.3. Three successive contours of the cleaving sea urchin egg illustrate the stationary rings S-S’. A is the initial center of the aster. The black circles represent particles attached to the surface. After Ishizaka (1958). The movements of the surface during cleavage may be summarized with reference to the stationary rings which define two distinct regions of the egg: the polar region which is essentially expansive and provides 80% of the new surface; and the furrow region which is essentially contractile, especially in the direction at right angles to the axis. The equatorial circle which becomes the furrow shrinks to zero. The importance of these results cannot be over-emphasized. Dan (1954a, b; Dan and Dan, 1940) has studied the movements of the surface following cleavage, and has found that particles which moved into the furrow region during cleavage move out again. This is strong evidence for the stretching of $he furrow region following cleavage, and further evidence for this view comes from his observations on the movements of the cortical granules.
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111. Theories of Cleavage What are the forces that give rise to the movements described above? Numerous theories have been offered (reviewed Dan, 1943b; Swann and Mitchison, 1958), and the current ones are : (1) Those that assume the forces to be associated with the internal structures of the cell, in particular the mitotic apparatus as in the theories of Gray and Dan. (2) The theory of Rashevsky which assumes the forces to arise due to diffusion. (3) Those that assume the forces to be associated with the cell surface. Marsland postulates the active region to be in a contractile furrow, whereas Mitchison and Swann suggest it to be at the expanding poles. Surface tension theories have found little favor in recent years, but Hiramoto has again invoked it. The theory of Chalkley refers mainly to amoebae. (4) The so-called “growth” theory (which is really a type of “cellplate” theory) of Selman and Waddington for the cleavage of the amphian egg (Section VII, G). A “cell-plate” theory has been put forward by Motomura for the sea urchin egg. Under “growth” theories m a y be included the cortical current theory of Chambers. A. THEORIES INVOLVING THE MITOTIC APPARATUS
1. Astral Growth Theory Gray’s (1924) astral growth theory conceives the asters as elastic spheres possessing a definite degree of elastic rigidity, and which can grow at the expense of the fluid cytoplasm. The continued growth of the asters resuks in the asters being pressed against each other in the equatorial plane “and the polar axis of the egg will increase in length as soon as the elastic force exerted by the asters is sufficient to overcome the tendency of the cytoplasmic and hyaline surface to resist change in form.” This increase in length causes fluid to flow towards the equator of the egg, and cleavage occurs. In support of his theory Gray has presented much evidence on the “close relationship that exists between the position and form of the asters with the psition and form of the cleavage furrow’’ (see Section VI, C, 2).
2. Spindle Elongation Theory Dan’s theory (Dan, 1943b; J. C. Dan, 1948) is based on four propositions: (1) An aster consists of a sphere of radiating spines formed of a gel. (2) The spindle possesses the capcity to elongate autonomously. (3) At the height of development of the aster, the spines of adjacent
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asters cross each other in the equatorial plane. (4) Astral rays are anchored to the cortex. The spindle elongates, and . when the asters are pushed apart and the crossing rays tend to dissociate, the equatorial surface included within the crossing range will be squeezed from both sides causing it to shrink.” Later stages of cleavage are brought about by a suction mechanism. This brief description does little justice to the very large number of observations Dan has made to support his theory, especially those on surface movements. I‘.
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3. Disczlssion The theories of Gray and Dan could be criticized on several grounds (see Swann and Mitchison, 1958), but here it is only necessary to point out that, since they depend on the active participation of the mitotic apparatus, they are unequivocally disproved by experiments in which cleavage has been observed in cells in which the mitotic apparatus had been destroyed or greatly modified (Beams and Evans, 1940 ; Swann and Mitchison, 1953) or removed (Hiramoto, 1956).
B. DIFFUSION-DRAGTHEORY Rashevsky and his collaborators (Rashevsky, 1948) have put forward theories of cell cleavage which in general involve considerable mathematical manipulation. However, a “theory that stands so much apart from all other m r k and totally neglects such observations and experimental evidence as is available is not easy to assess . . .” (Swann and Mitchison, 1958). Although the original theory (Rashevsky, 1948) has recently been somewhat modified, and new suggestions made in terms of “stresses in gels” (Rashevsky, 1952; Isenberg, 19533, a brief description of the original theory will be given as an indication of the approach. Because of the continual metabolism in cells, flows of different substances from regions of high concentration to regions of low concentration are continually occurring. This flow is generally assumed to occur by diffusion, and this process is slowed down by the resistance of the medium in which the diffusion occurs. It is suggested that this interaction between diffusing substances and the medium results in a complex set of forces acting throughout the cell. On this basis, and assuming the cell to behave as a liquid drop in which only surface tension forces act at the surface, systems of “diffusion forces” are considered which could lead to cleavage. An essential feature of the theory is the assumption that any deviation of the shape of a cell from that of a sphere is due to, and can only be maintained by, diffusion forces. No evidence has been offered to support this view. The further
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assumption by the theory that surface tension forces are dominant at the cell surface is not valid (see Section IV). Also no evidence is presented as to the magnitude of the diffusion forces, which may well be far too small to produce any significant effect on the cell at all. Further, the theory requires that the cell elongates before cleavage, and as Swann and Mitchison (1958) have pointed out, cells can cleave when elongation is prevented. They also point out that the sole experimental evidence in favor of the theory (Buschbaum and Williamson, 1943) may be expected from many theories.
C. GROWTH THEORIES A cell-plate theory has briefly been put forward by Motomura (1950) and is based on the appearance (in sections) of a row of vacuoles in the plane of the cleavage furrow. He has suggested that these give rise to the new cell wall. Also, Selman and Waddington (1955) have suggested that their theory which involves growth and which was developed to account for the cleavage of the amphibian egg may be applicable to the sea urchin egg. The main objection to any cell-plate or growth theory is that such theories require that the new surface area formed at cleavage be due to "growth" or formation of surface de novo, whereas in the cleavage of the sea urchin egg the new surface is formed by stretching the existing surface (Section 11). Such theories are thus inherently incapable of accounting for the surface movements during cleavage. Further, an egg can cleave even when there is an oil dxvp in the equatorial region (Chambers, 1938a), and electron microscopy has failed to reveal any modified cytoplasm ahead of the furrow (Mercer and Wolpert, 1958). These results seem to contraindicate the building up of a cell wall in the furrow region. Cortical currents have on occasion been invoked to account for cleavage (Chambers, 1938a), but how these could give rise to changes in form is not clear. Although such currents may play a role, they probably represent passive flow of the interior of the cytoplasm in response to surface movements. The detailed movements of endoplasmic particles during cleavage of the sea urchin egg (Hiramoto, 1958) seem to confirm this view.
D. SURFACE FORCE THEORIES 1. Cortical Gel Contraction Theories A theory based on the contraction of an equatorial band of gel has been put forward by Lewis (1942, 1951), but the most detailed theory involving a contractile ring is that of Marsland and Landau (1954). This is based on the cortical gel contraction hypothesis which ". . . assumes that
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the furrowing potency in animal cells depends on the structural state (and hence contractile capacity) of the gelated cortical cytoplasm in the furrow region.” Evidence for this arises mainly from the observed relation between the gelational state of the protoplasm in the furrow region (“gel strength”) (Section IV, B, 3) and the “furrowing potency” as measured by the hydrostatic pressure required to block cleavage. The initial deformation of the spherical cell is postulated to result from the initiation of a contraction in a rather broad band of the cortical gel in the equatorial region. The width of this band diminishes sharply, and the adjacent portions of the furrow cease to contract and may show passive stretching. In later stages of cleavage “. . . the geometrical configuration of the dividing cell is such that more and more cortical protoplasm becomes incorporated in the wall as the furrow deepens. Thus more and more cortical gel, possessing an unexpended fund ot contractile energy, is brought into an operative position as the furrow deepens. Therefore, in the final stages of furrowing, to complete the cleavage it is only necessary to assume that the region of active contraction shifts from the trough of the furrow to the side walls, first to the region immediately adjacent to the trough and later to a more peripheral site, somewhat removed from the trough. In this way, the gel at the very bottom of the trough, having performed its contractile function, could undergo solation, clearing the way for the approach and final fusion of the cell membrane which severs the stalk between daughter cells. Moreover, the residual gel in the walls of the furrow immediately adjacent to the trough, although no longer capable of further contraction, merely by retaining its rigidity, could transmit the force from the still contracting peripheral part, and thus bring the cell membrane into contact at the centre of the cleavage stalk . (see Fig. 4). I n a more recent paper (Marsland, 1956) it is suggested that as . . a prelude to division, before the cell elongates o r displays any definite furrow, a structural gradient must develq in the plasmagel sys-
. .”
‘I.
A
B
FIG.4. Diagram to illustrate the cortical gel contraction theory at an initial ( A ) and a late ( B ) stage of cleavage. 1 Indicates the region that is fully contracted and that is being pushed and is solating; 2 is the actively contracting region; 3 is the region subjected to stretching; and 4 is the region of solation.
MECHANICS AND MECHANISM OF CLEAVAGE
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tem, which initially seems to be strongly and uniformly set throughout. Cytokinesis is initiated, apparently, by a localised solation which weakens the cortical gel structure at each pole” and this allows the equatorial region to contract. Also, more emphasis is laid on the passive stretching of the polar regions. 2. Expanding Membrane Theory The expanding membrane theory of Mitchison and Swann (Swann, 1952 ; Mitchison, 1952 ; Swann and Mitchison, 1958) had its origin from consideration of the structure of the cell surface and the changes in birefringence of the cell during cleavage.
.’\ 3 4 FIG.5. Diagram to illustrate the expanding membrane theory. The broken line indicates the limit of diffusion of the X-substancz from the daughter chromosomes (C). After Mitchison (1952).
It is based on two fundamental assumptions: (1) that the cortex or structural layer of sea urchin egg membrane contains a looped protein structure and can expand actively in area; and (2) that the substance (X-substance) which causes this expansion is released from the two groups of daughter chromosomes in anaphase. The postulated process of cleavage is shown in Fig. 5 and is considered to occur in two stages. The first stage occurs when the X-substance reaches the poles of the eggs. It reaches these first, since each of the diffusion centers is nearer the polar surface than the equatorial surface. Assuming a slight initial tension at the surface, the expansion of the poles would release this tension at the poles, allowing the equatorial region to contract and produce first an elongation and then a furrow. The second stage starts when the furrow walls come in contact with the X-substance.
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The wall starts to expand, and the furrow is pushed inwards to complete the process of cleavage. This theory has been modified recently (Swann and Mitchison, 1958), and it is now suggested “. . . that most of the furrow wall behaves passively and is pushed inwards by active expansion at the rest of the surface . . .” and “. . . that it becomes plastic in the later stages, as a result, it may be supposed, of the high concentrations of the diffusing substance.” Support for the oheory came primarily from changes in birefringence of the membrane and from the movement of surface particles, in particular, the wave of linear expansion (see Section 11, B), which fit in well with the idea of polar expansion, but the modifications proved necessary due to Dan and Ono’s (1954) later report that there is no expansion in the area of the furrow region. It should be pointed out that in their most recent paper the authors have suggested the possibility of linking expanding membrane and growth theories, since both may involve a type of molecular disorientation. The wealth of experimental data provided by the authors in their attempts to verify their theory will be discussed in various other sections.
3. Astral Reluxation Thewy This is the new theory that I wish to propose. It is presented at this stage in the anticipation that the other theories will be shown to be unsatisfactory. The theory is not really new in that it incorporates features common to other theories, especially Marsland’s (1956) most recent version of the cortical gel contraction theory. The theory is based on three main postulates: (1) Prior to cleavage there is a uniform tension in the cell membrane. (2) Cleavage is initiated by relaxation of the membrane in the polar regions which expand in area and allow the furrow region to constrict and divide the egg in two. (3) The areas of membrane that will relax are determined before cleavage by the asters or astral centers. The asters differentiate the region of the membrane closest to them (Fig. 9). A detailed account is given in Section VI. The theory thus combines the polar expansion of the expanding membrane theory with the contraction in the furrow region of the cortical gel contraction theory. The most novel feature is the suggestion that it is the asters that differentiate the polar regions and that it is in this way that the plane of the furrow is defined. A theory for the cleavage of the amoeba based on similar mechanical principles has been put forward by Chalkley (1935) (see Section VII, F), and a theory based on contraction and relaxation has been suggested for the egg of Tubifex (Lehmann, 1946; Huber, 1947).
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IV. Mechanical Properties of the Cell Surface Before discussing the above theories it is necessary to consider the mechanical properties of the cell surface-a problem that I hope to consider in detail elsewhere-as well as the changes in these properties during cleavage. These properties are most important for the assessment of surface force theories. A. CELLMEMBRANE The surface of the cell was for a long time regarded as being similar to that of a liquid drop at which only surface tension forces were active. This concept was clearly disproved by Cole (1932), who showed the cell surface has elastic properties-i.e., the resistance of the surface to deformation increased with the extent by which it was deformed. This has been confirmed by Mitchison and Swann (1954a,b). Also, the forces acting at the cell surface have been shown to be small-of the order 1 dynehm. (Harvey and Danielli, 1938; Harvey, 1954). Further, in contradistinction to the surface of a liquid drop, the cell surface has been shown to have a definite structure with at least two main componentsthe plasma membrane and the plasmagel or cortex. The plasma membrane is a well-studied component, especially as regards its role in permeability, and its thickness is about 100 A. (Danielli, 1951). The presence of the cortex, a relatively thick gel layer underlying the plasma membrane, has been demonstrated in the sea urchin both by its ability to prevent the displacement of granules held in it (Brown, 1934; Marsland, 1939) and by microdissection (Chambers, 1938a; Hiramoto, 1957). By the former method the thickness of the cortex of the sea urchin egg had been found to be about 1.6 p (Mitchison, 1956a) and by the latter method 3 p (Hiramoto, 1957). Values of 6 p have also been reported (Marsland and Landau, 1954). Mitchison’s value of 1.6 p will be used in this paper. The cortex is generally assumed to be responsible for the structural and mechanical properties of the cell membrane both because of its relatively greater thickness, and because its solation by high hydrostatic pressure is associated with the rounding up of the cell and the loss of shape associated with cleavage or amoeboid motion (reviewed Marsland, 1956). Some further evidence for its structural role comes from microdissection (Chambers, 19384. Mitchison (1952) also ascribes the major structural properties to the cortex. He defines the membrane to include both the plasma membrane and the cortex, and this meaning is also adopted here. The molecular structure of the membrane will be considered later (Section VIII, B). It must be remembered that it is by no means clear what contributions
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to the mechanical properties are made by the cortex and the plasma membrane, and there may be a tendency to underestimate the contribution of the latter (Wolpert, l%O) The discovery of the elastic and structural properties of the surface has led to some neglect of those properties which gave rise to the “liquid-drop” concept, and the fluid nature of the outermost layer of the cell has been emphasized by Chambers (1938b). Examples of this type of behavior are: (1) Lack of wrinkling when the cell is deformed. (2) Readiness of the surface to be drawn out into strands which become beaded and eventually separate into drops. (3) Coalescence of the cell with an oil drop. (4) The formation of new surface by cell fragments. ( 5 ) The ability of the membrane of a sea-urchin egg to adapt itself to a reduction in volume of the cell by one half, when the cytoplasm was sucked out (Horstadius et al., 1950). These examples emphasize the “dynamic” nature of the cell surface. The mechanical behavior of the membrane thus presents, in a sense, a paradox, since it has properties associated with both the surfaces of liquids and elastic membranes. It is essential that neither of these aspects should be overlooked. This is particularly important when mechanical analogies are drawn between the cell and rubber spheres. Nothing has so far been said of the extracellular layers, e.g., the hyaline layer. Though they can modify considerably the shape of a cleaving egg, they are not essential for cleavage (Gray, 1931) and will not be considered further. B. METHODS OF MEASUREMENT
.
Many measurements of mechanical properties of the cell surface have been made, but their interpretation is not always easy, and inconsistencies between results probably arise from the fact that different methods measure quite different properties (see also Mitchison and Swann, 1955). Not all mechanical properties of the membrane are equally useful for assessing theories of cleavage. Probably the most important are the tension in the membrane and Young’s modulus, since it is these that will determine the form of the cell. Young’s modulus is a measure of the ease with which the membrane may be deformed. It is defined as the stress (force/unit area) required to produce unit strain. A clear distinction must be drawn between those methods of measurement in which the force required to produce a deformation of the membrane is measured and which provide information about Young’s modulus and the tension in the membrane, and those in which the ease with which granules in the membrane may be displaced is measured. These two types of measurement need bear no relation to one another and, as shown below, do not appear to be simply related.
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1. The Cell Elastimeter The most detailed studies of the elastic properties of the surface have been made by Mitchison and Swann (1954a,b), who have devised a most elegant method. The method consists in applying a small negative hydrostatic pressure to the cell surface by means of a micropipette and measuring the deformation produced-the slope of the pressure/deformation curve being called the “stiffness” of the cell membrane. The “stiffness” is shown to be directly proportional to the Young’s modulus of the membrane. However, the “stiffness” also increases if there is tension at the surface, and the separation of the relative contribution of Young’s modulus and the tension presents considerable difficulty. (It should be noted that the tension at the surface is directly related to the internal pressure (see Section IV, C).) This difficulty was partly resolved by them by measuring the “stiffness” of rubber ball models of dimensions similar to that of the sea urchin egg, viz. diameter 100, thickness 1.6 under varying conditions of internal pressure. This enables the relative contributions of the two factors to the “stiffness” to be assessed. From other considerations (see below) Mitchison and Swann concluded that the maximum internal pressure of the unfertilized egg was 93 dynes/cm.2 and rhus, from the model experiments, that the Young’s modulus lay between 0.54 x lo4 dynes/cm.2 for this internal pressure, and 1 X lo4 dynes/cm.2 for no internal pressure.
2. Compression This method was introduced by Cole (1932), who compressed a single sea-urchin egg by means of a gold microbeam. H e demonstrated the elastic properties of the surface and showed that the forces at the surface were s m a l l - a b u t 0.2 dynes/cm. when the egg was compressed 50% of its diameter. From extrapolation of the pressure deformation curve he suggested that there was an internal pressure in the unfertilized egg of 40 dynes/cm.2, but Mitchison and Swann (1954b) have shown that this conclusion is not justified. 3. Particle Displacement Marsland and Landau (1954) have modified Brown’s (1934) method to determine what they have called the “gel strength” of the cortex. The method essentially consists in determining the time required to displace granules from the cortex by centrifugation. This method has been criticized by Wilson (1951) on the basis that the majority of the granules are located in the cytoplasm rather than the cortex; and by Mitchison and Swann (1954b), who point out that it is uncertain just what physical property is being measured, since the method
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is really one for viscosity and not for elasticity. It may also be noted that the cortex has special properties in relation to the granules, in that granules migrate under cortical influence (Allen and Rowe, 1955). It would seem that whatever Marsland is measuring, it is not directly related to the elastic properties of the surface. The basis of his method is the measurement of the time required for a given displacement, under constant force. I t must thus be a measure of the viscous or plastic properties of the cortex. These, for any system, are not directly relatable to other properties of the membrane, e.g., Young’s modulus, tension, yield strength, or failure strength, though empirical relations may be established. For example, Marsland has shown that “gel strength” is related to furrowing potency. However, it does seem that “gel strength” is not related to either the tension in the membrane or the Young’s modulus. This can be seen by comparing the “gel strength” with the “stiffness” of the membrane obtained with the cell elastimeter. The comparison may be made in two cases: (1) The gel strength” rises with temperature whereas “stiffness” falls (Marsland, 1956 ; Mitchison and Swann, 1954b). (2) The changes from fertilization to cleavage give quite different results as is seen in Figs. 6 and 7. The sharp rise in “gel strength” to “a new order of magnitude” after fertilization (not shown in Fig. 6 ) is not reflected by the “stiffness” measurements. There can be little doubt that “gel strength” is not a measure of these properties which determine the ease with which the membrane can be deformed and rhus determine the shape of the cell. “Gel strength” is a property of the cortex, but its significance is quite unclear. Wilson’s (1951) method measuring the “rigidity” is to determine the centrifugal force required to displace the granules from the cortex within a given time. This method probably is related to both viscosity and shear strength of the cortex, and similar difficulties to those found with “gel strength” arise in the interpretation of its significance. 4.
Cell Elongation The degree of elongation of the cell caused by centrifugation has been used to measure the “tension at surface” (Raven, 1948). The interpretation, however, is not simple, especially if there are changes in the degree of stratification of the cell interior. Harvey. (1931) has used centrifugal force to pull an egg into two halves and has calculated the maximum tension in the unfertilized sea urchin egg to be about 0.2 dynes/cm. Norris (1939) has stretched the membrane of sea urchin eggs with microneedles and has obtained values similar to those obtained with the cell elastimeter.
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C. MECHANICAL ANALOGIES Having examined methods of measuring the mechanical properties of the membrane, how may the membrane be regarded? What mechanical analogy would be most appropriate? The membrane is clearly elastic and since the sea urchin egg has a large ratio of diameter to membrane thickness, it might be expected to behave as a thin-walled elastic sphere. However, Mitchison and Swann (1954a), on the basis of their measurements with the cell elastimeter, came to the conclusion that the sea-urchin egg is to be regarded, mechanically, as a thick-walled rather than a thinwalled elastic sphere. This contention must be carefully examined. The distinction between a thick-walled and a thin-walled sphere is that resistance to deformation in the former case depends both on the resistance to flexure (bending) and to the tangential extension and compression of the wall, whereas in the latter case the resistance depends only on the tangential strains, and resistance to flexure may be neglected. Thus, for example, a thick-walled sphere could have considerable resistance to compression even though there was very little extension (and hence increase in tension) of the wall. On the other hand, the resistance to compression of a thin-walled sphere arises almost entirely from the extension, and hence increased tension of the wall. Mitchison and Swann arrived at their conclusion from a comparison of the pressure deformation curves found for the eggs with that to be expected from pure surface tension, as well as with that for a thin-walled elastic sphere. This led them to believe that the cell membrane behaves like neither of these two systems but rather like a thick-walled sphere. “It is concluded that the membrane is sufficiently thick to resist deformation by virtue of its own rigidity, resembling therefore a tennis ball rather than a rubber balloon or a fluid drop. . . .” By rigidity they apparently mean the resistance to flexure or bending. This conclusion, which is of considerable importance to any considerations of mechanics, does not appear to be valid (Wolpert, 1 W ) . It is agreed that the cell surface does not behave mechanically as the surface of a liquid drop or of a thin-walled balloon. What is questionable is that a sphere whose ratio of diameter to thickness is greater than 60-which is the case for the sea-urchin egg-can be regarded as a thick-walled sphere. So large a ratio is normally indicative of a thinwalled sphere, and that this is unequivocally the case is demonstrated from Mitchison and Swann’s (1954a) tests on model balls. In Fig. 11 of that paper they have plotted the change in stiffness with wall thickness of the balls. For the range of thicknesses for which the ratio of diameter to thickness is about 60 the stiffness increases linearly with wall thickness.
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This is incompatible with the idea that the sphere is thick-walled, since if it were, then the stiffness should increase as the cube of the thickness, because resistance to flexure is proportional to the cube of the thickness. (This condition is approached as the thickness increases.) Resistance to extension-the case relevant to a thin-walled sphere-is directly proportional to wall thickness. The sea urchin egg membrane is thus to be regarded as a thin-walled elastic membrane, and flexure of the membrane may, in general, be neglected. This conclusion is strengthened by the fact that the cell probably maintains a constant volume when deformed. It can be shown that the amount of water driven out when the cell is compressed, say SO%, is negligibly small. This means that a cell which is normally spherical, such as the sea urchin egg, must always undergo an increase in surface area when deformed. Thus if the cell be considered as similar to a fluid filled elastic ball, deformation will be mainly resisted by tension in the membrane due to stretching. These considerations also permit the application of the equation P = T'/r' Trr/t" to the cell membrane where P is the difference in pressure across the membrane, T and r the principal tensions and radii of curvature. Tensions in the membrane may thus bring about changes in cell shape. As regards the tension at the membrane in the noncleaving egg, there is no reason to doubt Mitchison and Swann's (1954b) conclusion that it is small. The possibility of tension at the membrane during cleavage is discussed in Section VI, B, 1. Finally, the fluid nature of the cell membrane should not be neglected, and mechanical analogies should be used with great caution.
+
D. CHANGES IN
THE
MECHANICAL PROPERTIES OF THE SURFACE DURING CLEAVAGE
Having examined methods for measuring the mechanical properties of the membrane, it remains to examine what information they yield as to the changes during cleavage. Before doing this two easily observable and very obvious qualitative changes should be noted, since they are common to a large variety of cells. They are the rounding up of cells prior to cleavage, and the bubbling and weakness at the poles. The rounding up of sea urchin eggs is less obvious since the eggs are initially almost spherical. Rounding up does, however, occur. The weakness at the poles was demonstrated by Just (1922), who found the poles burst first when the eggs were placed in hypotonic sea water, and was confirmed by niicrodissection (Chambers, 1938a). It is also of interest that granules are held more firmly in the cortex in the furrow region (Marsland, 1939).
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The first measurement of change in surface properties during cleavage was by Brown (1934), who measured the “gel strength” of the cortex. H e found a sharp increase prior to cleavage. Similar measurements, but in much greater detail, have been carried out by Zimmerman et al. (1957). Their results are illustrated in Fig. 6, and show an increase in “gel strength” some time prior to cleavage, followed by a decrease associated with cleavage. A correlation has been established between “gel strength” and “furrowing potency” as measured by the hydrostatic pressure necessary to block cleavage. For example, some chemical agents and an increase in temperature cause an increase in “gel strength.” Both of these also increase the “furrowing potency” (Marsland, 1956). As indicated in Section IV, B, 3 the significance of “gel strength” measurements is by no.
100
7I 10
50% CLEAVED
. 20
30
40
50
I.
60
,I 70
80
90
TIME AFTER FERTILIZATION (MINI
FIG.6. The changes in the “cortical gel strength” of the sea urchin egg (Arbacia) following fertilization. The “gel strength” is measured by the time (in seconds) to displace pigment bodies from the cortex at a centrifugal force of 41,000 g and at a pressure of 8,000 lb. per square inch. After Zimmerman et al. (1957).
means clear, and they thus do not provide information on the forces acting at the membrane during cleavage. However, they are possibly an indication of some physico-chemical property of the membrane. It was Danielli (1952) who first demonstrated that there were changes in the resistance to deformation in the membrane of the sea urchin egg during cleavage. H e observed that if a thin coverslip was allowed to rest on sea urchin eggs so that the eggs were slightly compressed, then just before cleavage the eggs showed a tendency to round up and raised the plate. After cleavage the eggs relaxed. H e suggested that these changes were due first to an increase of the tension at the surface, followed by a lowering of the tension. Using a modified form of the apparatus used by Cole (Section IV, B, 2) I have compressed single sea urchin eggs by 50% and have confirmed Danielli’s observation on single eggs.
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With the cell elastimeter, Mitchison and Swann (1955) have measured the changes in “stiffness” of sea urchin eggs from fertilization to cleavage (Fig. 7). Their results show a sharp increase in “stiffness” during anaphase and a sudden drop at the start of cleavage which parallels Danielli’s observation, but they have interpreted this as being due to changes in Young’s modulus due to changes in “molecular orientation.” Recently I have extended Mitchison and Swann’s observations, using their elastimeter, by making more detailed observations on the change in “stiffness” during the period of cleavage. This has been done by doing
71)-
60-
ul
2!
5
. L . Y I
k
50-
40.
t k
30-
2010 -’
$‘ :
‘\
r\
a u
15
-_- m
/
-
FIG.7. “Stiffness” changes of the membrine of the sea urchin egg (Psammechinus miliaris) in calcium-free sea water following fertilization. The “stiffness” is in dynes/cmZ/p and is corrected for an egg diameter of 1OOp and a pipette diameter of 50 p. After Swann and Mitchison (1958).
“constant pressure runs” on the egg during cleavage. This involves sucking out a small bulge at the start of cleavage and observing the change in the deformation without altering the pressure as cleavage proceeds. The most important result has been the difference in behavior when the bulge is sucked out at the polar or furrow region. When it is the polar region that is observed, the deformation increases progressively during cleavage. However, in the furrow region the deformation decreases and, depending on the pressure, may even lead to the seal between egg and pipette being broken, due to the continued contraction in the furrow region. This clearly shows that during cleavage the “stiffness,” as measured at the poles, decreases and that in the furrow region increases. (These results will be published in detail elsewhere.)
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I t thus appears that, prior to cleavage, there is an uniform increase in “stiffness” over the surface of the egg, and that during cleavage this “stiffness,’ continues to increase in the furrow, whereas it falls at the poles. These changes are associated with the rounding up of the cell before cleavage and a weakness at the poles during cleavage. A final point relates to the thickness of the cortex. No significant change in thickness appears to occur during cleavage (Mitchison, 1956a; Hiramoto, 1957). E. STRUCTURAL CHANGES The birefringence of the cell membrane will reflect the structural state of the membrane, and this will be altered by any forces acting on it. The surface of the unfertilized egg is negatively birefringent with respect to the radius, and changes occur both at fertilization and cleavage (Monroy, 1945 ; Mitchison and Swann, 1952). On fertilization the birefringence disappears, but gradually returns, and by anaphase it again reaches a maximum. At cleavage the birefringence initially decreases at the poles and increases slightly in the furrow region. The decrease at the poles then spreads to the furrow, so that at the end of cleavage the birefringence of the whole surface is weak. The scattering by the membrane of vertical incident light shows a similar pattern of changes (Mitchison and Swann, 1952). It is ‘worth noting the similarity between the optical changes at the beginning of cleavage and those of the linear movements of the particles. Also, the increase in birefringence and light scattering at anaphase corresponds with the increase in “stiffness” at that stage. Corresponding changes in structure have been found by electron microscopy. Mercer and Wolpert (1958) have found that during cleavage a new layer, much thinner than the cortex, appears immediately below the surface. This layer is the only one to correspond with the contour of the cleaving egg, and this, together with its greater development in the furrow region, would suggest that it is connected with the structural properties of the membrane. Its distribution during cleavage is rather similar to the contracting cortical gel as postulated by Marsland and Landau (1954).
V. Discussion of Surface Force Theories Any theory that purports to explain the mechanics of cleavage must be able to account for the changes in form, surface configuration, mechanical properties, and optical properties that have been described in preceding sections. These may be restated briefly. (1) The polar regions expand and the furrow regions contract. (2) The “stiffness” of the membrane increases just before cleavage and falls away during cleavage. This fall
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in “stiffness” occurs mainly at the poles, whereas the stiffness in the furrow region continues to rise. (3) The cell rounds up before cleavage and during cleavage is weakest at the poles. (4) There is an increase in birefringence at the surface before cleavage. During cleavage it decreases, this decrease starting at the poles and spreading to the furrow. A. EXPANDING MEMBRANE THEORY This theory will be examined in two parts-that relating to the mechanics of cleavage, and that relating to the role of the chromosomes in determining the plane of cleavage (Section VI, C). Although the theory is well able to account for. certain aspects of cleavage-for example, the wave of eKpansion starting at the poles and the accompanying optical changes-severe difficulties are met when the mechanics of the process are considered. (1) The theory cannot account for the contraction in the furrow region, especially that which occurs at right angles to the cell axis. Since the theory is based on the release of an agent causing expansion, it is extremely difficult to see how contraction could occur, especially since the relaxing substance diffusing from two centers would be expected to have its highest concentration in the furrow region. Also, since the relaxing substance is to be released from the telophase chromosomes, how are those cases in which cleavage occurs in the absence of chromosomes (Hiramoto, 1956; E. B. Harvey, 1951) to be explained? (2) It is by no means clear why an active expansion of the membrane at the poles should lead to furrow formation (Marsland and Landau, 1954), and Swann and Mitchison (1958) have acknowledged this. (3) The theory does not offer any explanation of the marked changes in stiffness that occur at cleavage, and this must be considered a serious defect. (4) Perhaps the strongest evidence against the theory arises from the fact that the sea urchin egg can cleave when there is tension at the surface. If there is tension at the surface during cleavage, then a mechanism based on pushing by the membrane cannot function since pushing requires the membrane to be under compression. The authors have recognized this and have gone to some trouble to show that there is no tension or internal pressure during cleavage (Mitchison and Swann, 1955). However, there are conditions under which cells have been observed to cleave and in which there was almost certainly tension at the surface. a. The experiments of Chambers (1938a, 1951) showed that a sea urchin egg would still cleave if stretched along its axis by microneedles.
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Judging from the photographs, the cell membrane has been stretched at least 50% and is unquestionably under tension. b. The cell can cleave while supporting a small plate, as in the Danielli (1952) experiment (see Section IV, D). Under these conditions the egg has been observed to cleave when compressed to half its diameter, in which case the membrane was stretched about 30%. Throughout cleavage the cell must support the plate by virtue of the tension in its membrane. c. In the constant pressure experiments I have carried oyt with the cell elastimeter (Section IV, D ) , the membrane was certainly under tension due to the sucking out of the bulge, and the cell cleaved whether this bulge was sucked out in either polar or furrow regions. These experiments seem to exclude any mechanism based on pushing in of the furrow. The determination of the plane of cleavage by diffusion of substances from the chromosomes, as suggested by the expanding membrane theory, will be considered in more general terms in Section VI, C. Here it is only necessary to reiterate that cells can cleave in the absence of chromosomes, and they cannot be a major determinant.
B. CORTICAL GEL CONTRACTION THEORY Much of the theory proposed by Marsland and Landau (1954) is concerned more with the nature of the cortical gel and its properties than with the mechanics of cleavage, but the theory, especially that briefly outlined by Marsland (1956), can account for many of the features associated with cleavage such as the polar weakness and particle movements, and I believe it to be essentially correct. However, the suggestion that in the later stages of cleavage the furrow is pushed in is open to objections similar to those raised against the expanding membrane theory. More important, the theory in its present form does not account for the changes in “stiffness” associated with cleavage, and it also fails to relate cleavage to mitosis, especially as regards the position of the cleavage plane. The essential mechanical aspects of the theory are incorporated in the astral relaxation theory.
C. SURFACE TENSION THEORIES Surface tension theories have fallen from favor since the discovery of the elastic and structural properties of the cell membrane. However, Hiramoto (1958) has suggested that surface tension forces may be involved in the final stages of cleavage. A detailed criticism of surface tension theories has been given by Danielli (1951), and they will not be given further consideration here.
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VI. Astral Relaxation Theory The theory will be considered first in relation to the mechanics of cleavage and then in regard to astral differentiation.
A. MECHANICS The essential features relating to mechanics of cleavage are that an increase in tension prior to cleavage causes the increase in “stiffness” and rounding up of the cell. A relaxation at the poles allows the furrow region to contract and accounts for the fall in “stiffness” as cleavage commences and also for the polar weakness of the egg. According to the theory, the increase in “stiffness” and rounding up of the cells prior to cleavage are due to an increase in tension in the membrane. There is a seventeen-fold increase in “stiffness” from the value of 3.4 dynes/cm.2/p deformation at the streak stage to the “stiffness” of 57 at anaphase (Mitchison and Swann, 1955). If this is due to an increase in tension only and the Young’s modulus does not change, then the increase in the tension required to produce this is 2.7 dynes/cm. This corresponds to an internal pressure of 1070 dynes/cm.2. These values were obtained by extrapolation from the experiments on model balls (Mitchison and Swann, 1954a) and have been checked by an analytical expression (unpublished). The value for the tension is a maximum value, and if there is an increase in the Young’s modulus, which is very likely, then the tension and internal pressure will be less. It has not been possible to separate the relative contribution of these two factors. If the Young’s modulus at anaphase is taken as lo4 dynes/cm.2, which is a reasonable value for the Young’s modulus before mitosis (Mitchison and Swann, 1955)’ the tension corresponds to a strain of 170%, but since there is no change in length during this increase in tension it may be likened to the isometric contraction of a muscle. For a membrane 1 . 6 thick ~ this tension is equivalent to a stress of 17 gm./cm.2 which is a plausible value if the membrane is at all similar to an actomyosin sheet (see Section VIII, A ) . The expansion at the poles is well established. The point to be made here is that it is the lower tension at the poles relative to the furrow region that causes this to occur. The fall in tension at the poles relative to the furrow allows the furrow to contract and can bring about the expansion at the poles. Unless the tension is higher in the furrow region it cannot contract, and the form of the cleaving egg is determined by the relative tensions at the pole and furrow. This may be illustrated by T”//‘ (Section IV, C) to an early applying the equation P = T f / # stage of cleavage, e.g., elongation. If the cell has the somewhat idealized
+
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form illustrated in Fig. 8, then applying the equation to the poles and furrow region separately, the internal pressure P = 2Tp/0.9R = Tf/O.9R where T p is the tension at the poles and Tf is the tension in the furrow at right angles to the egg axis. Thus Tf = 2Tp and shows that the tension at the poles is one-half that in the furrow. A continued fall in tension at the poles relative to the tension in furrow would bring about cleavage. That such changes occur is suggested by the behavior of these regions during constant pressure runs with the cell elastimeter (Section IV, D). The changes in “stiffness” are thus most easily understood in terms of changes in tension. It must be emphasized that this discussion is not dependent on the cause and nature of the fall in tension at the poles, and this is discussed in -F-
P
FIG.8. Idealized diagram of the form of the sea urchin egg at elongation. P and F indicate the polar and furrow regions. R is the initial radius of the egg. The linear expansions in the furrow region during later stages of cleavage (Fig. 2 ) are probably due to the passive stretching of this region, and this may be associated with contractions perpendicular to the egg axis.
B. DISCUSSION OF MECHANICS Having suggested how changes in tension could bring about cleavage, it is necessary now to consider in more detail those points which are contrary to the view of other workers.
1 . Increase in Tension It is necessary to consider first the and Swann (1955) that the increase increase in Young’s modulus without Young’s modulus are closely related,
possibility suggested by Mitchison in “stiffness” could be due to an a change in tension. Tension and and the distinction between them
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may perhaps be better appreciated by considering two adjacent elements in the membrane. An increase in tension implies that there is an increase in the internal forces tending to move the elements toward each other; an increase in Young’s modulus means that there is an increase in the external force required to move the elements a small distance away from each other due, perhaps, to some change in the internal forces. It can thus be seen that an increase in tension could give rise to an increase in Young’s modulus. I n the case in which a compressed egg rounds up there can be no doubt that there has been an increase in tension in the membrane and an increase in attraction between two adjacent elements, and it is hard to imagine that a similar situation does not occur in the uncompressed egg. Moreover, it is not easy to explain the rounding up of the egg in terms of changes in Young’s modulus. Mitchison and Swann (1955) have gone to some lengths to demonstrate that during cleavage there is no tension in the membrane. They have investigated this in two ways-by measuring the increase or decrease in “stiffness” as the egg is swpllen or shrunken in solutions of different tonicity, and by determining the minimum shrinkage required to cause wrinkling. The former method gives somewhat equivocal results, and the latter method indicates an internal pressure of 19 dynes/cm.2 after fertilization and 500 dynes/cm.2 at late anaphase-which does in fact indicate an increase in tension of the membrane. They suggest that these are maximum values. It would seem that both methods are of uncertain value, since they assume the cell to behave as a simple elastic ball when placed in solutions of widely varying tonicity which will almost certainly affect the properties of the membrane. To cause wrinkling of the anaphase eggs the egg had to be shrunk 9.7%,’which required a change in tonicity from 1.08 to 1.74. I t is almost impossible by this method to demonstrate that the membrane is considerably stretched. These results cannot be regarded as evidence against an increase in tension, and, if anything, they tend to support it since the value of the internal pressure at late anaphase, viz. 500 dynes/cm.2 obtained by the shrinkage method, is only one-half the maximum postulated by the astral relaxation theory, viz. 1070 dynes/cm.2. Direct evidence for tension in the furrow is the contraction of this region against an applied pressure in the constant pressure runs with the elastimeter described above. Further direct evidence for tension in the membrane during cleavage comes from Chambers’ ( 1938a) microdissection experiment in which he burst one of the blastomeres of a cleaving egg. The remaining blastomere shrank in size like a gradually deflating balloon forcing its contents through the furrow region. Sichel and Burton (1936) on the basis of the rate of deflation calculated the tension to be 0.09 dynes/cm.2
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when cleavage was almost complete. Mitchison (1953) has attempted to repeat this experiment without success. These p i n t s lend support to the increase in tension explanation, and whereas the tension theory can form a rational basis for a theory of cleavage, no explanation has been offered in terms of changes in Young’s modulus. In regard to the increase in “stiffness” prior to cleavage, it has been argued that if the egg surface is to be passively stretched by a contractile ring in the furrow, it seems unlikely that the egg would make cleavage more difficult for itself by increasing its surface rigidity (Swann and Mitchison, 1958). However, the essential feature of this theory is that the “stiffness” falls the moment cleavage commences at just those portions of the egg being stretched.
2. The Contractile Ring An objection that has been raised against any theory involving a contractile ring has been that it requires that the circumference at the furrow should contract to nothing (Mitchison, 1952). This, however, cannot be regarded as an objection, since the particle movements clearly show that this is what occurs. Rather it is a problem that must be explained in terms of the physico-chemical properties of the membrane. A similar phenomenon is associated with the movement of amoebae, whose contractile tail may be likened to a furrow-insofar as its contraction is associated with “solation” of the membrane. Also, associated with the contraction of the furrow, there is the accumulation in the furrow region of granules originally evenly distributed throughout the cortex (Dan, 195413). This accumulation is apparently due to contraction and solation of the cortex. Mitchison (1953) has objected to a contractile ring theory on the basis that the furrow will pass through a needle inserted parallel to the egg axis. If there were a contractile ring, it is suggested that a gaping wound would appear. However, this experiment probably only demonstrates the fluid properties and dynamic nature of the membrane. The observations of Scott (1%) on the cleavage of eggs under slightly abnormal conditions support both the idea of tension at the surface and contraction in the furrow. H e has observed flow between blastomeres and the contraction of the neck joining the daughter cells.
3. Structural Changes The interpretation of the changes in birefringence is a most complex problem (see Swann and Mitchison, 1951). However, Dan and Okazaki (1951) have found that the birefringence of the membrane is increased by tension and that it also apparently depends on the thickness of the
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membrane. I n terms of Mitchison’s (1952) conception of the membrane (Section VIII, B) a tightly looped configuration will correspond to a high birefringence and the extended state of the protein chains to a low birefringence. With reference to both these interpretations, the rise in birefringence at anaphase would correspond to an increased tension or contracted state of the membrane and the decrease in birefringence, starting at the poles, to the reduction in tension and expansion. However, the structure causing birefringence has not yet been unequivocally identified (Section VIII, B) and the changes in birefringence may well be associated with the development of the new layer demonstrated with the electron microscope. This layer, which is most strongly developed in the furrow region, could account for the changes in both the optical and mechanical properties of the membrane during cleavage.
C. POLAR DIFFERENTIATION Whatever system of forces gives rise to cleavage, there can be no doubt that the polar region of the membrane behaves differently from the furrow region during cleavage. This has been amply illustrated above. What is the origin of this differential behavior? There can be little doubt that it is the mitotic apparatus that impresses the plane of cleavage on the cell, the cell almost always cleaving so as to bisect the spindle or the line joining the astral centers. The close connection between mitosis and cleavage has been stressed by Swann (1952), and it is one of the merits of the expanding membrane theory (Section 111, D, 2) that it attempted to relate mitosis and cleavage. The problem is to determine which component of the mitotic apparatus is responsible for the differentiation. The astral relaxation theory suggests that it is the asters, and evidence in support of this view will be given below. The exclusion of the spindle and chromosomes as active agents in the differentiation rests partly upon the fact that eggs can cleave in the continued absence of both (E. B. Harvey, 1936, 1940). The expanding membrane theory suggests that it is the chromosomes that play the major role in this determination. However, as pointed out already, cleavage of sea urchin eggs can occur in the absence of chromosomes. Moreover, the chromosomes are particularly unsuited for differentiating the membrane since, as will be shown, the differentiation occurs before anaphase. The chromosomes at this time are located in the center of the egg, and diffusion of any substance from them will affect all portions of the egg membrane equally. The astral relaxation theory postulates both that the asters differentiate the membrane and that this differentiation occurs before cleavage commences.
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1 . Precleavage Diflerentintion Evidence for the precleavage differentiation comes from those experiments in which the mitotic apparatus was destroyed by chemical agents just before cleavage commenced and yet cleavage occurred. (It must be noted that its earlier destruction blocks cleavage.) This has been done using colchicine (Beams and Evans, 1940; Swann and Mitchison, 1953) and podophyllin (Cornman and Cornman, 1951). Hiramoto (1956) even removed the whole mitotic apparatus with a micropipette before cleavage, and yet the egg cleaved. From these studies it appears that differentiation has occurred by the beginning of anaphase. This is a most important result and has received neither sufficient emphasis nor recognition. It is further illustrated by the experiments of E. B. Harvey ( 1935), which also demonstrate very beautifully that it is the mitotic apparatus that determines the plane of cleavage. Harvey shifted the mitotic apparatus by centrifugation at various times before cleavage and found that a furrow always appeared in relation to the original position. This furrow usually was abortive, and a new furrow appeared in relation to the new position of the mitotic apparatus. She also found that this cleavage furrow came in first from the side closest to the mitotic apparatus. More evidence for the precleavage differentiation of the cell is illustrated by the more firmly held granules in the furrow region (Marsland, 1939), and the differential behavior of poles and furrow region prior to cleavage in hypertonic sea water (Motomura, 1950) and in certain chemical agents (Kuno, 1954a).
2. Role of the Asters The role of asters in cleavage was clearly pointed out by Wilson (1901) especially in relation to the cleavage of etherized eggs in which asters were reduced in size. The cleavage furrows were in direct relation to the position and size of the asters. The role of the asters was further emphasized by Gray and Dan, and they played a fundamental role in their theories of cleavage. Though these theories are not acceptable (see Section 111, A, 3 ) , much of the associated data is most relevant, and this is particularly true as regards Gray’s theory, in which asters are assumed to have a weakening influence on the surface. His conclusions are best given by direct quotation (Gray, 1931) . “The most direct proof that the asters form an active part of the cleavage mechanism is provided by the fact that any irregularity in the size of position of these structures is invariably accompanied by an irregularity in the form and position of the cleavage furrow. “In order that cleavage should occur in echinoderm eggs it is essential that there should be two asters each of .which must be located within a
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short distance of the cytoplasmic periphery. A single aster near the periphery will deform the surface of the cell but it will not produce a cleavage furrow; this . . . is illustrated by the observations of Wilson (1901). Whenever there are two asters present equal in size and whose rays extend to the periphery of the cell, a cleavage furrow will form between them; if there are three asters there will be three cleavage furrows ; if there are four asters there will be four cleavage furrows. Similarly if two asters (of adequate size) are present-but one is larger than its mate-then unequal cleavage results. If the line joining the centre of the two asters does not pass through the diameter of the egg by the time the astral rays reach one side of the egg, then the cleavage furrow develops first on that side . . . all these phenomena can be observed in the natural cleavages of various types of cell. That the irregularity of the form and position of the asters is the cause and not the result of irregular cleavage is suggested by the fact that similarly irregular cleavages can be induced to occur in Echinus eggs by experimental means” (Gray, 1924). The role of asters in determining the position of the cleavage furrow is also demonstrated by the cleavage of enucleate eggs (E. B. Harvey, 1936). I n these eggs neither spindles nor chromosomes were present, but there were well developed asters, and the furrows developed in relation to them. Lorch (1952) has also described the development of cleavage furrows between the asters of enucleated eggs. When cleavage is unequal then the isolated mitotic apparatus shows corresponding differences in the size of asters (Dan and Nakajima, 1956).
3. Mechanism of Astral Action The astral relaxation theory postulates that it is the asters that differentiate the membrane closest to them as in Fig. 9, so that these regions will relax. Thus the poles expand and the furrow contracts. The sta-
P
FIG.9. Diagram to show the differentiation of the polar regions of the sea urchin egg by the asters prior to cleavage. The limit of the influence of the asters is indicated by the broken line. S-S’ indicates the stationary rings, A is the center of the aster, P is the polar region, and F the furrow region.
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tionary rings (Section 11, B) would be defined by the limits of the astral action. (For the structure, composition, and formation of asters, see Mazia, 1956.) How is this achieved? I n general terms, the problem is to find a mechanism such that the differentiation is greatest the closer the membrane is to the astral center. Since the furrow region is, in principle, acted on by both centers, the mechanism must be such that the influence falls off in a manner such that this double action is offset by the closer proximity of the asters to the poles. From Fig. 9 it may be shown that if the differentiating power fell off according to an inverse square law and if the double action is taken into account, the furrow will be the least differentiated region. Although the whole membrane may be influenced by the asters, the polar regions are relatively more differentiated. An obvious possibility whereby differentiation could be achieved is by regarding the asters as having a morphologically distinct limit and only those portions of the membrane in direct contact being differentiated. A mechanism of this type was suggested by Gray (1924). Direct contact of asters with the membrane has been suggested (Dan 1943b, Dan and Nakajima, 1956), but Swann (1951a, b) has shown that at metaphase the asters are only 15 p in diameter and thus still 15 p away from the membrane. Since, as pointed out in Section VI, C, differentiation has occurred by metaphase, it would seem that differentiation by direct contact is ruled out. However, the precise morphology of the asters in relation to the membrane is not quite clear. Also the asters may modify the cytoplasm adjacent to them and so indirectly influence the membrane. Another possibility is that there is a substance arising at the centrioles which can bring about changes at the cell membrane. Simple diffusion of such a substance is an unsuitable mechanism, since it will tend to become uniformly distributed throughout the cell. However, if as this substance diffuses it becomes bound to the cytoplasm, it is easy to see how the changes would be restricted and would be greatest at the poles. This seems to be the most attractive hypothesis, since current ideas on aster formation are based on the idea of a substance diffusing from the centriole (Mazia, 1956). It is possible, then, that the aster is the physical expression of such a substance which brings about structural alterations in the cytoplasm as it diffuses and thus restricts its own distribution. Possible chemical changes are considered in Section VIII, A. Whatever mechanism is involved, one thing is very clear. The dumbbell shape of the pair of asters is the ideal geometrical form for bringing about a differentiation between poles and furrow region. This dumbbell form is well illustrated in the isolated mitotic apparatus (Mazia and Dan, 1952; Rustad, 1959).
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D. MITOSISAND THE COORDINATION OF CLEAVAGE The main events in cleavage are the differentiation of the poles, the increase in tension, and the relaxation of the poles. These events are obviously associated with mitosis. Thus the increase in tension starts at the beginning of anaphase and the decrease at the start of telophase, and the problem is how cleavage and mitosis are linked. Swann (1952) has stressed the possibility of nuclear control and the correlation between nuclear and cytoplasmic changes, but, as has been pointed out, cleavage can occur in the absence of chromosomes. It would thus seem that both mitosis and cleavage are linked through a master process which may be associated with cytoplasm. The possibility does exist that the master process can be influenced and regulated by the chromosomes, and some evidence for this is that it is almost unknown for eggs to cleave before the separation of the chromosomes at the beginning of anaphase. There is some further evidence for the influence of the chromosomes on cleavage. Boveri (see Wilson, 1924) found that in eggs containing several asters, permanent cleavage furrows were only formed across chromosome-bearing spindles. Also cleavage furrows in enucleated eggs are often not permanent (Lorch, 1952). For a more detailed discussion of the relationship between various cell organelles and cleavage, especially the origin of the centrioles and their importance to cleavage, see Briggs and King (1959). The problem of the control of cell division as a whole is discussed by Swann (1957, 1958). E. ENERGY REQUIREMENTS The astral relaxation theory enables an estimate to be made of the work done during cleavage. Simplifying assumptions must be made, and the value of the energy should only be considered to represent an order of magnitude. Two approaches are : (1) The work done is assumed to be due to the furrow contracting to nothing against a constant tension equal to the value in the membrane just prior to cleavage, i.e., 2.7 dynedun. This assumption implies that the tension in the furrow does not alter when the furrow contracts and that it is in equilibrium with the tension at the poles at each stage in cleavage. If the furrow region is taken to be 5 p wide for an egg 100 p in diameter, then the work done is 4.2 X loe6 ergs. This is probably more like an upper limit to the energy required. This calculation assumed that the energy for the initial isometric contraction of the membrane is negligible, since it involves no mechanical work. (2) The work done is taken as equivalent to stretching the membrane
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28%, i.e., the increase in surface area during cleavage. If the Young’s modulus is taken as lo4 dynes/cm.2, which is the value of the Young’s modulus before mitosis (Mitchison and Swann, 1955), then for an egg 100 p in diameter the energy required is 8 X dynes/cm.2. If the true value lies somewhere between these two, then the equivalent chemical energy is about 10-lo moles ATP/egg if the egg is 25% efficient in its use of chemical energy. It must be remembered that if a special molecular mechanism is involved in the expansion of the poles, e.g., the unfolding of protein chains, then these calculations, especially the latter, no longer hold, and the energy required may be much less.
VII. Application of Astral Relaxation Theory t o Other Cells and Forms of Cleavage It is proposed here to consider how far it is possible to apply the astral relaxation theory to cells other than the sea urchin egg, and especially to those forms of cleavage which are not both equal and symmetrical as is the case for the sea urchin egg. It is intended to demonstrate the applicability of the theory to polar body formation, eccentric cleavage of eggs, the cleavage of tissue cells and amoebae, and the unequal cleavage of the grasshopper neuroblast. Polar lobe formation will also be dealt with. It will be seen that the theory is unable to account for the cleavage of the amphibian egg. While it is not possible to test the theory critically for each caseand this does not seem particularly necessary-it is necessary to examine the extent to which the special features associated with each type can be accounted for. The types of cells and forms of cleavage chosen are by no means comprehensive, but it is hoped that they are representative of a wide range. The most notable omissions are the insect egg, where cleavage is of a special type, viz. the nucleus divides a number of times before cytoplasmic division is brought about; and the cleavage of the highly differentiated protozoa. (For further discussion on the cleavage of eggs, see Waddington, 1956). Two features common to the cleavage of a wide variety of cells are worth noting (see Wilson, 1924) : (1) The cell usually rounds up prior to cleavage, and (2) the cleavage furrow in general bisects the line joining the poles of the spindle and develops first on the side closest to the spindle. The rounding up of the cell before cleavage possibly has more than merely a mechanical significance. It is most important for cells which in interphase have an irregular form, e.g., amoebae and tissue cells. The equal division of such cells would be extremely difficult if rounding up did not occur.
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There are few measurements relating to changes in the mechanical properties of the membrane of eggs other than sea urchins. Using a particle displacement method Wilson ( 1951) has measured the changes occurring in Chaetopterus, and Raven ( 1948) has studied cell elongation due to centrifugation in Limnaea. However, because of the great difficulties in interpreting such studies (see Section IV, B) they will not be considered further. The following considerations must thus rely more on indirect evidence with regard to the mechanics of cleavage, and greater attention will be given to the role of the asters in determining the plane of cleavage. A. POLAR BODYFORMATION Polar body formation (Fig. 10) is associated with the maturation division of eggs and results in a small cell being formed, usually at the
I
FIG.10. Diagram to show polar
2 body formation. At metaphase (1) only the
surface between S and S’ will be differentiated by the asters. C indicates the chromosomes. P is the polar body. After Wilson (1924).
animal pole of the egg. Polar body formation is an extreme case of unequal cleavage, the ratio of the diameters of the two resulting cells being more than 10. Also it is one of the few cases in which the cleavage plane does not bisect the metaphase spindle. An explanation for these two phenomena is to be found in the relation of the asters to the surface. If the asters differentiate the surface, only a very small area of the surface adjacent to the one aster would be expected to relax (Fig. 10). This is supported by Chambers’ (1917) . . . “impression that the polar body is due to the local weakening in the consistency of the surface where an internal pressure causes the cytoplasm to Aow out.” This seems to constitute very good evidence for the astral differentiation theory. Some
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evidence for an increase in tension at polar body formation comes from the formation of small polar lobes at the same time, and this is suggestive of an increase in internal pressure (Section VII, B). The intimate association of the aster with the cortex to form a “continuous gel” has been described by Chambers ( 1917). The relation between the mitotic apparatus and the pos:tion of the cleavage furrow at polar body formation is further demonstrated by shifting the maturation spindle by centrifugation away from its eccentric position. Under such conditions the egg can be made to cleave so that the polar body is the same size as the egg (Conklin 1917; Clement, 1935).
B. POLAR LOBEFORMATION The cleavage of certain eggs is modified by the pushing out of a large pseudopodium-like body just before the first division, which is known as the “polar lobe.” The cleavage runs so that the whole of this lobe becomes incorporated into one of the two daughter cells and the process is repeated through several of the later divisions. This phenomenon has some relevance to the theory propounded here. The relation of the lobes to cleavage and polar body formation is well described for the marine snail Ilyanussa (Morgan, 1933). Their formation involves considerable expansion (Dan and Dan, 1942), and the simplest explanation is that they are formed at that region where the membrane relaxes. An increase in internal pressure in the egg would tend to “blow out” such a region. The relaxation may take the form of a reduction in Young’s modulus when the rest of the surface is stimulated to contract at cleavage. This interpretation would account for the size of the lobe associated with first cleavage reaching its maximum just before cleavage commences. According to the astral relaxation theory, this is when the internal pressure is at its maximum. C. ECCENTRIC CLEAVAGE Eccentric cleavage, in which the furrow comes in from one side before the other, is typical of many eggs, especially those which are yolky. In ctenophore eggs, furrowing is from the one side only. Eccentric cleavage is always associated with the displacement of the mitotic apparatus from a central position, and the furrow always originates on the side closest to it. Such cleavages may be induced in sea urchin eggs by shifting the mitotic apparatus (E. B. Harvey, 1935). Dan (1958) studied particle movements in eggs that cleave eccentrically, viz. the heart urchin and clam, and using Ishizaka’s method (Section 11, B) investigated the existence of “stationary rings.” These rings were found (Fig. l l ) , but their position was less well defined. What is of particular interest is that
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the angle between these stationary rings bore a relation to the eccentricity of the spindle. (Eccentricity is defined as the ratio: the distance from the center of the spindle to the surface/the diameter of the cell.) Although the results show a considerable scatter, they do suggest a linear relationship between the t w w t h e angle increasing with the eccentricity. Dan has interpreted this as support for his spindle elongation theory.
FIG.11. Diagram of the stationary rings. S-S', in the Astriclypeus egg :..>wing the effect of the eccentricity of the mitotic apparatus. A is the center of the aster. After Dan (1958). This relationship is capable of explanation in terms of the position of the asters. Thus the eccentric position of the aster in Fig. 11 would lead to the area that would be differentiated to relax, being shifted in the same direction. Assuming the same differentiating power of the asters as in the symmetrical case, this will define stationary rings which are inclined to each other. This result seems strongly to support the idea of astral differentiation. Dan and Dan (1947a) have laid considerable emphasis on the bending of the spindle in such eccentric cleavages in support of their theory, but the simplest explanation seems to be that this bending is due to the incoming furrow. The particle movements show in general a similar pattern of movement to that found in the sea urchin egg (Dan and Dan, 1947a). Many observations have been made on the behavior of surface perforations (Dan and Dan, 1947b). The more passive nature of the vegetal half of the egg is suggested, since perforations there block the furrow and prevent cleavage, whereas those in the animal half are drawn into the furrow.
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D. TISSUE CELLS The cleavage of chick fibroblasts in tissue cultures gives the appearance of being typical of a wide variety of tissue cells. The cleavage of the fibroblast is well described by Strangeways (1922) (see also : newt, Boss, 1954; Hughes and Preston, 1949 ; mouse, Fell and Hughes, 1949). The main features of fibroblast cleavage are : the cells withdraw their pseudopodial projections and tend to round up ; after anaphase the cell begins to elongate, and this is associated with a diminution of the diameter of the cell in the equatorial region but not at the poles ; and the appearance at the poles of a series of blunted “blisters” which appear and disappearknown as “bubbling.” With further development of the furrow fine pseudopodia, characteristic of noncleaving cells, develop at the poles, and the cells move apart, which leads to the snapping of the cytoplasmic bridge. It should be noted that the locomotion of tissue cells bears many resemblances to processes occurring during cleavage. I t is highly probable that the same mechanism for membrane motility is involved in each case. The rounding up of the cells may be accounted for in terms of an increase in tension, and the membrane certainly contracts. However, for tissue cells, adhesion to other cells and to the surface on which they move must be important in determining the form of the cell, and loss of adhesion may also play a role in the rounding up. The bubbling at the poles can be accounted for by relaxation there, and polar weakness has been demonstrated by microdissection in grasshopper spermatocytes (Chambers, 1938a). The astral relaxation theory thus seems to be able to account for the mechanics of cleavage of such cells. Also Mota (1959) has concluded that the cleavage is due to the constriction of a gel-like ring in the furrow region. Asters are not nearly so obvious in fibroblasts as in the sea urchin egg, but their presence is demonstrable in polarized light (Hughes and Swann, 1948). Kawamura (1959) has described the asters in grasshopper spermatocytes. Also, Carlson (1952) has found, by microdissection, fine fibers connecting the spindle poles to the surface in grasshopper neuroblasts. That the position of the furrow is determined by the asters (as indicated by the position of the spindle poles) has been shown by Roberts and Johnson (1956), who have found that in the multispindle testicular cells of the beetle the initiation of furrows occurs between spindle poles whether connected by a spindle or not. These authors also conclude that the spindle poles play a role in determining the position of the furrow. Further evidence for the astral relaxation theory will be given when the glycerol extracted models of Hoffmann-Berling are considered ( Section VIII, A ) .
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That the chromosomes do not play a role in determining the position of the cleavage furrow is suggested by: (1) Hughes and Fell (1949) have reported abnormal mitoses brought about by nitrogen-mustards in which chromosomes were left behind in the furrow region without affecting cleavage. (2) In several cells (newt tissue, neuroblasts) the chromosomes fill almost the entire cell during mitosis and cleavage, and it does not seem possible for an agent released from them to differentiate a specific portion of the cell surface. Boss (1955), however, has described bubbling of the cell surface associated with proximity of the chromosomes.
E. UNEQUAL CLEAVAGE OF A NEUROBLAST Unequal cleavage is rare in tissue cells but has been studied in the cleavage of grasshopper neuroblasts, which cleave to form a large daughter neuroblast and a smaller cell that will become the ganglion. The following description is from Roberts (1955) (Fig. 12)
3
E 2 4
5
6
FIG.12. The cleavage of the grasshopper neuroblast into a large daughter neuroblast and a small ganglion cell. The spindle is represented by the broken line and the chromosomes are diagrammatic. In 1 the cell is at metaphase, and 2-6 show anaphase and telophase. DP is the pole of the daughter neuroblast, G P is the pole of the ganglion cell, El indicates the first polar expansion, and E2 the second polar expansion. After Roberts (1955).
At metaphase the spindle is small and eccentric in position, reaching the cell membrane at only one pole. This region invariably becomes the daughter neuroblast upon completion of division. “At anaphase the spindle extends from cell pole to cell pole and the cell elongates.” The first evidence of the beginning of the constriction process is a clearly visible
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expansion of the cell membrane at that pole which will give rise to the daughter neuroblast. Later a second expansion occurs at the ganglion cell pole, and the constriction furrow is established between the waves of expansion as they approach each other. Subsequent advance of the furrow divides the cell. Roberts has shown that the relative volumes of the ganglion and neuroblast cell are related to the time interval between the two expansions-the greater the interval, the farther the first expansion advances toward the ganglion pole before the establishment of the constriction furrow at its meeting with the second wave. H e regards this as strong evidence in favor of the expanding membrane theory, but suggests that if “the polar expansion is initiated by the diffusion of an X-substance, its origin should probably be attributed to the interaction of centromeres and spindle poles with the fibres of the achromatic figure during the entire period of anaphase movements of the chromosomes.” The application of the expanding membrane theory to this cell is subject to similar objections to those raised in the discussion of the sea-urchin egg (Section V, A) . The main point here is that it cannot account for the contraction of the furrow region. This limitation is recognized by Roberts. The astral relaxation theory, applied to this cell, gets over these difficulties and is also able to explain the differences in size of the daughter cells in terms of astral differentiation. Thus, the earlier and greater expansion at the pole of the future neuroblast cell may be regarded as being a relaxation due to the close proximity of the spindle pole to this surface at metaphase, and hence its being differentiated to relax to a greater extent than the ganglion cell pole. The position of the cleavage plane is defined by the relaxation of the poles. Carlson ( 1952) has carried out some very beautiful microdissection experiments on the dividing cell which include experiments on the relation of the plane of cleavage to the position of the spindles and the chromosomes, and which add further support to the astral relaxation theory. Carlson concluded that “the polarisation of the neuroblast is largely independent of the spindle and chromosomes ; probably it is determined by the cell cytoplasm, the plasma membrane, the pressure of surrounding cells or a combination of these.” However, his results do not seem to altogether bear this out. His main experiments are : (1) Rotation of the spindle at metaphase by 125” resulted in an outpushing of cytoplasm at the site where the ganglion cell would normally have formed. This soon disappeared and the cell cleaved in relation to the new position of the spindle. This demonstrates the precleavage
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differentiation of the ganglion cell, and that the cleavage plane can be altered by rotation of the spindle. (2) Rotation of the spindle so that its side lay toward the ganglion cells caused pseudopodia-like projections to appear adjacent to the ganglion cells, and cleavage did not occur. Later, the cell degenerated. However, his Fig. lO(A-F) does seem to show an attempt of the cell to divide in relation to the new position of the spindle. This shows that if the spindle is rotated to a position too much at variance with that which it normally occupies, cleavage cannot occur. This does support Carlson’s conclusion. (3) Rotation of the spindle through 180” does not alter the cleavage pattern although the chromosomes go to daughter cells different from those for which they were destined. Polarization is certainly not due to differences in the chromosome groups. (4) If the chromosomes and spindle are pushed to one end of the cell at middle anaphase, cleavage is still normal. This shows very clearly precleavage determination of the furrow and the absence of chromosomal influence on the membrane. (5) If, at metaphase, the spindle is displaced from its eccentric position such that it lies centrally, the cell undergoes equal division. This is a striking demonstration of the astral differentiation theory, since the astral centers now lie at equal distances from the poles, and hence cleavage is equal. F. AMOEBA The cleavage of Amoeba proteus is described by Chalkley (1935, 1951). When about to cleave, the cell rounds up and becomes studded with fine pseudopodia, this change of form being associated with prophase. With the approach of the daughter chromosomes to the cell surface during anaphase, elongation of the cell occurs together with protrusion of large pseudopodia at the poles. “At the equator of the cell the interior layer of the more rigid cytoplasm contracts and the plasmagel can be seen to break away and become fluid.” This involves cytoplasmic flow toward the poles as the equatorial region constricts, and pseudopodia form at the poles. This flow is not regular, and an alternate backward and forward flow through the bridge is seen. The furrow narrows to a fine bridge which is finally severed by the oppositely directed locomotion of the daughter amoebae. Chalkley has suggested a mechanism of cleavage based on Mast’s (1926) theory of amoeboid motion. Mast’s contention is that both the locomotion and the form of an amoeba are due to the elastic properties and tension of the plasmagel. Pseudopodia are pushed out where the strength of the plasmagel is lowest. On this basis, and assuming that the daughter
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chromosomes can weaken the plasmagel, Chalkley’s theory is that the rounding up is associated with the equalizing of the tension in the plasmagel. With the approach of the daughter chromosomes to the cell surface, the plasmagel at the poles is weakened and the “greater elastic strength of the plasmagel at the equator will result in gradual contraction of this region.” As cleavage progresses the locomotory mechanism is brought into play, and the final bridge is broken by the cells moving apart. This theory is almost identical to the astral relaxation thecry. Further support for the theory comes from two sources: (1) I have found that the behavior of a cleaving amoeba compressed by a plate is similar to that of the sea urchin egg (see Section IV, D) (unpublished). (2) Goldacre (1952) has developed a simple test with anesthetics for showing up that region of the membrane that is contracting. This region is at the tail during normal locomotion and in the furrow during cleavage. Goldacre and Larch (1950) have suggested that amoeboid cleavage is due to oppositely directed locomotion of the daughter cells. Chalkley’s theory assigns the cause of the relaxation of the poles to the approach of the daughter chromosomes. These presumably give off a relaxing substance that spreads by diffusion. (The daughter chromosomes are very close to the surface during cleavage.) It is uncertain whether the amoeba does have asters, and the cleavage of this cell is often regarded as being of the anastral type. Asters could, if present, account for the relaxation. EGG G. AMPHIBIAN At the present time it is not possible to account for the cleavage of the amphibian egg in terms of the astral relaxation theory, the main reason being that in the cleavage of this egg the new surface is not formed by stretching the existing surface but, instead, a new cell wall is formed. This is one of the main conclusions of the detailed study of the cleavage of the newt’s egg by Selman and Waddington (1955). The initial stages of cleavage of this egg involve merely a “dipping-in” of the furrow at the animal pole, which only later opens out and then shows white unpigmented cortex in the new furrow. Study of the movement of pigment granules showed that though there were contractions parallel to the furrow, and expansion at right angles to it (both of the order of lo%), there was no evidence of any net change in the original surface area. This, together with the appearance of the unpigmented cortex in the furrow, suggested that the new membrane was formed de novo beneath the surface clear of the egg. Cytological investigation of cleaving eggs revealed r r . signs of changes occurring in the sub-cortical cytoplasm in positions below and ahead of the furrow, whose progress could be seen on the surface of
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the egg. The modified cytoplasm lay only in the plane of the cleavage furrow, midway between the daughter nuclei produced by the mitosis, and perpendicular to a line joining the two nuclei.” It was thus “. . . concluded that the new unpigmented cortex, by which the daughter blastomeres remain in contact after cleavage, is first formed as a sheet of gel (which in later stages can be seen to be a double layer) which grows downwards by a process involving gelation at its lower edge.” Observations were also made on the changes in shape of the egg, which tends to flatten under gravity. There was a very distinct tendency to round up before cleavage and to relax when cleavage started, and these changes in shape corresponded to the changes in “stiffness” as measured with the elastimeter. The “stiffness” rose and fell in a way similar to the sea urchin egg. Selman and Waddingtun suggest that cleavage is brought about by the . . has increased to about one-sixth formation of the gel wall, when it of the cross-sectional area of the egg, extending downwards from the animal polar surface, it contracts.” This initially leads to the “dippingin” of the furrow. Further growth of the gel wall, and its contraction as soon as it is formed, lead to the completion of cleavage. It is suggested that the rounding-up of the egg and the changes in stiffness are due to either changes in Young’s modulus or in the thickness of the cortex, and the latter interpretation is preferred since the “decrease in rigidity of the entire cortex takes place precisely in the period in which the new subcortical gel is forming.’’ The suggestion is made that sol-gel transformations may be involved, together with streaming movements, by which new material would be transferred from the existing cortex to the new. A theory based on the expansion or “growth” of ‘a very narrow region adjacent to the furrow has been put forward by Schechtman (1937). This theory must be regarded as being improbable since the demonstration of the de mvo formation of the furrow walls. The astral relaxation theory is also unsatisfactory in this respect. However, the changes in the mechanical properties of the membrane would seem to be more simply interpreted in terms of changes of tension rather than thickness or Young’s modulus, for reasons similar to those given for the sea-urchin egg above. The significance of these changes are not clear, but it should be noted that for “dipping-in” to occur, the tension in the furrow must be greater than in the adjacent membrane, and the relaxation at the start of cleavage may be associated with this. Selman and Waddington do not attempt to relate cleavage to mitosis, and in particular to the factors that determine the position of the new gel wall. It would seem that the asters play a dominant role, since asters alone, in the absence of chromosomes and spindles, are capable of organiz-
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iiig cleavage furrows (Fankhauser, 1934 ; Gross, 1936). It is possible that the new gel wall starts to form where the astral influence on the membrane is lowest.
VIII. Biochemistry of Cleavage The biochemical problems associated with cleavage have so far been avoided in order that they should not be confused with the mechanics of cleavage. It is intended to review briefly some of the available data, especially insofar as they can provide an explanation of the mechanisms postulated by the astral relaxation theory. The main problems are those connected with the molecular structure of the cell membrane. More specifically the problems are : ( 1) the differentiation of the polar regions of the membrane, (2) the mechanism involved in both the contraction and relaxation of the membrane, (3) the mechanism whereby the membrane contracts and “dissolves.” Explanations on either chemical or molecular levels are as yet lacking for any of these phenomena, and our ignorance in this sphere is probably the most serious hindrance to a fuller understanding of cleavage. The most detailed studies on the membrane have been carried out on the red blood cell, but since this is a rather specialized cell and never cleaves, attention here will again mainly be confined to the membrane of the sea urchin egg. (For a review of the energy sources for cell division see Swann, 1957, 1958.) The most promising approach to these problems comes perhaps from the analogy that can be drawn between the behavior of the cell membrane and muscle, and this will be considered first. ANALOGY A. THEMUSCLEPROTEIN The demonstration by Hoffmann-Berling and Weber (1953) of the fundamental similarity between motility of muscle and cell movements, especially those involved in cell division, is one of the most exciting developments concerned with cleavage in recent years. Hayashi (1953) had also suggested this possibility. Hoffmann-Berling (1954) has prepared glycerol-extracted models of tissue culture cells which, on the addition of adenosine triphosphate (A T P ) , exhibit movements characteristic of that stage at which they were extracted. Thus cells extracted in early telophase constrict in the furrow region and show in general the characteristic movements of telophase. These cell models have many properties similar to glycerol-extracted muscles and actomyosin threads (Hoffmann-Berling and Weber, 1953; Weber, 1955). (1) A T P initiates the contraction, is the energy source, and is split ,during contraction. (2) Traces of magnesium ions, and in certain cases calcium, must be present. (3) If the concentration of A T P
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is increased much above the optimum (about 10-2M), substrate inhibition occurs and the magnitude of the contraction falls off steeply. (4) Contraction can be inhibited by naturally occurring factors which are known as relaxing factors. These factors raise the level at which substrate inhibition can occur. They are inhibited by calcium. (5) Certain poisons such as mersalyl and germanin which block -SH groups, can inhibit contraction. Finally, a contractile protein similar in behavior to actomyosin has been extracted from tissue cells (Hoffmann-Berling, 1956). The “furrowing” of the telophase models is due to the furrow region contracting much more than any other portion of the cell, and HoffmannBerling has shown that this region is less sensitive to substrate inhibition and to inhibition by germanin. Also, its optimum pH for contraction differs from the rest of the cell. H e has suggested that this is due to the A T P optimum on the furrow region being higher than elsewhere in the cell, which may be due to the concentration of the relaxing factor being higher at the poles than in the furrow. The behavior of these models in regard to the differential behavior of poles and furrow regions corresponds extremely well with the astral relaxation theory, which postulates precisely the difference between polar and furrow region found in the models. The postulated differentiation of the polar regions before cleavage commences is demonstrated by the “furrowing” of cells extracted in early anaphase (Hoffmann-Berling, 1954). A property peculiar to the cell models and not found in muscle is the ability to undergo active elongation. This is particularly clearly seen in the elongation of the spindle at anaphase. This movement can occur without splitting A T P and can also be brought about by inorganic polyphosphates. It should be noted that the models also show bubbling at the poles which is suggestive of active expansion of the membrane. It is thus very tempting to assume that the cell membrane has properties similar to muscle proteins or actomyosin threads, but much more evidence is required to establish this view, since it is not yet established that the membrane is the active agent in cell models. Results from living cells are as yet equivocal. Some evidence is available that the A T f concentration plays an important role in determining cell form and cell movements of tissue cells (Lettrk, 1952 ;LettrC and Schleich, 1954). Marsland and his collaborators (Landau et al., 1955 ; Zimmerman et al.. 1957) also have some indirect evidence for the role of ATP. They found that A T P increases the furrowing potency (see Section IV, B, 4) of several marine eggs, and that para-chloromercurobenozoate and mersalyl, which are known to inhibit reversibly the contraction of certain actomyosin-like systems, lower the furrowing potency. Zimmerman et al. (1957)
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have suggested that “. . . the metabolic energy from the A T P system of the egg initially goes into the formation of the cortical gel structure, and then later appears as mechanical energy, when the gel contracts during the furrowing process.” The observations on which I based my conclusions that A T P plays a direct role in cleavage (Wolpert, 1958) have now been found to be artifacts. Thus evidence from cells in vim, though suggesting a role for ATP, cannot yet confirm the presence of a system similar to the “actomyosin” system described for cell models. If such a system were in fact present, then the relationships between A T P concentrations, relaxing factors, and calcium represent a remarkably versatile system for causing relaxations and contractions, and schemes to account for the astral relaxation theory are easy to construct. It is tempting to make use of Mazia’s (1959) finding that the isolated mitotic apparatus binds calcium very strongly. The removal of calcium from the poles by the asters could result in the relaxing factor no longer being inhibited there. As a final point in the comparison between the membrane and actomyosin threads, it is of interest to compare the maximum tension of 17 gm./cm.2 required by the astral relaxation theory (Section VI, A ) to be developed in the membrane, with the value of 12 gm./cm.2 obtained by Portzehl (1952) for actomyosin threads. They are at least of the correct order. B. STRUCTURE OF THE CELLMEMBRANE
It has already been pointed out (Section IV, A ) that the cell membrane comprises at least two structural components-the plasma membrane and the cortex. The molecular structure of the plasma membrane has been the subject of much study, and it probably consists of a bimolecular lipid layer about 60 A. thick, with protein absorbed on either side (Danielli, 1951). The cortex has received much less attention, and the main contribution to the study of its molecular structure is that of Mitchison ( 1952). Mitchison has pointed out that all the cell membranes studied with the polarizing microscope show a combination of positive form, and negative intrinsic, birefringence. In order to account for this he has suggested that the cortex consists mainly of looped protein chains in which the micellar axis is tangential and the molecular axis is radial. Mitchison has also stressed the possibilities for active expansion inherent in such a structure, and although active expansion has been shown to be unsatisfactory in providing the mechanical force required in cleavage (Section V, A), the unfolding of the protein chains may well play a role in the fall of tens:on and expansion at the poles. The structure seems well suited to explain the bubbling movements observed in glycerol-extracted cell models ( Sec-
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tion VIII, A) . Unfortunately no attention has yet been given to the possibilities for contraction of such a membrane. Further, it is not certain that it is the cortex that is the cause of birefringence. The role of lipids in causing birefringence of the membrane has been stressed by Ohman (1945, 1947), and lipid solvents have been shown to lower the birefringence (Runnstrom et al., 1943). Minganti (1954) has shown that phospholipase destroys the birefringence whereas trypsin does not. These results would appear to demonstrate the role of lipids in maintaining the structural integrity of the membrane (Mitchison, 1956b). Thus the origin of the birefringence of the membrane is still disputed. It must also be remembered that it is not yet proven that the cortex is the active component in the membrane. Nothing corresponding to the cortex has been described in electron micrographs (Afzelius, 1956 ; Rothschild, 1958; Mercer and Wolpert, 1958).
C. ACTIONOF CHEMICAL AGENTS There have been a large number of investigations into the effect of various chemical agents on cell division though these have rarely been directed to solving problems specific to cleavage. The effect of chemical agents on the mehanical properties of the membrane and the studies of inhibition have thus far yielded little information. Of more interest is the differential response of the surface. 1. Mechanical Properties of the Membrane Studies on the effect of chemicals on the mechanical properties of the membrane (Kriszat, 1953, 1954; Mitchison, 1956c, Wilson and Heilbrunn, 1952; Zimmerman et al., 1957) are not easily related to cleavage or to each other. This is because studies on noncleaving cells are not necessarily applicable to those cleaving. Also, different methods measure different properties (Section IV, B). Much of the confusion as regards the effect of calcium on the membrane probably arises for this reason. Thus, although it is widely held that calcium strengthens the cortex, Mitchison ( 1 9 5 6 ~ )found that calcium did not affect the “stiffness” of the membrane of sea urchin eggs as measured with the cell elastimeter. Wilson and Heilbrunn ( 1952), using a particle displacement method on Chuetopterzcs, found an increase in “rigidity” on the addition of calcium.
2. Inhibition of Cleavage Whereas the inhibition of mitosis and the process of division as a whole has been widely studied, the search for specific inhibitors of cleavage has hardly been begun. In general, the inhibition of mitosis inhibits cleavage, but such an inhibition is no indication that the agent is affecting cleavage.
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As has been pointed out (Section VI, C) , cleavage is dependent on mitosis and especially the development of the mitotic apparatus, and it is only to be expected that the inhibition of mitosis will also inhibit cleavage. An inhibitor of cleavage but not of mitosis is easily identified, and Chalkley ( 1935) has reported that alizarin sulfonate, which inhibits amoeboid locomotion, if applied to amoebae at prophase completely inhibited cleavage but allowed mitosis to proceed. Also certain acids and bases were found to affect cleavage whereas nuclear division was relatively unaffected (Chalkley and Daniel, 1934). Something approaching the specific inhibition of cleavage is also found when low concentrations of mitotic inhibitors, especially narcotics, are applied to marine eggs. These block cleavage but allow mitosis to proceed, and this leads to the formation of multinucleate eggs (Wilson, 1901; Kuno 1954b). However, in all these cases the mitotic apparatus is much reduced, and cleavage is probably blocked for this reason. Thus with etherized eggs, Wilson found that cleavage furrows developed in direct proportion to the development of the asters. This interpretation is supported by the fact that if such agents are only applied at the start of cleavage, then inhibition does not occur-the exception being a detergent (Monogen) which is apparently specific in its action on the surface (Kuno, 1954b). A similar interpretation is probably applicable to the observations of Hughes ( 1952) that adenine, 2,6-diaminopurine, and benzimadazole inhibit the cleavage of chick tissue culture cells but allow reconstruction of the daughter nuclei. A method for testing the specific action of a chemical on cleavage is to apply it at the beginning of cleavage or at a stage at which damage to the mitotic apparatus will not block cleavage. (For sea urchin eggs this stage is at anaphase (Section VI, C, 2) .) I have carried out a series of such tests using a wide variety of agents (unpublished). The only substances that gave indications that were suggestive of a specific action in blocking cleavage were detergents. These have too general an activity to be of value. If specific cleavage inhibitors of known chemical action could be found it may provide a clue to the mechanism of contraction. The action of quinones on the cleavage of the Tubifex egg has been investigated by Huber (1947). 3. Differential Behavior of the Surface The differential behavior of the poles and furrow as regards structural properties, particle movements, and birefringence has already been described. A chemical approach to this problem has been made by Kuno
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(1954a), who placed eggs in various agents just prior to cleavage and found a differential behavior between poles and furrow region. In general, substances which easily react with lipids, such as wasp venom (containing leoithinase) , Monogen (detergent), and digitonin, attacked the poles preferentially, whereas protein precipitants, such as sodium tungstate, attacked the furrow region. H e thus suggested that protein predominates in the furrow region during cleavage. It is of interest that sodium tungstate attacked the egg differentially before cleavage commenced, which again demonstrates precleavage differentiation of the surface.
D. “DYNAMIC”PROPERTIES OF THE CELLMEMBRANES Investigation of the structural properties of the cell membrane has led to a tendency to ignore its “dynamic” and fluid properties, which have been referred to (Section IV, A ) . The reconciliation of these two aspects is a most important and difficult problem. The most significant dynamic properties that are relevant to cell cleavage are the ability of the membrane to contract away to nothing, the ability to undergo considerable changes in form without wrinkling, and the problem of the formation of new surface at cleavage. Although Marsland and his collaborators (reviewed Marsland, 1956) have given particular attention to the possibility of gel-sol transformations of the cortex, and though they have much evidence that such transformations are possible, this does not provide an explanation of how or why a contracting gel can solate. Goldacre and Lorch (1950) have suggested a theory related primarily to the contraction of the amoeba’s tail. They suggest that the protein molecules in the membrane are in the extended form and can form a crosslinked gel. When the gel contracts they become folded, breaking the links, and the gel is converted into a sol. Another mechanism has been suggested by Anderson (1956a, b), who has also reviewed much information relevant to the “dynamic” properties . . that the sol-gel transformaof the membrane. Anderson suggests tions in the cytoplasm may be best described in terms of reversible cross,,, linking, not of unrolled poly-peptide chains but of beaded chains . and he has developed these ideas in terms of charges on the colloidal particles being the controlling factors. These ideas are very attractive for explaining some properties of the cell membrane. H e conceives the cortex as mainly composed of a calcium complex which is in equilibrium with the cortex constituents in the adjacent cytoplasm, these constituents being the potassium and sodium salts. On this basis, he suggests, it is possible to explain the effect of calcium increasing the viscosity of the cortex-it
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is due to its displacing the K and Na and hence causing gelation. His conception of the cortex as being in dynamic equilibrium with the cytoplasm is most useful. Such concepts are also relevant to the formation of the new membrane that must occur when the cell cleaves (Section 11). It is often suggested that this formation of membrane must involve special mechanisms such as transfer of material or building up of a new gel. However, the “dynamic” properties of the membrane described here and in Section IV, A suggests that the membrane may be in some sort of equilibrium with the cytoplasm. The formation of the surface may merely be a phenomenon of a type occurring under certain physical conditions, as, for example, described by Anderson (1957). However, at this stage we have little idea of how cell membranes, in general, increase in extent as, for example, when a cell grows. To explain in molecular terms the changes that the cell membrane undergoes during cleavage is a most important task for the future, both for the problem of cleavage and for the general problem of cell motility. IX. Summary and Conclusions
1. The cleavage of the sea urchin egg involves an increase in area of the surface of about 28%, and this new surface is provided by the stretching of the existing surface. The polar region of the egg is essentially expansive and initially provides 80% of the new area, and the furrow region is mainly contractile-the equator contracting to nothing. 2. Theories which are based on the mitotic apparatus providing the forces that cause the cell to cleave, viz. the astral growth theory and the spindle elongation theory, are invalidated by the ability of cells to cleave in the absence of a mitotic apparatus. The forces that give rise to cleavage are located at the cell surface. 3. The cell membrane has elastic properties, and the cell elastimeter provides a most satisfactory method of measuring these properties. The significance of “gel strength” measurements of the cortex is uncertain, but they are not simply related to the elastic properties of the membrane. It is shown that the surface of the sea urchin egg can be treated as a flexible elastic membrane. Attention is drawn to certain fluid or “dynamic” properties of the membrane. 4. The mechanical properties of the membrane associated with cleavage of the sea urchin egg are: a. An increase in “stiffness” (as measured with the cell elastimeter) prior to cleavage and a tendency for the cell to round up. b. A fall in “stiffness” at ‘the start of cleavage which occurs mainly at the poles. 5. The expanding membrane theory, which is based on the active
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expansion of the poles (due to the release of an X-substance from the chromosomes) bringing about cleavage, is rejected. The main reasons are that it cannot, since it only postulates expansion, account for the contraction of the furrow, and because the egg can cleave when the membrane is under tension. A mechanism that relies on pushing cannot function when there is tension in the membrane. That part of the theory that suggests that the chromosomes play a central role in cleavage is also rejected, since the egg can cleave in the absence of chromosomes. 6. The cortical gel contraction theory which postulates active contraction of the furrow can account for many features, and its essential mechanical features are included in the astral relaxation theory. 7. The astral relaxation theory is the theory presented here. The main features are: (a) The differentiation of the poles of the cell by the asters before cleavage starts. (b ) Uniform tension at the surface preceding cleavage. (c) The relaxation in tension at the poles, relative to the furrow, due to the astral differentiation. This allows the furrow region to contract and the polar region to expand. This theory is shown to be able to account for the changes in the mechanical properties of the membrane during cleavage as well as the relation between mitotic apparatus and the plane of cleavage. The energy for cleavage is estimated to be about ergs per egg. 8. The astral relaxation theory can account for polar body formation, which may be regarded as an extreme case of unequal cleavage, and also for the eccentric cleavage of certain eggs. It is also consistent with observations on the cleavage of tissue cells and is particularly well able to explain the unequal cleavage of the grasshopper neuroblast. I t conforms in general with an early theory for the cleavage of the amoeba. The theory is unable to account for the cleavage of the amphibian egg, since the new surface in this egg is provided de novo. 9. The cleavage of the glycerol-extracted models of tissue cells shows the similarity between the movements involved in cleavage and the motility of muscle, especially in regard to the presence of a system in which A T P and a relaxing factor can, together with calcium, provide a most versatile system for contractions and relaxations. The behavior of the models corresponds very well with the astral relaxation theory, since it has been suggested that cleavage of the models depends on the concentration of a relaxing factor being higher at the poles. 10. A brief review is given of some properties of the cell membrane and on the effect of chemical agents on cleavage. Specific inhibitors of cleavage, as distinct from mitosis, are largely unknown. There is evidence for chemical differences between polar and furrow regions during cleavage. 11. It would seem that for the better understanding of the processes
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involved in cleavage the most important problem is the elucidation of the motility of the cell membrane in chemical and molecular terms.
ACKNOWLEDGMENT I would like to thank Professor J. F. Danielli, F.R.S., for much advice and assistance, and Dr. D. M. A. Leggett for valuable discussions on certain mechanical problems. The work has been carried out while in receipt of a Nuffield Biological Scholarship, for which I am most grateful.
REFERENCES Afzeluis, B. (1956) Exptl. Cell Research 10, 257. Allen, R. D., and Rowe, E. C. (1955) Biol. Bull. 109, 344. Anderson, N. G. (19%) Quart. Rev. Biol. 91, 169. Anderson, N. G. (1956b) Quart. Rm. Biol. 91, 243. Anderson, N. G. (1957) J . Cellular Comp. Physiol. 49, Suppl. 1, 221. Beams, H. W., and Evans, T. C. (1940) Biol. Bull. 79, 188. Boss, J. (1954) Exptl. Cell Research 7 , 443. Boss, J. (1955) Exptl. Cell Research 8, 181. Briggs, R., and King, T. J. (1959) In “The Cell” (J. Brachet and A. E. Mirsky, eds.), Vol. I, p. 537. Academic Press, New York. Brown, D. E. S. (1934) J . Cellular Comp. Physiol. 5, 335. Buschbaum, R., and Williamson, R. R. (1943) Physiol. Zool. 16, 162. Carlson, J. G. (1952) Chromosoma 5, 199. Chalkley, H. W. (1935) Protoplasma 24, 607. Chalkley, H. W. (1951) Ann. N. Y. Acad. Sci. 61, 1303. Chalkley, H. W., and Daniel, G. E. (1934) Protoplasma 21, 258. Chambers, R. (1917) J. Exptl. 2001.aS, 483. Chambers, R. (19384 I. Cellular Comp. Physiol. 12, 149. Chambers, R. (1938b) Am. Naturalist 72, 141. Chambers, R. (1951) Ann. N. Y . Acad. Sci. 61, 1311. Clement, A. C. (1935) Biol. Bull. 69,403. Cole, K. S. (1932) 1. Cellular Comb. Physiol. 1, 1. Conklin, E. G. (1917) I. Exptl. Zool. !B, 311. Cornman, I., and Cornman, M. E. (1951) Ann. N . Y. Acad. Sci. 51, 1443. Dan, J. C. (1948) Physiol. 2001.9, 191. Dan, K. (1943a) J. Fac. Sci. Tokyo Univ. IV 6, 297. Dan, K. (1943b) J. Fac. Sci. Tokyo Univ. IV 6,323. Dan, K. (1954a) Embryologia 2, 99. Dan, K. (1954b) Embryologia 2, 115. Dan, K. (1958) J. Exptl. Biol. I, 400. Dan, K., and Dan, J. C. (1940) Biol. Bull. 78,486. Dan, K., and Dan, J. C. (1942) Cytologia (Tokyo) 18,246. Dan, K., and Dan, J. C. (1947a) Biol. Bull. DS, 139. Dan, K., and Dan, J. C. (1947b) Biol. Bull. 93, 163. Dan, K., and Nakajima, T. (1956) Embryologia 8, 187. Dan, K., and Okazaki, K. (1951) J. Cellular Comp. Physiol. 88,427. Dan, K., and Ono, T. (1954) Embryologia 2, 87. Dan, K., Yanagita, T., and Sugiyama, M. (1937) Protoplasmu 28, 68. Dan, K., Dan, J. C., and Yanagita, T. (1938) Cytologia (Tokyo) 8, 521. -
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Danielli, J. F. (1951) I n “Cytology and Cell Physiology” (G. H. Bourne, ed.), p. 50. Oxford Univ. Press, London and New York. Danielli, J. F. (1952) Nature 170, 496. Fankhauser, G. (1934) J. Exptl. 2001.67, 349. Fell, H. B., and Hughes, A. F. (1949) Quart. J. Microscop. Sci. 90, 355. Goldacre, R. J. (1952) Symposia SOC.Exptl. Biol. 6, 128. Goldacre, R. J., and Lorch, I. J. (1950) Nature 168, 497. Gray, J. (1924) Proc. Cambridge Phil. SOC.Biol. Sci. 1, 164. Gray, J. (1931) “A Textbook of Experimental Cytology.” Cambridge Univ. Press, London and New York. Gross, F. (1936) Quart. I. Microscop. Sci.79, 57. Harvey, E. B. (1935) Biol. Bull. 69, 287. Harvey, E. B. (1936) Biol. Bull. 71, 101. Harvey, E. B. (1940) Biol. Bull. 79, 166. Harvey, E. B. (1951) Ann. N. Y . Acad. Sci. 61, 1336. Harvey, E. N. (1931) Biol. Bull. 61,273. Harvey, E. N. (1954) I n “Protoplasmatologia” (L. V. von Heilbrunn and F. Weber, eds.), Vol. 11, E.5. Springer, Wien. Harvey, E. N., and Danielli, J. F. (1938) Biol. Revs. Cambridge Phil. SOC.lS, 319. Hayashi, T. (1953) Am. Naturalist 87, 209. Hiramoto, Y. (1956) Exptl. Cell Research 11, 630. Hiramoto, Y. (1957) Embryologia 3, 361. Hiramoto, Y. (1958) I . Ezptl. Biol. SS, 407. Hoffmann-Berling, H. (1954) Biochim. et Biophys. Acta 16, 332. Hoffmann-Berling, H. (1956) Biochim. et Biophys. Acta 19, 453. Hoffmann-Berling, H., and Weber, H. H. (1953) Biochim. et Biophys. Acta 10, 629. Horstadius, S., Lorch, I. J., and Danielli, J. F. (1950) Exptl. Cell Research 1, 187. Huber, W. (1947) REV.suisse zool. 64, 63. Hughes, A. F. (1952) Exptl. Cell Research 3, 108. Hughes, A. F., and Fell, H. B. (1949) Quart. J. Microscop. Sci. 90, 397. Hughes, A. F., and Preston, M. M. E. (1949) J. Roy. Microscop. Sci. 69, 121. Hughes, A. F., and Swann, M. M. (1948) J. Exptl. Biol. 26, 45. Isenberg, I. (1953) Bull. Math. Biophys. 16, 73. Ishizaka, S. (1958) J. Exptl. Biol. 35,3%. Just, E. E. (1922) Am. I. Physiol. 81, 505. Kawamura, K. (1957) Cytologia (Tokyo) 22, 337. Kriszat, G. (1953) Exptl. Cell Research 6, 420. Kriszat, G. (1954) Ezptl. Cell Research 7 , 103. Kuno, M. (1954a) Embryologia 2,33. Kuno, M. (1954b) Embryologia 2,43. Landau, J. V., Marsland, D. A., and Zimmerman, A. M. (1955) J. Cellular Comp. Physiol. 45, 309. Lehmann, F. E. (1946) Rev. suisse 2001. eS, 475. LettrC, H. (1952) Cuncer Research la, 12. LettrC, H., and Schleich, A. (1954) Protoplamu 44,314. Lewis, W. H. (1942) In “Structure of Protoplasm” (W. Seifriz, ed,), p. 163. Iowa State Press, Ames, Iowa. Lewis, W. H. (1951)’ Ann. N. .Y . .Acad. Sci. 61, 1287.
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Lorch, I. J. (1952) Quart. J. Microscop. Sci. SS, 475. Marsland, D.A. (1939) J. Cellular Comb. Physiol. l3, 15. Marsland, D.A. (1956) Intern. Rev. Cytol. 6, 199. Marsland, D.A.,and Landau, J. V. (1954) J. Exptl. Zool. 126,507. Mast, S. 0. (1926) J. Morphol. and Physiol. 4, 347. Mazia, D. (1956) Advances in Biol. and Med. Phys. 4,69. Mazia, D. (1959) I n “Sulfur in Protein” (R. Benesch et al., eds.), 367. Academic Press, New York. Mazia, D., and Dan, K. (1952) Proc. Natl. Acad. Sci. U.S. XI, 826. Mercer, E.H., and Wolpert, L. (1958) Exptl. Cell Research 14,632. Minganti, A. (1954) Exptl. Cell Research 7, 1. Mitchison, J. M. (1952) Symposia SOC. Exptl. Biol. 6,105. Mitchison, J. M. (1953) J. Exptl. Biol. SO, 515. Mitchison, J. M. (1956a) Quart. I. Microscop. Sci. 97, 109. Mitchison, J. M. (1956b) Exptl. Cell Research 10,309. Mitchison, J. M. (1956~)1. Exptl. Biol. SS, 524. Mitchison, J. M.,and Swann, M. M. (1952) I. Exptl. Biol. A ! S, 357. Mitchison, J. M., and Swann, M. M. (1954a) I. Exptl. Biol. Sl,443. Mitchison, J. M., and Swann, M. M. (1954b) 1.Exptl. Biol. 31, 461. Mitchison, J. M., and Swann, M. M. (1955) J. Exptl. Biol. 33, 734. Monroy, A. (1945) Experienfia 1, 335. Morgan, T.H. (1933) J. Exptl. Zool. 64,433. Mota, M. (1959) Ezptl. Cell Research 17, 76. Motomura, I. (1950) Sci. Repts. T6huku Imp. Univ. Fourth Ser. 18,255. Norris, C. H. (1939) J. Cellular Conrp. Phydol. 14, 117. Ohman, L. (1945) Arkiv 2001.AS6 (7) 1. Ohman, L. (1947) Arkiv Zool. A S (11) 1. Portrehl, H. (1952) Z . Naturforsch. 7b, 1. Rashevsky, N. (1948) “Mathematical Biophysics.” Univ. Chicago Press, Chicago, Illinois. Rashevsky, N. (1952) Bull. Math. Biophys. 14,293. Raven, C. P. (1948) Biol. Revs. Cambridge Phil. SOC.a3, 333. Roberts, H.S. (1955) I. Exptl. Zool. lS0,83. Roberts, H.S., and Johnson, N. S. (1956) Biol. Bull. ll0,335. Rothschild, Lord (1958) Quart. J. Microscop. Sci. B9, 1. Runnstrom, M. J., MonnC, L., and Broman, L. (1943) Arkiv 2001.AS6 (3), 1. Rustad, R. C. (1959) Exptl. Cell. Research 16,575. Schechtman, A. M. (1937) Science 66,222. Scott, A. (1946) Biol.Bull. 91,272. Selman, G.G.,and Waddington, C. H. (1955) J. Exptl. Biol. 32,700. Sichel, M.,and Burton, A. C. (1936) Biol. Bull. 71,397. Strangeways, T.S. P. (1922) Proc. Roy. SOC.B94,137. Swann! M. M. (1951a) I. Exptl. Biol. 28,417. Swann, M. M. (1951b) J. Exptl. Biol. 28,434. Swann, M. M. (1952) Symposia SOC.Exptl. Biol. 6,89. Swann, M.M. (1957) Cancer Research 17,727. Swann, M. M. (1958) Cancer Research 18,1118. Swann, M. M.,and Mitchison, J. M. (1951) Progr. in Biophys. and Biophys. Chem. 2, 1. Swann, M. M., and Mitchison, J. M. (1953) J. Exptl. Biol. SO, 506.
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Swann, M. M., and Mitchison, J. M. (1958) Biol. Revs. Cambridge Phil. SOC.SS, 103. Waddington, C. H. (1956) “Principles of Embryology.” Allen & Unwin, London. Weber, H. H. (1955) Symposia SOC.Ezptl. Biol. 9,271. Wilson, E. B. (1901) Arch. EnbickZungsmech. Organ. lS,353. . Wilson, E. B. (1924) “The Cell in Development and Inheritance.” Macmillan, New York. Wilson, W. L. (1951) J . Cellular Comp. Physiol. 38,409. Wilson, W. L.,and Heilbrunn, L. V. (1952) Biol. Bull. 103, 139. Wolpert, L. (1958) Nature 181, 716. Wolpert, L. (1960) Symposium Royal Physical Society, Edinburgh. Zimmerman, A. M., Landau, J. V., and Marsland, D. V. (1957) 1. rcllrtlnr Comp. Physiol. 49, 395.
The Growth of the Liver with Special Reference to Mammals F. DOLJANSKI Department of Experimental Medicine and Cancer Research, Hebrew University, Jerusalem, Israel
I. Introduction ...................................................... 11. Development of the Liver during Embryonic and Postnatal Growth of the Organ ..................................................... A. The Anatomical Development of the Liver .................... B. Weight Changes in Liver Growth ............................ C. Postnatal Liver Growth ...................................... D. Mitotic Activity ............................................. E. Polyploidy ................................................... F. Nucleo-Cytoplasmic Ratio .................................... G. Binuclear Liver Cells ........................................ H. Changes in Chemical Composition ............................ 111. Liver Growth Response to Various Conditions in the Body ............ A. The Effect of Pregnancy and Lactation on Liver Size ........ B. The Effect of Tumors ....................................... C. The Effect of Diet ........................................... D. The Effect of Various Substances ............................ IV. Liver Regeneration after Partial Hepatectomy ..................... V. Concluding Remarks and Summary ............................... Acknowledgments ................................................. References .......................................................
Page 217
218 218 218 222 223 224
m 228
229 234 234 234 235 235 236 236 238 239
I. Introduction In this review an attempt is made to characterize the normal growth pattern of the liver in morphological and in biochemical terms. Much information on its chemical composition in the adult animal and during the periods of growth has been derived from the wide use of the liver in biochemical studies. The function of the liver has been extensively studied and was recently reviewed by Popper and Schaffner (1957). While the metabolic process occurring in the liver are manifold, its primary function lies in the transformation and storage of nutritive substances in such physical and biochemical forms as can subsequently be used by the rest of the body on the one hand, and in the conversion of waste metabolites into excretable substances, on the other. It is remarkable that, whereas in most organs various functions are carried out by morphologically different types of cells, in the liver, an enormous number of functions are performed by one cell type only. This may indicate the retention of a primitive condition, as also the ability of liver cells to multiply readily corroborates a primitive property. 217
218
F. DOLJANSKI
11. Development of the Liver during Embryonic and Postnatal Growth of the Organ
A. THEANATOMICAL DEVELOPMENT OF THE LIVER The structure and anatomical development of the adult vertebrate liver was in recent years reinvestigated by Elias (1955a) from three-dimensional reconstructions of histological sections. H e suggested that liver parenchyma is a “continuous mass of cells tunnelled by a labyrinth of lacunae in wh,ich the network of sinusoids is suspended”; and he proposed the term “muralium” for this system of walls. The adult vertebrate liver is remarkably uniform in structure, and though its embryological development shows much variability (Elias, 195Sb)-there are as many as 12 fundamentally different types of embryonic development-in the adult they all lead up to one strudural uniformity. The liver develops in close association with the yolk sac. In amphibians there is a direct conversion of part of the yolk mass into liver substance, and the vitelline veins are identical with the primitive liver sinusoids. From this evidence, Elias assumed that the primary function of the liver in the adult is identical with that of the yolk sac in yolkpossessing embryos.
CHANGES IN LIVER GROWTH B. WEIGHT The growth of an organ may be studied as an independent unit, by recording its actual weight at any given time. Thus, the curve of the weight will represent the function of time, from which the specific growth rate for any chosen time interval may be calculated, i.e., f ( t ) = 1 dw/w dt, where t is time and w is weight. Another way of observing weight changes during growth is to relate the weight of the organ to that of the whole body. The study of the relative weight of an organ, i.e., organ weight per body weight, would give an indication of possible changes in the organ: body relationship during growth ; moreover, it would be a suitable method for comparative studies of different species, age groups, or experimental groups. The embryonic and the postnatal growth of the liver has been described in the past, but data available are not sufficient to present the rate of growth through all stages from its first appearance as an organ in the embryo, through postnatal development, to senescence. The increase in liver weight during embryonic growth of the rat is reported by Williamson (1948), and his data for each day are summarized in Table I.
219
GROWTH OF LIVER WITH REFERENCE TO MAMMALS
As can be seen, the liver tripled its weight in the course of 6 days. However, these data as well as others showing the changes in absolute liver weight with increasing age are not instrumental in determining whether the changes in growth rate occurred in decreasing and increasing spurts, as assumed by Schmalhausen and Brody, or else by gradual decrease, according to Minot (Needham, 1931a). T o determine a steady decrease or a fluctuation in the growth rate of an organ, a more accurate test of the hypothesis concerning the inflection point is needed. The inflecTABLE I IN LIVER WEIGHT DURING EMBRYONIC GROWTHOF THE RAT THEINCREASE Gestation
Body weight
h Y
g.
Liver weight mg.
No. of animals
16 17 18 19 20 21
1.5 1.9 2.6 3.2 3.5 4.9 5.3
107 152 189 226 236 302 302
7 6 14 9 14 7 5
22
tion point observed by some workers may merely be the result of the difficulty of accurately determining the animal’s age, or of normal biological variability. On the other hand, the changes in the relative weight of the liver of various species during embryonic development provide more information on the character of liver growth. Table I1 shows the relevant data. As appears from Table 11, in most mammals examined, the relative liver weight decreases during embryonic growth. Chick and pigeon, on the other hand, show a steady increase. The decrease in mammals is partially due to the involution of hemopoietic islands in the liver during embryonic phase. Little information is available on the hemopoietic function of the chick liver (Kingsbury et d.,1956). In mammals, as mentioned above, involution of the hemopoietic tissue is one factor responsible for the decrease in relative liver weight; other causes of this phenomenon are indicated by Dick (1956a) in his very interesting study on sheep. He showed a fall in the relative number of hepatic cells per 100 g. body weight during fetal sheep liver growth, and correlated this fall with the decreasing rate of growth of the fetus. His hypothesis was that to maintain a given body growth rate, a definite number of liver cells is required, in other words, that the relative number of hepatic cells in the fetus is determined by the functional requirements of body growth and metabolism.
Animal
TABLE I1 THERELATIVE LIVERWEIGHTDURING EMBRYONIC GROWTH Stage of embryo and liver weight=
References
Days of gestation 16
Rats
17
18
Relative 5.3 7.2 6.0 8.0
- 3.5 7.1 8.0 5.2 6.8 ,6.8
liver 6.5 7.3 6.7 7.3
21
22
weight 5.9 4.9 6.7 6.2 6.0 5.5 - 5.9
5.7
20
Days of gestation 97 104 120
72
Sheep
19
Relative 7.2 5.0 10.3 8.5 8.3 6.3
-
-
Dumm, 1943 Williamson, 1948 Geschwind and Li, 1949 Givol, 1957
EIr
LI
z
142
v)
liver weight 4.0 3.7 2.3 8.0 6.5 4.7 6.0 4.8 3.2
K
CI
Carlyle, 1945 Turco, 1953 Dick, 19%
Length of the embryo in mm. Pig
15
25
58
12.3
15.9
108
125
191 242
Relative liver weight
0
Liver weight per 100 g. body weight.
15.9
8.4
6.2
r
3.3
2.7
Lowrey, 1911
TABLE I1 (Continued) ~
Stage of embryo and liver weight Stage of development
Animal Man
11 mm. stage
2-3 month
References
4 months
At birth
Jackson, 1909
7.5
TI
3z
0
Relative liver weight
4.85
0
q
r
5.0
3
5.23
%
E
Incubation day Chick
5
7
9
10
11
12
13
14
15
16
17
18
2 m
19
w
Relative liver weight
0.66
-
M
0.93 1.62 - 1.88 - 1.95 - 2.36 - 2.66 - 2.55 - 1.36 1.56 1.74 1.87 1.76 2.01 2.12 2.28 2.03 2.06 1.83
Kaufman, 1930 Weston, 1956
m"w
n M
e 0
Incubation day Pigeon
5
7
9
10
11
12
13
14
15
16
17
Relative liver weight
1.28 1.33 1.47 - 1.66 - 1.85 - 2.17 - 2.56 Kaufman, 1930 For dog (Latimer and Cordet, 1948) and cat (Latimer, 1934) only the curves of the relative liver weights are available.
Fz z
F
rn
h)
N
c
222
F. DOLTANSKI
C. POSTNATAL LIVERGROWTH Most papers on that subject deal with the relationship between body and liver weight (Jackson, 1913; Hatai, 1913; Addis and Gray, 1950). The latter authors showed that within age groups a constant relation prevails between liver weight and body weight, making it possible to predict liver weight from a given body weight. POST-NATAL L I V E R GROWTH IN THE RAT
7t
n E e -l n
t
g 5 -
.-
I. f
L Y
4-
.? 3
4
-
21-
Birth
dw dtw
2
3
4 5 Age weeks
6
7
8
SPECIFIC GROWTH RATE
Q7
I
1
t =days w=liver weight gm.
I
‘1
I
1
2
3
I
I
I
4
5
6 7. Weeks
1
I
1
8
9
FIG.1. Postnatal liver growth in the rat (upper) and specific growth rate (lower). (According to Givol, 1957.)
Jackson ( 1913), Donaldson (1923), and Givol ( 1957) have shown that there is an increase in the relative weight of the liver in rats at the weaning period. I n this connection Donaldson points out that “the phase of rapid growth of an organ coincides with the coming of the organ into full function.” Figure 1 shows the growth curve of the rat liver, and here an acceleration can be observed in the specific growth rate during weaning. The relative liver weight of various species is given in Table 111. In
GROWTH O F LIVER WITH REFERENCE TO MAMMALS
223
most cases this amounts to 2-476 of the body weight. In animals which continue to grow throughout life (e.g., fish, rat) the liver continues to grow, retaining its characteristic adult relative weight, with a tendency to decrease in old age. I n animals with determined growth, liver weight reaches its typical adult level with no further increase. TABLE I11 RELATIVE LIVERWEIGHT IN VARIOUS ANIMALS Liver weighta Animal Dogfish Ram esculenta Salamander Dairy cows Sheep Hogs Horses Pig Dog
Rat Guinea Pig Elephant Monkey Man a
(%)
Reference
4.02 4.0 8.02
Kellicott (1908) Welcker and Brandt (1908) Welcker and Brandt (1908) Brody (1945) Brody (1945) Brody (1945) Brody (1945) Lowrey (1911) Brody (1945) Brody (1945) Brody (1945) Brody (1945) Brody (1945) Brodv (1945)
1.31 1.85 1.B 1.12 1.38 4.2 4.8
3.4 0.94 2.45. 2.84
Liver weight per 100 g. body weight.
D. MITOTICACTIVITY During embryonic and early postnatal development, the liver grows mainly by cell multiplication, as has been demonstrated both by estimating the mitotic activity of liver cells at various periods and by the deterniination of the increase in the total cell population. At early stages there is high mitotic activity which declines almost to zero at different ages for various species (see Table I V). All data reported in this Table are based on very small samples giving no analysis of the variation in the mitotic indexes at various ages, such variations occurring even in strictly standardized conditions. Therefore, Fig. 2, showing an increase in the total cell population, constitutes a better indication of the proliferative activity of the cells. I t is of interest to note that McKellar (1949), Tier and Ravanti (1953), and Givol (1957) found in the liver of the 21-day-old rat a mitotic activity peak concomitant with the peak of relative liver weight preceding the shift in the ploidy distribution of liver cells. T h e diurnal variations in liver mitotic activity were described in the young male mice (Jackson, 1959) and in the adult regenerating rat liver (JaiTe, 1954).
224
F. DOLJANSKI
Mitotic activi,ty in the adult liver is mitosis per 10,000-20,OOO liver cells was Marble (1937), and 0.1% according to Swick et al. (1956) calculate the average to be at least 150 days.
extremely rare. Merely one found according to Brues and Leblond and Walker (1956). life span of the adult liver cell
TABLE IV MIT~TIC ACTIVITY OF HEPATIC CELLSDURING POSTNATAL GROWTH Animal
Age and Mitotic Activity
Reference
Age in days Rat
2
Newborn
1
-
7
14
2
Mitotic index" per lobuleb 12 33 28 11 1 1
23
25
41
59
118 McKellar
92
(1949)
1
Age in days
7
2
Rat
14
21
28
32
42
60 Givol (1957)
46
Mitotic index
1.3 2.1
7
Rat
12
4.5
26
21
0.5
2.9
0.3 1.2
Age - in days 28 42 70
84
0.4
150
Ravanti
No. of mitoses/100 microscopic fields
7
1610 8 1 2 1 2 1 2 10 10 10
2 1
2 7
1.
0 0 0
0 0 0
0 0
0
Age in days Pig
Newborn
2
7
20
35
66
86
125
229
0.5
0.0
0.0
White (1939)
Mitotic index
0.5 0
b
.
4.3 3.0 1.7 0.3
0.3
Mitotic index-number of mitoses per lo00 cells. With colchicine administration.
E. POLYPLOIDY The liver belongs to the group of organs composed of cells of a variable size, probably representing ascending degrees of ploidy. The increase in size of liver cells during developmental growth was observed as early as 1911 by Plenck. The first quantitative analysis of nuclear size distribution in liver cells was made by Jacoby (1925), who showed that the maxima of the multimodal curve of nuclear volume distri-
GROWTH O F LIVER WITH REFERENCE TO MAMMALS
225
bution have a ratio of an almost geometrical progression-1:2 :4:8. H e called the individual maxima nuclear classes, Class 1 being the basic class occurring in most somatic tissues, while the others are formed by duplication of previous class. Most nuclei of livers of newborn mammals belong to the first class, but in adult mammals (mice, rats, men, and probably others) the majority of liver cells are of the second class. The liver nuclei of fish, amphibia, and echidna show the first class only. Jacoby therefore concludes that higher nuclear classes are characteristic of mammalian liver.
FIG.2. Increase in the number of liver nuclei during development growth. A. Embryonic chick liver from the 9th to the 19th day of incubation (Weston, 1956). B. Postnatal rat liver (Fukuda and Shibatani, 1953).
The presence of nuclear classes in the liver was later confirmed by many writers, the most detailed statistical examination having been made by Helweg-Larsen (1952), who confirmed the results for mouse liver cells. Further research on three different lines : (1) chemical determination of DNA content ; (2) spectrophotometric estimation of DNA content of liver nuclei; and (3) chromosome counts in dividing cells, provided evidence that nuclear classes correspond to ascending degrees of ploidy. Chemical analysis accompanied by nuclear counts showed that average adult rat liver cells contain about 30% more DNA than a typically diploid
226
F. DOLJANSKI
cell, as, e.g., a kidney cell (Harrison, 1951; Frazer and Davidson, 1953; Thompson et ul., 1953). Spectrophotometric measurements of DNA in nuclei (Swift, 1950; Leuchtenberger et ul., 1951; Thompson and Frazer, 1954 ; Naora, 1957 ; Laquerriere, 1957) have shown adult liver nuclei of rat, mouse, and man to contain different amounts of DNA in a ratio of approximately 1 :2 :4. The fact that various authors found different percentage distributions of each of these cell classes may be ascribed to the variety of strains used. Direct counts of chromosomes were made by Clara (1931), D’Ancona (1941), Biesele (1944), and recently by Marquardt and Glass ( 1957), Glass ( 1958), and Mortreuil-Langlais ( 1958). Marquardt and Glass examined the liver of newborn and of 6-day-old rats, as well as the adult regenerating liver. They found that, whereas in the newborn and the 6-day-old rat the liver shows most mitotic figures with the diploid chromosome number (and a small percentage of haploid, polyploid, and aneuploid metaphases) , in the adult 6-months-old regenerative liver about half the metaphases were of various degrees of polyploidy, mostly tetraploid; in the liver many cells with aneuploid number of chromosomes can be found in each of the ploiding groups (Glass, 1958). Lison and Valeri (1958) suggest that the constancy of the DNA content per nucleus is not absolute as is generally considered. The time at which polyploidization occurs in liver development was studied from nuclear diameter measurements in mice (Helweg-Larsen, 1952; Siess and Stegmann, 1950) ; by spectrophotometric methods in rats (Naora, 1957) and man (Swartz, 1956) ; and by methods of chemical determination (Fukuda and Shibatani, 1953). Both Siess and Stegmann (1950) and Helweg-Larsen (1952) agree that in the mouse from the age of 3 weeks there is no further increase in the number of hepatic cells, which remains constant, yet the average nuclear and cellular volume increases in direct proportion to the logarithm of liver weight. Thus, while the authors show that at the age of 3 weeks a considerable development of the higher nuclear classes takes place, they do not give their percentage distribution during the development. Naora (1957) in his quantitative analysis of the development of polyploidy in the rat, as referred to above, showed that at the weaning period (corresponding approximately to a body weight of 30 g.) there is a marked decrease in the number of diploid nuclei with a concomitant appearance of tetraploid cells. I n the liver of the 50 g. rat, the number of diploid and tetraploid cells is about the same. Thereafter, the percentage of tetraploid cells increases, while that of diploid cells continues to decrease, falling to the characteristic aduLt distribution. When body weight reaches 100 g., a small number of nuclei appear which have a higher DNA content than that in tetraploid nuclei. These seem to grow in frequency as the mature
GROWTH O F LIVER WITH REFERENCE TO MAMMALS
227
rat continues to grow; similar results were obtained by Alfert and Geschwind ( 1958). No significant changes in the ploidy distribution were found in the liver of old rats (24 to 27 months) as compared to younger ones ( 12 to 14 months) (Falzone et al., 1959). In man, up to the age of 6 years the liver cells are diploid only. The tetraploid cells become established at the age of between 11 and 14 years. At about the age of 20, octoploid cells appear, and these three classes coexist in the liver throughout adult life (Swartz, 1956). Fautrez and Laquerrikre (1957) found only one nuclear class in the human liver probably of the diploid type. However, only two livers were examined. It may therefore be concluded that the mammalian liver contains 3040% of polyploid cells, which develop during postnatal liver growth. The turning point of this development is the weaning period in mice and rats, and puberty in man. The mechanism of polyploid formation is reviewed by Swartz (1956) and further discussed by Himes et al. (1957) and Phan and David (1958). The factors influencing the formation of polyploid liver cells, and the physiological significance of the latter, are not known. Helweg-Larsen (1952) was the first to show that their process of formation depends on the presence of growth hormone. In mice with hereditary anterior pituitary hypoplasia there is no polyploidy development, and treatment by growth hormone restores polyploidization. In rats, whose growth is inhibited by caloric restriction to 50 g. body weight, no formation of polyploid cells takes place in the liver (Naora, 1957). I t a u l d , consequently, be assumed that the effect of growth hormone on the polyploidization of liver cells is mediated by dietary factors. A further evidence in this direction: suckling rats, rapidly growing due to increased milk supply, show polyploidization shift at an earlier age (Kennedy and Pearce, 1958). Whereas development of liver plyploidy in the young growing animal and the slowly growing adult rat (Bass and Dunn, 1957) is affected by growth hormone and/or food supply, no such effect can be found in the adult regenerating liver (Geschwind et al., 1958 ; Doljanski and Novogrodski, 1959).
F. NUCLEO-CYTOPLASMIC RATIO The constancy of the nucleo-cytoplasmic volume ratio in the interphase cells was first stated by Hertwig (1908) on the basis of observations in unicellular organisms, but not on tissues of multicellular ones. Fankhauser (1945) assumed it for polyploid cells in amphibia. Volkmann and Marck (1943) were the first to describe an elaborate method of three-dimensional measurements of separated tissue cells. Marck (1943) determined the nucleo-cytoplasmic ratio in the liver of Ram
228
F. DOLJANSKI
temporaria and found it to remain unchanged during liver starvation (1: 10). Changes in the nucleo-cytoplasmic ratio of the developing mouse liver and the regenerating rat liver were studied by Iversen and Thamsen (1956), and in the fetal sheep liver by Dick (1956b), both using the Chalkley method. Kaufman (1930), Siess and Stegmann (1950), and Helweg-Larsen (1952) carried out similar studies by other methods. All came to the conclusion that there are changes in the nucleo-cytoplasmic ratio during development. In their studies on mice, Siess and Stegmann (1950) stated, however, that the ratio becomes constant in the period after weaning. Contrary to these findings, Iversen and Thamsen (1956) showed that the nucleo-cytoplasmic ratio continuously decreases in the first months of life with a tendency to increase toward advanced age. I n the sheep embryo the turning point in the nucleo-cytoplasmic ratio was found at the 29-mm. stage and Dick (1956b) suggested that the change may be correlated to the appearance of glycogen in the liver at this stage. In conclusion it may be said that the nucleo-cytoplasmic ratio in hepatic cells undergoes alterations during growth. However, there is no answer to the question whether the nucleo-cytoplasmic ratio remains constant or changes when the cells reach a higher ploidy class.
G. BINUCLEAR LIVERCELLS Miinzer (1923) was one of the first to determine the percentage of binuclear hepatic cells in the liver. Each species has its typical percentage of binuclear cells which varies under different conditions. Pfuhl (1930) critized the method used by Miinzer (counting cells from histological slides) and proposed a correction formula, since some Of the binuclear cells were not recognizable in sections. Miiller (1937) elaborated Pfuhl’s formula. Bohm (1934) developed another technique for counting, using fixed preparations of groups of separated liver cells, and found that the results obtained by the two methods were corresponding. St. Aubin and Bucher (1952) used liver cell smears to study the development of binuclearity in the growing postnatal rat liver. They showed that the percentage of binuclearity is low in the fetal liver and increases rapidly during the first week of life to reach about 30% of the total hepatic cell population in the adult rat (Table V ) . The two nuclei of binuclear cells are of about the same size and have the same staining properties (Wilson and Leduc, 1948). The size of each pair of nuclei varies, as does the size of the nuclei of mononuclear cells, according to Jacoby’s nuclear classes. Both nuclei divide simultaneously as in multinuclear cells in general.
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TABLE V DEVELOPMENT OF BINUCLEARITY IN THE RATLIVER Ane Percentage of binuclear cellsa Embryo 7 & 0.2 4 weeks 47 f 1.6 5 weeks 58 f 1.0 6-7 months 31 & 0.4 2 years 35 & 0.4 a Counted in Wistar rats. The Harvard strain has a lower percentage of binuclearity. ~~
~~~
The origin of binuclear cells is discussed by Wilson and Leduc (1948). In agreement with Beams and King (1942) they conclude that binuclear cells arise from mononuclear cells with the suppression of the cytoplasmic division. The further suggest that as the growing liver develops, there is an increasing suppression of mitotic activity: “The first step is a delay, and finally the failure of cytoplasmic division resulting in binuclearity. The next step is the failure of the spindle to form, so that the prometaphase proceeds to telephase without division of cytoplasm. Finally, in endomitosis, the chromosomes may form and double without breakdown of the nuclear membrane.”
H. CHANGES I N CHEMICAL COMPOSITION It is difficult to evaluate the scarce and varied information on the chemistry of liver growth, since different methods have been used to express the results. Usually, only one or two of the constituents have been analyzed, and no over-all picture could be obtained. The recent method to express results of tissue analysis in relation to the DNA content of the nucleus is obviously the most advantageous and is now widely accepted. This reference method, however, is complicated in the liver by the existence of polyploid cells. Therefore, Harrison (1953a) suggested that the reference base line be the amount of DNA in a typically diploid nucleus, instead of the content of DNA in an average liver cell. This suggestion was based on the assumption that the nucleo-cytoplasmic ratio is constant in the various polyploid cell classes, and that the cells represent merely doubIing of the basic diploid cell. The chemical composition of the liver cell as determined by Harrison is given in Table VI. Abercronibie and Harkness ( 1951) and Wilson ( 1958) stressed the point that the chemical characterization of the “mean” liver cell represents also the other, approximately 40% of nonhepatic cells contained in the liver. (The chemical data should therefore be taken with this reservation.)
-
230
F. DOLJANSKI
TABLE VI COMPOSITION OF RATLIVER CELLS A . Average Composition of the Diploid Liver Cell of Adult Rats Substance pg.) Water Protein (10-4 pg.) Glycogen pg.) Neutral lipids (10-4 pg.) Phospholipids (10-4 pg.) RNA (10-4 pg.1 DNA (10-4 pg.) Potassium (10-9 pg.) Iron (10-7 pg.) Copper (10-8 pg.) Zinc (10-7 pg.)
Male 12.5 4.35 1.17 0.41 0.76 0.26 0.06 2.16 3.5 1.1 1.1
Female 10.3 3.56 0.87 0.13 0.59 0.24 0.06 1.87 9.6 1.2 0.8
B. Percentage Composition of the Adult Rat Liver Cell Substance Water Protein Glycogen Neutral lipids Phospholipids RNA DNA
Male (%) 61.5 21.6 5.8 2.0 3.8 1.3 0.3
Female (%) 63.6 21.9 5.4 0.8 3.8 1.3 0.4
1. Water The percentage of water in the liver is low when compared with that in other visceral organs (Lowrey, 1913). Nevertheless, water and proteins make up about 80% of liver weight and are mainly responsible for weight changes during growth. Changes in water content during fetal liver growth in rats have been described by Dumm (1943), and in guinea pigs by Flexner and Flexner (1950), who distinguished between extraand intracellular water. Whereas in the guinea pig, in the fetal stage of liver growth water concentration decreases, intracellular water remains constant. The same was found in the rat, except for an increase in the short period between the 19th and 21st days of gestation when glycogen appeared in the liver. During postnatal liver growth in the rat, water concentration per unit liver weight continues to decrease, reaching the adult value at 4 weeks (Givol, 1957) ; however, no determination is available regarding the intracellular water changes for this period. Lowry et d. (1946) found no appreciable changes in the water content of the hepatic cell at various ages, including old age (rat), and concluded thjit a given cell type, regardless
GROWTH OF LIVER WITH REFERENCE TO MAMMALS
231
of age, has a composition with respect to certain of its major constituents, which is restricted within narrow limits. Needham (1931b) suggested that the decrease in water concentration during organ growth is the result of a decrease in the amount of primitive connective tissue and lymph, occuring at this time. A decrease in the amount of hemopoietic tissue in the liver during growth could also affeot water concentration in the embryonic liver. 2. Changes in D N A , R N A , and Proteins In the embryonic and the postnatal liver the chemical constituents increase in amount (per organ) with the increase in liver weight, at either corresponding or differing rates. In the embryonic chick liver the DNA synthesis proceeds in waves of alternating high and low rates; the peaks in the rate at which new cells are produced are delayed by 1 day in relation to those of DNA synthesis. The DNA concentration decreases during embryonic development in the chick (Weston, 1956), and in the rat (Geschwind and Li, 1949). The diminution in the vascular spaces (filled with nucleated erythrocytes in the chick) and the involution of the hemopoietic islands (in mammals) are probably the main causes of this decrase. The changes in DNA, RNA, and protein during early postnatal rat liver development are described by Fukuda and Shibatani (1953) in a study in which DNA, RNA, and protein were determined in the whole organ, in their concentration per unit weight, and in their amount per cell (Table VII) . If these recent results are combined with those of Naora (1957), who used the spectrophotometric measurements of nuclear DNA content, then several stages in the biochemical development of the liver can be visualized (Fig. 3). It appears likely that in the suckling rat the liver cells are diploid with a high nucleo-cytoplasmic ratio (assumed from the small amount of protein per cell). With the beginning of weaning (21 days) the cell protein content increases, and the nucleo-cytoplasmic ratio decreases. Some tetraploid cells start to form, but their number is too small to contribute to a change in the chemical composition. Toward the end of the weaning period more and more polyploid cells appear, reaching on the 31st day about 50% of the total hepatic cell population. In this period, characterized by a fall in the nudeo-cytoplasmic ratio, the RNA content of the hepatic cells increases, reaching the high value of the adult liver cell. Simultaneously polyploidization occurs. It would be interesting to investigate the possibility of interrelation between these two phenomena. In the adult, slowly growing rat, the total DNA content of liver can
TABLE VII DNA, RNA, PROTEIN, AND CELLNUMBER OF RATLIVERTISSUE IN POSTNATAL GROWTH@
'
Age days
10 21 31 41
weight (g.)
~i~~~ cell weight number (g.) X 10-6
Quantity per liver (W.1 DNA-P RNA-P P N
Concentration (mg./lOO g. liver) DNA-P RNA-P P N
12 0.3 168 0.098 0.17 4.95 33.3 25 0.98 445 0.2'23 0.50 19.4 23.6 50 2.6 668 0.614 234 52.1 23.6 100 5.7 1060 1.17 4.84 112.0 20.7 80 200 8.1 1270 1.44 6.65 195 17.7 182 340 12.0 1790 2.02 9.21 276 16.8 b6.l 2.5 7.0 9.4 9.4 5.7 11.0 8.9 a According to Fukuda and Shibatani. 1953. For male rats. b Variationcoefficient (standard deviation calculated in per cent of mean). * RNA-P/DNA-P ratio.
58.4 52.2 85.4 85.3 81.5 77.5 5.3
1.67 l.% 2.01 1.98 2.41 2.31 8.0
Quantity per cell
(w.1 DNA-P RNA-P
5.9 5.09 9.27 11.1 11.4 11.4 2.4
10.4 11.6 33.5 46.7 52.5 52.5 9.3
PN
29.3 45.6 78.0 103 154 155 13.2
RNA-P* DNA-P
1 1.76 3.62 4.21 4.59 4.62 11.0
U 0
5 u1
2!
GROWTH OF LIVER WITH REFERENCE TO MAMMALS
233
be predicted for each age group from body weight (Campbell and Kosterlitz, 1950). 3. Glycogen The beginning of glycogenic function in embryonic growth is reviewed by Needham (1931~).In all mammals, glycogen synthesis starts approximately in the last third of the gestation period, at which time an inter-
m m aa
:
DNA
I
RNA
0
PRCrmN
10 DAYS
31 DAYS
I
I
I
1
80 DAYS FIG.3. The postnatal development of the liver cell. (Based on data of Fukuda and Shibatani, 1953, and Naora, 1957.)
change of glycogenic function with the placenta takes place. The liver begins to store glycogen, its concentration reaching high levels (6-11 g./100 g. liver) at term (Corey, 1935; Dumm, 1943; Weston, 1956; Givol, 1957). In the rat glycogen is used up during the first days of life, its concentration falling to almost zero. A few days later, normal concentration is being resumed (Givol, 1957). I n the adult rat liver the ratio
234
F. DOLJANSKI
of glycogen per cell is higher in the male than in the female (Campbell and Kosterlitz, 1950 ; Harrison, 1953a).
4. Lipids The embryonic liver stores lipids, their concentration increasing to high values toward term, as seen in the guinea pig (Imrie and Graham, 1920; Hard et al., 1944) and in the mouse (Deane, 1944a). In the first few hours after birth the carbohydrate store is depleted. For some days to follow, the large store of fat is used, until 1-2 weeks later the adult liver lipid concentration is reached. In conclusion it may be said that the morphological development and differentiation are accompanied by a chemical development of the hepatic cell, and that at a certain stage (about the age of 50 days in rat) the cell reaches its “chemical maturation” (Moulton, 1923). 111. Liver Growth Response to Various Conditions in the Body
Apart from its developmental growth, the liver increases in mass, in response to a number of processes occurring in the body. Various conditions can lead to the growth of liver above its characteristic adult level. This type of growth coulld be termed “additive” growth, to be understood in terms of increase in either cell number, and/or total organ DNA, and/or total organ protein content. A. THEEFFECT OF PREGNANCY AND LACTATION ON LIVER SIZE Pregnancy induces an increase in relative liver weight and liver protein content in rats (Boikelmann and Sheringer, 1932) accompanied by an increase in the nucleoprotein concentration (Davidson and Waymouth, 1944). Campbell et al. (1953) showed that during the second week of pregnancy in rats and mice, but not in guinea pigs, there is a lO-lS% increase in the total DNA content of the liver, an increase in the total RNA content and concentration, and a decrease in glycogen concentration. During the second and third week of laotation, the liver increases both in respect to its total DNA and protein content. The effect is probably due to greater food consumption rather than to any endocrine changes, specific to lactation (Kennedy et al., 1958). OF TUMORS B. THEEFFECT
In the presence of tumors there is an increase in the ratio of liver weight to body weight or to body length (Yeakel, 1948; Kelly and Jones, 1950; Annau et d.,1951; Begg et d.,1953; Rodriguez and Cerecedo, 1955). Increase in liver weight is accompanied by proliferation of liver cells (Annau et d., 1951) and an increased turnover of liver DNA in the
GROWTH OF LIVER WITH REFERENCE TO MAMMALS
235
presence of a neoplasm (Kelly and Jones, 1950). There is no correlation between the size of the t u m r and the rate of increase in liver size. It is suggested that the liver enlargement is associated with the protein anabolism in the growing tumor (Yeakel, 1948). Wolf and Klemperer (1955) have shown that the weight and volume of liver cells in leukemic patients is considerably increased: 25.1 mg. X per cell in leukemic patients in persons not affected by the disease. as against 10.8 mg. X C. THEEFFECT OF DIET Liver weight and composition are very sensitive to changes in the amount and composition of food. At starvation, whereas all liver constituents decrease at variable rates, the total DNA content, i.e., the total number of liver cells, does not change, at least for 1 week (Harrison, 195313). Resumption of feeding after starvation or a restricted diet will induce mitosis in the hepatic cells in mice. Liver weight was not examined (Leduc, 1949). The mitotic activity after refeeding is much higher in young than in adult mice. A high protein diet elicits an increase in liver proteins (Walter and Addis, 1939) and a 37% increase in rat liver weight. The initiation of mitosis in liver cells by intraperitoneal injection of pulped liver, kidney, or egg yolk (Wilson and Leduc, 1947) may be of the same nature. It appears that food and/or protein supply result in increased liver weight and liver cell mitosis. D. THEEFFECT OF VARIOUS SUBSTANCES Various substances injected into the body induce liver cell proliferation and growth. These substances can be divided into two groups : (1) those which cause damage to the liver and a mitotic response d the reparative type (as e.g., CC14-Tsuboi et al., 1951) and (2) those which cause no visible morphological damage to the liver. The response of the liver after injury is reviewed by Himsworth ( 1954). The second group includes substances such as thiourea (Doljanski et al., 1956) ; mramine (Wilson and Leduc, 1950) ; large doses of thyroxine (Walter and Addis, 1939; Sternheimer, 1939) or thyroid powder (Guggenheim et al., 1958) ; trypan blue (Deane, 1944b) ; liver extracts (Tier and Ravanti, 1953); and others. Injection of large doses of thiourea, for example, elicits a 30% increase in the relative liver weight accompanied by multiplication of liver cells, increased RNA concentration, and loss of glycogen. The total amount of DNA is increased proportionally to liver weight. No visible morphological damage can be detected, and all effects are reversible. Feeding mramine to mice for prolonged periods causes liver enlargement and mitosis. Thyroxine administration
236
F. DOLJANSKI
in pharmacological doses induces liver growth, mitosis, and decrease in glycogen concentration, and the ef€ect is obtained before the initial rise in oxygen consumption. The effect of these substances on the liver could mean a restorative response to a damage as yet invisible. This, however, is unlikely as there is a liver weight increase above the characteristic adult level. A more plausible explanation is that these substances involve increased liver activity (detoxification).
IV. Liver Regeneration after Partial Hepatectomy Since growth of the liver after partial hepatectorny was recently reviewed by Harkness (1957), the present discussion will be restricted to the comparison between restorative and the developmental and “additive” growth. As in the latter two types of growth, so in liver restoration two phases of growth can be differentiated : (1) the acute phase where cell multiplication is the main factor, and (2) the period of s!ower growth where cell enlargement (probably the result of polyploidization) mainly contributes to liver growth (Beams and King, 1942; Yokoyama et al., 1953; Thompson and Frazer, 1954 ; LaquerriPre, 1957). The restoratively growing liver is characterized by a low percentage of binuclear cells (St. Aubin and Bucher, 1952; Sulkin, 1943), typical also of the embryonic liver. The early changes in composition of the regenerating liver are characterized by an increase in RNA and lipid concentration, and a decrease in glycogen and protein concentration (rat). Some of these features are also observed in the young postnatal liver as well as in the liver enlarged during pregnancy, and in the liver after thiourea treatment. I n determining the typical changes involved in the various growth responses, it is difficult to decide whether they are specifically associated with the mult‘plication of hepatic cells, or whether they result from liver function influenced by altered metabolic conditions.
V. Concluding Remarks and Summary The foregoing review has been made in an endeavor to describe and compare developmental, restorative, and “additive” liver growth, the latter meaning a controlled increase in liver mass above its normal adult level. During development the liver shows a “negative specific acceleration” with possibly some short periods of positive acceleration, e.g., in the weaning period. The latter, of crucial importance in liver differentiation, is associated with increased mitotic activity and the initiation of polyploidization of the
GROWTH O F LIVER WITH REFERENCE TO MAMMALS
237
hepatic cells. Further studies of this particular period might throw more light on the factors concerned with this organ’s differentiation. Polyploidization of liver cells at a particular stage of development is dependent on the presence of growth hormone and/or adequate fmd supply. The physiological significance of this process is not clear. A polyploid celI differs from a diploid one not merely quantitatively by the multiplication of its constituents, but also by the change in ratio between nucleus and cytoplasm. If it is assumed that the nucleo-cytoplasmic volume ratio is constant in the higher grades of ploidy cells, then the ratio of nuclear surface to cytoplasmic surface must change, or vice versa. [Mass is roughly proportional to volume ( 13), and not to surface area ( lz) .] In either case the nucleo-cytoplasmic relation is altered. Besides these quantitative variations, differences in the physiological function of cell-readiness to divide should be considered. Polyploid cells divide less readily than diploid ones (Marquardt and Glass, 1957). As polyploidization occurs at a certain stage of development, it could be suggested that in the liver a polyploid cell represents a further step in differentiation, and becomes functionally a more efficient cell. It is also possible that the liver is progressing toward a general type of cellular differentiation in organs, in which there is a separation into proliferating stem cells and cells with specific functions. Polyploidization occurring at the period of increased rate of body growth (in mice and rats at weaning, and in man at puberty) could be a means to meet an increased demand on the liver, which in turn would initiate increased efficiency. In most animals the adult liver constitutes about 2 to 4% of their body weight. It is relatively larger in carnivores and smaller in herbivores. In omnivores it is of intermediate size (Popper and Schaffner, 1957). Adult liver grows in response to various conditions in the body. Its relative weight can increase by 10-30oJo.This figure indicates the range of physiological flexibility in the growth of the organ. It also indicates that, for certain functions at least, there are no liver reserves. In comparing the different liver growth processes several common features come to lighlt. Two phases can be recognized, the first of a high growth rate achieved mainly by cell multiplication, the second phase of slower growth, by polyploidization. The actively growing liver has a low percentage of binuclear cells, their number increasing when growth rate declines. During active growth there is an increase in RNA and a decrease in glycogen concentration, and at times (during developmental and restorative growth) and increased lipid concentration. There is no change in the intracellular water. Dietary changes greatly influence liver weight and composition in the adult, but have no effect on the total cell number.
238
F. DOLJANSKI
One of the major problems of growth and restoration is the mechanism by which characteristic organ size is maintained. A factor in the serumeither a stimulant or an inhibitor-which might control liver growth is indicated in several works dealing with liver regeneration. Experiments undertaken to investigate such possibilities by injection of blood or serum from animals after partial hepatectomy showed that there is a humoral factor which initiates growth and mitotic activity in the liver (Friedrich and Zaki, 1954; Stich and Florian, 1958; Orlando, 1957; Adibis et al., (1958) ; Smythe and Moore, 1959). However Glinos (1958) and Kohn (1958) who carried out experiments on similar lines could not confirm these Observations. Experiments carried out by Christensen and Jacobsen, 1949; Bucher et al., 1951 ; Wenneker and Sussman, 1951 ; Orlando et al., 1957, with the aid of parabiosis also indicated the existence of a humoral factor. On the other hand, these results could not be confirmed by Islami et al. (1959) who found no effect of the regenerating partner on the normal one, conversely in inhibiting effect of the latter on the former. The discrepancies in the above-mentioned experiments might be due to the use of the mitotic index as a criterion for growth stimulation or inhibition. The mitotic index without knowledge of mitotic time is of limited value for determination of growth. The determination of changes in the total hepatic cell population (by cell count and/or DNA determination) would constitute a far better mean to obtain clear-cut results. In general it can be stated that most authors agree that there is a humoral factoc involved in the regulation of liver growth. This factor could be either a stimulant, a-an inhibitor. The latter possibility has been advocated by Glinos, who suggested on the basis of a large number of experiments that the inhibiting factor could be plasma proteins (Glinos, 1958). If it is assumed that the plasma proteins synthesized by the liver are used by various tissues, then liver size is regulated by the requirement for body proteins. This assumption tallies with the hypothesis of Dick (1956a), according to which the rate of body growth determines the total number of hepatic cells during developmental growth of the liver. It is further supported by the observations on increase in liver size during pregnancy and during tumor growth. On the other hand, liver also grows in response to an increased food supply. It could thus be concluded that liver size, within its genetic limits, is determined by a balance between the demands of the body for its products, and the amount and composition of nutritive supplies the liver has to metabolize.
Acknowledgments I wish to extend my thanks to Dr. M. Abercrombie, Dr. A. T. Dick, and Professor J. Gross for their constructive criticism of this paper, and Mrs. J. Meron for her editorial work.
GROWTH OF LIVER WITH REFERENCE TO MAMMALS
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158. Kingsbury, J. W., Alexanderson, M., and Kornstein, E. S.
(1956) Anat. Record 124, 165. Kohn, R. (1958) Exptl. Cell Research 14,228. Laquerriere, R. (1957) Nature 180, 1199. Latimer, H. B. (1931) Scritti biol. 9,313. . Latimer, H. B., and Cordet, R. L. (1948) Growfh 12,285. Leblond, C. P., and Walker, B. E. (1956) Physiol. Revs. 86, 255. Leduc, E.H. (1949) Am. J . Anut. 84,397. Leuchtenberger, C., Vendrely, R., and Vendrely, C. (1951) Proc. Natl. Acad. Sci. u. s. 57, 33. Lison, L., and Valeri, V. (1958) Acta Hisfochem. (Jena) 6, 337. Lowrey, L. G. (1911) Am. J. Anat. 12, 107. Lowrey, L. G. (1913) Anut. Record 7, 143. Lowry, 0.H., Hastings, A. B., McCay, C. M., and Brown, A. N. (1946) 1. Gerontol. 1, 345. McKel!ar, M. (1949) Am. I . Anat. 86,263. Marck, E. (1943) 2. Zellforsch. mikroskop. Anat. Sa, 557. Marquardt, H., and Glass, E. (1957) Chromosoma 8,617. Mortreuil-Langlais, M. (1958) Compt. rend. soc. biol. 162, 1485. Moulton, C. R. (1923) J . Biol. Chem. 67,74. Miiller, H. G. (1937) 2. mikroskop. onut. Forsch. 41,294. Miinzer, F. T. (1923) Arch. mikroskop. Anat. u. Entuicklungsmech. 98,249. Naora, H. (1957) J. Biophgs. B i d e m . Cjttol. 3, 949.
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Cytological Studies on the Affinity of the Carcinogenic Azo Dyes for Cytoplasmic Components YOSHIMI NAGATANI
Biological Institute, Faculty o f Literature and Science, Yariiaguchi Uriiversity, Yamagucki, Japan Page 243
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I. Introduction . . .. .. .. . . . .-......... . .. . . . .. . . . . . . . .. . . . . . .. .. . .. . 11. Methods for Demonstrating the Cellular Affinity of Carcinogenic Azo Dyes for Cytoplasmic Components . . .. . ... . . .. . . . . . . . . . . . . . . .. . . . A. Procedure for Fixed Preparation ....... .. . . . . . . .. . . .. .. . . . . .. B. Procedure for Vital Staining Preparation . . . . . . . . . . . . . . . . . .. . 111. Affinity of the Carcinogens for Cytoplasmic Components of Amphibian Erythrocytes . . .. . . .. .. .. . ... .. . . .. . ... . . .. . . ... .. . . . . . . . . . . . .. . . A. Classification of Cytoplasmic Granules in Amphibian Erythrocytes B. The Golgi Body of Amphibian Erythrocytes ... , . ... . . . ... .. .. C. Mitochondria of Amphibian Erythrocytes . . . . . .. . . . . . . . . .. . . . .. IV. Mitochondria of Mammalian Differentiated Erythrocytes . . . . . . . . . . . V. Affinity of Carcinogens for Amphibian Somatic Cells .. . .. . . .. . .. .. . A. General Architecture of the Golgi Apparatus Demonstrated by Silver Impregnation Method in Amphibian Somatic Cells .. . . B. Pathological Increase of Argentophil Substance . . .. . .. . . . . . . . C. Two Kinds of Argentophil Substances of Different Quality ... D. Two Kinds of Argentophil Substances of Different Quantity ... E. The Golgi Apparatus with Electron Miscroscopy .. . . . . . . . . . . . . F. General Architecture of Mitochondria .. ... . .... . . . .. . . . . . . . . . G. Stainability of Cellular Components with OYG Method . . . . . . . . H. Staining Mechanism of OYG Method . .. . . . . ... ... . .. . .. . . . . . . . I. Estimation of Hydrochloric Acid in the OYG Method . . . . . ,. . J. Chemical Natures of Carcinogens . .. . . .. . . , .. .. . .. . . . . . .. .. . . VI. Conclusion .. ........... . . .. ... .... . .. . ... ..... ...... . .. . . .. .. .. .. Acknowledgments ........ .................................... .... References . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . .
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244 245 245 246 246 249 249 255 257 258 269 270 272 275 277 288 292 2% 299 305 306 306
I. Introduction Since it was discovered by Yoshida (1932)) Sasaki and Yoshida (1935), and Kinoshita ( 1937) that o-aminoazotoluene (AAT) and p-dimethylaniinoazobeiizene (DAB) cause hepatoma in the rat, these hepatic carcinogens have been studied by several groups of workers in order to discover the reason why the cancer occurs. For this purpose, it is necessary to make clear the affinity of these carcinogens for the cytoplasmic components. Masayama and Sat0 (1947), Miller and Miller (1947)) and Fukuoka and Nakahara (1953) established a method to visualize DAB changed red by the acid in the liver. Miller and Miller (1952) proposed the so-called “protein deletion)’ theory as the etiology of certain cancers 243
244
YOSIIIMI NAGATANI
from the fact that the liver of rat fed the hepatic Carcinogen DAB contains amino azo dyes bound tightly to a cellular protein (Miller and Miller, 1947). I n addition, in regard to the binding protein, the carcinogen was found in the “h” fraction or in tyrosine by Sorof et al. (1951, 1958), Wirtz and Arcos (1958), Kusama and Terayama (1957, 1958), and Terayama et al. (1958a, b). However, it seems that these observations have not been extended beyond determining the affinity of the carcinogen for the liver. I t is equally important to determine where the carcinogens become localized within the cells. For this purpose, Motomura (1947) first established the OYG method, by which the carcinogens within the cells were changed to dark red in locust germ cells and sea urchin eggs, for the dye in the cells is yellowish and is not suitable for microscopic observation ; then he observed that the mitochondria and granules perhaps corresponding to the Golgi bodies were stained with AAT and DAB. Using his method, the author has studied chiefly amphibian cells (Nagatani, 1948, 1949, 1950, 1951, 1952, 1953, 1954a, b, c, 1955, 1956a, b, c, 1957a, b, 1958a, b c, 1959a, b ; Nagatani and Nakao, 1957). These investigations were undertaken in order to make clear which cytoplasmic components are stained with the carcinogens, and why the staining occurs. 11. Methods for Demonstrating the Cellular Affinity of Carcinogenic
Azo Dyes for Cytoplasmic Components Although it is necessary for the present studies to employ a species in which tumors are induced by carcinogens, it is more important to make clear what cytoplasmic components have an affinity for the azo dyes. Amphibian cells, therefore, were employed in the present study, because they are stainable with the carcinogenic azo dyes and are an especially valuable form for cytological studies, since they are much larger than those of most other species. These facts have made it possible in the present study to work out the topography of the cytoplasmic components much more completely than has been possible with mammalian cells. Triturus pyrrhognster (Boie) was mainly used, and several species of the frog, Rana japonica, R. nigromaculata, R . rugosa, Bufo vtrlgaris, Rhacophorus schlegeli, and other species, were also employed for the purpose of comparison. The carcinogenic azo dyes used by the author are o-aniinoazotoluene and p-dimethylaminoazobenzene. Since oil yellow C and oil yellow J are other names for the former and oil yellow D and oil yellow F N are also other names for the latter, the special granules stained with these dyes were tentatively named oil yellow granules (OYG), and this method was also named the “OYG Method” by Motomura (Nagatani, 1954a).
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This method is based upon the discovery that these carcinogens were changed to dark red with Mayer’s reagent, which is well known as an alkaloid reagent. The original OYG method for carcinogens (in which materials were previously fixed with acidified Mayer’s reagent) has been improved by the author by changing the fixation as follows :
SPECIAL REAGENT : Acidified Mayer’s reagent. Mayer’s reagent containing 1% hydrochloric acid by volume. Azo dyes. o-Aminoazotoluene or p-dimethylaminoazobenzene saturated in 1% hydrochloric acid.
PROCEDURE : FOR FIXED PREPARATION A. PROCEDURE 1 . Smeared Preparation for Hemocytes The smears are thoroughly air-dried for 24 hours before further fisation. The smears are stained with the azo dyes for 12-24 hours after fixation with formalin vapor, or Susa’s or Da Fano’s fluid. After rinsing for a short time, a small amount of acidified Mayer’s reagent is placed on the stained preparation and a cover glass is immediately dropped on it. The preparations may now be examined. The positive staining is dark red or reddish violet. 2. Sectioned Preparation a. Carbowax-Section Method. The materials are fixed in Susa’s or Da Fano’s fluid for about 6 hours. After rinsing for about 3 hours, the materials are transferred first to Carbowax 1500 without dehydration, next to the mixture of equal parts of Carbowax 1500 and 4o00, and finally to Carbowax 4000, at 55°C. for about 1 0 minutes each. Sudden cooling is preferable in making the wax block. The sections of 2 to 4 p thickness are floated on distilled water, and the Carbowax is removed from the section by changing the distilled water at least five times. These sections are then treated as for the smeared preparation. b. Parafin-Section Method. The paraffin-section processed by routine methods are stained as above. B. PROCEDURE FOR VITAL STAINING PREPARATION 1. Smeared Preparation for Hemocytes The blood cells of the animals which are stained vitally with the carcinogens are smeared as in routine methods. After drying for a short time, a small amount of acidified &layer’s reagent is placed on the slide without further fixation and a cover glass is immediately dropped on it.
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YOSHIMI NAGATANI
2. Sectioned Preparation a. Carbowax-Section Method. The tissues stained vitally with the carcinogens are fixed in Swats or Da Fano’s fluid for about 6 hours. The sections treated with the Carbowax method are mounted in acidified Mayer’s reagent after the Carbowax is completely removed from the sections by changing the distilled water. b. Parafin-Section Method. The fixed tissues treated as above are sectioned by routine alcohol-xylene method. The sections which are brought down to water are mounted with acidified Mayer’s reagent. Various cytochemical methods and the classic stainings were employed in order to make clear what cellular constituents are stained with these carcinogens. 111. Affinity of the Carcinogens for Cytoplasmic Components of
Amphibian Erythrocytes A. CLASSIFICATION OF CYTOPLASMIC GRANULESI N AMPHIBIAN ERYTHROCYTES By using the phase contrast microscope and various staining methods, two kinds of granules are recognized in newt erythrocytes: one kind is coarse granules, measuring 1 to 2 p, the other is rod-shaped granules, measuring 0.3-0.4 x 0.5 p. The former are observed as refracting granules with the phase contrast microscope in the fresh state (Fig. l ) , and as dark violet granules with Susa-Giemsa method (Nagatani, 1957b). They are easily stained with various basic dyes and appear first in the cells after vital or supravital staining with neutral red (Fig. 2) ; therefore, they were named tentatively “basophil granules” (Nagatani, 1958b). From their shape and staining reactions, they appear to be identical with the Chvonzatoide Kiigelchen described by Meves ( 1911), the segregation apparatus by Dawson (1928, 1930, 1933), the stigma by Nittis (1930), and B-granula by Yasuzumi et al. (1934). However, in the larval stage, these are distributed throughout the cytoplasm, and in the mature erythrocytes of the adult some of them show a tendency to appear close to the nucleus, and therefore two kinds of the basophil granules are recognized from their distribution : dispersing basophil granules (D-BG) and clustered basophil granules (C-BG). C-BG were observed in 14 individuals of 54 adults, and their appearance was independent of the sex (Nagatani, 1958b). The latter are also classified into two kinds of granules on the basis of their distribution and shape. Although they are not recognizable with the usual methods, they are specifically stained with the OYG method (Figs. 4,s). These granules in the orthochromatic erythrocytes of adult newts are faintly observable with the phase contrast microscope even in
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247
the living state. But they are more clearly recognizable with the phase contrast microscope when the preparations are treated with either hydrochloric acid (Fig. 6) or acidified Mayer’s reagent (Fig. 7) after fixation with formalin vapor only (Fig. 8). In the preparation, however, which is treated with the OYG method, they stained reddish violet and are clearly recognized in the cytoplasm, which is stained pink. They consist of two kinds: one is a granular form distributed throughout the cytoplasm, and the other is filamentous, forming chains distributed at about the central part of the cytoplasm and apparently giving rise to more or less extensive
FIG. 1. Phase contrast photomicrograph of fresh erythrocyte of the newt. ( X 300, enlarged approximately 3.5 x .) (From Nagatani, 19586.) FIG.2. Photomicrograph of erythrocytes of the newt stained vitally with neutral red, showing basophil granules and neutral red spheres. (x 300, enlarged approximately 3.5 x.) FIG.3. Photomicrograph of erythrocytes of the newt stained vitally with neutral red, showing the increase in the apparent size of the neutral red spheres. (x 273, enlarged approximately 2.4 x.)
networks. Since it has been found that they undergo morphological changes coincidentally with the appearance of hemoglobin, they also seem to be the preexisting granules. They were named tentatively “oil yellow granules” (OYG) by Motomura (Nagatani, 1954a). Accordingly two kinds of the OYG, the filamentous OYG (F-OYG) and the granular OYG (G-OYG) , are recognized in mature erythrocytes. Under examination with vital and supravital stainings, a large number of globules and spheres are conspicuously observed in the cytoplasm besides the basophil granules and the oil yellow granules (Fig. 2). They have a tendency to increase in apparent size as they are stained more and
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YOSHIMI NAGATANI
more heavily (Fig. 3 ) , while this is not detected with various cytochemical methods. Those stained vitally disappear after treatment with SusaGiemsa’s method; this is in contrast to the situation with the basophil granules. The OYG do not normally stain with Susa-Giemsa’s method, but after the nucelus is stained, they break up into short chains and disintegrate into granules, and then they frequently become stained. The reticular structures appear after the nucleus is stained and the OYG have
FIG.4. Photomicrograph of erythrocytes of the newt showing typical filamentous mitochondria. They were stained with the carcinogen after formalin vapor fixation and treatment with acidified Mayer’s reagent. ( x 420, enlarged approximately 2.4 x .) FIG.5. Photomicrographs of erythrocytes of the newt stained with the carcinogen after Da Fano’s fixation; a, basophil cells where mitochondria are grouped in the perinuclear region ; b, polychromatic cells ; c, orthochromatic cells showing typical filamentous mitochondria. ( x 390, enlarged approximately 2.4 x.)
disintegrated. From the fact that these structures are not detected with any cytochemical methods, it is supposed that they are caused by the degeneration of the cytoplasm. They may be identical with the Golgi apparatus described by Sinigaglia ( 1910), OberfEuchmnetz by Meves ( 1911) , the Golgi apparatus by Avel (1924), the reticular basophilia by Nigrelli (19B), the surface structure by Nittis (1930), and the internal reticulation by Shinagawa (1955) in their shapes and distributions. However, they are not the preexisting granules but are the granules formed by the
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action of the dye ; therefore, they were named “neutral red spheres” and “neutral red reticula” respectively. B. THEGOLGIBODYOF AMPHIBIANERYTHROCYTES The basophil granules are easily stained with the OYG method, and they seem to be rich in RNA and in shape resemble the granules observed with the ultraviolet microscope by Koga (1940) and Yamada (1953) as prominent granules in newt erythrocytes. They have argentophil and osniiophil natures, and are detected with vitamin C (Sosa, 1948), alkaline phosphatase (Takeuchi, 1943), organic phosphate ( Serra and Lopes, 1945), and calcium salt tests (CrCtin, 1924), while they are negative to the cytochromoxidase test by Graff and LoelCs method and the succinic dehydrogenase test by Semenoff (1935), as shown in Table I. They are also positive to phospholipid tests, and seem to consist mainly of sphingomyelin. On the basis of the chemical properties and shapes mentioned above, it seems to be reasonable to suggest that these granules may be identical with the Golgi bodies described by Worley (1943, 1951), Baker (1949), Montagna (1950, 1951), Hill and Bourne (1954), and Zeros (1951), although their properties and shapes differ from the so-called Golgi apparatus in the somatic cells. Pollister (1939) pointed out that the Golgi apparatus was not determined in amphibian erythrocytes ; the Golgi apparatus, however, shown by Dawson (1928, 1930, 1933), De Roo and Uffoed (1930), and Takagi (1936) is strikingly similar to the basophi1 granules. The Golgi apparatus, however, noted by Avel (1924), Bhattacharya and Brambell (1925), and Jordan (1925) seems to include the neutral red spheres and other artificial components besides the basophil granules ; on the other hand, the Golgi apparatus shown by Kurashige (193Oa, b) may be the oil yellow granules. From these facts it is assumed that the basophil granules are the special granules containing the Golgi substances. The Golgi substance will be discussed in the following section of this chapter. Accordingly if the basophil granules are the Golgi bodies, it is true that the carcinogens have an affinity for the Golgi bodies, since the basophil granules are not only stained with the OYG method after smearing, but also are detectable with vital staining for carcinogens, although, according to h4iller and Miller (1947), no bound dye was found in rat tissues such as erythrocytes in which DAB does not induce tumors. OF AMPHIBIAN ERYTHROCYTES C. MITOCHONDRIA
The reason why the patterns of the special granules stained with the OYG method were tentatively named the oil yellow granules was that they closely resemble the Chondriokonten described by Meves (1911) and
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YOSHINI NAGATANI
TABLE I REACTIONS OF CYTOPLASMIC GRANULES TO STAINING AND CYTOCHEIIICAL TESTSIN AMPHIBIANERYTHROCYTES” Cytoplasmic granules* Cytochemical staining
NRS NRR DBG CBG GOYG FOYG
Phase contrast microscopy Fresh material Formalin vapor fixation Previous treatment with 1N HCI Vital staining Neutral red Subsequent fixation with formalin vapor Susa-Giemsa method R N A detection Toluidine blue, pH 7.4 7.0 6.4
5.8 Previously treated with 1N HC1 (20 min.) RNase (60 min.) RNase (120 min.) Hot water (120 min.) PAS reaction Neutral polysaccharide test Acid polysaccharide test Mitochondria staining Supravital staining with Janus green B H’s hematoxylin method Golgi apparatus demonstration Da Fano’s silver impregnation Mann-Kopsch’s osmium impregnation Lipid detection Baker’s acid hematein test Previously treated with pyridine test chloroform ether ethanol Digestion with trypsin Plasma1 test
+ + -
+ + -
+ + + + + +
+ + +
-
-
+
+ + +
+
+ + ++ ++
-
-
+ +
+ +
+ +
-
+ + + -
++
-
+ + ++
+ + +
+
+ + + + ++
-
+
++
+
+
+
+
+
+
+
+
+ + +
+
+ + +
+
+
+
From Nagatani (1958b). NRS, neutral red spheres; NRR, neutral red reticula; DBG, dispersing basophil granules ; CBG ; clustering basophil granules ; GOYG, granular oil yellow granules ; FOY G, filamentous oil yellow granules. b
25 1
CYTOLOGICAL STUDIES WITH CARCINOGENS
TABLE I ( c o n t i w e d ) Cytoplasmic granulesb Cytochemical staining OYG method Previously treated with ethanol acetone ether
Sudan black staining Chlorophyll staining
NRS NRR DBG CBG GOYG FOYG -
-
-
-
++ ++ -+ +& ++ ++
+
+
+
&
-
+
A
* -
Okamoto’s test (A-test) (Ba-test) (Bb-test) Protein detection Tyrosine test Arginine test Calcium phosphate test with alizarin red Alkaline phosphatase test Vitamin C demonstration Respiratory enzyme reaction Peroxidase reaction Cytochromoxidase reaction Dehydrogenaze reaction Supravital staining with tetrazolium
-
-
-
-
-
+ + - + + - + -+ + + + + + + + + + +
the Golgi apparatus by Kurashige (1930a, b), but differ from the mitochondria described by Dawson ( 1930) and Takagi ( 1936). Accordingly the term of oil yellow granules must be used until it becomes clear what the oil yellow granules are. With regard to the morphological changes of the OYG with the maturation of erythrocytes in the adult newts, the OYG in immature cells having a basophil nature are of a granular form or in the form of a short chain around the nucleus (Fig. 5a) and then they are easily recognized even in the living state. The OYG in immature cells having a polychromatic nature undergo progressive changes and become condensed at one side of the nucleus (Fig. 5b), and finally some of the granules develop into long filamentous OYG as shown in Figs. 4 and 5c (Nagatani, 1954a, 1958b). Similar changes are also observable in the differentiating erythrocytes of developing anura ( Nagatani, 1954b) : the primitive erythrocytes appearing in the external gill stage tend to become flattened oval forms developing from the spherical form with a decrease in their yolks, as observed by Freidsohn (1910), Goda (1929), Togari (1938), and Cameron (1941), and then the OYG dispersing throughout the cytoplasm
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Y OSH IM I NAGATAN I
(Fig. 9a, b) tend to gather around the nuclei (Fig. 9c, d ) . The traiisformation of the OYG in the erythrocytes at the internal gill stage is closely similar to the changes of the OYG of maturing erythrocytes of the adult newts. But the OYG at this stage become short filaments segmented into rods of approximately the same diameter (Fig. 9e, f, g). From the results observed in the changes of the OYG, it is possible to assume that all of the cells in the primitive line do not disintegrate nor are they replaced by the new cells in the definitive line, although some of them will be translated into the definitive line, contrary to Goda (1929) and Cameron (1941 ) ;
FIGS.6, 7, and 8. Phase contrast photomicrographs of newt erythrocytes showing the action of acid treatment for mitochondria. Fig. 6. Cells treated with hydrochloric acid after formalin vapor fixation, showing mitochondria. Fig. 7. Cells fixed with acidified Mayer’s reagent. Fig. 8. Cells fixed with formalin vapor alone showing no mitochondria. ( X 420, enlarged approximately 2.4 x.)
thus the erythrocytes of the tadpole stage will be led from at least two kinds of hemopoietic centers. The transformation of the OYG of the maturing erythrocytes at the lung stage is also similar to that of the tadpole stage, but the OYG of
FIG.9. Photomicrographs of the erythrocytes of developing amphibia, showing evolution of mitochondria ; a and d, primitive cells of Rhacophorus schlcgeli arborco; c, e, and f, primitive cells of R. scklegeli schlegeli; b, g, and h, primitive and definitive cells of Polypedates buergeri; i, definitive cells of Rana catesbiano; a and b, cells showing scattered granular mitochondria; c, d, e, and f, cells showing granular and short chainlike mitochondria grouped near the nuclei ; g and h, cells showing chainlike mitochondria ; i, cells showing filamentous mitochondria ; e and f, cells showing intermediate form between primitive and definitive lines. ( x 390, enlarged approximately 2.4 x.)
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254
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Y O S R I M I NAGATANI
the mature erythrocytes at this stage become long filaments forming chains ; on the other hand, the number of the granules forming the chains is less than that of the immature cells (Fig. 9h, i). From these facts, it seems to be characteristic that the transformation of the OYG is not a regressive change but is a change with a single peak. In other words, the polychromatic erythrocytes contain the largest amount of the OYG. Comparing these results and the observations by Cowdry (1917) in the bone marrow of the rabbit, Takagi (1931) and Dawson (1936) in the chick embryo, and Rojas and De Robertis (1936) in amphibia, it seems reasonable to suggest that the OYG may be mitochondria, although the shapes of the mitochondria observed with hematoxylin staining differ from those of the OYG (Nagatani, 1954a, b). However, from the transformation of the OYG in the developing amphibian erythrocytes, it is supposed that the OYG may have no relation to hemoglobin formation, contrary to the opinions of Cowdry (1924). From the fact that the OYG in the amphibian erythrocytes are not stained vitally with neutral red and in injured cells they frequently are stained (Nagatani, 1958b), the OYG seem to have the nature of the introduced granules of Dawson (1929) and the pre-Golgi S t h t of ~ ~ Hirsch (1939). These special granules in the amphibian erythrocytes become weakly stained with the OYG method under narcosis, and with increasing anesthesia the filamentous structures tend to break up into short chains and disintegrate further into granules of approximately the same diameter ; then these granules are closely similar to the granular OYG (Nagatani, 1951). From this fact, two characteristic properties of the OYG can be recognized. The first is the disintegration of the OYG, and the second is the diminution of the stainability. The disintegration of the OYG is similarly observed in erythrocytes of newts in which the abdominal cavity has been injected with ammonium chloride (Nagatani, 1958b) and is also observed in the erythrocytes containing the special bodies of the frog (Nagatani, 19%). On the basis of these facts, it is believed that the OYG have the nature of mitochondria examined by Scott (1916) in the pancreatic acinar cells of the mice and by Ostrouch (1932) in the gastric cells of the rat. It seems reasonable to conclude that the diminution of the stainability with the OYG method suggests that the OYG contain the lipids. In experiments using fat solvents, the OYG become negative with the OYG method after pyridine or acetone extraction, and are only weakly stained with the OYG method after ether extraction. In addition, the OYG react positively to other cytochemical methods for the phospholipids such as Baker’s
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method (1946), etc., as shown in Table I. By using Okamoto’s test (Okamoto et al. 1952), it is supposed that the lipid of the OYG may be lecithin. In addition, the OYG react positively to respiratory enzyme tests. From the observations of Baker (1946), Bloom and Wislocki (1950), Goddard and Seligman (1952), and Hogeboom and Schneider (1955), it seems to be reasonable to conclude that the OYG are mitochondria. Thus the mitochondria of the amphibian erythrocytes consist of at least two kinds of granules, for the results of the reactions for the respiratory enzymes vary between the F-OYG and the G-OYG, although the F-OYG are produced by the G-OYG. Kuff and Schneider (1954) and Paigen (1954) observed that all of the mitochondria in the same cell do not always show the same ferment reaction.
IV. Mitochondria of Mammalian Differentiated Erythrocytes Another reason why the author has hesitated to conclude that the special granules stained with the OYG method are mitochondria, is that in large numbers of the mammalian orthochromatic erythrocytes without any cytoplasmic inclusions such as basophil punctation or Howell-Jolly’s bodies, a small number of the granules were also recognized which resembled the OYG in the amphibian erythrocytes, as shown in Figs. 10, 11, and 12 (Nagatani, 1948), although the mitochondria are totally absent in the nonnucleated red blood cells of normal adult human blood, according to Cowdry (1915), Cowdry (1917), Bloom and Wislocki (1950), and Watanabe (1956). In the smeared preparations, by using the fuchsin S or iron-hematoxylin methods, these dyes are combined so tightly with the mammalian erythrocytes that the granular components become invisible in the cells and these dyes resist the decolorizing action of acid alcohol. On the other hand, by using Baker’s acid hematein test, the cells fixed with formalin vapor after smearing are unstained, while the cells in tissue sections are strongly stained with it, as observed by Baker 1946) and Bloom and Wislocki (1950), and then any granular component is not recognized in the cells with either test. However, in cases where the cells are stained by these methods with preliminary treatments with 1% hydrochloric acid, HzSO1-Millon’s reagent, or acidified Mayer’s reagent after formalin vapor fixation, some weakly stained colored granules are observed within the cells. In shape and distribution these granules resemble the granules stained with the OYG method. The reason why these granules appear is not attributed to the staining but the action of the acidity, because no granules are recognized even with the phase contrast microscope in erythrocyte fixed with
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formalin vapor (Figs. 13a, 14a), but after erythrocytes are treated with acidified Mayer’s reagent, 1% hydrochloric acid, or with H2S04-Millon’s reagent, some granules appear in these cells (Figs. 11,13b, 14b). These granules are then positive to the OYG method and are colored with
FIG. 10. Photomicrograph of human orthochromatic erythrocytes showing the stainability of mitochondria with the OYG method. (x 300, enlarged approximately 3.5 x.) FIG.11. Phase contrast photomicrograph of human definitive erythrocytes treated with hydrochloric acid after formalin fixation, showing mitochondria. (x 300, enlarged approximately 3.5 x.) FIG.12. Photomicrograph of mice-mature erythrocytes stained with the OYG method, showing mitochondria. ( x 273, enlarged approximately 3.5 x.)
routine mitochondria1 methods of staining. From the measurements of ihe per cent of absorption of the cytoplasm treated in various ways (Table II), it is supposed that the acidity moderately removes hemoglobin which is conspicuously stainable (Nagatani, 1957a). TABLE I1 CHANGES OF PRESERVATION SHOWN BY PERCENTOF ABSORPTION IN VARIOUS OF FORMALIN-FIXED HUMAN ERYTHROCYTES AFTER-TREATMENTS Treatment
Absorption of cytoplasm 0 10 M I
a
Without further treatment 1N HC1 solution Acidified Mayer’s reagent H,SO,-MilIon’s reagent Per cent of absorption is given by ( l-I/Io)
I
(%)a
%
I
21 19 16 10
x
100.
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It seems reasonable to conclude that the mammalian orthochromatic erythrocytes contain mitochondria which are barely detectable, but are easily recognizable with the OYG method.
FIGS.13 and 14. Phase contrast photomicrographs of human orthochromatic erythrocytes showing the relationship between the appearance of mitochondria and the action of acid. Figs. 13a and 14a. Cells fixed with formalin vapor alone, where no mitochondria are visible. Fig. 13b. The same cells as those shown in Fig. 13a, treated with acidified Mayer's reagent, and Fig. 14b, the same cells as those shown in Fig. 14a, treated with hydrochloric acid. Figs. 13b and 14b show mitochondria. (Fig. 13a, b, x 450, enlarged approximately 3.5 x ; Fig. 14a, b, x 300, enlarged approximately 3.5 x.)
V. Affinity of Carcinogens for Amphibian Somatic Cells
.
Motomura ( 1947), who first employed the carcinogenic azo dyes AAT and DAB on cyto-staining in smeared locust spermatocytes, found that these azo dyes colored the mitochondria and the Golgi apparatus. These dyes also specifically stain the mitochondria and the Golgi body of amphibian heniocytes (Nagatani, 1954a, b, 1958b, c) . The reticular Golgi appartus as detected by the silver method in amphibian intestinal epithelia and the filamentous mitochondria as figured
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by previous workers in either intestinal or hepatic cells were not recognized by using these dyes (Nagatani, 1956b). Accordingly, some questions will still remain to be settled before the author starts the study of the affinity of the carcinogenic azo dyes for the intracellular components. What is the Golgi apparatus? What are filamentous mitochondria ? Which factors create the filamentous mitochondria ?
A. GENERAL ARCHITECTURE OF THE GQLGIAPPARATUS DEMONSTRATED BY SILVER IMPREGNATION METHODIN AMPHIBIAN SOMATIC CELLS The controversial structure of the silvered Golgi apparatus is seen in the form of a more or less dense network consisting of anastomosing strands of blackened crescents, rings, granules, or masses, each of which is reconstructed from the blackened band (B) enveloping the argentophobic reticulum, blackened simple (SS) or double (DS)strands lying across the argentophobic reticulum, and the blackened mass ( M ) which is encircled completely or incompletely by the argentophobic reticulum, as shown in Figs. 15 and 16. These patterns are also recognized in the figures shown by Holmgren (1914), Penfield (1920, 1921), Da Fano (1921), Jasswoin (1925), Brown (1936), De Robertis (1940), Sosa (1949), Adamstone ( 1952, 1958), and Adamstone and Taylor (1952). Accordingly the argentophobic reticulum and the silvered Golgi apparatus have a close relationship to each other as shown in Fig. 16c. T o find how the silvered Golgi apparatus is formed, both under- and over-silvering were tried, although, according to Cowdry 1924), variation in the thickness of the strands is often the result of faulty technique. If the blackened Golgi apparatus is the preexisting organoid, it must appear as a reticular apparatus during the ‘various silverings even with a faulty technique. Under insufficient silvering, however, the special cytoplasm which exists adjacent to argentophobic reticula is colored diffusely brown, where the silvered image forming SS or DS is recognizable (Fig. 16b), and the special cytoplasm has a tendency to become a silver image forniing B or M with the increase of the time of silvering. Under overimpregnation, on the contrary, the reduced silver granules come to be scattered and are distributed throughout the cell, and under much longer silvering they cannot be observed (Nagatani and Yoshimoto, 1959b). I n addition, if the silvered Golgi apparatus is a preexisting organoid, the reticular organoid must be observed even after removing the reduced silver with cyanide. Since neither reticular structure nor reticulum-like structure can be distinguished under these conditions of observation, while argentophobic reticula and other cellular inclusions are easily observable with the phase contrast microscope (Figs. 17, 18), it has been
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FIG.15. Photomicrograph of amphibian spinal ganglion cell, showing the silvered Golgi apparatus with Da Fano’s method. ( X 300. Print enlarged approximately 2.7 x .) (From Nagatani and Yoshimoto, 1959b.) FIG.16. a. Photomicrograph of newt abdominal gland cells, showing the silvered Golgi apparatus with Da Fano’s method. ( X 300. Print enlarged approximately 4.0 x.) (From Nagatani and Nakao, 1957.) b. Photomicrograph of newt abdominal gland cells showing the Golgi apparatus demonstrated with imperfect silvering. ( x 300. Print enlarged approximately 4.0 x.) c. Diagram illustrating the relation between the reticula and the silver deposit sites (slightly modified after Nagatani and Yoshimoto, 1959b). B , blackened band; SS, blackened simple strand; DS, blackened double strands ; M,blackened mass.
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YOSHIMI NAGATANI
suggested that the reticular Golgi apparatus is not considered as the specific cellular component szti generis. 1 . Argentophobic Reticulum The argentophobic reticula found in abdominal gland cells or in ganglion cells treated with the silver method are similarly recognized in amphibian
FIGS. 17 and 18. Photomicrographs illustrating the relationship of the silver deposit site and the Golgi substance stainable with methylene blue. Figs. 17a and 18a. Photomicrographs showing classic Golgi apparatus. Figs. 17b and 18b. Phase contrast photomicrographs showing the cells with silver removed from the specimen in Figs. 17a and 18a, where no apparatus is recognized, although the reticula are visible. Figs. 17c and 18c. The same cells as those shown in Figs. 17b and 18b, stained with methylene blue, where the special cytoplasm facing the reticula is stained with it. (Figs. 17a, b, c, amphibian spinal ganglion cells from Nagatani and Yoshimoto (1959b) .) ( x 300, enlarged approximately 4.7 x. Figs. Ma, b, c, abdominal gland cells of the newt, x 300, enlarged approximately 3.6 x.)
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hepatic, intestinal, and pancreatic cells, as well as in mouse prostate cells. The reticula are 0.3 to 0.5 p in width. They are distributed three-dimensionally throughout the cytoplasm, so they are typically studded with lacunae. The facts described below leave no doubt that the reticula are not artificial structures but are the preexisting elements ; however, the relation between the reticulum and the plasniosin reported by Bensley (1938)
FIGS.19, 20, and 21. Phase contrast photomicrographs, except Fig. 20b, of isolated cells, illustrating the reticula. Fig. 19. Spinal ganglion cells of the frog. ( x 390, enlarged approximately 2.6 x.) Figs. 20a, b. Intestinal cells of the newt. ( x '390, enlarged approximately 2.8 x.) Fig. 21. Hepatic cells of the newt. ( x 300, enlarged approximately 2.6 x .)
remains unknown; they are similarly observable in cells fixed in 1% osniic acid or in cells treated with the freezing-drying method, or even in fresh preparations with the phase contrast microscope (Figs. 19,20,21). In addition, the organoid fails to be stained either with basic dyes or with acidic dyes, but the reticulum is more acidophil in immature cells than in mature cells (Nagatani, 1958a, 1959a, b ; Nagatani and Nakao, 1957 ;
.
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YOSHIMI NAGATANI
Nagatani and Yoshimoto, 1959b). Similar structures are also recognized in cultured nerve cells (Nakai, 1956). The argentophobic reticulum in amphibian ganglion cells closely resembles the neurofibril noted by Beams and King (1932), Deitch and Murray (1956, 1957), Maximow and Bloom (1952), and Nakai (1956), but it is not regarded as the neurofibril since a similar structure is also recognized in various cells mentioned above. It is also possible to compare it in shape with the canalicular system noted by Beams (1931), Bensley (1910, 1951), O’Leary (1930), and Simpson (1941), the Type I canal by von Bergen (1904), the netlike structure by Adamstone and Taylor (1953), and the filamentous structure by Yamada (1953). Most of their opinions are based on the idea that its structure is a negative image of the Golgi apparatus. These ideas, however, may be applicable to explain the band-type image only. The argentophobic reticulum coincides also with the stable system of communicating space of the Golgi apparatus recognized by Ram6n y Cajal (1915) and to the osmiophobic core reported by Honjin (1956) and Lacy and Challice (1957), judging from its morphology. Moreover, the reticulum may be the same as the Golgi apparatus reported by Adamstone (1952) and Adamstone and Taylor (1952, 1953), since the mitochondria are almost always associated with the reticulum as observed by them. From the fact, however, that the Golgi apparatus exists in special areas while the reticulum is recognized throughout the cytoplasm, it seems that the reticulum is not one constituent of the Golgi apparatus and that this is not the negative image of the Golgi apparatus, although the relation between the silvered Golgi apparatus and the argentophobic reticulum is very intimate as mentioned above. According to Wilson (1925), all observations based on the reticular theory were in substantial agreement that minute granules or mitochondria are often scattered along the threads or collected at the wdes of the nerwork. The phenomena in which the mitochondria show a tendency to collect at the angles or nodes where two or more of the hyaloplasmic lamellae meet were closely imitated in artificial oil emulsions by Biitschli (1892). According to Wilson (1925), with less perfect fixation the alveolar spheres break up or run together to a varying degree while the hyaloplasm coagulates in the form of a more or less continuous network, and it seems certain that many of the so-called reticular formations in protoplasm, as described by earlier observers, arise in this way (also Frey-Wyssling, 1953, 1956). When examined in fresh isolated amphibian intestinal cells, the lacunae, studded with the argentophobic reticula forming a network which is observed at the supranuclear region, contain the liquid drops corresponding
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to alveoli (Fig. 20a, b). Accordingly those regions are possibly comparable to the vesicular structure of Fischer (1899), and, in the sectioned preparation, these regions are observed as reticular forms ; thus they appear somewhat paler than the rest of the cytoplasm as observed by Hibbard and Lavin (1945) and Simpson (1941). The interstices of these liquid drops, however, may be the network noted by Wilson (1925) but this is not the reticulum itelf. The reticula exist within this network corresponding to the ground cytoplasm. If the reticulum is the network noted by Wilson (1925), it must be a membranous form and must continuously envelop the liquid drops. But the reticula incompletely envelop the drops as shown in Figs. 20 and 21. These structures observed at the supranuclear region of amphibian intestinal cells are similarly recognized at the perinuclear region of amphibian hepatic cells (Fig. 21). The tomograms shown in Figs. 22a to 22f are taken at one-micron intervals, and they show that the interalveolar cytoplasm is not the reticulum, but that the reticulum exists in the interalveolar cytbplasm. With this architecture, the mitochondria ought to be along the reticulum as observed by a large number of previous workers. Likewise, the cytoplasmic granules such as the secretory granules in hepatic cells may exist along the reticulum. Accordingly the cytoplasm of amphibian hepatic cells appears to be the granular acidophil cytoplasm which forms a network, as noted by Kendall ( 1949). The reticulum in a living state is too stable to be displaced by centrifugation for 2 hours at 16,000 r.p.ni. as shown in Fig. 23a, and b. Since the Golgi substances exist adjacent to such a stable reticulum, the displacement by centrifugation of the Golgi apparatus can not be as remarkable as reported by Brown ( 1936). On the other hand, the distal part of the intestinal and ganglion cells seems to be sponge-like and is possibly comparable to the reticular structure of Fischer (1899) ; thus the reticula at the distal parts are studded with the lacunae in which is contained the continuous cytoplasm. 2. The Golgi Substance Although the term Golgi apparatus is generally given to the metalimpregnated figure, it has various names such as Golgi body (Baker, 1949, 1953a), Golgi complex (Bourne, 1951; Dalton and Felix, 1956a,b, c, 1957), Golgi element (Baker, 1949), Golgi material (Dalton, 1952 ; Dalton and Felix, 1954 ; Elftman, 1954a, b) , Golgi reticulum (Moussa, 1956), Golgi structure (Oberling, 1959), Golgi system (Gatenby and Roth, 1958), and Golgi substance (Dalton, 1953; Dalton and Felix, 1952, 1953, 1954, 1956~).According to Cowdry (1924), the word “apparatus” is
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YOSHIM I NACATANI
unfortunate because it carries with it the idea of a mechanism of a rather mechanical type. De Robertis et al. (1955) also noted that it seemed more appropriate to give the name of Golgi substance or Golgi complex to a material which had special staining properties. Cowdry ( 1924) remarked
I
I
FIG.22. Tomograms of hepatic cells
tne newt, illustrating the relation between the reticula and interalveolar interstices, stained with the carcinogen after formalin vapor fixation with post-chroming. The reticula are not visible in a, but are slightly visible in b and e, and clearly visible in c and d. ( x 390, enlarged approximately 2.4 x.) ot
that the Gdgi apparatus was an area of the cytoplasm frequently of reticular form. According to Gatenby (1951), the Golgi apparatus consists of a network of argentophil substance lying in the cytoplasm. Dalton and Felix (1952, 1953, 1954) also employed the term Golgi substance in the
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sense that it is the cytoplasm adjacent to vacuoles. So it is supposed that Dalton’s area is possibly comparable to the interstice itself which is present between the alveoli. Nevertheless, according to Schneider et al. ( 1933), and Schneider and Kuff ( 1954), Dalton’s Golgi substance isolated by the fraction method contains yellow pigment. In experiments on fresh material, the yellow pigment carotenoid is found in most of the alveoli at the juxtanuclear area of the intestinal absorbing cells of heptic cells and in the lipoidal granules of the ganglion cells. Similar pigment is also found in mitochondria by
FIG. 23. Photomicrographs of newt hepatic cells showing the stability of the reticula on centrifugation, illustrating that the reticula are unaffected, while the nuclei and most of the granules are moved toward the heavy pole; a, methylene blue stain after Susa’s fixation ; b, unstained preparation after Susa’s fixation. ( x 300, enlarged approximately 2.4 x.)
Gatenby (1919) in Limnaea, by Bourne (1935), by De Robertis (1940) in amphibian hepatic cells, and by Bensley (1947) in hepatic cells of guinea pig. The Golgi substance, however, referred to in this paper is the substance itself which is located around the reticulum, appearing in the interstices, and is detectable with cytochemical methods; however, Baker (1949, 1951, 1953a,b,c, 1954, 1955, 1957b) remarked that in chemistry nothing is known of any Golgi substance, and therefore his interpretation of the Golgi substance differs from that noted by Dalton and Felix (1952, 1953, 1954). To find what the Golgi substance is, various cytochemical methods were tried. From these results, two kinds of Golgi substances are regarded as existing : one is the substance associated with silver deposition, the argentophi1 substance, and the other has no association with it.
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Y O S H I M I NAGATANI
The inaterials remaining after the reduced silver has been removed by cyanide, are stained with methylene blue (Figs. 17c, 1&). The areas where silver had been deposited are strongly stained with it, and the peripheral parts of the reticulum where the first silver deposits appeared are conspicuously stained with it. The patterns stained with methylene blue resemble none of the forms commonly ascribed to the Golgi apparatus of the classic method (Figs. 17a, 18a), but there can be little doubt of their identity. In addition, in the perparation where the blackened Golgi apparatus was not formed after treatment with 1N solution of hydrochloric acid or with pepsin, the areas where the silver is deposited are only weakly stained with methylene blue. From these facts, it seems to be reasonable to consider that the substance colored with methylene blue is the Golgi substance and is removed by treatment with hydrochloric acid or pepsin (Nagatani and Yoshimoto, 1959b). The Golgi substance in ganglion cells is found to be refractory to staining with methylene blue at p H values below 3.6, while it is stainable at p H values higher than 4.8. It has an absorption of 260 mp. (Fig. 24a, ti) ; therefore, contrary to Hibbard and Lavin (1945), examination of the microphotographs showed that most cells have a peculiar tone variation in the Golgi zone. According to Okamoto (1954), RNA can combine with the Ag ion of silver nitrate. From these facts, it seems to be reasonable to suggest that the ribonucleoprotein is possibly the argentophil substance. In Paramecium, on the other hand, the crystals composed of calcium phosphate (Kudo, 1955) are easily blackened by Da Fano’s method, and are remarkably stained with alizarin red by Cretin’s method (Nagatani and Morihara, 1958). By using Cretin’s method, alizarin red conspicuously stains the Golgi substances in amphibian intestinal cells (Nagatani, 1959b) or in ganglion cells (Nagatani and Yoshimoto, 1959~).The Golgi substance is also found to be strongly positive to ammonium phosphomolybdate test (Serra and Queiroz Lopes, 1945) (Figs. 28b) as well as to the uranium test for detecting organic phosphate (Fig. 33). From the fact that the silver techniques by K6ssa (1901) and Gomori (1952) are based on the reduction of calcium phosphate, it is supposed that the argentophil nature of the Golgi substance is possibly attributed to phosphate in the Golgi substance, although Baker (1951, 1953a, b, c, 1954, 1955, 1957a, b) pointed out that the so-called Golgi techniques were not reliable either morphologically or histochemically, and Lison ( 1953) remarked that silver impregnation was capricious, and Bensley (1951) also noted that other factors obviously exist besides the chemical properties of the reacting components.
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Accordingly, the existence of phosphatase in the Golgi substance will be expected. Bourne (1943) and Emmel (1945) found that in the coluninar cells of the jejunum, alkaline phosphatase was localized in the Golgi region. Deane (1947) and Deane and Dempsey (1945) also detected both alkaline and acid phosphatase in the Golgi apparatus in various cells. The author also observed the presence of alkaline and acid phosphatase in the Golgi substance of the ganglion cells (Nagatani and Yoshimoto, 1959a).
FIG.24a. Ultraviolet photomicrographs of a section of newt hepatic cells fixed with Susa’s, showing the special cytoplasm facing the reticula absorbing ultraviolet rays at 260 mp. Fig. 24b. Hepatic cells of a starving newt. ( X 610, enlarged approximately 3 x.)
From the fact that the edge of the reticulum, where the silver deposits first, reacts intensely to various cytochemical tests, it seems reasonable to suggest that the Golgi substance is possibly concentrated at the edge of the reticulum. The formation of various individual silver images such as B, SF, DF, and M, which form the Golgi apparatus, have possibly an association with the quantity and quality of the Golgi substance, since the quantity of the alveolar interstitial Golgi substance varies within wide limits according to the functional activity of the cells. Thus the observation of the so-called Golgi apparatus with phase contrast illumination will fail to come up to our expectation unless a different optical path exists betwen the Golgi substance and the rest of the cytoplasm, although the Golgi apparatus can be observed according to Brice et al. (1946), Oettle (1948a, b), Baker (1949), and Beams and Tahmisian (1953) in germ cells, and Baker (1949), Gatenby (1953), Gatenby and Moussa (1948), Ihnuma (1952), Ihnuma and Yanagisawa ( 1953), Adamstone and Taylor (1953), Thomas (1952), Dalton and Felix (1954), and Tagaki and Masuda (1956), Tagaki and Tagawa (1957) in somatic cells.
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YOSHIMI NAGATANI
The phase microscopic observation, however, was not useful for the reticular Golgi apparatus in amphibian intestinal cells (Nagatani, 1956b). Tarao (1939, 1940, 1943a, b) established the idea that the Golgi lipids were in a masked state, from the fact that the Golgi apparatus has not been detected by lipoidal staining, although the Golgi apparatus had been supposed to be composed of phospholipids by a large number of other workers, i.e., Cowdry ( 1924), Tarao (1939, 1940, 1943a, b, 1953a, b) , MoniiC ( 1948), Baker ( 1949, 1954), Bourne ( 1951), Elftman ( 1954a, b) , Schneider and Kuff ( 1954), and Nagatani and Nakao ( 1957). In experiments on the abdominal gland or ganglion cells using Nile blue sulfate or Tarao’s method, Nile blue is found not in the alveoli, or in the reticulum itself, but in the substance which exists in the cytoplasm facing the reticulum. These blue patterns coincide with the positive images of Baker’s acid hematein or Sudan black tests. It seems reasonable to suggest that the Golgi substance is colored with these methods. The blackened Golgi apparatus, however, suffers no change after ether or acetone extraction, although Okamoto’s test carried out on the sections suggests the probable presence of sphingomyelin in the Golgi substance (Nagatani and Nakao, 1957 ; Nagatani and Yoshimoto, 1959b). From these facts, it is supposed that phospholipids in the Golgi substance are independent of the Golgi net formation. According to Gersh (1949) and Dalton and Felix (1954), the Golgi apparatus gives a positive PAS test. Lasfargues and Di Fine (1950) and Tagaki et al. (1956, 1957) also reported that the Golgi apparatus was stained metachromatically. In an examination of amphibian hepatic cells (Nagatani, 1958), abdominal gland cells (Nagatani and Nakao, 1957), ganglion cells ( Nagatani and Yoshimoto, 1959b), and intestinal cells (Nagatani, 1959b) as shown in Fig. 28a, the Golgi substances are also stained metachromatically with toluidine blue. According to Dempsey and Wislocki (1955), the polysaccharide is argyrophil in nature. It is supposed, however, that the polysaccharide in the Golgi substance has no relation to the silver image formation, because it is possible that the polysaccharide is hydrolyzed by nitrate in Da Fano’s fixative (Nagatani, 1958~). Moreover, in the Golgi substance of amphibian spinal ganglion cells, cytochromoxidase, peroxidase, and succinic dehydrogenase are also detected (Nagatani and Yoshimoto, 195%). Goddard and Seligman (1952) also reported that the Golgi apparatus of rat thyroid gland cells contained skcinic dehydrogenase.
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B. PATHOLOGICAL INCREASEOF THE ARGENTOPHIL SUBSTANCE Ram6n y Cajal (1915) noted that the Golgi apparatus of spinal ganglion cells disintegrates after section of their axons ; Penfield ( 1920, 1921) also observed the retispersion and the retisolution. Following the interruption of the axons, it is concluded that the central part of the cytoplasm of the amphibian spinal ganglion cells becomes compact by vacuolation of the peripheral part; these lacunae finally become rather small. It seems a remarkable change that basophilia decreases and argentophil property in-
FIG. 25. Photomicrographs of spinal ganglion cells of frogs, showing the increased Golgi substance after sectioning their axons. Fig. 25a. Cells showing massive blackened Golgi apparatus. Fig 25b. Cells with moderately removed reduced silver from the specimen in Fig. 25a, where reduced silver remains observable around the reticula. ( x 390, enlarged approximately 4.7 x.)
creases in the cytoplasm. Comparing the Golgi apparatus in the pathological cells after nerve section with the control, the former is too black for details of the cell to be recognized (Fig. 25a), while the latter shows the classic image (Figs. 15, 17a, 18a). If such a blackened section is left in the cyanide solution for shorter periods of time (Fig. 25b), or if the periods of silvering of the ganglion under the aforementioned conditions are shortened, the silver image resembling the Golgi apparatus described by Ram6n y Cajal is observed in the central region of the cells, and one looking like the Golgi apparatus as figured by Penfield is also recognized a t their peripheral layers.
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The disintegration and the retispersion of the Golgi apparatus follows the pattern which might be expected if the Golgi apparatus is considered as a preexisting organoid. These increased argentophil substances are strongly stainable with alizarin red and also react intensely to organic phosphate tests (Nagatani and Yoshimoto, 195%). Accordingly it is naturally concluded that the Golgi substance increases in quantity after nerve section, since the so-called Golgi apparatus is nothing but the reduced figure of the Golgi argentophil substance.
C. Two KINDSOF ARGENTOPHIL SUBSTANCES OF DIFFERENTQUALITY The controversial Golgi substance of the intestinal cells has been investigated at the juxtanuclear region. In experiments on intestinal cells of amphibian fed 10 hours previously, the remarkable blackened image (Fig. 26a) occurs at the distal portion when the periods of silvering are very short, and the form differs from the classic silvered image observed at the supranuclear region (Fig. 26b). In the preparation after 48 hours silvering, the former is colored brown and the latter is blackened. Although there are cases in which blackened images appear at both supranuclear and distal portions, in many cases either the Golgi image at the distal area or the Golgi image at the juxtanuclear area is observed, depending on the periods of silvering. As shown in Fig. 27a and b, the areas which have an affinity for silver coincide with the areas which have an affinity for osmium. From the results obtained by general methods, it is recognized that the supranuclear region looks like a vesicular type because of the existence of many large alveoli that appear yellowish, and resemble the Golgi bodies of Baker (1949), and the distal region looks like the sponge-like type of Fischer (1899). Therefore it is supposed that the Golgi substance at the juxtanuclear region exists around the reticulum, corresponding to the Golgi negative image of Adamstone (1958) and observed between the FIG.26. Photomicrographs of newt intestinal cells showing two kinds of silvered images; a, silvered Golgi apparatus at distal parts; b, silvered Golgi apparatus at juxtanuclear regicns. In some cells the Golgi images at distal parts are visible. ( X 390, enlarged approximately 2.4 x.) FIG.27. Photomicrographs of intestinal cells of newt larvae, showing the relation between argentophil and osmiophil areas ; a, silver impregnation; b, osmium impregnation for 6 days. (x 390, enlarged approximately 2.4 x.) FIG.28. Photomicrographs of newt intestinal cells showing the Golgi substance detectable with cytochemical methods ; a, methylene blue (pH 4.6) stain ; b, organic phosphate detection with Serra’s method. ( x 390, enlarged approximately 2.4 x.)
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alveoli, and that the one at the distal part occupies, more or less, the lacunae formed by the reticula. Accordingly, the supranuclear silvered image must be the reticular form and the distal silver image must be the massive form. So it is supposed from their localization that the reticular Golgi apparatus by Ostrouch (1932) and Dalton (1953) and the massive Golgi apparatus by Hibbard and
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Laviii (1945) are comparable with the former, while the massive Golgi apparatus shown by Gersh (1949) and the mitochondria at the distal part in the rat gastric cells shown by Ostrouch (1932) are comparable with the latter. The architecture of the intestinal cells, however, changes with varying functional activity in the cells, and the Golgi substances occupy specific positions which are essential for their proper physiological function. At the resting stage, the supranuclear region becomes the reticular form as a result of the presence of little alveoli; then the silvered Golgi image is small or absent. Ram6n y Cajal (1915) also observed that, in the goblet cells of the intestine, the Golgi apparatus became greatly hypertrophied with the beginning of the formation of secretory products. Moreover, polysaccharide or RNA tests react more intensely with the distal region than with the supranuclear region (Fig. 28a), while calcium salt or organic phosphate tests similarly react intensely in both regions (Fig. 28b). From these facts, it is naturally concluded that the argentophil substances appearing in different areas of the intestinal cells are also the Golgi substances, although neither of them is identical qualitatively with the silver reaction (Nagatani, 1959b).
D. Two KINDSOF ARGENTOPHIL SUBSTANCES OF DIFFERENT QUANTITY In the amphibian hepatic cells treated by Da Fano’s method, the perinuclear Golgi apparatus as figured by Cramer and Ludford (1926) is hardly recognizable, while the bandlike Golgi apparatus facing the bile canaliculi as figured by Nassonov ( 1926), Pollister ( 1939), Tarao ( 1939), and Fawcett ( 1955) is easily demonstrated (Nagatani, 1956b). The Golgi apparatus observed in both ,regions, however, is also associated with the argentophobic reticulum as in other cells (Figs. 29, 30). Since the perinuclear region of the hepatic cells appears to be an alveolar system and the region facing the bile canaliculi appears to be a reticular system, these FIG. 29. Photomicrograph of newt hepatic cells showing the silvered Golgi apparatus. ( x 420, enlarged approximately 2.4 x.) FIG. 30. Photomicrographs of newt hepatic cells showing two phases produced by different periods of osmium impregnation; a, impregnation for 3 days; b, impregnation for 7 days. ( x 390, enlarged approximately 2.4 x.) FIG. 31. a. Photomicrographs of newt hepatic cells showing that the Golgi substances are positive to PAS reaction. b. Cells showing Golgi substance digested with saliva. ( x 300, enlarged approximately 2.3 x .) FIGS.32 and 33. Photomicrographs of newt hepatic cells showing the Golgi substance detectable with cytochemical methods; Fig. 32, Toluidine blue (pH 4.6) stain; Fig. 33. W n i u m nitrate method. (Fig. 32, X 300, Fig. 33, x 390; enlarged approximately 2.3 x .)
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two areas are possibly comparable to the juxtanuclear and the distal region or the lateral borders of the nucleus of the intestinal cells. The silvered images appearing in different areas of hepatic cells, however, are not seen to be in the same condition as the Golgi apparatus of the intestinal cells. Figs. 24a and 32 show that the materials stained with nuclear dyes absorb ultraviolet radiation of wavelength 260 mp as shown by Opie and Lavin (1946). The tests for polysaccharide (Fig. 31a, b) and RNA (Figs. 24a, 32) react more intensely at the bile canalicular region than at the perinuclear region, while the tests for calcium salt with alizarine red and organic phosphate with uranium nitrate (Fig. 33) react more intensely at the perinuclear region that at the bile canalicular region. Pollister (1932) also observed that alizarine red stained the bile canalicular region. From these observations and the results of cytochemical tests, it is concluded naturally that the reason for difficulty in recognizing the silvered image at the perinuclear region of the hepatic cells is that the quantity of argentophil substance is too small to produce submicroscopic nuclei of silver. Dalton and Felix (1956c), with the electron microscope, also observed that in hepatic cells the double membranes were relatively few in number. On the other hand, it is supposed that the bandlike Golgi image facing the bile canaliculi is attributed to the overlapping figures of the elongated massive silvered image (Nagatani, 1953a, 1959a). As the architecture of the hepatic cells undergoes a change corresponding to the varying functional activity as the intestinal cells change, the chemical component of the Golgi substances in both areas undergoes changes in quantity as well as in quality. When the cells suffer sucli &anges the reticula also change in their distribution. Accordingly the trdmformation of the Golgi apparatus observed by Cramer and Ludford (1926) in mouse liver cells may be attributed to the chemical change of the Golgi argentophil substance and mechanical displacement of the reticula ; Pollister (1932), however, denied such a transformation. In view of the above facts the most reasonable conclusions to be drawn from available data are that the silvered Golgi apparatus differs, at least in the somatic cells, from the impregnated Golgi bodies reported by Hirsch (1939), MonnC (1939), Worley (1943, 1944, 1946, 1951), Thomas (1947, 1952), Baker (1949, 1951, 1953a, b, c, 1954a, b, 1957a, b), Montagna (1950, 1951), Bourne (1951), Zeros (1951), Shafiq (1953, 1954), Hill and Bourne ( 1954), Shafig and Casselman (1954), Casselman (1955), and Christie (1955). These authors, although differing in details of interpretation, presented evidence suggesting that the Golgi bodies exist in the living cells as groups or rows of droplets which are considered to be at least partly lipoidal in nature. The silvered Golgi apparatus also differs from the
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reticular structure resulting from phospholipids distributed throughout the cytoplasm during fixation (Palade and Claude, 1949a, b). In addition, the silvered Golgi apparatus is not the impregnated reticular Golgi apparatus as regarded by Bensley (1910), Jasswoin (1925), Tanaka (19329, Pollister ( 1939), Pollister and Pollister ( 1957), Tarao ( 1939, 1940, 1943a, b, 1953a, b), Simpson (1941), Gatenby (1951), Gatenby and Moussa (1948), Gatenby et d. (1955, 1958), Gatenby and Roth (1958), Gersh (1949), Sosa (1948, 1949, 1951, 1952), Dalton (1952, 1953), Dalton et al. (1950), Dalton and Felix (1952, 1953, 1954, 1956a, b, c, 1957), Wallgren (1951), Adamstone (1952, 1958), Adamstone and Taylor (1952, 1953), Schneider et al. (1953), Schneider and Kuff (1954), Lacy (1954a, b, c, d, 1955, 1956a, b, 1957), Lacy and Challice (1956, 1957), Sjostrand (1956), Sjostrand and Hanzon (1954a, b), Fawcett ( 1955), and Zetterqvist ( 1956). They presented evidence which supports the view that the classic form of the Golgi apparatus in fixed material is a rather close approximation to its form in living cells. The Golgi apparatus, however, is the apparent figure of the impregnated argentophil Golgi substance which exists diffusely around the reticulum, but is not the figure which was produced by silvering the surfaces of the globules at the Golgi area as reported by Weatherford (1932). The author’s idea that the silvered Golgi apparatus is really the argentophi1 substance existing around the reticula, is applicable not only to the gland cells observed in the reticular Golgi apparatus but also to the amphibian nerve cells in which the Golgi apparatus is of the dictyosome type. In other words, the fundamental dissimilarity between the “kontinuierlichen type” and the “diskontinuierlichen type” of Hirschler (1927) does not need to be stressed, for the two types of Hirschler are observed in developing cells, and the reticula which control the silvered image are also changed during maturation, as remarked by Nagatani and Yoshimoto (1959b). Accordingly, it seems to be a natural result that the Golgi apparatus revealed by the electron microscope in germinal cells (Minamino, 1955 ; Pollister and Pollister, 1957; Gatenby et al., 1958; Gatenby and Roth, 1958) conspicuously resembles that in somatic cells.
E. THEGOLGIAPPARATUS WITH ELECTRON MICROSCOPY By using the electron microscope, Dalton and Felix (1954) found that the Golgi substance (apparatus) is composed of a horseshoe-shaped group of large vacuoles, the lamellae arranged concentrically around the vacuoles, and the small granules intimately associated with the lamellae. The lamella is possibly comparable to the y membrane of Sjostrand (1956) or the agranular reticula of Palay and Palade (1955), although Palay and Palade
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hesitate to identify their agranular reticula with the Golgi apparatus. Similar results have also been reported by Fawcett (1955) in hepatic cells, Gatenby et al. (1955) in the choanocytes of the marine sponge, Beams and Tahmisian (1953), Minamino (1955), and Grass6 and Carasso (1957) in germinal cells, Haguenau and Bernhard ( 1955), Watanabe ( 1956, 1957), and Nagumo (1959) in hemocytes and tumor cells, Lacy (1957) in nerve cells, and Whaley et al. (1959) in maize root cells. Dalton and Felix (1954) noted that it would appear reasonable to suggest that the lipid components are located in approximately the same position as the lamellae and granules visualized with the electron microscope, from the fact that reduced osmic acid was found in the cytoplasm adjacent to the vacuoles. By using the electron microscope, in order to determine as precisely as possible where the silver is deposited, Lacy and Challice (1956) also observed that the heavy deposition of silver was regarded as revealing the chromophil region of the Golgi apparatus. The Golgi apparatus, however, observed with the electron microscope by many workers shows no boundary membrane such as mitochondria have. From these facts, it seems that the structure revealed with the electron microscope is not the structure of the Golgi apparatus but is the structure of the specific cytoplasm in which are contained the Golgi substances that have an argentophil nature. Accordingly, observations by Sjostrand and Hanzon (1954b) with the electron microscope in the mouse pancreatic exocrine cells and by Wang (1958) in the cat thyroid gland cells, indicating that the structure of the Golgi apparatus does not change during the secretion cycle, seem to be well established. Honjin (1956) and Lacy (1957) similarly concluded that the chromophi1 component of the Golgi apparatus corresponds to a system of paired membranes which usually enclose the inner dense substance, and the chroniophobic component corresponds to a substance lying within small dilations of the paired membrane. Honjin (1957) remarked that the Golgi vacuoles were too small to be resolved by the light microscope, but many aggregations of them could be resolved, and these appeared as the osmiophobic canal. This observation, however, serves to explain the idea that the Golgi apparatus consists of two systems, one a chromophil wall and the other a chromophobic core. But it is definitely concluded that the chromophobic core is not a component of the Golgi appartus and is nothing but the reticulum. The electron microscopic observations of the reticulum of the hypertrophy of the so-called Golgi apparatus under pathological conditions are open to further research.
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F. GENERALARCHITECTURE OF MITOCHONDRIA I t is well known that the mitochondria show a great lability and are readily altered into the filamentous form from the granular form by the action of various factors such as the state of nutrition, the type of food administered (Noel, 1923), digestion (Rojas et al., 1934), starvation (Shibatani and Fukuda, 1954), and season (Takahashi et d., 1957). On the other hand, Pollister (1932), in the observation of amphibian hepatic cells, noted that the forms such as short rods, granules, and spheres of chondrioconts were artifacts and the long slender chondrioconts were most characteristic and oriented along the axis which united the blood capillaries with the bile capillaries. Pollister (1941) suggested again that the orientation depends upon the direction of the currents of diffusion within the cells and would be intimately linked to the submicroscopic structure of the fundamental cytoplasm. On the other hand, Bourne (1951) noted that the typical form of mitochondria was small rods or granules in the cells of some organs. According to Fawcett (1955), the mitochondria in the liver cells of the rat appear as plump rods or slender filaments randomly oriented in the cytoplasm depending upon the physiologic state of the animal. Claude (1941) also observed, by using biochemical analysis, that the small particles in the hepatic cells of the guinea pig fed normally behave very much like the small particles in the hepatic cells of the guinea pig fasted 72 hours. It is necessary to determine what the true mitochondria are, because, in newt hepatic cells, two kinds of granular elements and three kinds of filamentous structures appear in the mitochondria1 stains using mordants, as far as the author has been able to find. Accordingly, the observations regarding the cellular components colored with lake stainings of newt hepatic cells will be mainly described.
1. Granular Mitochondria Two definite kinds of granules are recognizable in amphibian hepatic cells. One type is the small rod-shaped granules, measuring 0.4-0.5 x O.fG1.5 p. This is therefore possibly comparable to the isolated small granules of Claude (1941), the isolated mitochondria, at least in part, of Hogeboom et al. (1948), and Hogeboom and Schneider (1955), and to the mitochondria observed with the phase contract microscope by Zollinger (1948) in their morphology. The other type is the large granules, globular in shape, ranging in size between 1.5 and 1.8 p (Fig. 34a, b) . This type is therefore possibly comparable to the plasts which appear abundantly after feeding (Noel, 1923), and to the secretory granules observed by Claude (1941).
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By using chemical analysis on fractionated cellular components, Claude (1941) showed that the large granules were specialized mitochondria, and were identical to the mitochondria reported by Bensley (1937) ; Claude thus agreed with the opinion of Noel (1923), who offered evidence to show that the large granules had their origin in a progressive transformation of the smaller mitochondria elements. Shibatani and Fukuda (1954) also noted that the typical pictures of the premortal enlargement of the mitochondria were observed in the livers of guinea pigs dying from experimental scurvy. Under mitochondrial lake stainings such as iron hematoxylin or hot fuchsin S methods, no significant differences exist between these two groups in staining reactions; moreover, the large granules are so similar that it is not possible to distinguish them on the basis of their stainability (Fig. 35a, b) or their shape from the large granules which appear to fill up the entire cytoplasm in starved liver cells. Nevertheless, methylene blue at p H 4.6 colored the large granules more intensely than the small granules ; on the other hand, the basophilia of the large granules suffered no remarkable diminution under the treatment with 1N solution of hydrochloric acid; This was Contrary to the effect on the small granules. This dissimilarity is also observed with the ultraviolet microscope (Olympus, Model MOB) : the small granules absorbed more intensely the maximum light at 260 mp than the large granules, although both of them are of relatively low absorbing power as shown in Fig. 24a and b. With Baker’s method, the small granules give a positive acid hematein test but are negative after the pyridine extraction test, while the large granules are not, as shown in Fig. 35c. The mitochondria of hepatic cells of Amblystomu, according to Bensley FIG.34. Phase contrast photomicrographs of newt hepatic cells showing large granules. The small granules presented throughout the cytoplasm are mitochondria, and they are visible as black bodies; a, fresh material; b, sectioned preparation after Susa’s fixation. (Fig. 34a, b, x 300; Fig. 34a, enlarged approximately 2.6 x ; Fig. 34b enlarged approximately 2.9 X.) FIG.35. Photomicrographs of newt hepatic cells showing the stainability of large granules with mitochondria1 method; a, hot fuchsin S stain after Susa’s fixation without post-chroming; b, Baker’s acid hematein test; c, hot fuchsin S stain after formalin fixation and pyridine extraction. ( x 420, enlarged approximately 2.4 x.) FIG.36. Photomicrographs of newt hepatic cells showing cytochromoxidase reaction. The small granules (mitochondria) are positive to these methods, while the large granules are negative. Lipoidal drops are also positive by the presence of lipids (Fig. 36b) ; a, fixed material; b, fresh material. (x 390, enlarged approximately 2.5 X.)
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and Gersh (1933), prepared with the freezing-drying method show no change with acetic acid, while, according to Bensley and Hoerr (1934), the isolated mitochondria in the hepatic cells of the guinea pig are soluble in ammonia. Baker (1957a) also observed that acetic acid at 5% was not necessarily destructive of mitochondria of mouse pancreatic cells. In the material fmed in fixatives containing acetic acid such as Bles’s for 24 hours or the material immersed in 5% acetic acid for 12 hours at room temperature after Baker’s formalin fixation, these two kinds of granules suffer no change in their stainability and shapes, while in the material treated in 1% ammonia for 5 hours, the large granules alone melt at their margins, and then decrease in their stainability. Under the cytochromoxidase tests carried out on crushed liver in 0.8% saline solution, the small granules appear to be dark blue, while the large granules are colorless as shown in Fig. 36a and b. The small granules are also more easily colored supravitally with Janus green B than the large granules, and finally decolorization occurs in the small granules. From the recent knowledge of the localization of the respiratory enzymes within the cells (Hogeboom et al., 1948; Hogeboom and Schneider, 1955), and the results of electron microscopic observations by Dempsey and Wislocki (1955) and by Fawcett (1955), it seems reasonable to suggest that the small granules are mitochondria, at least in the newt hepatic cell, while the large granules are strikingly similar to the small granules under commonly used indirect methods. Similar problems arise also in other amphibian cells. Cowdry (1914) found that the mitochondria and the lipoidal granules were similar, and the author is sure that Beams (1931) regards the lipoidal granules as the mitochondria. Nevertheless in the experiments on amphibian spinal ganglion cells, the mitochondria appearing in a rod shape can be distinguished from the proper1 granules and the lipoidal granules, for the mitochondria are less colored than the proper granules, and much less so than the lipoidal granules (Nagatani and Yoshimoto, 1959b). 2. Filamentous Mitochondria The question must be considered why three kinds of filamentous structures appear. a. F;ilamentous Structure Caused by Combination of Large Gratrules. When the large granules are lined up together and too much dye is used, they appear to be the fatty filamentous form (Fig. 37a, b). These 1 These
(195%).
special granules were named proper granules by Nagatani and Yoshimoto
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fatty filamentous structures are also possibly comparable, at least in part, to the filamentous mitochondria shown by Noel (1923), and to the fatty rod mitochondria shown by Bensley and Gersh (1933) and Ludford (1935). Although these appear to be homogeneous structures having a bright refringence (Fig. 38a), and to have rounded ends under phase contrast (negative-high) , it is found under phase contrast (negativemedium or positive-medium) that these structures are composed of a chain of some granules as shown in Fig. 38b. If it is accepted as true that
FIG. 37. Photomicrographs of newt hepatic cells showing filamentous structures caused by the presence of large granules; a, fuchsin S method; b, acid hematein test. ( x 390, enlarged approximately 2.4 x.) FIG.38. Phase contrast photomicrographs of newt hepatic cells showing that fatty filamentous structures consist of large granules ; a, phase contrast microscope (Negative-High) ; b, phase contrast microscope (Negative-Medium). (x 390, enlarged approximately 2.4 x.)
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the large granules are not the true mitochondria, these patterns therefore must be considered not to be characteristic of the filamentous mitochondria, because they appear when violent staining is employed after previous chromatization. As for the lining of the large granules, it seems to be attributed to the fact that some large granules distributed throughout the cytoplasm are closely pressed together to form chains by the development of fat globules and, contrary to the opinion of Noel (1923), not because of any property of their own since such patterns often appear in the cells which contain many large fat globules. b. Filamentous Structure Caused by Presence of Special Cytoplasm. In amphibian hepatic cells blunt filamentous structures are frequently recognized when the cytoplasm facing the reticula and the small granules arranged along the reticulum (Fig. 39a) and, in some cases, the large granules are strongly stained with phenol-fuchsin S after Champy’s fixation and post-chroming (Fig. 39b, c). In some areas of the same section, the sharp filaments which resemble the blunt filaments in size are recognized independently of the heat of dyeing. These patterns are possibly comparable to the mitochondria shown by Jasswoin (1925) in proximal convoluted tubules of kidney of Triton, by Bensley and Gersh (1933) in Amblystoma hepatic cells, by Ludford (1935) in cultured fibroblast of chick embryo, and by Takahashi et al. (1957) in various anuran hepatic cells. In experiments to determine whether this filamentous structure is the colored image of the preexisting organoid or not, it appears that this structure consists of the special cytoplasm and the granular elements, for the small granules resist the decolorizing action of sodium acetate for a longer period than the special cytoplasm, as shown in Fig. 39e and f, and any preexisting filamentous organoid is not observed in the decolorized area even with the phase contrast microscope. Similar stained patterns are also recognizable even under methylene blue staining without postchroming after Susa’s fixation, while these patterns disappear after postchroming. These filamentous structures are clearly observed even after Champy’s fixation without any subsequent treatments, and after fixation
FIG.39. Photomicrographs of newt cells stained with mitochondria1 methods showing filamentous structure caused by the presence of the special cytoplasm facing the reticulum; .a, b, and e, hepatic cells stained with routine acid fuchsin; c, hepatic cells stained with Baker’s acid hematein test; d, intestinal cells stained with routine fuchsin S stain; f, the same cells as those shown in .e, decolorized with sodium acetate, showing that the special cytoplasm is differentiated with sodium acetate, while the small granules (mitochondria) resist it. (x 390, enlarged approximately 2.4 x.)
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with Baker’s fornialin or Susa’s fluid with post-chroming. From these facts, it is supposed that these filamentous structures are not true filainentous mitochondria but are the artificial images caused by the postchroming. According to Gatenby (1950), the main point of the mitochon-
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drial staining is to avoid washing out the mordant too much when the slides are being transferred from the iron-alum to the hematoxylin. Baker (1958) remarked that Altmann (1894) himself admitted that the differentiation was difficult. According to Sato (1956), the key point in obtaining good mitochondria is probably the period of fixation. It is supposed that these ideas may have been developed from the fact that the mitochondrial stainings are based on their acid-fast property ; therefore, it is doubtful whether the mitochondria1 stainings with the mordants are the methods to recognize or produce mitochondria. The method applied in almost all of the cytochemical methods is not a regressive method but a progressive one, because it reveals the natural shape as well as the characteristic properties. Therefore the author cannot agree with Cowdry (1924) that in the matter of interpretation it must be remembered that imperfect preparations will often convey a false impression of a reduction in the amount of mitochondria and of the mitochondria being more granular in shape than they actually are in the living tissue. If these filamentous structures are the filamentous mitochondria, these structures must be recognized with the phase contrast microscope, and colored supravitally with Janus green B, and detected with various respiratory enzyme reactions. But these methods show only the granular mitochondria, never any filamentous structure. Therefore, it is naturally concluded that these filamentous structures are not the filamentous mitochondria. On the other hand, it is also supposed that the patterns of the small granules which show that they lie along the reticula, which appear when the dyes of the special cytoplasm are removed from the filamentous structures, are not the filamentous mitochondria formed by their properties. In regard to the arrangement of the small granules, it is supposed that this is not a property of the granules themselves but a secondary pattern caused by adhesion to the reticula, for their arrangement is irregular and they occur at wider intervals than the filamentous mitochondria, F-OYG, of amphibian erythrwytes. Accordingly the arrangement of these small granules shows no filamentous mitochondria. In amphibian ganglion cells, the specikl cytoplasm facing the reticula is also recognized with phenol-fuchsin S staining as the filamentous mitochondria-like structure, although mitochondria of these cells are identified as the rod form (Nagatani and Yoshimoto, 1959b). The filamentous mitochondria shown by Takeichi (1931) in rabbit prostate cells and by Adamstone (1958) in rat jejunum cells seem to be the overstained special cytoplasm. The mitochondria of the intestinal cells, according to De Robertis et al. (1955), are filamentous in the apical zone and at the sides of the nucleus, and granular in the basal cytoplasm. However, from the
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architecture of the intestinal cells and the relation with the granular mitochondria and the reticula, it is supposed that the filamentous mitochondria at the juxta-nuclear region or at the lateral borders of the nucleus niay be the apparent image caused by the overstaining of the special cytoplasm and the small granules which lie along the reticula, as shown in Fig. 39d (Nagatani, 195913). It is, therefore, naturally concluded that the granular mitochondria lying along the reticula show the apparent filamentous mitochondria by overstainability of each of the granular mitochondria and the stainability of the cytoplasm facing the reticulum. This special cytoplasm, at least in part, coincides with the area where the silver deposits. Accordingly the pattern of the pathological condensed mitochondria in rat gastric cells shown by Ostrouch (1932) is similar to the
FIG.40. Photomicrographs of newt hepatic cells stained with fuchsin S showing filamentous structure caused by stainability of the reticula; a, formalin fixation with post-chroming; b, formalin fixation with pyridine extraction. ( x 396, enlarged approximately 2.4 x.) silvered Golgi apparatus at the distal part of amphibian intestinal cells. Takagi (1959) 'also found that the Golgi filaments which are possibly comparable to the author's special cytoplasm facing the reticula were detectable with Janus green B, and therefore the distinction between the mitochondria and the Golgi filaments became difficult. c. Filamentous Structure Caused by Presence of Reticulum. The reticula, when overstained, look like the filamentous mitochondria (Fig. a), and they are comparable with the patterns drawn by previous workers (Pollister, 1932 ; Maximow and Bloom, 1952) ; under imperfect staining, they are stained on their surface alone. In the latter preparations, they look like canals which are composed of chromophil walls and a chromophobic core. In any case the small granules are recognized around the reticula. These are strikingly similar to two kinds of mitochondria, black and white, shown in figure by Guilliermond (1919) in the spermatophytes of Narcissus poeticus. Similar patterns are recognized in cells stained
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with fuchsin S after formalin fixation and subsequent pyridine extraction without previous chroniing (Fig. 4Ob) ; moreover, they appear as blue patterns with methylene blue after formalin fixation and subsequent pyridine extraction. From these facts, it is supposed that these structures resemble the mitochondria reported by Bensley and Gersh (1933) in the hepatic cell of Ambdystoma. These structures, however, are not the mitochondria but the reticula themselve, because the localization of the respiratory enzymes is not detected within them, and they are recognized as the reticula with the phase contrast microscope. These various patterns mentioned above are not those produced by artificial factors as reported by Pollister (1932) ; they are, rather, the stained images of characteristic components within the cells, because each of the various patterns is recognized in the cells, depending upon the location of the cell within the hepatic lobule as observed by Fawcett (1955) in the rat liver cells, although these various patterns are observed more or less even in the same cell, as shown in Fig. 39c. Moreover, the appearance of various patterns has no relation to the difference in fixation caused by the size of the tissue blocks, at least in amphibian hepatic cells, although Cowdry (1924) noted that it is important to use small pieces of tissue not more than 3 mm. in thickness. Accordingly it seems to be reasonable to suggest that the appearance of these various patterns is attributed to the morphological and physiological states of the cells. Therefore, the transformation of mitochondria in the hepatic cells should be recognized in the same cells. Thus the reason why Bensley and Gersh (1933) reached the conclusion that the application of water and of salt solution to the mitochondria was not always followed by the same results seems to be that they observed various kinds of cells. From these discussions, it is naturally concluded that the true mitochondria are those of granular form having a rod shape, and the so-called filamentous mitochondria figured by Scott (1916), Noel (1923), Nassonov (1926), Ostrouch (1932), Duthie (1935), Shafiq (1953), and Christie (1955) are the figures apparently caused by overstaining of the small granules and other cytoplasmic components. The mitochondria revealed FIG. 41. Photomicrographs of newt hepatic and intestinal cells stained with the carcinogen, after Susa's fixation (Carbowax-section method) : a and b, hepatic cells showing that granular mitochondria are visible as black bodies, while large granules are not stained. The Golgi substance at the region facing bile canaliculi is more intensely stained than that at perinuclear region; b, cells of starving newt; c and d, intestinal cells sbawing that the Golgi substances at the distal region as well as at the juxtanuclear region are easily stained with them. (Fig. 41a, x 200; Fig. 41b, c, x 300; Fig. 41d, x 390; enlarged approximately 5.1 x.)
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with the electron microscope by Sjostrand (1953), Sjostrand and Hanzon (1954a), Dempsey and Wislocki (1955), Fawcett (1955); and Dalton and Felix (1956a, 1957) and the isolated mitochondria by Novikoff (1956, 1957), Novikoff et al. (1956), and Randall (1957) are of the
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rod form, although Porter et al. (1945) and Palade (1952) observed slender mitochondria with the electron microscope. Accordingly the mitochondria in the somatic cells are the small granules to be treated in the following sections. Therefore, the problem of the relation between the filamentous structures and the true mitochondria is as important as the Golgi controversy. G. STAINABILITY OF CELLULAR COMPONENTS WITH OYG METHOD It is well known that o-aminoazotoluene (AAT) and p-dimethylaminoazobenzene (DAB) are lipoidal dyes, and thus the smear method used for germ cells (Motomura, 1947) and for blood cells (Nagatani, 1954a, b, 1955, 1956a, 1957a, b, 1958b, c) is useful for the purpose of finding the affinity of these carcinogens for the cytoplasmic components, but the paraffin-section method is not. Therefore the Carbowax section method was employed for this purpose (Nagatani, 1956b, c) .
1. Stainability of Cellular Components in Carbowax-Section The cellular components stained strongly with the OYG method are the lipoidal bodies in the hepatic (Fig. 41a), intestinal (Fig. 41c, d), and ganglion cells (Fig. 42c) ; thus these granules are observable as reddish violet granules. The fat globules that look like vacuoles in the hepatic cells are not stained. Although the proper granules of the ganglion cells or the large granules of the hepatic cells are weakly stained with it, the mitochondria are stained red or reddish violet. In the OYG method, the mitochondria are the granules of rod shape and they lie in close proximity to the reticulum, but are not filament forms as observed in the mitochondrial lake stainihgs. In starved hepatic cells, the rodlike mitochondria react positively to the OYG method, while the large granules are refractory to staining with it as observed in normal cells (Fig. 41b). The reticula in any cells fail to be stained with the OYG method studied so far, while the cytoplasm facing the reticula is feebly stained pink, but is stained red at the Golgi zone. The patterns (Fig. 41c, d ) which the cytoplasm at the distal region as well as the juxtanuclear region of amphibian intestinal cells showed with carcinogen staining are comparable to the patterns of silver impregnation (Fig. 2Oa, b). The pattern of the abdominal gland cells stained with the OYG method (Fig. 42a) resembles the silver image (Fig. 42b). Therefore it is supposed that the Golgi substance gives a positive reaction to the OYG method, and the Golgi substances at the juxtanuclear region and the distal region are stained with the carcinogens in almost the same degree. In ganglion cells, the Golgi substance lying along the reticula and the increased Golgi substance after nerve-section also react
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positively to it (Fig. 42c, d), while in the hepatic cells, the Golgi substance facing the bile canaliculi is more intensely stained with it than the Golgi substance at the nuclear region.
~~
FIG. 42. Photomicrographs showing the relationship between the areas stainable with the carcinogen and the argentophil areas; a and b, abdominal gland cells of the newt (from Nagatani and Nakao, 1957) ; c and d, frog spinal ganglion cells after section of their axons; a and c, Carbowax-section method of OYG method; b and d, silver method. Fig 42c. Cells showing that increased Gold substances are stained with carcinogen. (Fig. 42a, b, x 300; Fig. 42c, d, x 390; Fig. 423, b, enlarged a p proximately 3.5 x ; Fig. 42c, d, enlarged approximately 2.5 x.)
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On the other hand, the karyoplasm is very weakly stained with it as observed by Motomura (1947),and the nucleolus is found to be refractory, although it is well known that the chromosomes are affected by these carcinogens (Amano and Ando, 1942; Tanaka, 1952, 1954, 1959; Tanaka and Kano, 1952 and Sasaki, 1958).
2. Stainability of Cellular Components in Parafin Section Comparing the paraffin section with the Carbowax section, both of which were stained with the OYG method after Da Fano’s, Susa’s, or Baker’s formalin fixation, the stainability of the lipoidal granules in the paraffin section decreases more conspicuously than in the Carbowax section, and the stainability of the mitochondria in the paraffin section is weaker than that of the mitochondria in the Carbowax section, but the weakness is not so remarkable as that of the lipoidal granules. On the other hand, the stainability of the Golgi substance, the proper granules of the ganglion cells, the large granules of the hepatic cells, and the nuclear components becomes stronger than that in the Carbowax section, and so the Golgi substance looks pink. The bile comes to react intensely positive to the OYG method in the paraffin section, although it is negative in the Carbowax section as shown in Fig. 43a, b, and c.
3. Intracellular Localization of Carcinogen in Vital Staining When 0.5 to 1.0 ml. of AAT saturated in olive oil is injected into the abdominal cavity of the newt, the newts’ tongues are stained yellow in about 5 hours after the injection, and then AAT is detected in their hepatic cells. The larvae keep their life with difficulty for about 7 days in saturated DAB aqueous solution and they become yellow. Comparing these methods with the paraffin-section method of the fixed preparation, the intracellular localization of the carcinogens is almost the same in the hepatic and intestinal cells, although the stainability of the cellular components is not stronger than that of the latter as shown in Fig. Ma, b, and c. In this vital staining, however, the Golgi substance and mitochondria are more clearly observed than in the latter, for the remaining cytoplasm is only weakly stained with it. Since the intracellular localization of the carcinogens in the paraffin section of the vital staining preparation (Fig. 44b) is similar to that in the Carbowax section of the vital staining prepFIG.43. Photomicrographs of newt hepatic and intestinal cells stained with OTG method (paraffin-section method), showing that such Golgi substances are more intensely stained than those in Carbowax-section method; a, hepa+ic cells of fatty newts; b, hepatic cells of starving newts ; c, intestinal cells of fatty newts. (x 390, enlarged approximately 5.5 x.)
CYTOLOGICAL STUDIES WITH CARCINOGENS
292
YOSHIMI NAGATANI
aration (Fig. 44a), it seems to be reasonable to suggest that the carcinogens in the cells are extracted with difficulty with organic solvents. These results may bear a relationship to the results of Miller and Miller (1947) who found that the bound dye in the liver of rats fed with DAB was not removed from the preparations by boiling organic solvents or by hot trichloroacetic acid. Any fundamental dissimilarity between AAT and DAB is not recognized in the three methods (Nagatani, 1959b).
H. STAINING MECHANISM OF OYG METHOD The findings that the reticular Golgi apparatus and the filamentous mitochondria are not detected with the OYG method, but the Golgi substance and the granular mitochondria are easily demonstrated with it, are similar to the results of tests for detection of calcium salt or organic phosphate. The stained pattern shown in Fig. 28b is conspicuously similar to one shown in Fig. 43c. These facts show that the carcinogens have a strong affinity for such cellular component such as mitochondria thus are related to the ideas of Woods and duBuy (1945) that mitochondria1 diseases could be considered as the underlying cause of many cancers. The data of Waddington and Goodhart (1949) who found by using the fluorescence microscope that in newt gastrulae lipochondria and liposomes showed the bright blue fluorescence of benzpyrene in molecular solution and that there was also occasionally a structureless blue fluorescence around the nucleus corresponding perhaps to the so-called Golgi apparatus ; have similar implications. This is also supported by observations with the electron microscope by Hashimoto et al. (1956), Tujimura (1957), and Onoe et al. (1959) which demonstrate that mitochondria of hepatic cells of rats fed on DAB show pathological changes. Accordingly, the question arises now : What chemical substances in these components have the affinity for the carcinogenic azo dye? 1 . Removal of Cellular Chemical Substances Comparing the results observed in the Carbowax sections with the ones observed in paraffin sections, it seems to be reasonable to suggest that these carcinogens stain some chemical substances in addition to the lipoidal substances, although Takagi (1953) remarked that the OYG method was lipid staining. Comparing the somatic cells using the section FIG.44. Photomicrographs of hepatic and intestinal cells of the newt stained vitally with the carcinogen. The Golgi substance and mitochondria are visible as the components which b v e affinity for the carcinogen; a and c, Carbowax-section method; b, paraffin-sectionmethod. ( x 390, enlarged approximately 5.5 x.)
CYTOLOGICAL STUDIES WITH CARCINOGENS
293
294
YOSHIMI NAGATANI
methods with the blood cells observed by the smear method, it will be naturally concluded that the chemical components of the mitochondria of somatic cells differ from those of amphibian erythrocytes, for the former are still positive to the OYG method even after fat extraction, while the latter become negative or stain only a little. In the paraffin section fixed preparation, the stainability of the cellular components with the OYG method decreases after treatment with the following reagents : 1N-hydrochloric acid at 60°C. for 10 minutes in order to remove the nucleic acids (Fig. 45a), citrate buffer of pH 4.6 for 24 hours in order to remove the calcium salts (Gomori, 1952), or ammonium molybdate-free molybdate reagent ( Serra and Querioz Lopes, 1945) at 10°C. for 2 weeks, followed by 2 days at 25°C. in order to remove the phosphate, and pepsin (Tarao, 1939) at 37°C. in order to digest the protein. From these results and the fact that the patterns with the OYG method are conspicuously similar to the patterns of organic calcium and organic phosphate detection (Figs. 28b, 43c), it is supposed that the carcinogens have an affinity for protein combined with such chemical substances in the Golgi substance and in mitochondria. On the other hand, the polysaccharide in the Golgi substance may not be stained with the OYG method, because the specific granules of amphibian basophil leucocytes are only slightly stained with it, although they seem to be polysaccharide-rich granules (Nagatani, 1958~)and because any difference is not recognized in the patterns stained with the OYG method after Da Fano’s fixation and Susa’s or formalin fixation. If the polysaccharides were stained with the OYG method, the stainability after Da Fano’s fixation would become weaker than that after Susa’s, for it is possible that the polysaccharides are hydrolyzed by nitrate in Da Fano’s fluid.
2. Decolorization Test In order to find the relationship between the carcinogens and the cellular substances excepting lipids, the paraffin section stained with the azo dye (saturated o-aminoazotoluene in 1% hydrochloric acid) is mounted in Mayer’s reagent after immersion in water, 5% sodium chloride, 10% sodium chloride, xylene, and toluene, respectively, for 24 hours. Any remarkable change is not recognized in the preparations extracted with these fat solvents and the control. The extractions with xylene and toluene yield similar results. Under sodium chloride treatment, bound dye is pretty well removed and then the mitochondria become difficult to observe, but the bound dye still remains as shown in Fig. 45b. Under water treatment on the other hand, the dye is more completely removed than in sodium ,chloride treatment, and then the section becomes fainter pink, but
CYTOLOGICAL STUDIES WITH CARCINOGENS
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the dye still remains in the Golgi substance and in the mitochondria (Fig. 45c) ; therefore, mitochondria are more recognizable than in sodium chloride treatment, although the hues are very feeble. From these results and the opinion of Shibatani (1948), it seems reasonable to suggest that the affinity of the carcinogens for the cellular components is to be attributed to adsorption rather than to the formation of salts (Nagatani, 1959a, b) ,
3. Effect of PH in Dye Bath Since the hydrochloride of AAT or DAB is water soluble (Schultz, 1931), these dyes are employed in 1% hydrochloric acid for the OYG method. It is supposed that the acid and basic groups of these dyes are very largely ionized in the hydrochloric acid as follows.
pDimethylaminoszobenzene:
Accordingly, it seems possible that AAT may have the color cation that general basic dyes have. If the mechanism of staining in the OYG method is attributed to polar adsorption, the stainability of the cellular elements with the OYG method will be affected by the pH of the dye bath. The materials are stained by using 1 ml. of AAT saturated in alcohol added to 50 ml. of McIlvain's buffers of pH 2.2, 5.6, and 7.0. No remarkable change is observed in the somatic cells, and their patterns are almost the / same as the control (pH 1.4). On the other hand, the stainability of the cytoplasm of amphibian erythrocytes increases as pH values of the dye bath increases. However, in experiments with eosin and methylene blue saturated in 1% hydrochloric acid, the former stains' the cytoplasm of amphibian erythrocytes fixed with fonnalin vapor and the latter feebly stains the nuclei. From these facts, it is supposed that the carcinogens are the special
296
YOSHIMI NAGATANI
dyes and the mechanism of staining with the OYG method cannot be explained by polar adsorption itself. (These results are unpublished work.)
I. ESTIMATION OF HYDROCHLORIC ACIDIN THE OYG METHOD It is supposed that 1% hydrochloric acid in the OYG method is valuable for several reasons. First, since AAT and DAB are dissolved in 1% hydrochloric acid, the affinity of the carcinogens for the cellular components with the OYG method may be the affinity of the chlorides of their carcinogens for them. The OYG method, however, may be necessary to make the precausal changes of the hepatoma clear, for according to Kinoshita (1937), hyperplasia of the hepatic cells can be observed in the rat fed chloride of DAB, although it does not develop into a hepatoma, and he suggested that the hyperplasia was caused by DAB, since DAB-HC1 is so labile as to convert easily to DAB ; the reason why the hepatoma could not be observed might be attributed to the fact that the quantity of the dye concentrated in the liver by his method was too little to develop into the hepatoma. In addition, the carcinogens combine tightly with the cellular components in the amphibian cells when AAT and DAB are employed for the vital stainings, although any hyperplasia of the cells is not recognized; moreover, any difference in the affinity of the azo dyes for the cytoplasmic components is not recognized in the vital stainings and the fixed preparations as mentioned above. According to Miller and Miller (1947), on the contrary, no bound dye was found in rat tissues in which DAB does not induce tumors, viz., small intestine, red blood cell, etc., nor in the livers of the species which are resistant to the carcinogenic action of DAB, although the livers of rats fed it have been found% contain amino azo dyes bound tightly to a cellular constituent. The significance of the contrary results, however, in the author’s experiments and those of Miller and Miller (1947) is open to further research. Second, the acidity of 1% hydrochloric acid has not only an action as an unmasking reagent (Ackerman, 1952), but also removes the cytochemical substances such as hemoglobin and polysaccharide so as to visualize the granular components within the cells for the demonstration of mitchondria in the erythrocytes. Therefore, mitochondria in amphibian erythrocytes are recognizable even by staining with eosin or methylene blue dissolved in 1% hydrochloric acid, although mitochondria are not stained with them. On the other hand, in experiments with eosin in buffer solution at various FIG.45. Photomicrographs of newt hepatic cells showing that bound carcinogen resists decolorization; a, hydrochloric acid treatment ; b, sodium chloride treatment ; c, water treatment. (x 390, enlarged approximately 5.5 x.)
CYTOLOGICAL STUDIES WITH CARCINOGENS
297
29s
YOSHIMI NAGATANI
pH values, mitochondria are not observable, for the cytoplasm is progressively stained as pH values increase. Therefore, the reason why mitochondria in somatic cells are easily detected with the OYG method may be attributed to such a mechanism. Shibatani (1951) also suggested that removal of RNA from the cytoplasm might be necessary for detecting mitochondria. Third, the presence of the hydrochloric acid has an influence upon the ionization of the protein within the cells. According to Miller and Miller (1947), DAB in the livers of the rat fed the carcinogen dye binds tightly to a cellular constituent, probably protein. In regard to the binding protein, the carcinogen (DAB) was found in the “h” fraction with electrophoretic analysis by Sorof et al. (1951, 1958) and Wirtz and Arcos (1958), and in tyrosine (Kusama and Terayama, 1957; Kusama et al., 1958; Terayama et al., 1958a, b). The fact that the cells digested with pepsin fail to be stained with the OYG method seems to agree with those data. It is supposed, however, that the carcinogens have no afinity for the protein itself but have an affinity for the organic phosphate binding to protein, for the dye bath of the OYG method has a pH of about 1.4. Therefore, the cellular constituents in such a medium will carry a hydrogen ion and are positively charged ; consequently the positively charged ion of the carcinogens cannot enter into the proteins in the cells nor into the phospholipids, since the I.E.P. of the cytoplasm is usually at about pH 4.6-5.0 (Seki, 1957) and that of the phospholipids is at pH 2.7 (FreyWyssling, 1953). Furthermore, the patterns resulting from staining with the OYG method strikingly resemble those obtained with tests for RNA, phospholipids, calcium salts, or organic phosphate. Therefore, the reason why the Golgi substance and mitochondria give a positive reaction to the OYG method seems to be attributed to the fact that the organic phosphates within the Golgi substance and mitochondria are hydrolyzed with hydrochloric acid in the presence of the carcinogens and then reduce the phosphocarcinogen precipitate. According to Claude (1941) and Palade and Siekevitz (1956), the mitochondria share with the microsomes a high content of phospholipids. Hultin (1956a, b, 1957) and Gelboin et al. (1958) observed that the carcinogen existed in the mitochondria or in microsomes. In regard to the affinity of the carcinogens for the cellular components, any fundamental dissimilarity between fixed preparations and vital staining preparations is not recognized. Therefore it seems reasonable to suppose that the hepatoma may have an association with phosphate metabolism, although Miller and Miller (1952) proposed the so-called “protein deletion” theory as the etiology of certain cancers. Fourth, the concentration of the 1% hydrochloric acid in the acidified
CYTOLOGICAL STUDIES WITH CARCINOGENS
299
Mayer’s reagent is necessary for the fixation. Mitochondria are not distorted by the hydrochloric acid in the process of the OYG method. Casselman and Jordan (1954) also noted that the hydrochloric acid was a suitable fixative. In addition, the hydrochloric acid is necessary for the revelation of the carcinogens. Mayer’s reagent is produced according to the following formula : 4KI
+ HgC1,
= 2KC1
+ K2Hg14
The carcinogens-HC1 in the cells are thus changed into reddish violet or dark red by the following formula : 2(C14H16N3C1)
+
K&g14
= (c14H16N3)2
Hgl*
+ 2KC1
From the facts that the carcinogens in the cells are not colored by Mayer’s reagent but are colored by the acidified Mayer’s reagent with hydrochloric acid, and that the molecular formula of AAT or of DAB is C14H16N3,it is supposed that the character of the colored material may be (C14HlBN3)2Hg14; therefore, the hydrochloric ion is necessary for the revelation of the azo dyes.
J. CHEMICAL NATURES OF CARCINOGENS To clarify whether the detection of lipoidal constituents and other constituents by the OYG method occurs because the carcinogens consist of various constituents or because the carcinogens can dissolve in lipoid as well as in other constituents, the carcinogens treated variously were analyzed with the spectrophotometer (Shimazu, Model QB SO). 1 . Solubility of o-Aminoazotoluene When AAT was added to the mixture of toluene and water, it dissolved into different layers. The dye in the toluene and in the water tentatively was named AAT/Tol and AATIH20. The remaining dye by evaporation of the toluene in AAT/Tol again dissolved in water (AAT/Tol/H20). The graph showing the transmission of AAT/Tol/H20 strikingly resembles that of AAT/H20, and the substances absorb strongly at about 380 mp as shown in Fig. 46a. When the toluene was added to the AAT solution saturated in 1% hydrochloric acid (AAT/HCl) , AAT/HCl in the hydrochloric acid dissolves again into the toluene (AAT/HCl/Tol), and so the toluene becomes orange, and then the hydrochloric acid solution becomes faint pink (AAT/HCl/Tol+HCl) . Comparing AAT dissolved in the toluene (AAT/Tol) with AAT/H20/Tol and AAT/HCl/Tol, these solutions have almost the same tendency of absorption, and they absorb at about 400 mp as shown in Fig. 46b. The graph showing the transmission of the AAT/HCl is striking similar to that of AAT/HCl/Tol+
300
0
%
YOSHJMI NAGATANI
s
‘NOISSIWSNVZII
0
rD
3: ‘ N O I S S I M S N V U
,
ii
I
.I .1
0
cy
FIG.46a. Graph showing the transmission of light through a layer 1 cm. thick of AAT/H,O and AAT/Tol/H,O. FIG.46b. Graph showing the transmission of light through a layer 1 cm. thick of AAT/Tol, AAT/ H20/Tol, and AAT/HCI/Tol. FIG.46c. Graph showing the transmission of light through a layer 1 cm. thick of AAT/HCl and AAT/ Hcl/Tol + HCI.
V,
2 $
; x m
z
302
YOSHIMI NAGATANI
HCl which is the remaining solution of AAT/HCl/Tol, and the solutions have very characteristic absorption spectra with sharply defined double peaks at about 340 mp and 500 mp as shown in Fig. 46c. From these facts, it is supposed that AAT itself is soluble in either fat solvents, water, or hydrochloric acid. Accordingly, the experiments described above leave no doubt that the cellular components are easily stained vitally with the azo dye (Nagatani, 1956c), and that the patterns stained with the OYG method are strikingly similar to those of vital staining preparations of the OYG method (Nagatani, 1959a, b)
.
FIG.47. Paper chromatographsof p-dimethylaminoazobenzene.
2. Solzcbility of Developed p-Dimethylaminoazobenzene When a spot of the DAB solution saturated in 1% hydrochloric acid is paper chromatographed by the n-butanol-acetic acid-water system in which the ratio of the each reagent is 6:3:2, 6:4:2, or 6:5:2, the dye changes easily from red to yellow at the origin, and subsequently passes slowly above without development. But in the developers (6:6:2, 6:7:2, . . 6:10:2), the dye passes slowly above without color change. When the front is reached at about 2-3 cm., the yellow dye begins to appear from the red spot as shown in 1 of Fig. 47. The yellowish dye goes up faster than the red dye; therefore, the colored band which consists of the yellow band (DAB-Y) and the red band (DAB-R) is observed when the front reaches about 3-5 cm. as shown in 2, 3, and 4 of Fig. 47, and subsequently the red band disappears by changing into yellow dye when the
.
303
CYTOLOGICAL STUDIES WITH CARCINOGENS
front reaches about 10 cm. as shown in 6 of Fig. 47. A similar phenomenon is also observed in AAT. DAB-Y as well as DAB-R changes to reddish violet with acidified Mayer’s reagent. DAB-Y and DAB-R which are cut off, each in turn, from the paper, are dried, and subsequently are extracted with some reagents. In the extraction with 1% hydrochloric acid, both solutions become red (DAB-Y/HC1, DAB-R/HCl) . The graphs showing the transmission of DAB-Y/HCl and DAB-R/HCl strikingly resemble that of DAB dissolved in 1% hydrochloric acid (DAB/HCl), and they have maximum absorption at about 515 mp as shown in Fig. 48a. In the extraction with toluene, the dye of DAB-Y is so easily extracted that the toluene becomes yellow (DAB-Y/Tol), while the dye of DAB-R is only slightly extracted (DAB-R/Tol) . The graphs showing their transmission resemble that of DAB dissolved in toluene (DAB/Tol) , and they absorb at 405 mp as shown in Fig. 48b. From these results, it is supposed that DAB-Y and DAB-R may be the same substance (Nagatani, 1959b). These chromatographed dyes are easily extracted with hydrochloric acid solution, but are extracted with difficulty with Susa’s or Da Fano’s fixatives. DAB-Y is more easily extracted with toluene, n-butanol, or ethanol than DAB-R, and DAB-R is more easily extracted with 10% TABLE 111 SOLUBILITY OF P-DIMETHYLAMINOAZOBENZENE IN PAPERCEROMATOCRAPHED YELLOWBANDAND RED BAND Solvent HCl, 1% Toluene rt-Butanol Ethanol NaC1, 10% HZO Susa’s Da Fano’s 0
+, soluble; -,
DAB-R
+++
- or - or ’
f f
+ ++ +++ 2
f
DAB-Y’
+++ +++ +
-
+++ f
- or
&
f
*
insoluble.
sodium chloride or water than DAB-Y, as shown in Table 111. From these results, it will be seen why the preparations stained with the carcinogens were only slightly decolorized with xylene and are more easily differentiated with water than with salt solution, and why the dyes in the cells stained vitally with the carcinogens remained so even in paraffin sections after Da Fano’s fixation (Nagatani, 1956~).
-.-.- DA B-R/HCE -.-.DAB-Y/Ha - .-.- DAWHCC
WAVELENGTH,
-._.MB-R/TBt -.-. - MB-Y/ TM
.-.
- -
DAB/TBe
WAVELENGTH,
mp
(a) (b) FIG.48. a. Graph showing the transmission of light through a layer 1 cm. thick of DAB/HCl, DAB-Y/HCl, and DAB-R/HCl. b. Graph showing the transmission of light through a layer 1 cm. thick of DAB/Tol, DAB-Y/Tol, and DAB-R/Tol.
CYTOLOGICAL STUDIES WITH CARCINOGENS
305
VI. Conclusion To find what cytoplasmic components have an affinity for the carcinogenic azo dyes o-aminoazotoluene and p-dimethylaminoazobenzene, the OYG method by which the carcinogens within the cells are revealed with Mayer’s reagent was tried with amphibian blood cells and somatic cells. Before this study the reliable forms of the cellular components such as the silvered Golgi apparatus and mitochondria were re-examined. The results obtained are as follows: 1. In amphibian erythrocytes two kinds of granules are recognized which are considered to be the Golgi bodies and mitochondria. Moreover, the Golgi bodies as well as mitochondria are divided into two kinds according to chemical nature and occurrence, but they have a strong affinity for the carcinogenic azo dyes. 2. Differentiated mammalian erythrocytes have mitochondria which have an affinity for the carcinogenic azo dyes. 3. The fact that the OYG method easily detects mitochondria in the erythrocytes is attributed to the removal of hemoglobin with hydrochloric acid in the dye bath of the OYG method. 4. The Golgi apparatus detectable with silvering is apparently caused by the fact that the Golgi argentophil substance becomes silvered. Since the Golgi substance is not found in the alveoli, or at the surfaces of the alveoli, but is found around the preexisting reticula appearing in the interstices of the alveoli at the Golgi area, the silvered images are dependent upon the forms of the lacunae studded with the reticula. 5. Since the argentophil Golgi substance, ribonuclear protein, calcium salt, and phosphate salt are detected around the reticulum, alkaline and acid phosphatase are demonstrated at the Golgi area. Although lipids and polysaccharides are also detected as the Golgi substance, they seem to have no relation to the formation of the blackened Golgi apparatus. 6. The argentophil Golgi substance in the intestinal cell is of two different qualities, and in the hepatic cells is of two different quantities. In the spinal ganglion cells after section of their axons, the argentophil Golgi substance increases. These argentophil Golgi substances have an affinity for the carcinogenic azo dyes. 7. The so-called filamentous mitochondria are apparently caused by overstaining of the true mitochondria and other cellular components. The true mitochondria are of the granular form appearing in rod shape, and have an affinity for these dyes. 8. The affinity of these dyes for the nuclear substance is less conspicuous.
306
YOSHTMI NAGATANI
9. The affinity of these cytoplasmic components for these dyes seems to be attributable to the fact that these components consist of phospholipids and phosphate salt and that the carcinogenic azo dyes are fat soluble, acid soluble, and water soluble. 10. The bound azo dyes in the cells are extracted with difficulty.
Acknowledgments The author wishes to express his hearty thanks to Prof. I. Motomura of the T6hoku University for his supervision throughout this work.
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Epidermal Cells in Culture' A. GEDEON MATOLTSY Department of Dermatology, University of Miami, School of Medicine, Miami, Florida
I. Introduction ..................................................... 11. Properties of Epidermal Cells in Vivo ........................... 111. Properties of Embryonic Epidermal Cells in Vitro ............... A. Organized Growth ........................................... B. Unorganized Growth ......................................... C. Wound Healing .............................................. IV. Properties of Postnatal Epidermal Cells in Vitro .................. A. Organized Growth ........................................... B. Unorganized Growth ......................................... C. Wound Healing .............................................. V. Remarks on Keratinization ....................................... Acknowledgments ................................................ References .......................................................
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I. Introduction The explantation of skin has been of considerable importance in the development of tissue culture. Ljunggren (1898) maintained human skin in vitro in ascites fluid, and proved its survival by reimplantation, only 13 years after the basic experiment of Roux (1885) in which chick medullary plate was cultured. Early progress included the work of Kreibich (1914), who explanted adult human skin on blood or peptone agar. This author observed amitotic cell division in the epidermis, and migration of epidermal cells over the cut surface of the dermis. Results significant in terms of growth, proliferation, and differentiation were obtained only after introduction of more refined technics, particularly the cover slip hanging drop method of Harrison (1912) and Carrel and Burrows (1911) ; the ingenious watch glass technic with a cellulose supporting net (Fell and Robinson, 1929; Shaffer, 1956) ; short term maintenance in buffer solution (Bullough and Johnson, 1951) ; and long term propagation of cell lines (Earle, 1948). In plasma-embryo extract mixture on cover slip, epidermal cells grow in both organized and unorganized fashion. In such cultures, properties 1 The experimental work, described in this paper, was started in the Dermatological Research Laboratories, Harvard Medical School and Massachusetts General Hospital, and continued in The Rockefeller Institute and University of Miami and was aided by grants from the National Institutes of Health (RG-3921,C-4036, 26-224) and the American Cancer Society.
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of epidermal cells of embryos as well as of adult organisms were successfully studied. When cultured on a plasma clot in a watch glass, embryonic epidermis develops in a manner very similar to that in vivo. This technic then provides an excellent opportunity to study by an in vitro method proliferation, differentiation, or keratinization of embryonic epidermis. Bullough’s flask technic, utilizing liquid medium, is most satisfactory for short term investigations of the proliferative capacities of adult skin. The long term technics of Earle provide opportunity to study the unique properties of a cell population under controlled experimental conditions for many months or years. It has been well documented that explanted skin carries with it the capacities with which it had been most concerned in its original location ; whether it was derived from an embryonic, newborn, or adult organism, it will tend to proliferate, grow, differentiate, or keratinize according to its major function in the site from which it was taken. For example, epidermis derived from a young embryo, which in vivo grows rapidly both in thickness and in area (lateral growth), will proliferate and differentiate rapidly in tissue culture. Similar events cannot be expected, however, in explants of an adult organism, since it is relatively slow growing in vivo, is of constant thickness, and lateral growth does not occur normally. The epidermal cells of adult skin therefore, under appropriate culture conditions, reveal but few mitoses and a slow but continuous process of differentiation. A perfect identity of in vivo and in vitro events, however, cannot be expected to occur. In the strictest sense, the explanted skin is altered by wounding (excision) and the artificial medium and environment affect its normal functions. An immediate sign of such an effect might be a general slowing of all tissue functions (lag period). Necrotic cells appear at the edge of the skin fragment soon after explantation. The surviving cells at the edge behave as they might at the edge of a wound, and start to migrate vigorously along the cut edge of the explant or move out into the medium. These migratory cells may maintain some of their inherent capacities for a period of time, but more often their morphological, chemical, behavioral, and developmental properties become somewhat modified. I n the central area of the explant, however, structural integrity of the epidermis is well maintained. This part of the explant rapidly adapts to .the new environmental conditions and continues to function relatively normally for some time. The properties of cells in vitro may best be understood when compared to their behavior in vivo. Therefore in the following paragraphs, some of the in vivo properties of the normal epidermis and of wounded epidermis are recalled for comparison and general information. Properties of epi-
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dermal cells in vitro will be reviewed with respect to organized growth, unorganized growth, and activities concerned with repair. Embryonic and post-natal epidermis are treated separately. Specific reference is made to epidermal cells derived from the chick, mouse, guinea pig, rat, rabbit, and the human. Other species are not included. 11. Properties of Epidermal Cells in Vivo
Structural and histochemical changes of the chick epidermis during development are illustrated in Fig. 1. It can be seen that the thin epithelial sheet of the early embryo rapidly proliferates and by the 6th day of embryonic life consists of two layers. The upper layer, the periderm, contains flat epithelial cells with oval nuclei oriented with their long axis parallel to the skin surface. The lower layer consists of cuboidal cells with spherical nuclei. This epidermis reacts weakly with the -SH reagent and appears isotropic in polarized light (Fig. 1, &day). Between the 6th and 14th days of embryonic life the epidermis thickens, the cell sheet underlying the periderm becomes multilayered and its cells tend to assume a columnar shape. The outer surface of the periderm now shows a strong -SH reaction, while the rest of the epidermis still appears isotropic (Fig. 1, 10-day and 14-day). Around the 16th day of embryonic life the intermediate epidermal cells begin to flatten into squamous cells. The cells of the periderm become extremely flattened and some of the nuclei degenerate. This periderm reacts intensely with -SH reagents and also reveals a very weak birefringence. The rest of the epidermis continues to be weakly -SH positive and appears isotropic in polarized light (Fig. 1, 16-day). The cornified layer is formed from the periderm and from some of the underlying cell layers between the 16th and 18th days of embryonic life. The cornified layer is about 4 to 5 cells thick on the 18th day and consists of anucleated horny cells. It shows an intense -SH reaction and also reveals moderate birefringence. The rest of the epidermis is moderately reactive with the -SH reagent and isotropic in polarized light (Fig. 1, 18-day). The epidermis assumes its final state between the 18th and 21st days of embryonic life. During this period it becomes thinner and develops a thick cornified layer which is strongly -SH positive and shows intense double refraction (Fig. 1, 20-day). Because the stratum corneum reveals a more intense reaction with the -SH reagent after treatment with a reducing agent, it can be assumed that the keratin structure is stabilized bonds at this stage of development (Matoltsy, 1958a). by a few -S-SFigure 2 shows the thickness of the epidermis in relation to days of development. The curve can be divided into two characteristic parts. The
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first part shows increases in thickness from the 6th to the 16th days in an S-shaped curve, characteristic of growth. The second part, starting on the 16th day, reveals a further increase for two days, followed by a decrease up to birth. These changes in thickness of the older epidermis coincide with the period of keratinization and appear as characteristics of the process (Matoltsy, 1958a). The epidermis of the chick, mouse, rat, or human embryo develops essentially the same way. The stage of periderm-cuboidal cells shown in Fig. 1, for 10-day chick embryo, is reached on the 12th day of embryonic life by rats and around the 4th month in humans (Pinkus, 1910). These dates are all close to half periods of the embryonic terms of these different species. A main difference in epidermal structure between avian and mammalian epidermis is the absence of stratum granulosum in the avian epidermis (Fell, 1957). In the mouse embryo the stratum granulosum develops on the 14th day, in the rat on the 18th (Hanson, 1947), and in humans around the 6th month of embryonic life. The characteristic increase in epidermal thickness followed by a decrease in late chick embryos, related to keratinization, takes place during the same period of late development in mouse embryos. In rat embryos it starts only on the 19th day of embryonic life (Hanson, 1947). Reduction in thickness of the epidermis before birth also was noted by Fraser (1928), Pic6n (1933), David ( 1934), Gibbs ( 1941) , and Gliicksmann ( 1945). In the embryonic epidermis, cell division is the function of the undifferentiated cells, i.e. the cells of the stratum germinativum and those of the periderm before they begin to keratinize. The differentiating cells of the stratum granulosum, stratum lucidum, or stratum corneum do not divide (Hanson, 1947). During the latter half of embryonic life, and at all times after birth the excessive epidermal cells shed from the surface. Their replacement is secured by newly formed cells in the germinative layer, which in turn differentiate into horny cells. Mitotic activity in the epidermis is rhythmic (Carleton, 1934 ; Blumenfeld, 1939). Mitotic frequency in mouse and rat epidermis is highest at 1 :00 p.m. (Bullough, 1948; Kiljunen, 1956). In human epidermis, mitoses are more frequent at night than in the morning hours (Cooper and Schiff, 1938; Cooper, 1939; Broders and Dublin, 1939). Mitotic frequency and the thickness of the epidermis is affected FIG.1. Survey of epidermal development in the scalp of chick embryos. Numbers indicate age in days. Left : Hematoxylin-eosin stained preparations. Middle : Demonstration of -SH groups by Barrnett and Seligman’s (1952) technic. Right : Unstained preparations in polarized light.
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by systemic factors, such as the hair growth cycle (Butcher, 1934; Chase et d.,1953) and hormones (H. F. Bullough, 1943;W. S. Bullough, 1950, 1953;Ebling, 1954). Mitotic rate is high in the infant mouse epidermis and it is lower at maturity. It increases during middle age and decreases in old animals (Bullough, 1949). Storey and Leblond (1951) and Bertalanffy (1957) find that in the rat or mouse epidermis 5.2% of the cells undergo division each day and the entire epidermis is renewed in 19.1 days. Renewal of the guinea pig foot pad epidermis was estimated at 42 to 45 days (Volkmann, 1950). 24
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FIG.2. Thickness of the epidermis (scalp) plotted against days of epidermal development. The epidermis of an adult human skin is renewed in 32 to 36 days (Volkmann, 1950). According to Fular (1953) during a 10-day period the average loss on the forearm is only 2.5 layers, and on the knees or elbows 12.5 layers. Pinkus (1954), assuming a mitotic duration of one hour, calculated that only one mitosis per 600 basal cells is theoretically required to replace one stratum corneum cell per day. His geometrical calculations reveal that an area on the surface covered by a single strongly flattened cornified cell is identical to an area of 25 cells in the basal layer, each of these basal cells being available to give rise by mitosis to a new cell for replacement. In view of the above, it is not surprising to find an extremely low mitotic activity in the adult human skin; mitotic indices are found to vary from 0.1 to 1.6 (Pinkus, 1957).
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The epidermis inhibits diffusion of fluids into the organism as well as prevents evaporation of body fluids to a considerably high extent. Blank (1952, 1953), Blank et al. (1957), and Szakall (1958) assume that the barrier is located in the compact lowest layer of the stratum corneum. Monash (1958) believes that the functions of a barrier are subserved by practically the entire thickness of the stratum corneum.
I
I
I
FIG.3. Summary tabulation of skin wounds made in chick embryos. Open bars indicate non-healing ; cross-hatched bars, healing in progress ; and solid bars, healed wounds. In the extensive literature on grafts, healing of the embryonic epidermis is primarily related to migration of epithelial sheets whose free margins move over the defect. Proliferation and differentiation of the emigrated epidermal cells follows secondarily and continues until perfect structural continuity is reestablished. Weiss and Matoltsy (1957, 1959) studied the healing of skin wounds in chick embryos and found that healing of the epidermis varies significantly with age of the embryos (Fig. 3, upper part). Migration of the epithelium to cover the wounds was absent during the
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first half of the embryonic period, whereas healing was normal in embryos wounded during the second half. It was also noted that wounds made during the first half of the embryonic term became covered by epithelium only after the embryo passed half term (Fig. 3, lower part). Non-healing was due to migratory failure rather than to any impairment of growth and proliferation of epidermal cells. The continued growth without migratory expansion caused the epidermal cells to pile up at the edge of the wound instead of spreading over the denuded surface. A full and satisfactory explanation has not yet been offered for the non-healing nature of the young epidermis. Layton et al. (1958) suggest that newly synthetized acid mucopolysaccharides may be partially responsible for the migratory stimulus affecting the epithelial cells. They assumed that failure of healing of epidermal wounds in the chick embryo prior to the midperiod of the embryonic term may be due to the lack of enzyme systems responsible for synthesis of acid mucopolysaccharides, or the embryo may not yet have cells of the type or maturity required for secretion of the particular sulfated polysaccharides. Healing of mammalian embryonic epidermis was studied by Hess (1954) in guinea pig fetuses from 37 to 48 days in age. It was noted that, when the skin on the vertebral column was severed in zctero, the epithelium made no attempt to migrate for 2 days. After this lag period, however, a marked migration of epithelial cells occurred, forming large epithelial tongues. After 4 days a large percentage of the embryos exhibited healed wounds. On the 5th day, remodelling of the hyperplastic epithelium started and persisted more than 12 days. The steps in healing, although more rapid, were essentially the same as in post-natal animals. Since Hess did not study wound healing in embryos younger than 37 days old, information is not available whether non-healing of epidermal wounds in young embryos occurs only in the chick or is also a property of other species. Epidermal wounds of adult organisms are rapidly healed by simple migration of epidermal cells from the intact portion of the epidermis. The characteristic morphological events of this process are as follows : The epidermal cells next to the wound border release their tight connections with the basement membrane. Subsequently, they migrate and remain in close contact at all times with the dermal surface. Migration continues until closure is complete. This is followed by proliferation, differentiation, and reconstitution of the normal architecture. In the case of larger epithelial wounds the number of mitotic cells increases in the more distant parts of the epidermis. These loci then supply epidermal cells for wound closure and for replacement of those cells which have
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previously migrated (Arey, 1932, 1936; Needham, 1952 ; Gillman and Penn, 1956 ; Chu, 1956 ; Bullough and Laurence, 1957). Pinkus (1952) and Lobitz and Holyoke (1954) noted that if the cornified epithelium of the adult human skin was removed by repeated stripping of the skin surface with Scotch tape, repair proceeds in the following manner. The first response occurs not in the upper part of the epidermis, near the damage, but in the basal layer, where the epidermal cells swell to almost twice their original size. This is followed by considerable unrest in all epidermal layers and by accumulation of glycogen in the basal cells in large quantities. A peak of mitotic activity is reached in the germinative layers of the damaged epidermis only after 48 hours. The newly formed cells are displaced to higher levels and in turn differentiate into parakeratotic cells. At a later time this parakeratotic layer sloughs off, and is replaced by normally keratinized cells. 111. Properties of Embryonic Qidermal Cells in Vifro
A. ORGANIZED GROWTH Organized growth of the chick epidermis in culture was observed by Strangeways and Fell (1926) when they studied differentiation of various tissues of the limb bud. The limb bud growing on a thick plasma clot was seen invested by a stratified coat which became progressively thicker and also keratinized as the age of the culture advanced. Under less favorable culture conditions, such as in a thin plasma clot, keratinization of the epithelium was slower. Miszurski (1937) placed a foot or the end of a wing-rudiment of the 6-day chick embryo on the surface of a clot. Although intense proteolysis occurred and the clot was liquefied within a few days, the fragment grew in volume during the first days. The epithelium proliferated intensely and the original two-cell thick epidermis changed into three-cell thick tissue at a total age of 8 days. At a total age of 11 days the epidermis further increased in thickness. The basal layer consisted of cuboidal or cylindrical cells above which were several layers of polygonal cells containing little chromatin and large nucleoli. The uppermost layer consisted of flattened cells having small, elongated, and darkly stained nuclei. Further development was abnormal because at the total age of 14 days the epidermal cells started to degenerate and only a thin basal layer remained alive. Miszurski also noted if the cut surface of the explant was not epithelized and connective tissue strands grew out into the medium, development of the epidermis was more like the normal and keratinization occurred as well. With the introduction of the watch glass technic (Fell and Robinson, 1929) excellent opportunities were created for investigation of in witro
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development of the chick epidermis. This technic was improved by placing a cellulose acetate fabric between the explant and the plasma clot (Shaffer, 1956). Since the fibroblasts rapidly grow into the frames of the fabric, the skin fragment is tightly bound to it. Accordingly, the explant does not curl and can be easily manipulated while it is bathed or transplanted to a new medium. Using this technic, Fell (1957) vividly demonstrated in vitro development of the epidermis in skin fragments derived from chick embryos at different ages. She noted that the epidermis of 7-day chick, consisting of a layer two cells thick, undergoes rapid proliferation and differentiation and at a total age of 13 days it is already keratinized. If the skin was taken from 13-day chick, the epidermis developed in a manner quite similar to that in vim. The epidermal cells seemed to proliferate at a normal rate and at a total age of 17 days the epidermis acquired a distinct stratum corneum, most similar to that in vivo. It was also noted that on further cultivation of this skin for a 28-day period the epidermis steadily increased in thickness and reached the stage of full development. If the skin was excised from 18-day embryos it remained healthy during a 20-day observation in culture, showed plentiful mitoses in the basal layer, and the stratum corneum progressively thickened. These observations of Fell indicate that development of the chick epidermis in culture progresses in a manner most similar to that in vivo, if the skin is taken from donors 13 or 18 days of age. Differentiation, however, is accelerated if the skin is derived from younger embryos, such as a 7-day chick. Matoltsy (1960) cultured skin fragments of 6-, 8-, lo-, 12-, 14-, and 16-day chick embryos in watch glasses by Fell and Robinson’s (1929) technic. Fell’s (1957) results were confirmed. The process of differentiation in the epidermis of 6- and 8-day skin fragments was noted to be accelerated, whereas fragments taken on the 12th, 14th, or 16th day of incubation reveal a development quite similar to that in viva The best correlation between in vivo and in vitro differentiation of the epidermis was seen when skin fragments of 10-day embryos were cultured. The results are shown in Fig. 4, revealing almost identical structures seen during in vivo development (compare Fig. 4 with Fig. 1). The only difference found was the persistence of the periderm in cultured skin, which keratinizes in vivo. Formation of keratinizing pearls in the epidermis of explanted tissue fragments was described by Fell ( 1929). Miszurski ( 1937) experimentally produced such pearls and studied the mechanism of keratinization. He stripped most of the covering epidermis of the limb bud of 6-day chick embryo and placed it into hanging drop culture. After 1 or 2 days the
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remnants of the epidermis were enwrapped from all sides by connective tissue. At various places on the limb bud fragment, small islands were formed. Within these islets the epithelium closed up on itself and formed either solid pearls or hollow cysts. In the very center of the islet some degeneration occurred at about the 4th day of culturing. In the next 2
FIG.4. Survey of is vitro epidermal development in scalp of chick. Uppermost picture shows the explanted epidermis of a 10-day embryo. Numbers on the other pictures indicate total age of the cultured tissue. or 3 days most of the islets consisted of multiple concentric layers of epithelial cells. The outermost layer corresponded to the basal layer of the epidermis. On the 11th day, at the total age of 17 days, mitoses occurred in many basal cells, (the intermediate cells were differentiating and the cells in the center kerdtinized. After a month the islets appeared normal without any trace of degeneration. Keratinization was complete
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and the mechanism apparently identical to that of the normal process. Litvac ( 1939) repeated Miszurski’s experiments and studied the distribution of -SH groups during keratinization. She noted that at a total age of 12 days a strongly positive -SH reaction was present in the keratinizing epithelium above the germinative layer. A few days later, when the first keratin lamellae appeared, the reaction still persisted above the basal layer as well as in the lamellae. At a total age of 20 days the germinative layer and the mature keratin were negative and the region above the germinative layer was weakly positive. She also noted that 10day-old keratin, formed in the culture, can be digested with pepsin but not with trypsin. A high degree of self organization capacity of chick epidermal cells in culture was ingeniously demonstrated by Weiss and James (1955) by preparing skin “dissociates” by Moscona and Moscona’s ( 1952) trypsinmechanical separation technic. The skin was stripped from the back of 7- to 8-day-old chick and dissociated into single cells. After centrifugation of the cell suspension, the sediment was cut into small pieces and planted on the surface of plasma clots in Maximow slides. At a total age of 10 days the dissociated cells reassembled. The epidermis showed an advanced stage of differentiation, revealing squamous cell layers three to four cells thick. The nuclei in the superficial layers of the epidermis began to fade at a total age of 11 days; and on the 12th day a conspicuous sheet of horny layer was present. I t was noted that the reaggregated cells formed not only a structurally normal epidermis but also pearls and cysts similar to those noted by Fell (1929) and Miszurski (1937) in epithelial islets. The intensity and time schedule of the steps taking place during reaggregation and differentiation varied somewhat with size and shape of the cyst or pearl formed. The capacities of epidermal cells were tested in “tissue aggregates” by Matoltsy (1960). The epidermis is very loosely attached to the dermis on the neck of 1Cday chick embryos, and can be readily stripped without trypsinization. Such epidermal strips were suspended in Earle’s solution containing 20% horse serum and continuously shaken for 48 hours. In some parts of the tissue aggregates the epidermal cells formed tubules with a well-developed basement membrane (Fig. 5). The epidermal fragments at the periphery did not differentiate but thickened greatly by proliferation of basal cells. In the center of the aggregates masses of round cells intermingled with masses of degenerated or autolyzed cells. These results indicate that the epidermis in the absence of dermis diverts from its normal course of development and its cells instead of forming keratin assemble into tubes.
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FIG.5. Upper picture shows aggregates of isolated chick epidermis, cultured in fluid medium for 2 days. Note tubule formation, intense downward proliferation of the lining epithelium and cytolysis in center. Lower picture shows tubules with basement membrane at high magnification.
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Champy (1914) and Drew (1922) noted that if the skin is cultured with a large amount of dermis, keratinization is frequent, whereas if epithelium was grown without fibroblasts, keratinization was less abundant. Accordingly, it was assumed that differentiation in epithelial tissues is conditioned and controlled by the stroma. The importance of the stroma in differentiation of epidermal cells was recently demonstrated by the thoughtful experiments of McLoughlin ( 1958a). The epidermis of 5-day chick was separated from the dermis by trypsin and placed on denuded limb, proventriculus, or gizzard mesenchyme as well as on heart mesenchyme. On limb mesenchyme the epidermis developed normally and formed keratinizing epithelium, whereas on proventriculus mesenchyme a mucin secreting epithelium developed at first, which later keratinized. On gizzard mesenchyme a mucus secreting epithelium was formed. If the epidermis on heart became surrounded by myoblasts, mucus secreting cysts were formed, whereas when it was in contact with fibroblasts keratinization occurred. McLoughlin (1958b) also noted that separated epidermis of 5-day chick cultured on agar coated cover slip is capable of differentiating into keratinizing epithelium. The isolated epidermis failed to grow on a glass surface without agar. The outstanding work of Fell and Mellanby (1953) and Fell (1957) on the metaplasia producing effect of excess vitamin A in cultures of embryonic chick skin was reviewed in great detail in Volume V I I of the International Review of Cytology, by Lasnitzki (1958). It also includes Weiss and James’s (1955) work with skin dissociates which were exposed to a single treatment of vitamin A. In recent studies Pelc and Fell (1960) attempted to identify metabolic changes of epidermal cells caused by vitamin A. Incorporation of SS6-DL-cystine and SS6-DL-methionine, C14leucine and C14-tyrosine, H3-thy&dine, C14-adenine, and Sa6-su1fate was studied using 2- or 3-day cultures of 13-day embryonic skin. Autoradiographic observations indicated that vitamin A stimulated the incorporation of adenine and thymidine into differentiating cells, whereas incorporation of cystine was greatly reduced throughout the entire epidermis and tyrosine was diminished in the upper layers. Maximow (1925) cultured rabbit skin, Hardy (1949) and Hanson (1950) mouse and rat skin on cover slips. Hiebert (1959) planted mouse embryo ski,n in watch glasses according to Shaffer’s (1956) technic. Early development of the rabbit epidermis was normal, abnormalities were noted only during keratinization. In mouse and rat skin explants on cover slips variations were noted in the rate of differentiation among cultures of the same age and parentage. In watch glasses, the 15-day-old mouse skin differentiated into a stratum basale, stratum spinosum, stratum
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granulosum and a thick stratum corneum. A week after explantation both spinous and granular cells keratinized. Chlopin (1932) cultured human skin derived from 2-month-old fetuses on cover slips. The epidermis, originally two cells thick, progressively thickened during the first week and changed into a multilayered tissue consisting of mitotically active basal cells, spinous cells with tonofibrils, and intercellular bridges covered by several layers of flat cells. The periderm persisted and was easily distinguishable. Around the 8th to 12th days the uppermost flat cells changed into nucleated, abnormally keratinized parakeratotic cells. Following this, all strata above the basal layer keratinized and the living portion of the epidermis finally consisted only of a narrow layer of basal cells. Chlopin concluded that the human embryonic epidermis in cultures undergoes an atypical differentiation and resembles normal keratinization only in part. Since the human embryonic epidermis in a 2-week period passed through stages which in vivo last for 2 to 4 months, it is evident that its differentiation was highly accelerated in vitro. B. UNORGANIZED GROWTH Epidermal cells emigrating from the explant have been studied by Carrel (1922). H e attempted to obtain a permanent strain of epithelium from embryonic chick skin, but did not succeed; after 2 or 3 weeks the cultures were always invaded and overgrown by fibroblasts. Similar fibroblastic invasion was noted in hanging drop cultures of embryonic human skin by Bornstein (1930). Drew (1922) was more successful in growing embryonic mouse or rat skin as he noted rapid and vigorous growth of epithelial membranes on cover slips. Chlopin (1932) using the skin of 2-month-old human fetuses observed that the epidermal cells prefer to migrate directly on the glass surface and liquefy the clot. In cases of extensive liquefaction, outgrowth of fibroblasts from the explant was hindered. The emigrating epithelial cells formed continuous membranes two to three cells thick by the 3rd or 4th day of culture. Emigration took place from both layers of the epidermis ; periderm and cuboidal cell layer. The epithelial membranes continued to grow both in thickness and in lateral direction after removal of the original explant. Some differentiation of cell constituents was noted, but the membranes never keratinized. Bassett et d. (1956) cultured 2- to 4-month-old embryonic skin from human fetuses with Evans and Earle’s (1947) long term culture technic in placental cord serum and filtered chick embryo extract. Three days after planting, luxuriant migration and proliferation of both epithelial and fibroblastic elements occurred. Both epidermal cells and fibroblasts migrated out from the skin fragment at the same time. It appears significant
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that during a &month observation period the epithelial cells did not form sheets, but rather migrated as individual cells. In the peripheral region of a skin explant, regardless of the technic used, some cells always migrate on the surface of the stroma. The encasing cells proliferate but usually do not differentiate in exactly the same way as cells do in the organized part of the epidermis. In the encapsuled region the basal cells often remain flat, and do not change into cuboidal or columnar cells. The cytoplasmic components may show limited differentiation. The outermost cells do not keratinize normally but resemble parakeratosis (Matoltsy, 1960). Complete encasement of a skin fragment may terminate its life, as fluids can no longer freely diffuse into the inside of the tissue. Miszurski (1937) found that epithelium even if not entirely keratinized prevents diffusion of nutritives and oxygen into explants and leads to cell degeneration, and cell death. A satisfactory explanation has not yet been presented on plasma liquefaction by epidermal cells in culture. Although it is quite clear that enzymes are involved in fibrinolysis, it is not known whether the enzymes are released by the living cells or only after the breakdown of the cells. In relation to this it is noteworthy to recall that Chlopin (1932) called attention to cytolysis occurring by the 3rd day in epithelial cells grown out of embryonic skin fragments. Fibrinolysis is not a specific property of epidermal cells. Other epithelia, such as the corneal, iris, lens, gall bladder, or pulmonary epithelium, thyroid cells, or cancer cells, etc. also show this phenomenon in culture (Matsumoto, 1918; Fischer, 1922, 1924; Erdmann, 1923; Chlopin, 1924 ; Policard, 1925 ; Ebeling, 1925 ; Lang, 1926). C. WOUND HEALING Healing of epidermal wounds in skin explants proceeds by a combination of unorganized and organized growth processes. Weiss and Matoltsy (1959) cultured skin of 6- to 8- or 10-day chick embryos on rayon acetate netting on top of a plasma clot in watch glasses for 4 to 6 days. After 1 day of culture, minute wounds were made with the tip of fine forceps in the center of the explants and marked by carbon grains. At a total age of 10, 12, or 14 days, contrary to the observations made in vivo (see Section I I ) , the epidermal cells vigorously migrated on the denuded dermal surface toward the center of the wound (Fig. 6A). On the 16th day of total age, the wounds were covered by hyperplastic and excessively keratinized epithelium (Fig. 6B). In the vicinity of the wound mitotic activity was increased. Intense cell proliferation in these regions yielded new cells far both normal differentiation and repair of the lesion. After wound closure, these processes were usually uncontrolled,
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as multiple layers of abnormally keratinized, parakeratotic cells were produced at a rapid rate. Healing of wounds in skin fragments of full term rat embryos in vitro was studied by Bentley (1935). He made circular wounds extending into the dermis. Spreading of the epithelium along the surface of the defect was noted after 18 hours. Proliferation occurred after 36 hours and the
FIG.6. Wounds in embryonic chick skin observed in dtro. A. Epithelium passed well beyond carbon grains marking the edge of the wound at the total age of 12 days. Skin was explanted on the 7th day of incubation. B. Hyperplastic and excessively keratinized epithelium, after wound closure. Skin was explanted on the 9th day of incubation and healing is shown on 16th day of total age.
area of the defect was fully covered and healed in 54 hours. If these results are compared with Hess’s (1954) in Vivo studies, it can be seen that the lag period in vitro is reduced and the process of healing is considerably accelerated.
IV. Properties of Postnatal Epidermal Cells in Vitro A. ORGANIZED GROWTH As mentioned, adult skin was successfully maintained in ascitic fluid by Ljunggren as early as 1898 and its survival proved by reimplantation in the donor. Half a century passed before this line of investigation was
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renewed and Ljunggren’s technic successfully improved. Medawar ( 1948a) based his technic on Parker’s (1936, 1937) observations that adult tissues need more oxygen to grow in culture than embryonic tissues and they adjust faster in a fluid medium than in a clot. He floated lightly vaselined skin fragments of adult rabbit skin on serum in an atmosphere of airpxygen mixture and kept them agitated at body temperature. By this technic, skin fragments were kept alive in a satisfactory condition for 6 to 8 days. After reimplantation of the cultured skin to a raw area of the donor’s chest, the skin adhered normally and widespread growth of strongly hyperplastic epithelium was noted 10 days later. In the epidermis of cultured skin, cell divisions were abundant and slightly more frequent at the thickened periphery of the fragment than elsewhere. Increased keratinization or parakeratosis was not noted. When skin slices were incubated in hydrogen, cell division was absent and migratory activity of epidermal cells failed (Medawar, 1947). The explants, however, did not die but survived for about a week. Their survival was proved by the vitality of the explants after reimplantation. Medawar (194Sb) also noted that the normal immunological reaction of skin is altered in vitro. Homologous skin grafts did not give the expected in vivo reaction when cultured in the immune serum. A simple and useful method for culturing human skin was recently devised by H. Blank et al. (1959) based on Chen’s lens paper technic (1954). Thin slices were cut with the Stadie-Riggs (1944) microtome from the prepuce of infants received immediately after surgery. A microculture slide with straight wall was entirely filled up with human serum, diluted with Hanks’ solution. Subsequently it was covered with lens paper and the skin slices placed on the center. Mitoses were seen in varying numbers in the epidermis even after 4 weeks. Although commonly the outermost layers of the epidermis degenerated, almost invariably a new stratum corneum was formed under it (Fig. 7A). Trowel1 (1959) used the skin of newborn rats and successfully maintained it for a short period in his TS-synthetic medium, containing amino acids, salts, glucose, and some other substances. The skin fragments were unaltered for 3 days on a tantalum wire grid in a well-oxygenated chamber. ~~
FIG.7. A. Human infant skin cultured for 3 days on lens paper by Blank et al. (1959) technic. Note mitosis and formation of a new horny layer. (Courtesy of Dr. E. W. Rosenberg.) B. Adult human skin cultured for 3 days by Lewis et al. (1949) technic. Note flattening and emigration of germinative cells and degeneration of the upper cells in the epidermis. C. Adult human skin after 3 days of culturing. Surface was stripped with adhesive tape prior to culturing. Note absence of degeneration in the upper cells.
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On the 6th day the basement membrane seemed to dissociate and the epidermal cells and the stroma cells intermingled. Differentiation was accelerated and a large amount of extra keratin was formed. Although differentiation continued its course, the epidermal cells did not multiply. Human and rabbit skin was also cultured in hanging drop preparations in a plasma clot and maintained for quite a long time. Bornstein (1930) cultured the skin of young humans and kept it alive for 90 days. Blazd (1931) used young rabbit skin. In agreement with Hsu (1952), Matoltsy (1956a) found that the adult human skin is viable only for 7 to 10 days in hanging drop cultures prepared according to the technic of Lewis et al. (1949). In such cultures the rete ridges gradually disappeared and the whole epidermis markedly flattened. In the basal layer the cells soon loosened up and started to migrate toward the periphery. Mitoses were not noted. The cells of the upper layers (corresponding mainly to the spinous cells) appeared slightly increased in size and showed a small, intensely stained pyknotic nucleus (Fig. 7B). In older cultures these same cells may change into strongly dehydrated cells with weakly staining nuclei, resembling the picture of parakeratotic cells. In hanging drop cultures of human skin, Pinkus (1932, 1938, 1954) also noted these degenerative changes of the upper epidermal cells. In subcultured skin he noted mitoses in basal cells in the central portion of the explant, even on the 12th day after explantation. The keratohyalin granules soon disappeared from the granular cells and Pinkus (1938) did not see them reformed. Matoltsy (1960) found that survival of epidermal cells of the adult human skin in plasma clot is greatly enhanced if the cornified epithelium is repeatedly stripped with adhesive cellophane tape prior to culture. In such cultures the cells above the basal layer remain viable for a longer period than in the unstripped skin (Fig. 7C). The studies with stripped and unstripped skin emphasize the possibility that, while the barrier of the epidermis is intact (Blank, 1952, 1953; Blank et d.,1957; Monash, 1958; Szakall, 1958) nutrients of the culture medium reach the epidermal cells mainly by passing through the dermal residue or by penetration from the sides of the explant at the cut edges. Requirements for the maintenance of epidermal mitoses in vitro were studied by Bullough and Johnson (1951). Fragments, 2.5 X 5.0 mm. in size, were cut from the thin peripheral segments of the adult male mouse ears and were incubated at 38°C. for 4 to 5 hours in saline-phosphate buffer solution. Glucose added in a final concentration of 0.02 M was found to maintain optimal numbers of mitoses, approximately 3 per cm. length of 7 p sections. Since in Vivo mitotic counts averaged 7 to 8 per cm. length, and in accordance with the results of Medawar (1947, 1948a),
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indicating the dependence of mitoses in rabbit epidermis in vitro on the oxygen tension, studies of the gaseous phase of the incubates were undertaken. Pure oxygen was found to increase the number of mitoses to 5 per cm., in contrast to 3.5 per cm. in the presence of 95% 0 2 / 5 % COz. The number of cells entering mitosis was said to be directly proportional to the oxygen concentration (Bullough, 1955). Further work by Bullough and associates was designed to test the roles of glycolysis and the Krebs cycle in providing energy for mitosis. Fructose, pyruvate, lactate, and certain Krebs cycle intermediates were said to substitute more than adequately for glucose in maintaining mitoses (Bullough, 1955). The stimulus of insulin and growth hormone on mitosis in vitro, plus the ability of the aforementioned substances to maintain mitosis, were interpreted to indicate a relative inefficiency in the transport of glucose across the cell membrane via hexokinase. These phenomena and the decreased ability of glycolytic substrates to support mitosis in the presence of inhibitors of glycolysis and the Krebs cycle were taken to indicate the dependence upon energy provided by these pathways for mitosis in vitro. The evidence that energy derived from these sources is of prime importance in maintaining in vitro epidermal mitoses was disputed in a series of investigations by Gelfant. Utilizing a similar technic of culturing fragments of mouse ear in saline-phosphate buffer solution he found optimal glucose concentration for maintenance of mitoses to be 0.002 M, and that only at this concentration did mitoses parallel oxygen tension (Gelfant, 1959a). The effects of low oxygen tension were attributed to loss of viability rather than an effect on mitotic ability. The sine qua non for the actual stimulus of mitosis by this method was stated to be the physical act of cutting (trauma) of the tissues, rather than any combination of glucose and oxygen (Gelfant, 1959b). The ability of fructose, pyruvate, and lactate to support mitoses in numbers equal to those in the presence of glucose was confirmed, but the synergistic effect of oxygen could not be demonstrated for the alternative substrates. Furthermore, the Krebs cycle intermediates citrate, a-ketoglutarate, and succinate could not be found to substitute for glucose in concentrations equal to the latter (Gelfant, 196Oa). Inability to reverse the effects of metabolic inhibitors with appropriate substrates, and the general toxicity and lack of specificity of these substances were thought to contradict the interpretations of Bullough of the role of glycolysis and the Krebs cycle in maintaining mitoses in vitro (Gelfant, 1960b). On the basis of these experiments, Gelfant proposed that in fact no direct evidence for the production of energy by the conversion of glucose in vitro has been obtained, and that the mechanism which might explain the singular effect of glucose on mitoses in vitt.0
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remains to be adequately documented. It must be stated, however, that evidence of many workers (reviewed by Brachet, 1957) indicates that an “energy reserve” is probably accumulated just prior to mitoses, derived from carbohydrate metabolism and oxidative phosphorylation. This period, preceding mitosis, was termed “antephase” by Bullough ( 1955).
B. UNORGANIZED GROWTH Unorganized growth of epidermal cells derived from postnatal skin was most often studied in hanging drop preparations. It is characteristic that a relatively long lag period passes before emigration of cells can be noted. Skin samples from various human subjects under identical culturing conditions revealed marked differences in lag period from 24 to 72 hours. Blazd (1931) noted that it is the germinative cells which primarily emigrate from the explanted epidermis ; fibroblastic growth is considered inhibited. Single cells appear at first on the cover slip which is followed by an outgrowth of epithelial tongues. The plasma clot around the tongues is often liquefied and single cells may float in it. Subsequently the tongues fuse and the explant becomes surrounded by a continuous multilayered epidermal sheet (Fig. 8). Microcinematographic observations show (Lewis et al., 1949 ; H u et al., 1951) that the cells which move out first from the fragment vigorously migrate in all directions and some of them even may reunite with the explant. The outgrowing tongues thicken fast and may form 20- to 30cell thick layers. The cells reveal a high degree of motility at their margins, commonly seen as an undulating membrane. I n some of these marginal regions many vacuoles were noted and related to pynocytosis. At the other regions highly mobile filamentous mitochondria can be seen migrating to and from the periphery revealing rotary and bending movements. Intercellular bridges are numerous in areas exposed to mechanical stress. The “outgrowth” area around the explanted adult human skin is formed by both emigrated cells and their descendants. “Growth” seems to occur rather by a continuous flow of epidermal cells from the explant than by intense mitotic activity of emigrated cells. Hsu (1952) noted that the cells in the “outgrowth” area gradually increase in size and that normal mitosis is a rare event (out of 240 preparations only 32 contained mitotic figures). Matoltsy ( 1960) also noted many degenerating and highly polyploid nuclei arrested in mid-phase in such cultures (Fig. 9). HSU’S (1952) detailed analyses show that prophases and metaphases are normal but only a few cells pass beyond this stage. The chromosomes either aggregate and become pyknotic or divide abnormally and form anomalous anaphases. Polyploid cells were quite frequent and some of these were in dividing stages.
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Failure of growth of epidermal cells emigrating from fragments of the adult skin in hanging drop cultures may be related to poor nutritional conditions and defects in nucleic acid synthesis. Hsu (1952) noted numerous degenerating nuclei in cells next to the explant, whereas in a distance more normal nuclei were seen. Feulgen-stained preparations
FIG.8. Adult human skin cultured in hanging drop preparations for 7 days, showing “outgrowth” of epidermal cells. showed that the cells next to the explant are faintly charged with nucleic acids. Hsu assumed that the first emigrating cells are under most favorable condition. They synthesize nucleic acid normally, move away from the explant, and may divide normally. The latecomers pass through a region which is deficient in nutrients, develop defects in nucleic acid synthesis, and therefore mitoses are abnormal. Parshley and Simms (1950) cultured skin in a large and thick clot in Carrel flasks. They found that growth of epidermal cells can be stimulated
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by addition of 216 solution, which contains salts, asparatic acid, and some other chemicals. Doljanski et al. (1942) used heart extract and found that it promoted epidermal growth. Ulloa-Gregori et d. (1950) and Allgover et al. (1952) tested the following substances for growth promoting activity : Autologous and homologous human serum, cord serum, serum
~~
-
~~~
FIG.9. Epidermal cells in the “outgrowth” area of explanted adult human skin. Note numerous highly polyploid nuclei in mid-phase.
from dogs on a fat deficient diet, serum ultrafiltrates and their residues, serum inactivated by boiling, plasma fractions of Cohn, human ascitic fluid, cattle and human hemoglobin and sanguinum, red blood cell and buffy coat suspensions, d-panthothenyl alcohol, chlorophyl, scarlet red, vitamin C, and 216 solution of Parshley and Simms (1950). While ascitic fluid and cord serum had a significant growth promoting effect, none of the others
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gave better results than human serum. Their test consisted of placing a fragment of adult human skin in hanging drop preparation in a medium of 1 drop of embryo extract, 1 drop of chicken plasma, and 4 drops of the test fluid. Gey et al. (1948) grew skin successfully in roller tubes at a low temperature of 31°C. for a 6-month period. During this time epidermal cells migrated from the explant and markedly proliferated. Transfer of pure epithelial growths was not successful. Long term culture technics in general seem to be more satisfactory for maintaining human epidermal cells. In such cultures, however, most workers find that the cells become modified. Bassett et al. (1956) cultured human autopsy skin in Carrel flasks under perforated cellophane. In the majority of cultures, both epithelial cells and fibroblasts migrated first from the explant. Later, growth of epithelial cells ceased and fibroblastic overgrowth started. Small isolated islands of epithelial sheets were noted for periods of 6 months. These showed a tendency to break up into fibroblast-like cells. Wheeler et al. (1957) planted human skin in roller tubes. After 30 days of incubation in one of the tubes, growth of a small epithelial colony was noted. On the 45th day this colony was successfully removed, subcultured, and kept alive. During a 9-month period an estimated 130 million cells have been grown from the original colony in 30 subcultures. These cells resembled “the cells of epidermal carcinomas of anaplastic type.” Perry et al. (1956), using the skin of a 65-year-old man, separated the epidermis from the dermis by trypsin. The isolated epidermis was then dispersed into single cells and planted in roller tubes. During early life of the culture the cells appeared spindle-shaped, later more like epithelial cells. This strain was also carried through a single cell culture (Perry et al., 1957). The plated cells formed a continuous sheet and did not reveal fibroblast-like changes. In well-oxygenated media, complete encasement of skin fragments by emigrating epithelial cells was described by Medawar (1948a) and Cruickshank and Lowbury (1952). The emigrated cells formed several layers on the surface of the dermis and proliferated intensely. Slow encasement of adult human skin fragments in hanging drop cultures is a common phenomenon. After 5 days usually only a single cell layer covers the free dermal surface (Fig. 1OA). On the 7th to loth day the structure of the epidermis may completely dissociate and the dermal fragment is covered by an even multilayered epithelium (Fig. 10B). Mitotic activity occurs neither in the epidermis nor in the epithelial sheet, covering the surface of the dermis, indicating that the encasement is elaborated by the initial cell population of the epidermis (Matoltsy, 1956b). Dermal islands also
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may be formed by penetration of cells into the dermis at various parts of the fragment (Fig. 1OC). This usually occurs where hair follicle or sweat duct remnants become disorganized. The cells of follicular or duct epithelium readily intermingle with the epidermal cells and become indistinguishable. As soon as the dermal fragment is encapsuled by a single layer of cells or a dermal island is formed, penetration of nutrients into
FIG. 10. Encasement of explanted adult human skin, in hanging drop preparations. A. After 5 days of culturing. B. After 7 days of culturing. C. Formation of dermal islands.
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the dermis is reduced. Dermal fragments covered by multilayered epidermal cells often become acellular and consist only of fibrous elements. This stage is followed by liquefaction of the dermis, and progressive death of the lining epithelium, too. C. WOUNDHEALING Healing of quite large wounds in fragments of cultured adult human skin was studied by Blocker et d. (1950). A circular opening was made in the center of the split-thickness graft with a sharpened 16-gauge needle. After culturing in hanging drop preparations for 5 days, the epidermal cells were seen to have migrated fairly symmetrically from the border toward the center of the wound and to have almost completely closed the opening. Healing of shallow and deep microwounds in skin fragments was studied by Matoltsy (1955) in hanging drop cultures. Microwounds were made with the tip of a surgical knife in the central portion of the skin prior to culturing. In small epidermal wounds, the neighboring basal cells perfectly restored the interrupted basal and suprabasal layers within a few days (Fig. 11A). Large epidermal losses were repaired by emigration of basal cells of rete ridges, next to the wound (Fig. 11B). When the cut penetrated the entire thickness of the explant and the edges of the wounds were not far apart, the migrating cells formed a bridge across the wound and reestablished continuity of the basal layer (Fig. 1lC). If the edges were far apart, even if the wound was filled with a plasma clot, the cells preferred to migrate on the dermis and failed to close the wound (Fig. 11D). Mitotic foci were not noted in the vicinity of the wound ; healing was elaborated by the original cell population of the explant. In the absence of proliferative activity, mobilization of the given cell population of the rete ridge regions appears as a compensatory mechanism, revealing the extremely dynamic nature of the epidermis. Matoltsy and Sinesi ( 1957) experimentally induced keratinization in the adult human skin by repeated stripping of the skin surface with Scotch tape prior to culturing. Since in the hanging drop cultures proliferation of cells is greatly inhibited, differentiation could be observed without proliferative activity. [ Compare with Pinkus’s ( 1952) and Lobitz and Holyoke’s (1954) in Vivo stripping studies, reviewed in Section 11.1 Histochemical and polarization optical studies demonstrate that in the stripped epidermis keratinization proceeded in situ. After 3 to 5 days of culture, the uppermost cells became flattened and lost their nuclear content. After 7 to 10 days, a new keratogenous zone developed above the basal layer, rich in -SH content (Fig. 12A). Above the keratogenous zone a
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thick horny layer was formed, revealing large quantities of -SH and -S-Sbonds (Fig. 12B) and showing intense birefringence (Fig. 12C). The intense double refraction and the occurrence of -S-Sbonds thus indicate that these upper epidermal cells had keratinized in situ and have
FIG.11. Healing of wounds in explanted adult human skin. A. Small epidermal wound on second day of culturing. B. Large wound on fifth day of culturing. C. Bridge formation. D. Failure of repair. developed a well-oriented and fully stabilized keratin. The progressive changes observed, provide valuable information about the capacities of epidermal cells in various layers, when considered from the viewpoint of keratin formation. The basal cells seem to lack the capacity to develop a keratin producing system. An adequate system for keratin synthesis de-
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velops efficiently in the suprabasal and spinous cells (differentiating cells). The upper cells appear to contain a satisfactory system not only for keratin synthesis but also for keratin stabilization. There are essential differences among epidermal cells at various levels, and they differ not only in mor-
FIG.12. Keratinization in adult human skin induced by removal of the cornified epithelium. In the absence of proliferative activity, in hanging drop preparations, keratinization proceeds in situ. A. Keratogenous zone develops above the germinative layer on the 7th day of culturing and reveals high -SH content. B. Above this, a thick horny layer is formed with abundant -S-Sbonds. C. The same region shows intense double refraction.
phological properties but also in capacities, such as synthesis or stabilization of keratin. Mitotic activity in the wound area (edge of explant) of mouse ear fragments was studied by Gelfant (1959b) in great detail. He used Bul-
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lough and Johnson’s (1951) short term technic. Mitotic cells were abundant at the cut edge, diminishing toward the uninjured portions of the ear fragment. When the cut edge was sealed with paraffin to prevent diffusion of nutrient and oxygen, the number of mitotic cells was somewhat reduced, but the mitotic gradient remained the same. Gelfant concluded that, although nutrients and oxygen are essential for proliferation, the primary impulse for mitosis is cutting (wounding). V. Remarks on Keratinization Keratinization is a notable property of the skin and in its initiation and maintenance both epithelium and dermal components appear equally involved. Fischer (1922, 1924) cultured pure epithelial cells from the iris of the chick embryo and noted keratinization in some parts, where the epithelium thickened. As a result of this observation, Fischer put forward the idea that keratinization occurs at places where the epithelium thickens and the cells are deprived of nutrients. This view has considerably influenced many investigators and the question was raised whether keratinization is degeneration or differentiation (reviewed by Pinkus, 1954). On the basis of tissue culture studies, Miszurski (1937) came to the conclusion that “keratinization is not a simple degenerative process, but is rather the final stage of a complicated process of differentiation in the covering epithelium.” Miszurski experimentally produced epithelial islets in the stripped chick limb bud and studied keratinization under poor nutritional conditions, at lowered temperature, in poor oxygen supply, and even in cultures kept in an atmosphere of nitrogen. He noted that keratinization was not promoted under any of these conditions and concluded : “All these facts contradict Fischer’s views accepting keratinization as a result of restrained nutrition of epithelium.” On the basis of the many experiments on epidermal differentiation which have been performed since the above views were formulated, one may now characterize the process of keratinization as follows. The principal role of the skin in vivo is the production of a highly resistant terminal product, the cornified cell. For this purpose some of the existing activities of the germinal cells of the epidermis increase in intensity (production of tonofilaments), but also new and specialized activities begin (production of keratohyalin, doubling of cell membranes). Existing activities become de-emphasized, certain cell constituents decrease in quantity (mitochondria), others entirely disappear from the cells (nuclei), Keratinization therefore is specialization, a form of cytodifferentiation, a process best characterized by Grobstein ( 1959) as “relatively stable, maturational changes of cellular properties which progressively concentrate the activ-
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ities and structure of the cell, or portions of it, in particular directions at the expense of others.” It is highly characteristic of this process of keratinization that once it begins it cannot be reversed, it will run its full course. Morphological, chemical, and biological properties of epidermal cells, related to keratinization, may be briefly reviewed as follows. The visual manifestations of keratinization, as seen in the electron microscope (Brody, 1959a,b; Charles, 1959; Charles and Smiddy, 1957; Horstmann and Knoop, 1958; Laden et al., 1957; Matoltsy, 1960; Mercer, 1958; Porter, 1954a,b; Odland, 1958, 1960; Selby, 1955, 1956, 1957; Weiss and Ferris, 19j4), is the appearance of cytoplasmic filaments in abundant quantities, such as the tonofilaments. As cytoplasmic density increases, the fine filaments tend to clump and form aggregates (Fig. 13D). Parallel with this a highly osmiophylic, fine granular substance (in mammals) condenses and gives rise to extremely small keratohyalin granules. These in turn grow in size by continued deposition of the fine granular substance (Fig. 13D). The cell membrane also undergoes changes as the original singlelayered membrane becomes a double membrane (Fig. 13B). At an advanced stage of keratinization mitochondria and nuclear substances decrease. Prior to the final stage the cells contain differentiation products in large quantities and some remnants of other cell constituents enclosed by a differentiated cell membrane. At the final stage the keratohyalin granules dissociate, the cell content fuses (Fig. 13C) and becomes dehydrated ; fibrous keratin takes its final form and place (Fig. 13A). In chemical terms, keratinization primarily means synthesis of proteins. Most investigators agree that the macromolecular precursors of the final horny component assemble in tonofilaments and keratohyalin granules (Derksen and Heringa, 1936 ; Derksen et al., 1937 ; Giroud and Leblond, 1951 ; Laden et al., 1957 ; Matoltsy, 19581, ; Matoltsy and Herbst, 1956a,b; Mercer, 1958; Rudall, 1952). Tonofilaments change their chemical and physicochemical properties as keratinization advances. Their -SH content may increase either by incorporation of -SH containing amino acids (Rothman, 1954) or by forming a complex with an interfibrillar component, high in -SH (Brody, 1959a,b). Formation of highly resistant cell membranes also requires attention. Their resistance toward moderately strong alkalies and reducing substances is higher than that of epidermal keratin (Lagermalm et al., 1951; Matoltsy, 1957; Matoltsy and Balsamo, 1955a). These cell membranes, together with the horny component of the cell appear to constitute a most efficient and resistant means of protection against physical and chemical agents, which may impinge upon the surface of the skin. Chemical investigations reveal a relatively
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simple composition of the terminal cell. The dry cornified epithelium of the human skin consists of 65% insoluble epidermal keratin, 10% soluble protein, 10% dialyzable substances, 7 to 9% lipids, and 5% cell membrane material (Matoltsy and Balsamo, 1955a,b). The germinative cells do not specialize during development, but remain in an undifferentiated state (Montagna, 1956). Specializing cells in the epidermis correspond to the uppermost spinop cells which are about to change into granular cells. In these, both mitotic and migratory activities diminish as specialization proceeds. They have lost the capacity to adapt to major environmental changes and are extremely sensitive to inadequate nutritional conditions. For instance, in hanging drop preparations, embedded in a plasma clot, they cease to produce keratohyalin and the nucleus readily becomes pyknotic (Pinkus, 1938, Matoltsy, 1956a). The onset of keratinization may be placed in the period elapsing between the last mitosis of a germinative cell and the beginning of increased tonofilament production or keratohyalin formation. The factors influencing the germinative cells to abandon mitotic activity and start specialized functions seem to be extrinsic rather than intrinsic. The important role of the dermal component of the skin in induction of keratinization is indicated by Champy’s ( 1914), Drew’s (1922), and McLoughlin’s ( 1958a) in vitro studies. They emphasize that in the absence of the stroma keratinization fails or is inhibited. In relation to this, Toro’s (1940) in vitro studies with pure strains of epithelia and fibroblasts are also noteworthy. These indicate that diff erentiative activity and organoid behavior are absent in pure strains but are present in a combination of the two. The exact role of stroma in keratinization and the mechanism by which the dermal component influences keratinization awaits future exploration. A biochemical mechanism may be expected to be involved in this, on the basis of Grobstein’s (1953) studies. H e noted if a 20 p-thick membrane, through which cells cannot pass, separated the isolated epithelial and niesenchymal component of the salivary rudiment, morphogenesis was induced and proceeded as in the case of direct contact. Tissue culture is a most powerful tool for exploration of the true character of epidermal cells. Its numerous technics yield a wide variety of experimentations. By its skillful use, morphological, chemical, beFIG.13. Manifestations of keratinization as seen in the electron microscope. A. Fibrous keratin. B. Double cell membrane of a horny cell in human epidermis. C. Transitional cell (arrow) between stratum granulosum and stratum corneum revealing mixing and desiccation of the cell content in rat epidermis. D. Keratohyalin granules and aggregated tonofilaments in a granular cell of the rat epidermis.
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havioral, or developmental properties of epidermal cells may be explored, even those that are often obscured in vivo. The limitless opportunities for variation of the culture conditions exemplify the excellence of the technic. Acknowledgments The author acknowledges the valuable help of Dr. Robert Crounse and Mrs. Margit Matoltsy in the preparation of the manuscript, and Mr. David Taplin in the photographic work. Some of the illustrations in this paper have already appeared elsewhere. The author wishes to thank The Williams & Wilkins Company, Academic Press Inc., The University of Chicago Press, and The Wistar Institute of Anatomy and Biology for the permission granted to reproduce Figs. 1 and 2 from J . Invest. Drrmatol. 31, 344 and 345 (1959) ; Figs. 3 and 6 from Develop. Biol. 1, 307 and 317 (1959) ; Figs. 8 and 10 from “Dynamics of Proliferating Tissues” (edited by D. Price, p. 37, University of Chicago, Chicago, Illinois, 1956) ; Figs. 11 and 12 from Atcat. Rccord 122, 584 and 585 (1955) and ia8, 65 and 67 (1957).
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Author Index Numbers in italics indicate the pages on which the references are listed at the end of the article. A Abercrombie, M., 229, 239 Aberg, B., 67, 75, 96 Ackerman, G. A., 296, 306 Acra, A. N., 78, 79, 93, 95 Adams, R., 7, 33, 93 Adamstone, F. B., 258, 262, 267, 270, 275, 284, 306 Addis, T., 222, 235, 239, 241 Adibis, 238 Adler, H. I., 57, 62, 92, 95 Afifi, A. K., 78, 79, 93, 95 Afzelius, B. A., 140, 141, 153,158, 208, 213 Albers, V. M., 37, 82, 99 Alexander-Jackson, E., 5, 7, 10, 17, 93 Alexander, J., 5, 7, 10, 17, 93 Alexanderson, M.,219, 240 Alfert, M., 227, 239 Allen, R. D., 178, 213 Allfrey, V., 157, 160 Allgover, M., 338, 348 Allsopp, C. B., 102, 123 Altman, R., 284, 306 Altmann, H. W., 157, 158 Alyea, H. N., 10, 26, 93 Amano, S., 290, 306 Ambrose, E. J., 102,123, 151,158 Anderson, C. E., 14, 100 Anderson, E., 156, 158 Anderson, N. G., 138,158,210, 211, 213 Anderson, T. F., 146, 161 Ando, T., 290, 306 AndrC, J., 140, 153, 156, 158 Annau, E., 234, 239 Aoki, T., 244, 298, 312 Arai, S., 292, 311 Arcos, J. C., 244, 298, 313 Arey, L. B., 323, 348 Arzac, J., 68, 84, 93 Atchison, E., 114, 128 Atkinson, W. B., 5, 9, 19, 21, 93 Atwood, K., 47, 93 Aue, J. C., 238,239 Auerbach, C., 101, 102, 115, 116, 117, 118, 123, 124
Aufsess, Alexandrine V., 75, 93 Avanzi, M. G., 102, 111, 112, 121, 124, 126 Avanzi, S., 121, 124,126 Avel, M., 248, 249, 306 Avery, A. G., 110, 125 A v i k N., 50, 531 54, 55, 76
B Backstrom, H. L. T., 10, 26, 93 Bahr, G. F., 139, 146, 159 Bairati, A., 139, 159 Baker, J. R., 71, 93, 249, 254, 255, 263, 265, 266, 267, 268, 270, 274, 280, 284, 306 Baker, R. F., 146, 161 Baker, W. W., 144, 159 Bal, A. K., 103, 106, 111, 112,134 Balsamo, C. A., 345, 346, 350 Baltus, E., 150, 159 Bang, F. B., 149, 159 Bangle, R., Jr., 77, 97 Barber, H. N., 32, 93 Barer, R., 149, 159 Barger, J. D., 5, 8, 16, 17, 93 Barka, T., 18, 43, 69, 76, 92, 93 Barnes, B. G., 140, 159 Barrett, R., 339, 349 Barmett, R. J., 158,159, 318, 348 B ~ c. H., ~m, 239~ ~ ~ Bass, A. D., 227, 239 Bassett, C. A. L., 329, 339, 348 Bassi, M., 121, 134 Battaglia, E., 102, 109, 121, 124 Bauch, R., 110, 112, 121,124 Baud, Ch. A., 149, 159 Bauer, A., 142, 159 Bauer, H., 3, 9, 68, 70, 73, 93 Beadle, G. W., 103, 124 Beams, H. W., 144, 156, 158, 159, 170, 191, 213, 229, 236, 239, 262, 267, 275, 276, 280, 306, 308 Beatty, A. V., 119, 133 Beaufay, H., 287, 310
353
~
354
AUTHOR INDEX
Becker, M. W. A., 109, 121,124 Bedell, W. C., 339, 351 Beermann, W., 139, 146,159 Begg, R. W., 234, 239 Behrens, M., 71, 95 Bellelli, E., 238, 241 Belt, W . D., 3, 78, 94 Bensley, C., 9, 94 Bensley, R. R., 261, 262, 265, 266, 278, 280, 281, 282, 286,306 Bentley, F. H., 331, 348 Berger, C. A., 113, 121,124 Bernhard, W., 139, 142, 143, 144, 159, 160,161, 276, 308 Bersin, K., 71, 95 Bertalanffy, J., 320, 348 Bertossi, F., 114, 125 Beutner, E. H., 11, 94 Bhattacharya, D. R., 249, 306 Bhattacharya, S. N., 32,42,43,68, 94 Bhattacharyya, N . K., 103, 104, 105, 118, 121, 134 Biddle, H. C., 10, 16, 17, 57, 94 Biesele, J. J., 117, 125, 226, 239 Bird, M . J., 102, 125,128 Bissett, K. A., 47, 94 Blacet, F. E., 9, 10, 94 Blaedel, W . J., 9, 10, 94 Blake, M . I., 33, 97 Blakeslee, A. F., 110, 125 Blank, H., 332, 348 Blank, I. H., 321, 334, 348 Blatt, A. H., 33, 94 Blazsb, A., 334, 336, 348 Bloch, D. P., 69, 94 Blocker, T. G., Jr., 338, 341, 348,351 Bloom, M. L., 255, 306 Bloom, W., 262, 285, 309 Blumenfeld, C. M., 318, 348 Bohm, J., 228, 239 Bornstein, K., 329, 334, 348 Bokelmann, O., 234, 239 Bolognari, A., 143, 159. ' Bopp-Hassenkamp, G., 146, 159 Borysko, E., 146, 149, 159 Boss, J., 199, 200, 213 Bourne, G. H., 249, 263, 265, 267, 274, 277, 306, 307,308 Bovey, R., 139, 159
275,
154,
116,
Bowen, C. C., 112, 136 Boyd, C., 332, 348 Boyland, E., 102, 125 Brachet, J., 106, 125, 143, 150, 157, 159, 336, 348 Braden, A. W. H., 75, 94 Bradfield, J . R. G., 91 Brambell, R., 249, 306 Branch, G. E. K., 6, 24, 40, 98 Brandt, T. A., 223, 241 Brandes, D., 158, 161 Bretschneider, L. H., 91 Brice, A. T., 267, 307 Briggs, R., 194, 213 Brock, R. D., 103, 125 Broders, A. C., 318, 348 Brody, I., 345, 348 Brody, S., 223, 239 Broman, L., 208, 215 Brown, A. N., 230, 240 Brown, D. E. S., 175, 177, 181, 213 Brown, D. M., 66, 94 Brown, R. H. J., 258, 263, 307 Brues, A. M., 224, 239 Bucher, N. L. R., 228, 236, 238,239,241 Bucherer, H. T., 39, 40, 94 Buckaloo, G. W., 24, 96 Buckley, S. M., 117, 125 Biitschli, O., 262, 307 Bullough, H. F., 320, 348 Bullough, W. S., 315, 316, 318, 320, 323, 334,335, 336, 343, 344, 348 Burchenal, J. H., 117, 125 Burgos, M. H., 50, 94 Burrows, M. T., 315, 349 Burstone, M . S., 3, 77, 78, 94 Burton, A. C., 188, 215 Burton, V., 5, 10, SO, 75, 96 Buschbaum, R., 171, 213 Butler, J . A. V., 102, 125 Butcher, E. O., 320, 349
C
268,
Cain, A. J., 73, 76, 94 Calder, 75 Callan, H. G., 139, 159 Camara, A., 103, 125 Cameron, J. A., 251, 252,307 Campbell, R. M., 233, 234, 239
355
AUTHOR INDEX
Canby, C. M., 339,351 Carasso, N.,276, 308 Carey, M.W., 9, 13,24,31, 94 Carleton, A., 318, 349 Carlson, J. G.,199,201, 202,213 Carlton, W.M.,116,125 Carlyle, A., 220, 239 Carr, J. G.,116, 124 Carrasco, C., 20, 95 Carrel, A., 315, 329,349 Casabona, U.,121, 125 Cassela, C., 73, 94 Cason, J., 7, 33, 94 Caspersson, T.,18, 19, 45, 57, 62, 67, 68,
69,70,94, 115, 150,159 Casselman, W. G. B., 2, 70, 75, 94, 274, 299, 307, 311 Castelengo, L., 277, 311 Catcheside, D. G.,66,94, 102,123,125 Cawley, E.P.,339, 351 Cazeneuve, M.P.,46, 94 Cerecedo, L. R.,234, 241 Cerroni, R. E.,67, 92, 94 Chace, E.M., 6,10, 24, 94 Chakravorti, A. K.,103,125 Chalkley, H.W.,169, 174, 202, 203, 209,
213 Challice, C. E., 262,275, 276,309 Chambers, R.,169, 171, 175, 176, 180, 184,
188, 1%,
197, 199, 213 Chambers, E. LA,92 Champy, C., 328, 346, 349 Chapin, R. M.,91 Charles, A., 345, 349 Chase, H.B.,320,349 Chaudhuri, M.,111, 121,134 Chautard, M.P.,6, 10,24,27,29,94 Chayen, J., 67, 92, 94 Chen, J. M.,332, 349 Chenoweth, M. B., 16, 24,96 Cheronis, N. D., 33, 94 Chlopin, N.,329, 330, 349 Christensen, B. G.,238,239 Christie, A. C., 274, 286,307 Chu, C. H.U., 24, 25,26,27, 31,78, 94 Chu, J., 323, 349 Clara, M.,226, 239 Claude, A., 146, 161, 275, 277, 278, 288, 298, 307, 311
Clement, A. C., 197, 213 Clermont, Y.,8, 75, 95 Cohen, A. I., 143, 159 Cohen, P.P.,244, 298,312 Cole, K.S.,175, 177, 181,213 Coleman, L. C., 4,8, 15, 94 Conant, J. B.,33, 94 Conger, A. D., 105, 122,125 Conklin, E. G., 197, 213 Conn, H.J., 11, 12, 13, 15, 94,98 Cooper, Z.K.,318,349 Cordet, R. L,221, 240 Corey, E. L,233, 239 Cornman, I., 111, 121,125,191,213 Cornman, M.E.,191, 213 Cortini, C., 110, 125
Cova, G.,110, 125 Cowdry, E. V., 254, 255, 258, 263, 264, 268,280,284,286, 307 Cowdry, N. H.,254,255,307 Cramer, W.,272, 274,307 CrCtin, A.,249, 307 Crick, F. H.C., 68, 71, 99 Crippa, A.,75, 94 Crocker, E. C., 6, 13, 25, 26, 27, 30, 57, 67, 94 Cruickshank, C.N. D., 339,349
D da Fano, C., 258, 307 Dalton, A. J., 263, 264, 265, 267, 268,
271, 274, 275, 276, 287, 307, 308, 309, 311 DAmato, F., 101, 102, 103, 104, 105, 108, 110, 111, 112, 121,125,126 Damianovitch, H.,21, 39, 94 Dan, J. C., 168, 169, 170, 191, 197, 198, 213 Dan, K., 166, 168, 169, 170, 174, 189, 191, 192, 193, 197, 198,213,215 D'Ancona, D., 226, 239 Daniel, G. E.,209, 213 Danielli, J. F., 73, 95, 123, 126, 175, 176, 181, 185, 207, 214 Danon, M.,146, 160 Darlington, C. D., 102, 114,126,127 Datta, A.,116, 122,128,134 Davenport, H.A., 10, 11,96 David, H.,227, 241
356
AUTHOR INDEX
David, L. T., 318, 349 Davidson, J. N., 226, 234, 239, 241 Davies, D. V., 75, 95 Davis, B. J., 50, 75, 98 Davis, J. H., 140, 159 Dawson, A. B., 246, 249,251, 254,307 Dawson, I. M., 139, 141, 159 De, D. N., 114, 120, 121, 135, 139, 144, 159, 160 Deane, H. W., 73, 95, 234, 235, 239, 267, 307 De Duve, C., 287, 310 Deeley, E. M., 63, 98 De Groodt, M., 139, 159 Deitsch, A. D., 262, 307 DeLamater, E. D., 5, 8, 16, 17, 47, 68, 93, 95 de Ley, J., 121, 131 De Marsh, Q. B., 153,160 Demerec, M., 119, 127 Dempsey, E. W., 73, 95, 267, 268, 280, 287, 307 Denighs, G., 91 Denues, A. R. T., 144, 150,159 Deriaz, R., 66, 99 Derksen, J . C., 345, 349 De Robertis, E., 146, 154, 159, 254, 258, 264, 265, 277, 284, 308,311 Derom, F., 139, 159 De Roo, G. I., 249, 308 DeTomasi, J. A., 4, 5, 8, 13, 95 Dettmer, N., 158, 159 Deufel, J., 122, 127 Deutsch, K., 91 Devine, R., 275, 308 Deysson, G., 102, 121, 122,127,131 Dick, A. T., 219, 220, 228, 238,239 . Dickinson, T. E., 234, 239 Di Fine, J., 268, 309 Di Stefano, H. S., 68, 69, 95 Dodson, E. O., 146, 159 Dolcher, T., 105, 127 Doljanski, F., 227, 235, 239 Doljanski, L.; 338, 349 Donaldson, H. H., 222, 239 Dondero, N. C., 57, 62, 95 Doring, H. H:, 122, 127 Doxey, D., 116, 127 Drew, A. H., 328, 329, 346, 349
Dublin, W. B., 318, 348 Du Buy, H. G., 292, 313 Duhamet, L., 110, 127 Dutnm, M. E., 220, 230,233, 239 Dunn, A. E. G., 91 Dunn, C . E., 227, 239 Durrsclmabel, K., 35, 95 Duryee, W. R., 146, 159 Dustin, P., 102, 112, 116, 118, 127, 133 Dustin, P., Jr., 110, 127 Duthie, E. S., 286, 308
E Earle, W. R., 307, 315, 316, 329, 339, 348, 349, 351 Ebeling, A. H., 330, 349 Ebling, F. J., 320, 349 Ehrenberg, L., 119, 127 Eidinger, D., 75, 96 Eigsti, 0. J., 110, 127 Elftman, H., 4, 10, 11, 19, 42, 68, 95, 263, 268, 308 Elias, H., 218, 239 Elvove, E., 8, 10, 24, 95 Ely, J. O., 10, 11, 19, 21, 68, 95 Emery, Arthur J., Jr., 12, 95 Emmel, V. M., 267, 308 Ennis, W. B., 121, 127 Entriken, J. B., 33, 94 Ephrussi, B., 121, 128 Erdmann, Rh., 330, 349 Erickson, J. O., 345, 350 Ericksson, R. O., 110, 128 Eshkol, Z., 235, 239 Estable, C., 139, 149, 159 Evans, T. ,C., 144,159, 170, 191, 213 Evans, V. J., 329, 339, 348,349,351 Ezell, D., 332, 334, 336, 350
F Fabergk, A. C., 119, 128 Fahmy, 0. G., 102, 128 Fairchild, L.M., 105, 122, 125 Falzone, J. A., 227, 239 Fankhauser, G., 205, 214,227, 239 Fautrez, J., 227, 239 Fawcett, D. W., 145, 159, 272, 275, 276, 277, 280, 286,287, 308 Feigl, F., 7, 25, 26, 27, 30, 67, 95
AUTIIOR INDEX
Felix, M. D., 263, 264, 265, 267, 268, 274, 275, 276, 287,307, 308,311 Fell, H. B., 199, 200, 214, 315, 318, 323, 324, 326, 328, 349,351 Ferris, W., 345, 351 Feulgen, R., 3, 4, 5, 6, 9, 10, 19, 24, 65, 66, 71, 92, 95 Ficq, A., 150, 157, 159 Fieser, L. F., 33, 95 Fieser, M., 33, 95 Fincke, Heinrich, 8, 24, 95 Findlay, G. H., 76, 95 Finholt, P., 10, 95 Fischer, A., 263, 270, 308,330,344, 349 Flemming, W., 149, 159 Flexner, J. B., 230, 239 Flexner, L. B., 230, 239 Flint, E. R., 27, 95 Florian, M. L., 238, 241 Ford, C. E., 117, 128 Francois, M., 6, 16, 24, 25, 37, 38, 95 Frankel, D. R., 322, 350 Franzl, R. E., 92 Fraser, D. A., 318, 349 Frazer, S. C., 226, 236, 239,241 Frehden, O., 12, 95 , Freidler, G., 322, 350 Freidsohn, A., 251, 308 Frey-Wyssling, A., 123, 128, 262, 298, 308 Friedrich-Freska, H., 238, 239 Fries, G., 8, 19, 20, 21, 36, 39, 40, 41, 42, 44, 57, 67, 68, 75, 89, 93, 96 Fiirst, K., 12, 95 Fujimoto, M., 29, 95 Fukuda, M., 66, 99, 225, 226, 231, 232, 233, 239, 277, 278, 312 Fukuoka, F., 243, 308 Fular, G., 320, 349 Fullam, E. F., 288, 311 Fuller, R. C., 112, 128 Fuse, Y., 292, 308 Fuson, R. C., 7, 99 G Gaeke, G. C., Jr., 93 Galinsky, I., 122, 128 Gall, J. G., 139, 141, 146, 147, 148, 152, 153, 156, 159, 160 Ganguly, D. N., 122, 128
357
Gatenby, J. B., 263, 264, 265, 267, 275, 276, 283, 308 Gates, R. R., 157, 160 Gavauden, P., 102, 128 Gay, H., 140, 146, 153, 154,160 Gayon, U., 91 Gelboin, H. V., 298, 308 Gelfant, S., 335, 343, 349 George, L. A., 11, 158, 160 Georgia, F. R., 91 Gerard, P., 73, 95 Gersh, I., 158, 160, 268, 272, 275, 280, 281,282,286,306,308,309 Gerthner, P., 345, 350 Geschwind, I. I., 220, 227, 231, 239 Gettler, A. O., 91 Gettner, M. E., 140, 156, 161 Gey, G. O., 339, 349 Ghose, A., 32, 94 Giacometti, G., 128 Gibbs, H. F., 318, 349 Giles, N. H., 102, 103, 119, 128,133 Gillman, T., 323, 349 Giral, E. H., 20, 95 Giroud, A,, 345, 349 Gisquet, P., 103, 104, 128 Givol, D., 220, 222, 223, 224, 230, 233, 235, 239 Glass, E., 226, 237, 239, 240 Glegg, R. E., 8, 75, 95, 96 Glick, D., 249, 266, 294,308,311 Glinos, A. D., 238, 239 Glover, P., 50, 75, 96 Gliicksmann, A., 318, 349 Goda, T., 251, 252, 308 Goddard, J. W., 255, 268, 308 Godman, G. C., 69, 94 Gold, H., 16, 24, 96 Goldacre, R. J., 117,128,203,210,214 Goldstein, L,157, 160 Goldstein, N. O., 92 Gomori, G., 73, 95,266,294,308 Goodhard, C. B., 292, 312 Goodspeed, T. H., 119, 128 Gopal Ayenger, A. R., 102,123 Gosselin, A., 110, 128 Gould, E., 321, 334, 348 Graham, S., 234, 240 GrassC, P. P., 276, 308
358
AUTHOR INDEX
Hatai, S., 222, 240 Hawkes, J. G., 110,129 Hayashi, T.,205, 214 Hayes, E.R.,3, 73, 78,94, 95 Heagy, F. C.,226, 241 Hediger, E.M.,16,24, 96 Heilbrunn, L. V.,208, 216 Helweg-Larsen, €I. F.,225, 226, 227, 228, 240 Herbst, F. S. M., 345, 350 Heringa, G. C.,345,349 Herskowitz, I. H., 146,160 Hertwig, G., 149, 160 Hertwig, R., 227, 240 Hess, A., 322, 331, 350 Hetherington, A. C.,19,21, 23, 25, 26, 27, 30, 38, 99 Hibbard, H., 263,266, 270,308 Hiebert, C. A.,328, 350 Hill, C. R.,249, 274,308 H Hillary, B., 69,96 Hadorn, E., 101, 129 Himes, M.,53, 79,96, 227,240 Hakansson, A., 103, 129 Himsworth, H. P.,235, 240 Haferkemp, M.E.,103,129 Hiraoka, T.,57, 62,96 Haguenau, F.,139, 142, 159,160,276,308 Hiramoto, Y.,166, 167, 168, 170, 171, Hair, J. B., 102,129 175, 183, 184,185, 191, 214 Hale, A. J., 2, 75, 95 Hirsch, G. C., 254, 274,308 Halevy, S.,235, 239 Hirschler, J., 275, 308 Hall, C. E.,138, 160 Hitier, H., 103, 104,128 Hall, W.T., 33, 97 Hormann, H., 8, 19,20, 21, 36, 39, 40, 41, Handa, D.T.,224,241 42, 44,57, 67,68,75, 89, 93,96 Hanhausen, E.,20, 95 Hoerr, N. L,280, 306 Hanks, J. H., 339,349 Hoffman, J., 227, 240 Hanson, J., 318, 328, 349 Hoffman-Ostenhof, O.,102, 104,105,129 Hantzch, A., 34, 35, 37, 95 Hoffman, R. S.,338, 349 Hanzon, V.,275, 276,287,312 Hoffmann-Berling, H., 1!2!9,205,206,214 Hard, W.L.,234, 240 Hoffpauir, C. L.,24, 96 Hardy, M. H., 328,350 Hofmeister, F.,137, 160 Harkness, R.D.,229,236,239,240 Hogeboom, G. H., 156,160, 255,277, 280, Harris, P.,139, 160 308, 309 Harrison, M. F., 226, 229, 234, 235, 240 Hohl, K., 119, 129 Harrison, R. G.,315, 350 Holmes, B. E.,66, 94 Harvey, E. B., 184, 190, 191, 192, 197, Holmes, S. G., 336, 350 214 Holmgren, E.,258, 309 Harvey, E. N., 175, 178,214 Holyoke, J. B.,323,341,350 Hashim, S. A., 69, 78, 95 Honjin, R.,262,276,309 Hashimoto, M.,292,308 Hooghwinkel, G. J. M., 67,75, 96 Hassenkamp, G., 139, 160 Horstadius, S., 176, 214 Hastings, A. B., 73, 95,230, 240
Grassman, W., 8, 19, 20, 21, 36, 39, 40, 41, 42,44, 57, 67,68,75,89,93,96 Gray, H., 222, 239 Gray, J., 169, 170, 176, 191, 192, 193, 214 Gray, L.H., 102, 128 Green, L.W.,9, 13, 24, 94 Greunlach, V. A.,114,128 Griesemer, R. D.,321, 334,348 Grobstein, C.,344, 346, 349 Gropp, A., 142, 159 Gross, F.,205, 214 Guareschi, 29, 69, 95 Guggenheim, K.,235, 239 Guiellermond, A.,285, 308 Guimochet, M.,121, 135 Gunthardt, H. M.,103,129 Gustafsson, A.,116, 119,127, 129 Guthrie, J. D.,24, 96 Guyhot, E.,146, 160
359
AUTHOR INDEX
Horstniann, E., 142, 143, 156, 160, 345, 350 Hossack, J., 139, 141,159 Hotchkiss, R. D.,3, 8,74,96 Hovanitz, W.,143, 160 Hsu, T. C.,334,336,337,350 Hu, F. N.,336, 350 Hubbard, J. C.,238, 240 Huber, W.,174, 209, 214 Hughes, A. F.,199, 200,209,214 Hughes, W.L., 151, 161 Hultin, T.,298, 309 Hunter, M. E.,47, 95 Huskins, C. L.,114, 129 Hutchinson, W.C.,226, 241 Hyatt, G. W., 339, 351 Hyde, B. B., 121, 129 Hymas, F. C.,11, 24, 97
I Ichikawa, T., 3, 29, 55, 77,78,100 Igolen, G.,121, 135 Ihnuma, M.,267, 309 Imrie, C. G.,234, 240 Innes, R. H., 234, 239 Isenberg, I., 170, 214 Ishizaka, S.,168, 198,214 Islami, A. H., 238, 240 Itikawa, O.,7, 18, 69,96 Iversen, S.,228, 240 Izard, C.,103, 104,128
J Jackson, B., 223, 240 Jackson, C. M.,221, 222,240 Jackson, E. L.,74, 96 Jackson, S.F.,138,160 Jacobsen, E.,238, 239 Jacoby, W., 224, 225, 240 Jaffe, J. J., 223, 240 James, R., 326, 328,351 James, T.W.,139,160 Jash, M.,121, 135 Jasswoin, G.,258, 275, 282,309 Johnson, J. R.,7, 33, 93 Johnson, M.,315, 334, 344,348 Johnson, N. S., 199,215 Jones, H. B., 234, 235, 240 Jones, R. P.,267,307 Jordan, B. M.,299, 307
Jordan, H. E.,249, 309 Jorpes, J. E., 67, 75, 96 Joseph, S.,149, 159 Josephson, K., 4, 6, 18,31, 32,96 Jurand, A., 91 Just, E. E.,180,214
K Kahler, H., 275, 307 Kamm, O.,33, 96 Kano, K., 290, 312 Kaplan, R., 112, 129 Karasaki, S., 154, 160 Karpachenko, G. D.,110,129 Kasamatzu, J., 246, 313 Kasten, F. H., 2, 5, 10, 12, 15, 22, 23, 37, 42, 43, 46, 47, 50, 54, 61, 63, 64, 66, 67, 68, 70, 71, 75, 76, 81, 82, 83, 84, 85, 86, 96 Kastle, J. H., 21, 32, 96 Kaufmann, B. P., 121, 131, 144, 150, 160 Kaufman, E.,235, 239 Kaufman, L.,221, 228, 240 Kautz, J., 153, 160 Kawamura, K., 199, 214 Keck, K., 102, 104, 105,129 Kellicott, W.E.,223, 240 Kelly, L. S.,234, 235, 240 Kendall, J. I., 263, 309 Kennedy, G.C.,227, 234,240 Kephart, J. E.,276, 313 Khoshoo, T.,120,132 Kihlman, B. A., 103, 110, 119, 120, 121, 129, 133 Kiljunen, A., 318, 350 King, R. L,229,236,239,262,306 King, T. J., 194,213 Kingsbury, J., 219, 240 Kinoshita, R., 243,296, 309 Kiriyama, M.,142, 143,162 Klemperer, P.,235, 241 Klingman, C., 61 Knoop, A.,142,143,156,160,345,350 Knutsson, B., 116, 132 Koch, A. L., 224, 241 Koga, S., 249, 309 Kohn, R.,238, 240 Kolb, C. L.,9,11, 15, 16, 19,20, 96 Koller, P. C., 102, 115, 116, 127, 129, 130
360
AUTHOR INDEX
Komoto, Y., 107, 132 Kornstein, E. S., 219, 240 Kostoff, D., 121, 130 Kosterlitz, H. W., 233, 234, 239 Kramm, David E., 9, 11, 15, 16, 19, 20, 96 Kreibich, K., 315, 350 Kriszat, G., 208, 214 Kudo, R. R., 266, 309 Kuff, E. L., 255, 265, 268, 275, 309, 311 Kuno, M., 191, 209, 210, 214 Kurashige, S., 249,. 251, 309 Kurnick, N. B., 2, 96 Kuroda, S., 244, 298, 312 Kurosumi, K., 144, 160 Kusama, K., 244, 298,309,312 Kusomoto, Y., 255, 311
La Cow, L. F., 103,130,134 Lacy, D., 262, 275, 276, 309 Laczynska, T., 105, 107, 130 Laden, E. L., 345, 350 Lafontaine, J. G., 143, 147, 148, 152, 160 Lagasse, A., 139, 159 Lagermalm, G., 345, 350 Landau, J. V., 171, 175, 177, 181, 183, 185, 206, 208, 214,215,216 Lang, F. J., 330, 350 Iaquerrihre, R., 226, 227, 236, 239, 240 Lasfargues, E., 268, 309 Lasnitzki, I., 328, 350 Latimer, H. B., 221, 240 Laurence, E. B., 323,348 Lavin, G. I., 263, 266, 271, 274, 308, 311 Layton, L. L., 322, 350 Lazarus, S., 80, 96 Lea, D. E., 102, 119, 130 Leblond, C. P., 8, .75, 95, 96, 224, 240, 320, 345, 349, 351 Leduc, E. H., 228, 229, 235, 240,241 Lee, C. S., 235, 241 Lee, W. A., 66, 67, 96 Lefevre, L., 12, 46, 96 Lehmann, F. E., 139,159,174, 214 Lessler, M. A., 2, 45, 66, 67, 68, 69, 70, 71, 96 LettrC, H., 101, 112, 121, 130, 206, 214
Leuchtenberger, C., 2, 63, 65, 67, 71, 75, 96, 226, 240 Levan, A., 102, 103, 104, 105, 106, 107, 109, 110, 112, 113, 118, 119, 120, 121, 122, 127,129,130,136 Lewis, S. R., 332, 334, 336, 341, 348, 350 Lewis, W. H., 171, 211 Lhotka, J., 10, 11, 96 Li, C. C., 231, 239 Lillie, R. D., 3, 8, 9, 15, 73, 74, 76, 77, 96, 97 Lindberg, J., 345, 350 Linnert, G., 102, 130 Linstead, R. P., 7, 33, 97 Liquier-Milward, J., 158, 160 Lison, L., 19, 24, 25, 26, 27, 30, 31, 32, 73, 74, 76, 78, 97, 226, 240, 266, 309 Litvac, A., 326, 350 Livingood, C. S., 336, 350 Ljunggren, O., 315, 331, 350 Lloyd, B., 275, 307 Lobitz, W. C., Jr., 323, 341, 350 Lona, F., 110, 130 Longley, J. B., 5, 8, 11, 15, 16, 17, 18, 20, 21, 29, 53, 75, 97 Lopane, F., 102, 130 Lorch, I. J., 176, 192, 194, 203, 210, 214, 215 Lorenz, C., 23, 24, 27, 29, 97 Lorenzo-Andreu, A., 121, 130 Lotfy, T. A., 102, 104, 105, 106, 113, 121, 130. 135 Loveless, A., 102, 115, 117, 128, 130, 131 Lowbury, E. J. L., 339,349 Lowman, F. G., 146, 151, 152,160 Lowrey, L. G., 220, 223, 230, 240 , Lowry, 0. H., 230, 240 Ludford, R. L., 272, 274, 281, 282, 307, 309 Lyford, E. F., 6, 10, 24, 26,100 Lythgoe, B., 66, 94
M McCarthy, K. S., 5, 7, 10, 17, 93 McCay, C. M., 230, 240 McClintock, B., 102, 131 McConnel, K. P.,336, 350 McDonald, M. R., 121,131,150,160 Macduffee, R. C., 146, 161
AUTHOR INDEX
McElvain, S. M., 33, 97 MacEntee, F. J., 57, 62, 97 Macfarlane, E. W. E., 121, 131 McGookin, A., 33, 97 McKellar, M., 223, 224, 240 Mackey, J., 116, 129 McLeish, J., 102, 114, 115,127,131 McLoughlin, C. B., 328, 346, 350 McManus, J. F. A., 3, 9, 74, 97 Mahn, H., 97 Majumdar, A., 111, 135 Malaprade, M. L., 24, 74, 97 Malheiros-GardC, N., 121, 131 Malone, J. D., 320, 349 Mangenot, G., 121, 131 Mann, F. G., 16, 97 Manginelli, A., 234, 239 Marble, B. B., 224, 239 Margolena, L., 9, 97 Margoliash, E., 235, 239 Mark, E., 227, 240, 241 Marquardt, H., 103, 104, 105, 107, 110, 131, 226, 237, 240 Marsland, D. A., 171, 172, 174, 175, 177, 178, 180, 181, 183, 185, 191, 206, 208, 210, 214, 215, 216 Masayama, T., 243, 309 MascrC, M., 102, 122,131 Massart, L., 121, 131 Mast, S. O., 202, 215 Masuda, H., 267, 268, 312 Mather, K., 138, 160 Matoltsy, A. G., 317, 318, 321, 324, 330, 334, 336, 339, 341, 345, 346, 350, 351 Matsumoto, S., 330, 350 Maurer, K., 97 Mautner, W., 50, 75, 98 Maximow, A. A., 262, 285, 309, 328, 350 Mazia, D., 138, 160, 193, 207, 215 Medawar, P. B., 332,334, 339, 350 Mehra, P., 120, 132 Meek, G. A., 149, 159 Meletti, P., 122, 132 Melin, C. G., 12, 13, 14, 15, 98 Mellanby, E., 328, 349 Mello-Sampayo, T., 121, 132 Mellon, M. G., 81, 97 Mencle, J. J., 92
361
Mercer, E. H., 171, 183, 208, 215, 345, 350 Merriam, R. W., 153, 154,160 Messing, A. M., 121, 131 Meves, F., 246, 248, 249, 309 Meyer, J. R., 101, 132 Michaelson, K., 119, 132 Micou, J., 157, 160 Middleton, G., 11, 24, 97 Miller, B., 14, 100 Miller, E. C., 243, 244, 249, 292, 296, 298, 308, 309, 312 Miller, J. A., 243, 244, 249, 292, 296, 298, 308, 309 Millican, R. C., 8, 15, 29, 53, 75, 97 Milovidov, P. F., 65, 97 Minamino, T., 275, 276, 309 Minganti, A., 208, 215 Mirsky, A. E., 4, 43, 68, 71, 98, 157, 160 Miszurski, B., 323, 324, 326, 330, 344, 350 Mitchison, J. M., 164, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 195,207,208,215,216 Moats, William A., 75, 97 Moffat, D. B., 80, 97 Mohler, E., 5, 9, 24, 44, 56, 97 Mollenhauer, H. H., 276, 313 Monash, S., 321, 334, 350 MonnC, L., 208,215,268,274,310 Monroy, A., 183, 215 Montagna, W., 249, 274, 310, 320, 346, 349, 351 Montezuma de Carvalho, J., 112,132 Mookerjea, A., 113, 114, 121,135 Moore, 238 Moore, F. J., 33, 97 Morard, J. C., 80, 97 Morales, R., 91 Moree, R., 7, 97 Morgan, T. H., 197, 215 Moriber, L., 53, 79, 96 Morihara, M., 266, 310 Mortreuil-Langlais, M., 226, 240 Moscona, A., 326, 351 Moscona, H., 326, 351 Moses, M. J., 81, 83, 97, 145, 146, 150, 152, 154, 157, 160 Mota, M., 104, 132, 199, 215
362
AUTHOR INDEX
Motomura, I., 169, 171, 191, 215, 244, 257, 288,290, 310 Moulton, C. R., 234, 240 Mounat, A., 103, 104,128 Moussa, T. A., 263, 267, 275, 308,310 Moutschen-Dahmen, Jean, 102, 106,132 Moutschen-Dahmen, Madeleine, 102, 106, 132 Mowry, R. W., 5, 8, 15, 29, 53, 75, 80, 97 Miiller, H., 24, 27, 97 Miiller, H. G., 228, 240 Miiller, H. J., 103, 132 Mukherji, R. N., 102, 135 Muldoon, H. C., 33, 97 Miinzer, F. T., 228, 240 Mulinos, M., 33, 98 Mulliken, S. P., 6, 10, 18, 19, 97 Muroya, K., 292, 308 Murray, M. R., 262, 307
N Nagatani, Y., 244, 246, 247, 250, 251, 254, 255, 256, 257, 258, 259, 260, 261, 262, 266, 267, 268, 270, 272, 274, 275, 280, 284, 285, 288, 289, 292, 294, 295, 302, 303, 310 Nagumo, S., 276, 310 Nakahara, K., 107, 132 Nakahara, W., 243, 308 Nakai, J., 262, 310 Nakajima, T., 192, 193, 213 Nakajima, Y., 292, 311 Nakayama, T., 244, 298, 312 Nakao, Y., 244, 259, 261, 268, 289,309 Nambiar, P. K., 121, 132 Naora, H., 69, 70, 81, 82, 83, 97, 99, 226, 2 3 , 231, 233, 240 Nassar, T. K., 80,99 Nassonov, D., 272, 286, 310 Natarajan, A. T., 112, 136 Nauman, R. V., 93 Navashin, M., 103, 107, 121, 132, 134 Nebel, B. R.; 120, 133 Needham, A. E., 323, 351 Needham, J., 231, 233, 241 Neff, R. J., 67, 94 Nelson, L., 158, I60 Nesbett, F. B., 73, 95 Nichols, C., 102, 103, 105, 132
Niggli, H., 101, 129 Nigrelli, R. E., 248, 310 Nilan, R. A., 103, 119, 129, 132 Nittis, S., 246, 248, 310 N d l , R., 277, 278, 281,282,286,310 Noelting, M. E., 46, 97 Noller, C. R., 32, 33, 98 Norris, C. H., 178, 215 Norris, K. P., 67, 94 Novick, A., 116, 132 Novikoff, A. B., 287, 310 Novogrodski, M., 227, 239 Nowinski, W. W., 264, 284, 308, 338, 351 Nybom, N., 116, 132 Nygren, A., 116, 132 0 Oberling, Ch., 142, 159, 263, 310 O'Brien, R. T., 158, 160 Odate, Z., 146, 152, 162 Odland, G. F., 345, 351 Oehlkers, F., 101, 102,132,133 btergren, G., 46,98,103, 111,133,136 OettlC, A. G., 267, 310 Ogura, Y., 7, 18, 69, 96 Ohman, L., 208, 215 Okamoto, H., 255, 266, 311 Okazaki, T., 189, 213 O'Leary, G. E., 262, 311 Olenov, J. M., 102, 133 Oleson, J. J., 8, 98 Ono, T., 166, 174, 213 Onoe, T., 292, 311 Opie, E. L., 274, 311 Orlando, E., 238, 241 Ornstein, L., 23, 43, 47, 50, 65, 75, 76, 81, 85, 86, 92, 93, 98 Osswald, G., 34, 35, 37, 95 Oster, K., 33, 98 Ostrouch, M., 254,271,272,285,286,311 Ota,Y., 146, 152, 162 Ott, M. G., 244, 298, 312 Otte, R., 24, 27, 98 Overend, W. G., 25, 31, 32, 66, 98 8ya, M., 277, 282, 312
P Pack, G. T., 238, 240 Paigen, K., 255, 311
363
AUTHOR INDEX
Palade, G. E., 158, 159, 275, 277, 280, 288, 298, 309, 311 Palay, S. L., 138, 146, 161, 275, 311 Palek, E. S., 143, 161 Pappas, G. D., 139, 141, 161 Parker, R. C., 332, 351 Parmentier, R., 118, 133 Parrot, D. M . V., 234, 240 Parshley, M . S., 337, 338, 351 Patt, H . M., 119, 133 Patterson, E. K., 109, 133 Patton, R. L., 120, 133 Paul, J., 24, 98, 331 Pauly, H., 23, 26, 27, 30, 98 Patau, K., 70, 71, 81, 98, 99 Peacocke, A. R., 66, 67, 96 Pearce, W . M., 227, 234, 240 P a r s e , A. G. E., 2, 3, 70, 73, 75, 76, 80, 98 Pease, D. C., 146, 161 Peeters, G., 121, 131 Pelc, S. R., 328, 351 Penfield, W. G., 258, 269, 311 Penn, J., 323, 349 Perry, V. P., 339, 351 Peterson, A. R., 12, 13, 98 Peto, F. H., 103, 107, 133 Pfuhl, W., 228, 241 Phan, N . V., 227, 241 Phelps, E. P., 24, 98 Philip, B., 345, 350 Philips, F. S., 117, 125 Pic6n, J . M . O., 318, 351 Pieragnoli, E., 238, 241 Pinkus, F., 318, 351 Pinkus, H., 320, 323, 334, 341, 344, 346, 351 Plaine, H. L., 119, 133 Plaut, W., 66, 68, 98 Plenck, H., 224, 241 Policard, A., 330, 351 Pollister, A. W., 23, 63, 65, 76, 85, 86, 98, 140, 156, 161, 227, 240, 249, 272, 274, 275, 277, 285, 286, 311 Pollister, P. F., 275, 311 Pomerat, C. M., 332, 334, 336, 338, 341, 348, 350, 351 Popper, H., 217, 237, 241 Porter, C. W., 6, 24, SO, 98
Porter, K. R., 138, 148, 161, 238, 311, 345, 351 Portzehl, H., 207, 215 Post, J., 227, 240 Poussel, H., 107, 133 Porter, K. R., 138, 148, 161 Prebus, A. F., 143, 161 Prescott, D. M., 152, 161 Preston, M . M . E., 199, 214 Price, J . R., 32, 93 Prud'homme, M., 5, 7, 11, 26, 37, 46, 5
Q Queiroz Lopes,A., 249, 266, 294,311 Quercioli, E., 111, 133
R Rafalko, J. S., 4, 7, 10, 98 Ram6n y Cajal, S., 262, 269, 272, 311 Randall, J . T., 138, 160, 287, 311 Rapoport, H., 7, 33, 94 Rapport, M . M., 92 Rasch, E., 65, 81, 83, 99 Rashevsky, N., 169, 170, 215 Ravanti, K., 223, 224, 235,241 Raven, C. P., 157,161,178, 196,215 Read, J., 119, 133 Rebhun, L. I., 141, 161 Rees, H., 103, 119, 133 Reinders, W., 11, 98 ResendC, F., 104, 133 Revell, S. H., 102, 115, 116, 117, 118, 131, 133, 136 Reynolds, 0. E., 234, 240 Rhoades, A., 116, 127 Richards, B. M., 2, 63, 67, 98, 99 Richter, G. H., 33, 98 Riggs, B. C., 332, 351 Riley, H . P., 119, 128, 133,134 Ris, H., 4, 43, 68, 71, 98, 144, 146, 147, 148, 149, 150, 151, 152,160,161 Ritter, H. B., 8, 98 Roberts, H . S., 199, 200, 201, 215 Robinson, R., 315, 323, 324,349 Robson, J. M., 101, 116,124 Rodriguez, N. U., 234, 241 Rojas, P., 254, 277, 311 Rollen, A., 121, 127 Rondoni, P., 121, 134 Rosen, G. V., 110, 128 '
364
AUTHOR INDEX
Rosenberg, E. W., 332 Rosenthaler, L., 91 Rosin, J., 7, 98 Ross, M. H., 10, 11, 19, 21, 68, 95 Ross, W. C . J., 117, 128 Rossenbeck, H., 3, 4, 5, 6, 9, 10, 19, 24, 65, 66, 95 Rossi, A., 110, 111, 134 Rosza, G., 144, 161 Roth, A., 234, 239 Roth, F., 332, 348 Roth, L. E., 263, 275, 308 Rothman, S., 345, 351 Rothschild, Lord, 208, 215 Rouiller, Ch., 140, 153, 158 Roux, W., 315, 351 Rowe, A. W., 24, 98 Rowe, E. C., 178, 213 Roy, M., 109, 135 Rudall, K. M., 345, 351 Rumpf, P., 19, 36, 39, 40, 41, 57, 67, 93, 98 Runnstrom, M. J., 208, 215 Ruska, 137 Rustad, R. C., 193, 215 Rutihauser, A., 103, 130, 134 Ryan, M. H., 121, 131
,.S Saez, F. A., 264, 284, 308 Sagami, S., 332, 348 St. Aubin, P. M. G., 228, 236, 241 Sanford, K., 339, 351 Sarkar, I., 121, 134 Sarkar, S. K., 110, 135 Sasaki, M., 290, 311 Sasaki, T., 243, 311 Sato, H., 243, 309 ' Sato, S., 284, 311 Saunders, B. C., 16, 97 Savage, R. E., 66, 68,98 Sawada, T., 142, 143, 162 Sax, K., 107; 134 Scanlan, J . T., 14, 15, 98 Scapa, S., 322, 350 Scarascia, G. T., 104, 134 Scarascia-Venezian, M. E., 104, 134 Schaechter, M., 47, 95 Schaffer, C., 91 Schaffner, F., 217, 237, 241
Schechtman, A. M., 204, 215 Scheringer, W., 234, 239 Scheuing, G., 3, 11, 19, 20, 21, 35, 36, 38, 39, 40, 42, 43, 45, 46, 62, 67, 68, 89, 100 Schibata, T., 255, 311 Schibsted, H., 6, 15, 19, 24, 29, 98 Schiff, A., 318, 349 Schiff, H., 2, 3, 4, 5, 6, 11, 34, 37, 98 Schimamoto, T., 255, 311 Schkarnikov, P. M., 107, 134 Schleich, A., 206, 214 Schmidt, J . G., 3, 22, 23, 24, 25, 26, 27, 29, 30, 31, 32, 99 Schneider, W. C., 156, 160, 255, 265, 268, 275, 277, 280, 308, 309,311 Schoetzow, R. E., 9, 13, 24, 94 Schooley, C., 227, 239 Schrader, F., 75, 96 Schultz, G., 295, 311 Schultz, H., 154, 161 Schultz, J., 146, 161 Schwartz, W., 158, 159 Scott, A., 189, 215 Scott, F. C., 8, 16, 99 Scott, J . F., 238, 239 Scott, W. J . M., 254, 286, 311 Scudder, H., 91 Sebruyns, M., 139, 159 Sedar, A. W., 144, 161 Segal, L., 16, 99 Seki, M., 298, 311 Selby, C. C., 138, 144,161,345, 351 Seligman, A. M., 255, 268, 308,318, 348 Seligman, J., 158, 161 Selman, G. G., 169, 171, 203, 204,215 Semenoff, W. E., 249, 311 Semmens, C. S., 27, 32, 99 Sen, S., 108, 114, 135 Senseney, C. A., 144, 159 Serra, J. A., 143,161,249, 266, 294, 311 Shaffer, B., 315, 324, 328, 351 Shafiq, S. A., 274, 286,311 Shanklin, W. M., 78, 80, 95, 99 Sharma (nCe Mookerjea), A., 110, 111, 134 Sharma, A. K., 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 114, 116, 118, 120, 121, 134, 135
365
AUTHOR INDEX
Shaw, G. W., 121, 135 Sheldon, H., 158, 161 Shelton, E., 307 Sher, I. H., 322, 350 Shibatani, A., 57, 62, 66, 69, 70, 83, 99, 225, 226, 231, 232, 233, 239, 277, 278, 295, 298, 311, 312 Shinagawa, C., 248, 312 Shindo, K., 277, 282, 312 Shreiner, R. L., 7, 99 Shock, N., 227, 233 Shoesmith, J. B., 19, 21, 23, 25, 26, 27, 30, 38, 99 Sichel, M., 188, 215 Siess, U., 226, 228, 241 Siekevitz, P., 298, 311 Simmonds, C., 91 Simms, H. S., 337, 338, 351 Simonet, M., 121, 135 Simpson, W. L., 262, 263, 275, 312 Sinesi, S. J., 341, 350 Singer, D., 235, 239 Sinigaglia, G., 248, 312 Sjostrand, F. S., 148, 161, 275, 276, 287, 312 Smiddy, J. G., 345, 349 Smith, D. E., 119, 133 Smith, H. H., 121, 135 Smith, L., 103, 129, 135 Smith, R. H., 143, 161 Smits, G., 67, 75, 96 Smyth, J. D., 267,307 Smythe, 238 Snell, C. T., 9, 24, 99 Snell, F. D., 9, 24, 99 Sorof, S., 244, 298, 312 Sosa, J. M., 249, 258, 275, 312 Sosson, C . E., 19, 21, 23, 25, 26, 27, 30, 38, 99 Sotelo, J. R., 139, 149, 159 Sparrow, A. H., 103, 116,132,135 Sparrow, R. C., 103, 135 Srb, A. M., 121, 135 Srinivsachar, D., 70, 99 Stacey, M., 66, 98. 99 Stadie, W. C., 332, 351 Stalfelt, M. G., 110, 135 Staple, P. H., 7, 15, 99 . Stedman, Edgar, 66, 99
Stedman, Ellen, 66, 99 Steffensen, D., 105, 135 Stegmann, H., 226, 228, 241 Steiglitz, J., 40, 99 Steinegger, E., 105, 110, 121, 130,136 Steinitz, L. M., 114, 129 Sternheimer, R., 235, 241 Stewart, T. D., 6, 24, 40, 98 Stich, H. F., 149, 161, 238, 241 Stock, C . C., 117, 125 Storey, W. F., 320, 351 Stotz, Elmer, 12, 95 Stowell, R. E., 5, 8, 37, 43, 71, 82, 99, 235, 236, 241 Strangeways, T. S. P., 199,215,323,351 Straube, R. L., 119, 133 Striebich, M. J., 275, 307 Stubbe, H., 122, 127 Sugihara, R., 142, 143, 162 Sugioka, M., 142, 143, 162 Sugiyama, M., 166, 213 Sulkin, N. M., 236, 241 Sullivan, B. J., 110, 121, 124, 136 Sussman, N., 238, 241 Suzuki, I., 292, 311 Swaminathan, M. S., 112, 136 Swann, M. M., 164, 169, 170, 171, 173, 174, 175, 176, 177, 178, 179, 180, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 199, 205, 214, 215, 216 Swanson, C., 150, 161 Swartz, F. J., 226, 227, 241 Swick, R. W., 224, 241 Swift, H., 2, 18, 21, 45, 62, 65, 67, 70, 71, 81, 83, 84, 98, 99, 140, 141, 153, 154, 156, 161 Swift, H. H., 226, 241 Szakall, A., 321, 334, 351
T Takagi, S., 249, 251, 254, 267, 268, 285, 292, 312 Tagawa, M., 267, 268, 312 Tahmisian, T. N., 267, 275, 276,306,308 Tajima, A., 246, 313 Takahashi, C., 292, 311 Takahashi, Y., 277, 282, 312 Takeichi, T., 284, 312
366
AUTHOR INDEX
V Takenaka, Y., 105, 136 Takeuchi, T., 249, 312 Vaarama, A., 112, 136 Tamura, R., 50, 75, 98 Valeri, V., 226, 240 Tanaka, H., 275, 312 Van Breeman, V., 144, 159 Tanaka, T., 290, 312 Vanderlyn, L., 106, 136 Tarabusi, T., 121, 136 van Duijn, P., 53, 54, 79, 99 Tarao, S., 268, 272, 275, 294, 312 van Houcke, A., 121, 131 Taylor, A. B., 258, 262, 267, 275, 306 Van Winkle, Q., 143, 161 Taylor, J . H., 66, 68, 99, 151, 157,161 Veddrely, C., 226, 240 Teece, G., 66, 99 Vendrely, R., 226, 240 Tennenbaum, E., 338, 349 Vercauteren, R., 121, 131 Terayama, H., 244, 298, 309,312 Verne, J., 73, 76, 99 Teruya, K., 244, 298, 309,312 Vincent, W. S., 150, 161 Thamsen, A., 228, 240 Vles, S. I., 11, 98 Therman, E., 114, 118, 136 Vogel, A. I., 7, 9, 33, 99 Thiersch, J . B., 117, 125 Voit, K., 3, 24, 71, 92, 95 Thiery, M., 139, 159 Volkmann, R., 227, 241, 320, 351 Thomas, 0. L., 267, 274, 312 von Bergen, F., 262, 312 Thomas, P. T., 116, 136 von Bitto, Bela, 6, 9, 24, 25, 26, 27, 30, Thompson, R. Y., 226, 236, 241 32, 94 Thorvik, A., 10, 95 von Buttlar, R. F., 23, 26, 27, 30, 98 Tier, H., 223, 224, 235, 241 von Khssa, J., 266, 309 Timofeeff-Ressovsky, N. W., 103, 136 von Pechmann, H., 24, 27, 97,98 Tjio, J . H., 103, 110, 112, 118, 119, 130, von Rosen, G., 122, 136 136 von Wettstein, V., 119, 127 Tobie, W. C., 4, 6, 7, 10, 14, 15, 19, 23, W 24, 25, 30, 31, 32, 99 Waddington, C. H., 169, 171, 195, 203, Toro, E., 346, 351 204, 215, 216, 292, 312 Togari, T., 251, 312 Wakonig, T., 111, 133 Tollens, B., 27, 95 Walker, B. E., 224, 240 Tolman, L. M., 6, 24, 99 Walker, P. M. B., 2, 63, 67, 98, 99 Tomlin, S. G., 139, 159 Wallgren, I., 275, 312 Trescot, T. C., 6, 24, 99 Walter, F., 235, 241 Tron, F., 93 Walters, M. S., 103, 136 Trowell, 0. A., 332, 351 Wang, A. B., 3, 99 Tsuboi, K. K., 235, 236, 241 Wang, L. S., 276, 312 Tujimura, H., 292, 312 Ward, R., 140, 156, 161 Turco, G. L., 2 0 , 241 Watanabe, Y., 255, 276, 312 Tyree, E. G., 119, 133 Watson, J . D., 68, 71, 99 Watson, M. L., 139, 140, 141, 148, 153, U 154, 156, 161 Uber, F. M., 119;128 Waymouth, C., 234, 239 Ueda, M., 255, 311 Weatherford, H. L., 275, 312 Uffoed, E. H., 249, 308 Weber, H. H., 205, 214, 216 Ulloa-Gregori, O., 338, 351 Wechsler, H. J., 110, 136 Upcott, M. B., 102, 127 Weedon, B. C . L., 7, 33, 97 Urbaine, M. G., 37, 99 Weidinger, A., 345, 349 Usieli, V., 235, 239 Weil, H., 35, 95
367
AUTHOR INDEX
Weiss, P., 321, 326, 328, 330, 345, 351 Welcker, H., 223, 241 Wennecker, A., 238, 241 Wermel, E., 19, 99 Werner, B., 67, 75, 96 Wertheim, E., 5, 7, 11, 15, 21, 100 Wessel, W., 154, 161 West, P. W., 93 Weston, J. C., 221, 225, 231, 233, 241 Whaley, W. G., 276, 313 Wheeler, C. E., 339, 351 White, A. V., 234, 239 White, E. G., 224, 241 Widstrom, G., 6, 18, 45, 57, 67, 69, 70, 100 Wieland, H., 3, 11, 19, 20, 21, 35, 36, 38, 39, 40, 42, 43, 45, 46, 62, 67, 68, 89, 100 Wiggins, L., 66, 99 Wild, F., 7, 9, 100 Williamson, M. B., 218, 220, 241 Williamson, R. R., 171, 213 Wilson, D. F., 144, 161 Wilson, E. B., 156, 161, 191, 192, 194, 195, 196, 209, 216,262, 263, 313 Wilson, G. B., 112, 136 Wilson, J. W., 228, 229, 235, 241 Wilson, M. E., 236, 241 Wilson, W. L., 177, 178, 1%, 208,216 Winbury, U., 234, 240 Wirtz, G. H., 244, 298, 313 Wischnitzer, S., 140, 141, 142, 146, 153, 154, 156, 161 Wislocki, G. B., 73, 95, 255, 268, 280, 287, 306, 307 Witkus, E. R., 113, 121,124 Wolf, R. L., 235, 241 Wolff, S., 119, 136 Wolffenstein, R., 26, 100
Woll, E., 114, 136 Wolman, M., 73, 75, 100 Wolpert, L., 171, 176, 179, 183, 207, 208, 209, 215, 216 Woodman, A. G., 6, 10, 24, 26,100 Woods, M. W., 292, 313 Woods, P. S., 66, 68,100, 151, 157,161 Worley, L. G., 249, 274, 313 Wunsche, F., 29, 100 Wyburn, G. M., 139, 141, 159 Wyckoff, R. W. G., 144, 161
Y Yakar, N., 121, 136 Yamada, E., 249, 262, 313 Yanagisawa, N., 267, 309 Yanagita, T., 166, 213 Yarbo, D. L., 14, 100 Yasuma, A., 3, 29, 55, 77, 78,100 Yasuzumi, G., 142, 143, 144, 146, 149, 161, 162, 246, 313 Yeakel, E. H., 234, 235, 241 Yokoyama, H. O., 236, 241 Yoshida, M., 82, 83, 100 Yoshida, T., 243, 311, 313 Yoshimoto, T., 258, 259, 260, 262, 266, 267, 268,270, 275, 280, 284,310 Young, E. M., 244, 298, 312
, z Zaki, F., 238, 239 Zalokar, M., 157, 162 Zelle, M. R., 57, 62, 92, 95 Zeros, N., 249, 274, 313 Zetterqvist, H., 158, 161, 275, 313 Zimmer, K. G., 103, 136 Zimmerman, A. M., 181,206, 208,21+, 216 Zinner, G., 27, 100 Zollinger, H. U., 277, 313
Subject Index Antirrhinicm, mutation in, 103 A Arrhenal, mutagenic properties of, 112 AAT, see o-Aminoazotoluene Arsenic, mutagenic properties of, 121 Acaritkarriocba, Feulgen reaction in, 67 Acenaphthene, mutagenic properties of, Asters, role in cleavage, 191-193 121 Astral relaxation theory of cleavage, 169, Acetaldehyde, Schiff reaction of, 7, 42, 57 174, 186, 212 Acetic acid, effect on Schiffs reagent, 17 applications of, 195-205 Acridine derivatives, mutagenic proper- Auramine 0, in Feulgen reaction, 58, 60ties of, 121 61 Acriflavine, Azine series, mutagenic properties of, 121 mutagenic properties of, 121 Azo dye(s), as Schiff-type reagent, 48, 51, 54-55 affinity for cytoplasmic components, -SO,, in Feulgen reaction, 60,63 243-313 Adenine, mutagenic properties of, 119-120 as cleavage inhibitor, 209 reagent, 245 -induced chromosomal breakage, 120 Azotoriprite, mutagenic properties of, 121 Adenosine triphosphate, effect on cleav- Azure A, as Schiff-type reagent, 47-48, age, 205-206 51 Aesculetin, mutagenic properties of, 111B 112 .4lcohols, Schiff-negative, 30 BAL, mutagenic properties of, 112 .4ldehydes, reaction with Schiff’s reagent, Bases, 25-27, 37-45, 77 failing to give Schiff’s reaction, 31 Alizarin sulfonate, inhibition of cleavage reaction with Schiffs reagent, 27 by, 209 Benzene, mutagenic properties of, 121 Alkaloid-induced chromosome breakage, Benzimadazole, as cleavage inhibitor, 209 110-112 Benzoquinone, mutagenic properties of, -4!h811 species, mutation in, 102, 104, 106, 118 108, 111 Benzpyrene, mutagenic properties of, 121 Alloxan-Schiff reagent, 29, 55, 77 Amino oxides, reaction with Schiff‘s re- Berberine, mutagenic properties of, 110111 agent, 25 o-Aminoazotoluene, scc also Oil yellow Bismarck brown Y, as Schiff-type reagent, 54-55 granules Bromine, reaction with Schiff’s reagent, as hepatic carcinogen, 243 29 mechanism of staining method, 295 Broinus hybrids, mutation in, 103 solubility of, 299-302 Aminohydroxyl compounds, detection Butyraldehyde, Schiff reaction of, 57 with Schiffs reagent, 3 C f3-Aminophenol-induced chromosome Caffeine, breakage, 118. 8-ethoxy-, mutagenic properties of, 106, Amoeba protetrs, cleavage of, 202-203 120 Amphibian egg, cleavage of, 203-205 mutagenic properties of, 110, 120-121 Annular complex, cytological definition, Calcium, 139 deficiency, effect on chromosomes, 105 Annuli, cytological definition, 138 effect on cleavage, 206 Anthracenes, mutagenic properties of, effect on cell membrane, 208 119-120 368
SUBJECT INDEX
369
Calcium phosphate, cytoplasmic detec- Chromatin, fuchsin stain for, 4-5, 7 tion of, 266 silver-Feulgen method for, 91 Cancer, photomicrography of tissue in Feulgen Chromic acid, mutagenic properties of, 121 reaction, 59-61 -Schiff reagent, 29 “protein deletion” theory of, 243, 298 Carbon tetrachloride, effect on liver, 235 Chromosome breakage, 101-136 Carcinogens, alkaloid-induced, 110-112 affinity for somatic cells, 257-304 anthracene-induced, 119-120 chemical nature of, 299-304 colchicine effect on, 107 cytoplasmic studies of, 243-313 coumarin-induced, 110-112 effect on erythrocytes, 246-255 drug-induced, 112 intracellular localization of, 290-292 dye-induced, 109-110 Cell (s), essential oil-induced, 112 argentophobic reticula of, 260-263 growth regulator-induced, 116 constituents, see under individual strucby heterocyclic bases, 120 tures hormone-induced, 113-116 in hybrids and polyploids, 103 elastimeter, 177, 182, 185, 211 elongation of, 178-179 mineral deficiency effects on, 105, 108 schematic representation of, 155 mustards-induced, 116-119 surface, mechanical properties of, 175nucleic acid and, 123 183 osmotic balance and, 107-108 Cell membrane, oxygen effect on, 119 birefringence, 183, 189-190, 208 by phenols, 116-119 calcium in, 210 pigment-induced, 110-112 changes during cleavage, 180-183 protective chemicals for, 119 chemical effects on, 208 purine-induced, 121 comparison to actomyosin, 205-207 R N A metabolism and, 106 compression of, 177 spontaneous, 102-106 cortex, gel strength of, 177-178 technical limitations in, 107-110 “dynamic” properties of, 210-211 temperature-induced, 107-108 gel strength of, 181 vitamin-induced, 116 mechanical analogies for, 179-180 water-induced, 108 particle displacement of, 177-178 X-ray induced, 107, 119 plasmagel of, 175 Chromosomes, properties of, 175-176 Balbiani rings of, 148 “stiffness” changes in, 182-183 chromonema of, 151 structure of, 183, 207-208 in cleavage, 185, 194, 200, 202-203, 212 surface theories and, 183-185 D N A of, 150-152 Young’s modulus of, 195 giant, 146-148 Ceroid, histochemical test for, 176 interphase, 143-144 Clmetoptencs, cleavage studies on, 1% lampbrush, 146-148, 150 Clrlairrydornoaas, stain for, 47 meiotic, 145-146, 150 Chloramine T, use with Schiff‘s reagent, mitotic, 144-145 77 morphology of, 151 Chloranil, mutagenic properties of, 121 osmium fixation of, 149 Chloroform, mutagenic properties of, 121 p-Chloromercurobenzoate, effect on RNA of, 150, 157 cleavage, 206 ultrastructure of, 143-148, 152
370
SUBJECT INDEX
Chrysoidine R, as Schiff-type reagent, Colchiczinz species, autotoxicosis in, 105 48, 51, 89, 90 colchicine from, 110 Chrysoidine Y-SO,, in Feulgen reaction, Contractile ring, in cleavage, 189 87-88 Cicer arietinum, heterochromatic pairing Coramine, effect on liver, 235 Coriphosphine, as Schiff-type reagent, 56 in, 116 Cortex, cell membrane, 175 Cleavage, 163-216 Cortical gel contraction theory of cleavof amphibian egg, 203-206 age, 185, 212 asters’ role in, 191-193 astral relaxation theory of, 169, 174, Coumarin, mutagenic properties of, 110112 186-202, 212 Cresyl blue, A T P effect on, 205-206 as Schiff-type reagent, 46, 90 biochemistry of, 205-211 -induced chromosome breakage, 109 calcium effects on, 208 Cresyl violet, as Schiff-type reagent, 18, cell-late theory of, 171 51 cell surface movement in, 168 Crocus, chromosome breakage in, 104 chemical effects on, 208-210 chromosomes’ role in, 185, 194, 200, Cyclohexylcarbamate, mutagenic properties of, 121 202-203, 212 Cytophotometry, 81 contractile ring in, 189 Cytoplasm, azo dye affinity for, 243-313 coordination of, 194 cortical gel contraction theory of, 171D 173, 185, 212 DAB, see p-Dimethylaminoazobenzene diffusion-drag theory of, 170-171 DDT insecticides, mutagenic properties eccentric, 197-198 of, 112 energy requirements of, 194-195 Deoxyribonucleic acid, expanding membrane theory of, 173detection with Schiffs reagent, 3, 18, 174, 184-185, 201, 211 28 of fibroblast, 199 Feulgen reaction of, 53, 57 geometry of, 165-168 in liver, 229-233 growth theories of, 171 Detergent, effect on cleavage, 210 inhibition of, 208-209 Diallyl sulfide, effect on onion mutation, membrane changes during, 180-183, 105 190-192 2,6-Diaminopurine, as cleavage inhibitor, of neuroblast, 200-202 209 polar body formation in, 1%-197 Diazofuchsin, as Schiff-type reagent, 46 polar lobe formation in, 197 Di (2 :I-epoxypropyl) ether, -induced precleavage differentiation, 191 chromosome breakage, 115 in sea urchin egg, 166-168 Diethyl ether, mutagenic properties of, “stationary rings” in, 197 106, 115 spindle elongation theory of, 169-170 Diffusion-drag theory of cleavage, 170 surface force theories of, 171-174, 185 Digitonin, effect on cleavage, 210 tension changes in, 187-189 Dimethane sulfonyloxybutane, chroniotheories of, 169-174 some breakage by, 106 Colchicine, p-Dimethylaminoazobenzene, see also Oil -induced chromosome breakage, 107, yellow granule method 110 as hepatic carcinogen, 243 mitotic destruction by, 191 as histochemical stain, 2 9 S m use in liver mitosis study, 224 solubility of, 302-304
371
SUBJECT INDEX
“Discontinuities,” cytological definition of, 138, 155 Doebner’s violet, 11, 36 as Schiff-type reagent, 46 Drugs, mutagenic properties of, 112 Dyes, mutagenic properties of, 109-110
E Electron microscopy, autoradiographic, 158 cytochemical, 158 nomenclature, 138-139 use in cell study, 137-162 Endoplasmic reticulum, cellular, 155 Epidermal cells, in culture, 315-351 irt vitro properties, 323-331 irt Vivo properties, 317-323 keratinization of, 324-326, 328-329 organized growth of, 323-329, 331-336 plasma liquefaction by, 330-331 postnatal, in vivo properties, 331-334 -SH positive, 317, 326 unorganized growth of, 329-330, 336341 vitamin A effect on, 328 wound healing in, 341-344 Epidermis, healing of, 321-322 human, culture of, 332-334 keratinization of, 321-328 mitotic activity of, 318-320 renewal of, 320 stratum corneum function of, 321 Ergastoplasm, cellular, 155 Erythrocytes, carcinogen studies on, 246-255 cytoplasmic granule staining of, 25025 1 Gold body of, 249 granules of, 305 mitochondria of, 249-257 oil yellow granules of, 246-255 Essential oils, mutagenic properties of, 112 Ethyl urethane, mutagenic properties of, 122 Ethylenediaminetetraacetic acid, mutagenic properties of, 121
Expanding membrane theory of cleavage, 184-185, 201
F Fatty acids, detection with Schiffs reagent, 3 Fats, mutagenic properties of, 112 Ferric oxide, use in polysaccharide detection, 80 Feulgen reaction, 28, 37, 43, 45-46, 50, 53, 64-74 acids in, 68-69 DNA absorption curve, 82 PAS reaction in, aldehyde-specific reagents, 79 photomicrographs of tissue in, 58 protein effect on, 70 reviews on, 2 with Schiff-type reagents, 54-55 silver, 91 species effect on, 71 theory of, 66, 69 Fibroblast, cleavage of, 199 Flavophosphine N, in Feulgen reaction,
60 Formaldehyde, Schiff reaction of, 40-41, 43, 57, 67, 92-93 Fuchsin, see also Schiff’s reagent absorptive curves of, 13 reaction with aldehydes, 2-3 spectrophotometric data on various batches, 14 0 Gallic acid, mutagenic properties of, 121 Gammexane, mutagenic properties of, 121 Germanin, effect on cleavage, 206 Glucose, effect on epidermal mitosis, 335336 Glucosides, Schiff-negative, 30 Glycogen, detection with Schiffs reagent, 3, 67, 92 in liver, 233-234 Glycolipids, reaction with Schiffs reagent, 28 1,2 Glycols, detection with Schiffs reagent, 3, 29, 74
372
SUBJECT INDEX
Glycoproteins, reaction with Schiff's reagent, 28 Golgi apparatus, 155, 263-268 argentophil material of, 258-268, 305 electron microscopy of, 275-276 enzymes of, 267-268 of erythrocytes, 249 lipids in, 268, 276 OYG staining of, 288 pathological increase of argentophil substance of, 269-270 phosphatase in, 267 pigment in, 265 reaction with Schiffs reagent, 28 Guaiacol, mutagenic properties of, 118 Guanidine nitrate, mutagenic properties of, 121 Guanine, mutagenic properties of, 120
H Hair, histochemical test for, 76 Halogens, mutagenic properties of, 121122 Hemocytes, smeared preparation for, 245246 Heparins, reaction with Schiff's reagent, 28 Hepatoma, phosphate metabolism in, 298 Histochemistry, Schiffs reagent in, 2829 Hordcrirn vrclgare, chromosome breaks in, 106 Hormones, effect on liver polyploidy, 227 mutagenic properties of, 113-116 Hyacinthus, mutation in, 103 Hyaluronic acid, Feulgen reaction of, 67 Hydrochloric acid, effect on Schiffs reagent, 17 Hydrogen peroxide, mutagenic properties of, 121 Hydroquinone, mutagenic properties of, 118 as SchifYs reagent preservative, 11 Hydroxyhydroquinone, muhgenic properties of, 118
I Ilyanassa, cleavage studies on, 197 Indoin blue, as Schiff-type reagent, 46
Indolylacetic acid, mutagenic properties of, 113-114 Indolylbutyric acid, mutagenic properties of, 113 Indolylpropionic acid, mutagenic properties of, 113 Isobutyraldehyde, Schiff reaction of, 57
K Karyoplasm, OYG staining of, 290 Karyotypes, hormone-induced clarification of, 113 Keratin, detection with Schiffs reagent, 3 Keratinization, 324-326, 328-329, 341-348 Ketone acids, Schiff-negative, 30 Ketones, reaction with Schiffs reagent, 27 Schiff-negative, 31
L Latltgrrcs, mutation in, 103 Leukemia, liver size in, 235 Lirnnaca, cleavage studies on, 1% Lipids, in liver, 234 reaction with Schiff's reagent, 29, 76, 78 Liver, anatomical development, 218 cells, binuclear, 228-229 composition of, 230 DNA content, 225-226, 229-233 nuclear classes in, 225 nucleo-cytoplasmic ratio, 227-228 polyploidy of, 224-227, 237 change in lactation, 234 change in pregnancy, 234 changes in chemical composition, 229234 diet effects on, 235 effect of tumors on, 234-235 effect of various chemicals on, 235-236 embryonic growth, 219-221 glycogen in, 233-234 growth in mammals, 217-241 lipids in, 234 mitotic activity of, 223-224
373
SUBJECT INDEX
postnatal growth of, 222-223 protein in, 231-233 regeneration after hepatectomy, 2% regulation of size, 237-238 RNA in, 231-233 water content of, 230-231 weight changes in growth, 218-221 weight of in various species, 223 Liver extracts, effect on liver, 235
Mucoproteins, reaction with Schiffs reagent, 28 Muscle protein analogy in biochemistry of cleavage, 205-207 Mustards, mutagenic properties of, 116119 Mutation, see also Chromosome breakage seed age and, 103-101 X-ray induced, 103
M
N
Magdala red, as Schiff-type reagent, 15, 53 Magenta, sce Fuchsin Magenta 11, structure of, 12 Magnesium deficiency, effect on chromosome breakage, 105 Malachite green, as Schiff-type reagent, 12 Maleic hydrazide, mutagenic properties of, 106, 114-115 Mannich reaction, 36, 41 Matrix, cytological definition of, 139 Mayer’s reagent, 248, 299 Mercury compounds, mutagenic properties of, 121 Methanol, Schiffs reagent for, 9 Methylene blue, mutagenic properties of, 109 stain for Golgi apparatus, 266 Microfibrils, definition of, 139 Microsomes, 155 Milk, bacteria detection in, 75 Mineral deficiency, effect on chromosome breakage, 108 Mitochondria, 155 filamentous, 280-288, 305 granular, 277-280, 305 oil yellow granules and, 255, 288 of stained erythrocytes, 255-257 structure of, 277-288 Monochromator, use in cytophotometry, 81-82 Morphine, mutagenic properties of, 121 Mucins, reaction with Schiff’s reagent, 28, 92 Mucopolysaccharides, reaction with Schiff’s reagent, 28 role in epidermal healing, 322
Naphthalene, mutagenic properties of, 121 Naphthalene acetic acid, mutagenic properties of, 113 8-Naphthoquinoline, mutagenic properties of, 121 8-Naphthoxyacetic acid, mutagenic properties of, 113 Narcotics, inhibition of cleavage by, 209 Neuroblast, 200-202 Neutral red -induced chromosome breakage, 109 as Schiff-type reagent, 46-47, 52 Ninhydrin-Schiff’s reagent, 29, 55, 77 Nothoscordrcm, mutation in, 103 Nuclear envelope, 155, 158 definition of, 138 ultrastructure of, 139-141, 149, 132-154 Nuclear membranes, definition of, 138 Nucleic acid, in chromosome breaks, 123 Nucleocytoplasm, relations of, 152-158 ultrastructure of, 137-162 Nucleolonema, 155, 158 definition of, 139 ultrastructure of, 142 Nucleolus, 155, 158 function of, 150 granules in, 142-143, 156 ultrastructure of, 142-143, 149 Nucleoplasm, ultrastructure of, 118 Nucleus, DPN-enzyme of, 156 RNA in, 156-157 ultrastructure of, 137-162
0 Oil yellow granule (OYG) method, for carcinogen study, 244
374
SUBJECT INDEX
for cellular components, 288-292 decolorization test in, 294-295 in erythrocytes, 247 hydrochloric acid estimation in, 296299 mechanism of, 292-296 pH effect on, 295-296 Oils, vegetable, mutagenic properties of, 112 Ocnothera, chromosome breakage in, 104 Orcein-induced chromosome breakage, 109-110 Oxygen, mutagenic properties of, 122
P Pneonia, mutation in, 103-104 PAS reaction, see Periodic acid-Schiff reagent Peracetic acid-Schiff reaction, 29 Performic acid-Schiff reaction, 29, 55 Periodic acid-Schiff reagent, 28-29, 74-76 Permanganate-Schiff reagent, 29 Phaseoks vulgaris, chromosome breakage in, 104 Phenanthrene, mutagenic properties of, 121 Phenols, mutagenic properties of, 116-119 Schiff -negative, 31 Phenosafranin-SO2, as Schiff-type reagent, 48, 51, 53-55, 86 Phenylacetic acid, mutagenic properties, 113 p-Phenylenediamine, mutagenic properties, 118 Phloroglucinol, mutagenic properties, 118 Phosphatase, in Golgi apparatus, 267 histochemical use of, 80 Phosphate metabolism, role in hepatoma, 298 Phosphates, mutagenic properties of, 121122 Phosphatides, reaction with Schiffs reagent, 28 Phosphine, as Schiff-type reagent, 56 Phospholipase, effect on cell membrane birefringence, 208
Phospholipids, affinity for azo dyes, 306 reaction with Schiffs reagent, 29 Phosphomanganate, mutagenic properties of, 122 Pigment-induced chromosome breakage, 110-112 Piperidine, reaction with Schiff's reagent, 27,32 Pisum, mutation in, 103 Plasma1 reaction, 71-73 of Schiff's reagent, 3 Plasmalogens, reaction with Schiffs reagent, 28,29 Podophyllin, mitotic destruction by, 191 Podophyllotoxin, mutagenic properties of, 110-111 Polysaccharides, reaction with Schiffs reagent, 27, 29, 80 Schiff -negative, 31 Propiolactone, mutagenic properties of, 121 Propionaldehyde, Schiff reaction of, 57 Proteins, detection with Schiff's reagent, 3, 29, 77-78 in liver, 231-235 Protoanemonine, mutagenic properties of, 110 Purines, mutagenic properties of, 120 Putrescin, mutagenic properties of, 104, 110 Pyridine, reaction with Schiffs reagent, 27, 32 Pyrocatechol, mutagenic properties of, 118 Pyrogallol, mutagenic properties, 118 as preservative for Schiffs reagent, 11
Q Quinoline, mutagenic properties of, 121 Quinones, effect on cleavage, 209
R Resorcinol, mutagenic properties of, 118 Reticulum, argentophobic, 260-263 Ribonucleic acid, cellular synthesis, 156-157 in liver, 231
375
SUBJECT INDEX
Rosaniline, leucosulfonic acid, 37, 44-45 para-, 35 structure, 12
S Safranin 0, as Schiff-type reagent, 46,49 Salicin, mutagenic properties of, 112 Salicylates, mutagenic properties of, 122 Schiffs reagent, see also Fuchsin acetaldehyde reaction of, 42 acids in, 5, 10, 16-18 acriflavine use with, 63-64 alloxan-, 29, 55, 77 charcoal purification of, 15-16 chemistry of, 1-100 chromic acid-, 29, 73 compounds not reacting with, 30-32 in DNA detection, 3, 18 dyes resembling, 6 5 6 Feulgen reaction of, 28, 56-71 glycogen detection with, 92 heat effect on, 21-22 histochemical reaction of, 28-22, 65-80 history of, 2-3 hydrolytic dissociation of, 22 kinetics of, 56, 62-64 lipid staining by, 78 Mannich reaction in, 36, 41 mucin detection with, 92 multiple staining by, 78-80 ninhydrin-, 29, 55, 77 peracetic acid-, 29, 76 performic acid-, 29, 53, 76 periodic acid-, 28-29, 74-76 permanganate-, 29 photometric studies of, 81-89 plasma1 reaction of, 71-73, 92 preparation methods, 4-11 preservatives for, 11 protein detection by, 77-78 “pseudoreaction” of, 32 radioactive, 76 reaction with aldehydes, 37-45, 57 abnormal, 38-39 alkyl-sulfonic acid theory of, 39-42 condensation theory, 37-38 Wieland and Scheuing theory of, 4245 reaction with bromine, 29
reaction with formaldehyde, 40-41, 9293 salt effect on, 16-18 sensitivity of, 11-22 specificity of,22-33, 89 stability of, 10-11 standardized, 11, 15 sulfur dioxide in, 19-21 ultraviolet-, 29 various compositions, 5-9 Scdla, mutation in, 103 Sea urchin egg, cleavage in, 166-168 Seed, aging, relation to mutation, 103104 Sodium tungstate, effect on cleavage, 210 Soybean extracts, chromosome breakage in, 104 Spermatocytes, nucleus of, 145 Spindle elongation theory of cleavage, 169 Spirit blue, as Schiff-type reagent, 53 Starvation, effect on liver, 235 Stratum corneum, function of, 321 Sugar alcohols, Schiff -negative, 31 Sugars, mutagenic properties of, 121 reaction with Schiff‘s reagent, 25 Schiff-negative, 31-32 Sulfa compounds, mutagenic properties of, 112 Sulfaminic acids, 36 Sulfhydryl groups, in keratinization, 317, 326, 341-345 Sulfur dioxide, in Schiff‘s reagent, 4-5, 19-21 Surface tension theories of cell cleavage, 185
T Tetrahymena, Feulgen reaction in, 67 iron effects on, 92 Theobromine, mutagenic properties of, 110, 120 Theophylline, mutagenic properties of, 110, 120 Thionine-S02, as Schiff-type reagent, 4647, 49, 52-53 Thiourea, effect on liver, 235 Thyroxine, effect on liver, 235
376
SUBJECT INDEX
Toluidine, mutagenic properties of, 109 as Schiff-type reagent, 47, 49 Tradescantia, chromosome studies on, 102 Trillium species, mutation in, 103 use in cytophotometry, 83 Triticum, mutation in, 103 Trypan blue, effect on liver, 235 Trypaflavin, mutagenic properties of, 121 Trypsin, effect on cell birefringence, 208 Tubifex, cleavage studies on, 174 Typogen brown, as Schiff-type reagent, 15, 53
U Uracil, mutagenic properties of, 120 Urethane, mutagenic properties of, 121
V Veratrine, mutagenic properties of, 110 Veronal, mutagenic properties of, 121
Versene, mutagenic properties of, 121 Vicia species, chromosome studies on, 103-106, 114, 151 Vitamin A, metaplasia from, 328 Vitamins, mutagenic properties of, 116, 121
W Wasp venom, effect on cleavage, 210 Water, mutagenic properties of, 108 Wound healing, 330-331, 341-344
X X rays, mutagenic properties of, 107, 119 Xylene, mutagenic properties of, 121 Xylaquinone, mutagenic properties of, 122
Y Young’s modulus, in cell cleavage, 176177, 186
2 Zenker’s fluid, use in cytophotometry, 84
Cumulative Index Volumes I-IX A Absorption, curves of cells, interpretation of, 11,
108-110 visible, use in cytochemistry, 11, 462-
465 Absorption histospectroscopy, 11, 455-468 A cetabwlaria, development of, nucleocytoplasmic relationships in, 11, 475-498 interspecific grafts, 11, 478 ff., 4%-497 morphogenic substances in, 11, 477-489 mode of action of, 11, 478-485 origin of, 11, 485-486 nucleate and anucleate parts of, 11, 477-
position in sperm, V, 443 sperm penetration and, VII,209 structural aspects of, VII,207-212 Acrosome reaction, V, 365-393 Actidione, effect on mitosis, IX,299-300 Action potential, sodium and, VIII, 463-
467 Active transport, in cells, IX, 132-133 minimal energy requirements for, 11,
437-441 transport potentials, 11, 436-441 Adductor muscle, VII, 297-298 Adenine, lymphocytes and, VII,260-261 Adenosine deaminase, isolated nuclei and,
VIII, 291, 331
478, 4954% nucleolus, functions of, 11, 497 nucleus effect on morphogenesis, 11,
477-489 Acetate, activation, cations and, VIII, 476 enzymatic, 11, 217-222 in plant systems, IV,179 Acetic acid, as fixative, IV,85-86 Acetobacter xylinum, microfibril synthesis in, VIII, 51 Acetone, as fixative, IV,86-87 Acetyl coenzyme A deacylase, isolated nuclei and, VIII,290, 337 Acetylcholine, VII, 70-92 Acetylcholinesterase, in erythrocytes, V,285-286 “neurosecretory” cells and, VII,364 in placenta, VI, 330 in thyroid, VI, 285-286 Aconitase, isolated nuclei and, VIII,291 Acrosome, VI, 96 chemical properties of, VII,209 degeneration of, V, 444-445 filament of, role, V,389-391 formation in flagellate sperm, V, 398-
429 functions of, V, 445-446 lysin of, V, 386-387 occurrence, V, 443
Adenosine phosphatase, isolated nuclei and, VIII, 292, 336 Adenosine triphosphatase, isolated nuclei and, VIII,292-293,336-337 Adenosine triphosphate, effect on amoeboid form, V, 221-223 effect on furrowing strength, V, 219-
221 enzyme activation and, VIII,373-375 intercellular material and, VII,634 ion movements and, VIII,470-471 magnesium and, VIII,369, 372 in nuclei, VI, 391-392 Adhesion, artificial substrate and, IX,211-220 of cells, IX, 187-255 contact guidance and, VII,399-400 of malignant cells, IX,195 morphogenesis and, IX,198-206 reticulocytes and, VIII, 157 role in metastasis, IX,206-211 specificity of, IX,204-205 wettability and, IX, 192-193 Adrenal gland, cell mitochondria, IX,237-238 lymphocytes and, VII,269-271,273 lymphopoiesis and, VII,247-248 nervous stimulation of, VIII,82 synaptic vesicles and, VIII,76
377
.
378
CUMULATIVE INDEX
- VOLUMES I-IX
Adrenal medulla, ascorbic acid content, chloroplast structure of, IV, 201, 203shock effect on, 11, 121 207 Adrenaline, flagellate, IV, 203-204 mitochondria of, IX, 238-239 chromatophores and, VIII, 203 A l l t h cepa, kinetochore structure in, effect on mitosis, IX, 301 VII, 125, 137-138 PAS-reaction of, VI, 223 Adrenochrome, effect on explanted tis- Alloxan, effect on glycolysis, IX, 302 sues, 11, 336-341 Ambinon, chick thyroid and, VII, 108 Amblystoma, kinetochore structure in, Adrenocorticotropic hormone, VII, 139 chromatophores and, VIII, 203, 204 Amine oxidase, isolated nuclei and, VIII, effect on mitosis, IX, 301 295, 322, 324, 327, 337 swollen mitochondria and, IX, 246 Amino acid (s) , Agapanthus urnbellatus, in bacterial cell walls, V, 32-33,47 chromosome organization in, IX, 152connective tissue and, VIII, 227-229 153 pachytene chromosomes of, IX, 95 effect on tissue cultures, 111, 34-42 Aggregation, selective, VII, 408 incorporation into nuclei, VI, 407-411; 0-Alanine, effect on tissue cultures, 111, VIII, 286, 326, 333 52 metabolism in nuclei, VI, 394-395 Albedo, background adaptation and, VIII, oxidation reactions of, VI, 217-219 pleomorphism induction by, IX, 54 180 Albumin(s), reticulocytes and, VIII, 150-151 cell refractive index, VIII, 417 sequence in collagen, VIII, 243-245 chloride ions and, VIII, 398 in spore formation, IX, 34 foreign, effect on kidney cells, IX, 256stains for, IX, 374-377 257 Amino acid oxidases, isolated nuclei and, mitochondria1 concentration of, IX, 254 VIII, 295, 337 nuclear membrane and, VIII, 329 fi-Aminobenzoate acetylase, isolated nupinocytosis and, VIII, 494 clei and, VIII, 296, 337 Alcian blue, “neucosecretory” cells and, fi-Aminobenzoic acid, effect on tissue cultures, 111, 52 “11, 358-360,369 P-Aminohippuric acid synthesis, isolated Alcohol, nuclei and, VIII, 296, 337 bone induction and, VIII, 267 Aminopolypeptidase, activity of, VII, 617 as fixative, IV, 86-87 Alcohol dehydrogenase, Amitosis, reticulocytes and, VIII, 162-163 Amoebae, inhomogeneity of, VIII, 358 phenanthroline and, VIII, 359-361 accumulation of material in, I, 141-146 zinc and, VIII, 355-361,380 chromosome study of, VIII, 85 Aldehyde dehydrogenase, cations and, divalent cations and, VII, 630-631 form, VIII, 476 Aldehyde-fuchsin, “neurosecretory” cells A T P effect on, V, 221-223 and, VII, 354-357 pressure and temperature effects on, Aldehydes, histochemistry of, VI, 231-233 V, 203-210 Aldolase, micrurgical treatment of, IV, 2-3 activation of, IX, 165 movement, V, 203-218;IX, 198-199 isolated nuclei and, VII, 293, 320, 324, pinocytosis and, VIII, 481-482,489 327, 329-332,336 Amoebocytes, IV, 31-75 Algae, Amphibia, cell walls of, VIII, 42-45 acrosome formation in, V, 404-406
CUMULATIVE INDEX
chromatophorins in, VIII, 204-205 development in, I, 165-193; IX, 203, 332 cyto-embryology of, IX, 321-367 nucleocytoplasmic relations in, I, 165193 quantitative changes in cytoplasm, I, 166-177 unbalanced chromosome combination in, I, 177-179 without chromosomes, I, 177-179 eggs, cleavage of, XI 203-205 differentiation in poly- and haploid, I, 173 surface coat of, IX, 342-343 embryos, gastrulation in, VI, 349-356 metabolism and determination in, VI, 349-361 perforated nuclear membranes in, I, 316 Amylase, localization in cells, I, 69 in nuclei, VI, 396-397; VIII, 2% Anastomoses, arterial, 111, 367-368 Androgen-estrogen balance, in cancer, VII, 97-98 Anemia, reticulocytes and, VIII, 136, 137, 145, 147, 163 Aneuploidy, amphibian, I, 177-179 Animals, cell nutrition and enzymic capacities in, I, 27-33; 111, 1-68 Annelids, acrosome reaction in, V, 380 Annulus, sperm, structural aspects of, VII, 223-224 Anodorzto, muscle of, VII, 297 ff. Anolis porcatus, color changes in, VIII, 181 Artomia, muscle of, VII, 300 ff. Anopheles A virus, VI, 181-182 Antibiotics, antimitotic activity of, IX,. 300 Antibodies, cell aggregation and, VII, 412-413 formation of, V, 19-20 . ' ; functions of transferred, in embtyos, v, 317-318 in &fro production of, VII, 283-284 histochemical studies with, V, 2, 4-7 fa
-VOLUMES I-IX
379
labeling compounds for, V, 2 lymphocytes and, VII, 280-283 Antidiuretic substance, VII, 375, 380 Antigen (s) , -antibody reactions, V, 8-10 bacterial cell walls as, V, 43-45 on cell surface, IX, 200-201 foreign, fate of injected, V, 10-11 histological changes and, VII, 278-279 native to tissues, detection of, V, 14-16 system of Paramecium aurelb, VI, 1-23 factors determining, VI, 7-13 Antimitotic agents, IX, 299-301 Antisera, preparation of, V, 2 APC Virus, VI, 182-183 Apyrase, cations and, VIII, 476 of mitochondria, IX, 162-163 Arback, eggs, divalent cations and, VII, 631-632 effects on division of, V, 211-216 hyaline layer, function of, VII, 589 phosphate uptake by, IX, 144-145 water,co#ent,of, KIII, 423434,426 . phosphate uptake by, IX, 144-145 Arca, muscle of, VII, 300 Arginase, in nuclei, VI, 400; VIII, 296297; 320, 330, 336 Arginine, stains for, IX, 374-375 Argin$e phgsphate, ion movement and, VIII, 470, Arteriosclerosis, elastase and, VIII, 232 Arthropods, chromatophores, VIII, 181201 Ascites tumor (s) , cells, miqurgical treatment of, IV, 3 in rats, cytology of, VI, 25-84 chromosome number in, VI, 29-31, 33-34 effect of drugs on, VI, 68-73 stemline cell theory of, VI, 26, 33, 54, 58, 78-82 Ascorbic acid, cd ,adreml medulla, shock e&ct, on, 11, 121 biosynthesis of, 11, 119-120 bound, 11, 114-117 in cell metabolism, 11, 122-126
380
CUMULATIVE INDEX
chemical properties, 11, 97-101 effect on tissue cultures, 111, 48-49 enzymes and, 11, 122-123 epidermal, I, 280 estimation of, 11, 88-108 function of, 11, 118-122 infrared spectrometry of, 11, 107-108 intercelhlar material and, VII,638 localization, cytochemical, 11, 112-113 and determination in root tips, 11, 110-113 intracellular, 11, 77-131 occurrence of, 11, 113-122 S36-sulfate metabolism and, VII, 188189 ultraviolet absorption of, 11, 101-102 ultraviolet microspectrophotometry of, 11, 108-110 in wound healing, VIII,236 Astacurans, chromatophorotropins and, VIII, 1%-201 Aster (s) , formation in Poa alpina, VII, 150-152 role in cleavage, X,191-193 Asteroids, acrosome reaction in, V, 370372 ATP, see Adenosine triphosphate Autolysis, osmotic pressure and, VIII, 408-409 Autoradiography, cytochemical applications of, IX,382 of nucleic acids, IV,260 techniques of, VII, 160-162 Avicula, muscle of, VII,300, 307 Axial fiber bundle, sperm, VII,217-230 Axonal flow, neurosecretion and, VII, 378-379 Azo dye(s), use in enzyme histochemistry, 111, 329358
'
B
Bacillaceae, endospore formation in, IX, 32-35 Bacillus anthracis, capsule formation studies on, IX, 8 Bacillus cerew, lipid granules in, IX,2629 Bacillus megabacterium, IX,9-34
- VOLUMES I-I?( Bacillus mycoides, spore germination in,
IX,34 Bacillus Polymyxa, spore germination in, IX, 34 Bacillus subtilis, IX,27-34 Bacteria, abnormal cell division of, IX,63-64 active transport in, IX,146-147 cell walls of, 11, 134-141 chemical composition, V, 25-50 enzymatic lysis, V, 36-38 chromosomes of, IX,86-88 CytOlOgY Of, I, 93-118; IV,103-114 comparison with higher forms, IV, 110-112 cytoplasmic membrane, 11, 141-142 ; IV, 108 differentiation of nuclei and mitochondria in protoplast, 11, 142-152 envelopes of, IV,116-117 Feulgen reaction in, IV,124-126 formation of giant and dwarf forms, IV, 110 granular inclusion in cells of, I, 100-101 growth and cell division in, I, 98-100 histochemistry of, IV,115-142 as hosts to bacterial viruses, 11, 152-153 isolation of structural elements of, IV, 134-138 L-foms of, IX, 52-53 nucleic acids of, IV,121-122 pathogenic, properties of, 11, 136-141 plasmoptysis of, IX,51-52 pleomorphism, IX,50-68 protein turnover in, V,57-62 reproductive methods of, I, 102-103 S-Rmutation of, IX,64 spheroplasts of, IX, 52 Bacterial cell, effect of culture on, IX, 1-76 Bacteriophages, see also Coliphages attachment to host cell, VI, 156-157 description of, VI, 155-156 electron microscopy of, VI, 155-164 growth of, I, 120-123, 126-133 particles of, VI, 160-163 reproduction of, I, 119-134 structure, biological significance of, VI, 158-160
CUMULATIVE INDEX
Bacteroides, L-forms of, IX, 67-68 Bacteroides amylogenes, polysaccharides of, IX, 44, 47 Barbiturates, lymphocytes and, VII, 269, 272 Barnea, muscle of, VII, 307 Basal filaments, ergastoplasm and, VII, 426-428 Basal membranes, intercellular matter in, VII, 594 Basement membrane, epidermal cells and, VII, 394-395 structure of, V, 486, 490 Basilar membrane, cochlear, structural aspects of, VII, 563 Basophilia, ergastoplasm and, VIII, 18-20 Basophilic cells, cytoplasmic, 11, 411 of epidermis, I, 280-281 3,4-Benzopyrene, cytoplasmic effects of, VII, 81-83, 86 effect on fetal lung, VII, 82 Beryllium, phosphatase and, VIII, 369 Betaine aldehyde oxidase, in liver cell nuclei, VIII, 530 Biological indicators, radiation and, VII, 23-42, 56 Biotin, effect on tissue cultures, 111, 51 Bipolar cells, synapses of, VIII, 79-80 Birds, acrosome formation in, V, 403-404 embryo, histochemistry of, VI, 289-341 oocytes, lipids of, IX, 315-316 Bladder, mucosa ossification of, VIII, 260-262 Blastocoel, formation of, IX, 343-344 Blastula, mesenchyme, IX, 348-351 Blood, reticulocytes in, VIII, 160-161 Blood vessels, caliber changes in, 111,367 effects of various agents on, 111, 368371 growth of, 111, 364-366 heterotropic ossification and, VIII, 268-269 S35-sulfate studies on, 179-180 Bobbins, basement membrane and, VII, 395-398 chemical nature of, VII, 397
-VOLUMES
381
I-IX
Bone, devitalized, osteogenesis and, VIII, 269-276 extracts, heterotropic ossification and, VIII, 262-267 formation, transparent chamber methods in studies on, 111, 375 histochemistry of, VI, 307-312 growth, dicarnitine and, VIII, 107-108 vitamin A excess and, VII, 98-103 . induction, cell contact and, VII, 416417 parathyroid hormone effect on, VII, 110-111 rudiments, VII, 111-115 S35 studies on, VII, 175-179 ultrastructure, I, 319-320 Bone marrow, reticulocytes in, VIII, 154-155 Bowman’s glands, 11, 304 Brachurans, chromatophorotropins in, VIII, 185-193 Brain, cation movement in, VIII, 410-411 Brilliant cresyl blue, reticulocytes and, VIII, 137-138, 141 Brush-border epithelium, pinocytosis and, VIII, 486-487 Bufo arenarum, spermatid nucleus in, VII, 199-201 Byssus, muscle of, VII, 298 C
Cadmium, metalloprotein and, VIII, 365-
368 Caffeine, glycolytic activity of, IX, 302 Calcium, active transport in photosynthesis, IV, 394-395 ATP and, VIII, 374-375 effect on acrosome reaction, V, 382 heterotopic ossification and, VIII, 256, 259, 267-268 in neoplasms, IX, 207-208 role in cellular adhesion, IX, 197, 207209, 216-218 role in morphogenesis, IX, 205-206
382
CUMULATIVE INDEX
Callinectes sapidus, chromatophorotropins in, VIII, 187-
188 color rhythms in, VIII, 190-191 Cambarellus shufeldti, chromatophorotropins in, VIII, 194,
196, 198-201 Cancer, calcium role in, IX, 415 cell characteristics in, IX, 161,206,211 mitochondria1 changes in, IX, 246-248,
260-261 “protein deletion” theory of, IX, 243,
298 Capsules, bacterial, IX, 40-48 Carassius auratus, melanophores of, VIII,
203 Carausius morosus, chromatophorotropins of, VIII, 201-202 Carbohydrates, effect on tissue cultures, 111,27-33 metabolism of, embryonic determination and, VI,
343-376 protein synthesis and, VI, 345 ff: Carbon tetrachloride, metal distribution and, VIII, 378-380 Carbonic anhydrase, in isolated nuclei, VIII, 297 zinc in, VIII, 353-354 Carboxypeptidase, zinc and, VIII, 354-355 Carcinogenesis, alkaline phosphatase and, 11, 273-274 chemical, IX, 297-298 genetic induction, IX, 211 radiation and, VII, 2-4, 10-11, 20-21,
36-39,56 Carcinogens, affinity for somatic cells, IX, 257-304 effect on organ cultures, VII, 79-121 Cartilage, electron microscopy of, VII, 594 formation studies on, 111, 375-376 heterotopic ossification and, VIII, 258 hypertrophic bone induction in, VIII,
277 Sakulfate studies on, VII, 169-174 Caryometric studies on tissue cultures, 111, 69-111.
- VOLUMES I-IX Catalase, in nuclei, VIII, 297-298,337 Cathepsin, in nuclei, VIII, 298-299, 322,
337 Cations, active transport of, VIII, 410-412 electron energy and, 11, 426-430 evidence for redox pump in, IV, 377-
396 in frog skin, IV, 385-387 in plants, IV, 394-395 in red cells, V, 296-298 in yeast, IV, 378-384 Cell ( s) , adhesion of, IX, 187-225,364 aggregation of, IX, 200-204 animal, nutrition of, 111, 1-68 amino acids and peptides, 111, 34-42 carbohydrates, 111, 27-33 enzymic capacities and, I, 27-33 inorganic substances, 11, 21-27 oxygen, 111, 33-34 role of hormones, 111, 53-57 autoradiographic analysis of, 11, 468-
469 bacterial, kinetic model of, V, 78-80 nuclei and chromosomes of, 11, 158-
177 protein turnover in, V, 59-62 bacteriophage-infected, VI, 163-164 biology, history of, I, 1-8 centrifugation of, IV, 5-6 chromophilic, V, 154-156,167 ciliated, free surface of, V, 485-489 concentration gradient of, IX, 150-154 concept of, VI, 85 ff. contact, VII, 391-423 division, see Mitosis and Meiosis enzyme processes in, IX, 129-186 epidermal, mitotic activity, I, 287-289 furrowing, see Cytokinesis Golgi bodies in, V, 447-448 large, analysis of, 11, 448-450 living, protein molecules in, I, 136-141 study of, I, 5-6 locomotion of, IX, 198-199 nonphagocytic, uptake of macromolecules by, V, 304-307 osmotic work, VIII, 408-410,422-426,
-
432-436’
CUMULATIVE INDEX
in amoeba, I, 141-146 in metazoa and other cells, I, 147152 pancreatic, ingestion in, V, 326-328 permeability to glucose, I, 70-85 potassium in, VIII, 449-450 refractive index, VIII, 417-418 secretion, V, 323-364 separation from solid tissues, IV, 3-4 small, intracellular analysis, 11, 450455 somatic, chromosome member in mammalian, 111, 177-197 surface, 11, 409-410; VII, 395-398 ultrastructure, electron microscopic studies of, V, 456-553 interpretation of, V, 521, 523-526 primary, I, 314-315 secondary, I, 316-321 uptake and transfer of macromolecules by, V, 304-307 virus-infected, VI, 134-139, 174-183 volumes, measurement, of, IV, 7-8 ; VIII, 413-418 water diffusion through, VIII, 438-442 Cell membrane, IX, 49-50 water permeability of, VIII, 440-442; IX, 132, 149 Cell walls, bacterial, as antigens, V, 43-45 chemistry of, IV, 135-136; V, 25-50 composition of, V, 25-50 demonstration of, IV, 116-117 enzymatic lysis of, V, 36-38 fractionation of, V, 26-27 isolation of, IV, 135-136 properties of, 11, 134-136; V, 28-35 structure of, V, 39-40 composition, VIII, 33-35, 42-45 synthesis of, VIII, 42-45 Cellobiose, histochemistry of, VI, 208 Cellular materials, microdetermination of, IV, 6-11 Cellulose, of cell walls, VIII, 33-35 structure of, VI, 195-1%; VIII, 36-39
-VOLUMES I-IX
383
Centrifugation, differential, scope and limitations of, 111, 239-254 in study of tissue enzymes, 111, 225-275 techniques of, 111, 226-239 Centrioles, bacterial, IV, 105-106 sperm neck and, VII, 214-216 structure of, VIII, 26-28 Centromere, see Kinetochore Chaetomorpha, microfibril orientation in, VIII, 57-58 Chelation, use in cell adhesion study, IX, 217-218 Chemotaxis, lymphocytes and, VII, 244245 Chicken, embryonic determination in, VI, 361366 S35 studies on embryos of, VII, 166167 Chitin, histochemistry of, VI, 208-209 . Chloride, erythrocytes and, VIII, 462 ionic equilibrium and, VIII, 451-453, 474 reticulocytes and, VIII, 149 Chloroplasts, IV, 197-217; V, 509-510 Choline, effect on tissue cultures, 111, 5253 Cholinesterase (s) , in neuromuscular junctions, IV, 335375 in nuclei, VIII, 299-300, 337 in organogenesis, VI, 301-303 Chromates, as fixatives, IV, 82-83 Chromatid bodies, in mammalian sperm, V, 428-429 in spermatogenesis, V, 442-443 Chromatin, chemical composition of, 111, 135-136 extended state of, 111, 134-135 Feulgen reaction of, 4-5, 7, 91 Chromatophores, IX, 2-4, 32 function of, VIII, 177-181 physiology of, VIII, 175-210 Chromatophorotropins, VIII, 181-206 Chrome-alum-hematoiq4in,“neurosecretory” cells and, VII, 340-342, 354362, 374
384
CUMULATIVE INDEX
Chromic acid, (Bauer) reaction, basis of, VI,200-201 Chromomeres, chromosomal, IX, 106-107 Chromonemata, IX,81-82, 84-86, 91 Chromosomes, alkaline phosphatase in, 11, 271 of ascites tumors, VI,25-84 bacterial, IV,106; IX,86-88 optical studies on, 11, 164-165 stains for, 11, 158-162 composition of, IX,77, 107-119 division of, VII, 143-144; X, 185, 194, 200, 202-203, 212 fragmentation in mutagenesis, IX,295297 giant, functional significance, 111, 161-164 structure of, 111, 131-173 isolation, 111, 134 lampbrush, IX,95-%, 107, 114; XI146148,150 maintenance of structural integrity of, IX, 113-116 movements, kinetochore and, VII, 146150 nucleic acids in, IX,108-113 number, in germ cells, 111, 178-182 reduction of, I, 18-21 in sifu, 111, 182-183 in somatic cells, 111, 177-197 in tissue cultures, 111, 183-186 variations in, nuclear DNA and, V, 179-182 organization of, VII, 152-155 ; IX, 77127 phase contrast and electron microscopy of, 11, 136 polynemic, IX,81-96 polytenic, IX, 96-98 radiation and, VII,45-47; IX,89 reproduction, I, 12-15 somatic, morphology of, VI,4-5 stains for, IX,110-111 structure of, IX,78-119 Chondriome, function of, IX,272 Chondroitin sulfuric acid, histochemistry of, VI, 210
- VOLUMES
I-IX
Chondrosarcoma, S35-sulfate and, VII, 187 Chrotogonus trachypterus, oocyte study Of, IX, 308-309 Cilia, kinety system of, VIII,102-103 Citrate, intercellular material and, VII, 633-635, 639 Citric acid (Krebs) cycle, 11, 202-204; VI, 448-451 and auxiliary enzyme systems, 11, 211217 Clostridium welchii, collagenase and, VII, 603-606 hyaluronidase and, VII,608 Cnidia, ultrastructure of, I, 319 Coaption, cell contact and, VII,412 Cochlea, structure of, VII, 542-563 ; VIII, 76 Cochlear duct, structure of, VII,543-567 Coenzyme A, 11, 215 ff. role in oxidative cell metabolism, 11, 225 Coenzymes, intracellular, formation, control of, 111, 291-295 Colchicine, effect on chromosomes, IX, 296, 299; X, 107, 110 effect on explanted tissues, 11, 339-340 lymphocytes and, VII,271-272 Coleoptera, acrosome formation in, V,43 Coliphages, see also Bacteriophages growth of, I, 123-126 Collagen, aging and, VIII,237-238 composition, VI, 216-217; VII, 596597; VIII, 226-230 distribution of, VIII, 230-231 electron microscopy of, VIII,240-241 enzymatic susceptibility of, VIII, 232234 fibers, VII, 592-595, 611-612; VIII, 239-242 heterogeneity, VIII, 242-246 histochemical identification, IX, 385386 mucoprotein complex of, VII,494-496 reconstituted, VII, 594 ultrastructure, VIII, 222-225
CUMULATIVE INDEX
Collagenase, activity of, VII, 603-605; VIII, 240241, 245 histochemical use, IX, 385 specificity of, VII, 604-605 “Collagenmucoproteinase,” VII, 611-612 Collecting tubule, VII, 522, 532 arched, VII, 522-532 connecting part and, VII, 522 Colloid, thyroid, VI, 265-270 Computers, use in enzyme kinetics study, IX, 175-176, 179 Conifers, microfibril studies on, VIII, 3637, 51-52 Conjugation, ciliary corpuscles and, VIII, 108-110 Connective tissue, see under Tissue Contact guidance, cell locomotion and, VII, 406-408 essential features of, VII, 399 selective, VII, 408 Copper sulfate, bone induction and, VIII, 268-269 Corneal mucopolysaccharide, histochemistry of, VI, 213 Cortisone, effect on lymphocytes, VII, 114-115, 247-248, 261, 269-271 effect on mitochondria, IX, 247 Corynebocterium diphtheriae, cell division in, IX, 24 metachromatic granules of, IV, 130-131 volutin granules in, IX, 16-18, 20-21 Crago, chromatophores of, VIII, 185 chromatophorotropins in, VIII, 193, 197 Cristae ampullares, innervation of, VII, 565-568, 570, 575, 578 Cristae, mitochondrial, IX, 229-231 Crustaceans, chromatophores of, VIII, 183-201 vesiculiform sperm of, V, 433-438 Cupula, S36-studies on, VII, 186-187 structure of, VII, 578 Cuspidaria, muscle in, VII, 300, 304, 310, 313, 327 Cyanide, lymphocytes and, VII, 269,272 Cyclophorase-mitochondria1 (C.M.) en-
-VOLUMES I-IX
385
zyme systems, 11, 202 ff. self-contained nature of, 11, 206-207 Cysteine, “neurosecretory” cells and, VII, 356-363, 369 Cysteine desulhydrase, in nuclei, VIII, 532 Cytochemistry, quantitative, critical review of, 11, 447 Cytochondria, see Mitochondria Cytochrome c, electron transfer to, IV, 169-170 in nuclei, VIII, 300-301, 320, 337 relation to mitochondrial cristae, IX, 240 Cytochrome oxidase, in nuclei, VIII, 301, 321-325, 337 in plant particles, IV, 170-174 Cytokinesis, V, 210-218 CytOlOgY, bacterial, IV, 103-114 freezing and drying techniques in, I, 35-63 Cytomembrane, of inner ear cells, VII, 555 intracellular folds, V, 482-484 of kidney tubule cells, VII, 490494,504 of mast cells, 111, 405 penetration of, I, 65-92 specialized regions of, V, 484 tubular invaginations of, V, 482 Cytophotometry, methods of, V, 173-177 in study of nuclear DNA, V, 171-197 Cytoplasm, azo dye affinity of, VIII, 1-31 basophilic component, V, 4974% components of, VI, 426-432, 453-460 concentration gradients in, IX, 150-154 enzymes in, VI, 390 ground substance of, V, 490-499 of mast cells, 111, 405 nucleic acid formation in, VI, 406-407 opaque particles of, V, 497 osmiophilic granules of, V, 499 structure of, VIII, 1-31 ultrastructure of, I, 320 study by electron microscopy, 11, 408-409 vesicles of, V, 497
386
CUMULATIVE INDEX
Cytoplasmic bodies, bacterial, IV, 130-138 multiplicity and heterogeneity of, 111,
250-258 Cytoplasmic membrane, bacterial, 11, 141-142;IVY108 demonstration of, IV, 116-117 electron microscopy of, VI, 99 ff. structure of, V, 490-494 Cytoplasmic organelles, VI, 86-88
D Degeneration, mitochondria and, VIII,
9-15 Dehydroascorbic acid, bacterial, estimation of, 11, 93 Deiters’ cells, organ of Corti and, VII,
550 Deoxyribonuclease, in animal cells, VI, 401-402 effect on cellular nucleoproteins, IX,
108-109 in isolated nuclei, VIII, 302, 325, 329,
330, 333-334,337
-VOLUMES
I-IX
somatic inconstancy and, 111, 188 species differences in, V, 172-173 synthesis during mitosis, V, 187-188 variations in, related to chromosome number, V, 179-182 nucleolar, IV, 285-286 in plant nuclei, 11, 39-41 Deoxyribonucleoproteins, of chromosomes, IX, 117-119 Dermis, elastic fiber production and, VIII, 239-240 “Detailed balancing” principle, IX, 170-
175 Determination, embryonic, VI, 343-376 Diaminopimelic acid, in bacterial cell walls, V, 33-35 of Gram-positive bacteria, V, 41 Diastase, use in identification of intracellular glycogen, VI, 205-206 “Diauxie” phenomenon, IX, 163 Dicarnitine, bone growth and, VII, 107-
108 2,6-Dichlorophenolindophenol, estimation of ascorbic acid with, 11, 88-90 Differentiation, 111, 277-327;VII, 31 Diffusion, in enzyme kinetics, IX, 146-
use in cytochemistry, IX, 110-113 Deoxyribonucleic acid, 11, 33-58 of bacteriophages, VI, 156-159 150 in chromosomes, IX, 88,112-116,295 Digestive tract, embryonic, histochemistry in homogenates, VIII, 334-335 of, VI, 312-320 lymphocytes and, VII, 247, 260-261, Diphosphopyridine nucleotide, 264, 267, 270 synthesis by nuclear preparations, 111, metabolism of cellular, VI, 409-415, 212-213;VIII, 288, 303, 329, 332,
419-420
nuclear, VIII, 316-319 cell division and, V, 187-191 constancy of, 111, 132-133;V, 177-
179 cytophotometric study on, V, 171-
197 during maturation, fertilization and cleavage, V, 191-193 effect of pathological changes on, V,
185-186. effect of physiological changes on, V,
182-185 in embryonic and differentiating tissue, V, 188-191 in interphase nuclei of normal tissue, V, 177-182
336 zinc metalloenzymes and, VIII, 355-
365 Disease, elastic tissue and, VIII, 219-221 reticulocytes and, VIII, 163-165 Distal convoluted tubule, anatomy of, VII, 512-522 Disulfide groups, stains for, IX, 373-374 Divalent cations, effect on, VII, 629-633 Diver balance, IV, 8 Donax, muscle of, VIII, 300,319 Donnan effect, erythrocytes and, VIII, 462-463 osmotic pressure and, VIII, 396-398 potassium and, VIII, 453-454
CUMULATIVE INDEX
Donnan theory, applicability to intracellular pH, V, 230-235, 262-265 Dosinia, muscle of, VII, 299,300, 307 Downey cells, lymphocytes and, VII, 242-243 Dreissim, muscle and, VII, 300, 307, 326 Drosophila species, chromosome studies on, IX, 97-101, 103-104 Drugs, action, sites of, IX, 301-302 effect on ascites tumor cells, VI, 68-73 metabolic function of, IX, 283-294 Ductus cochlearis, see Cochlear duct Dyes, affinity to tissue structure, I, 237241
E Ebner’s glands, 11, 301 Echinoderms, cyto-embryology of, IX, 321-367 Echinoids, acrosome reaction in, V, 366371 Egg (s 1,
activation of, VI, 107-127 avian, uptake of macromolecules by, V, 307-309 enzymes in, 111, 277-291 fertilization of, VI, 107-127 histochemistry of, VI, 292-295 interaction with spermatozoa, I, 257263 micrurgical treatment of, IV, 3 RNA in cytoplasm of, VI, 292 ff. sea urchin, studies on, VI, 107 ff. Ehler’s Danlos syndrome, elastic tissue and, VIII, 532 Elastase, activity of, VII, 606-607 Ehler’s Danlos syndrome and, VIII, 220-221 physiological significance of, VIII, 232233 specificity of, VIII, 220, 233-234 staining of,’VIII, 216, 218, 386 Elastic fibers, aging and, VIII, 238-239 distribution of, VIII, 230-231 “elastomucoproteinase” and, VII, 611612 models of, VIII, 225-226
-VOLUMES I-IX
387
production from collagen, VIII, 239242 X-ray diffraction studies of, VIII, 241242 Elastic tissue, in disease, VIII, 219-221 staining of, VIII, 215-219 Elastin, chemical composition, VIII, 226-230 chemical properties of, VII, 598-599 enzymatic susceptibility of, VIII, 232234 formation of, 111, 422-423 X-ray diffraction of, VIII, 223-224 Elastomucin, definition of, VIII, 213-214 “Elastomucoproteinase,” elastic fibers and, VII, 611-612 Electron energy, active transport of cations and, 11, 420430 secretion of €I ions + and, 11, 425-426 Electron microscopy, of cellulose, VIII, 36-37 of elastic fibers, VIII, 222-223, 240241 of Golgi apparatus, VI, 98-105 mucoprotein-collagen complex and, VII, 594-596 of nucleic acids, IV, 258 of thyroid, VI, 266-270 of tissue sections, I, 305-322; 11, 403407 cytopathological findings, I, 320-321 dehydration and embedding, 11, 405407 effect of fixation, I, 313-314; 11, 404405 reliability of, VIII, 3-4 reticulocytes and, VIII, 141-142 ultrastructure of cells derived from, V, 456-553 interpretation of data, V, 521, 523526 of viruses, VI, 129-191 Electron transfer, in plants, IV, 169-174 Electron transport system, 11, 225-226 of muscle mitochondria, V, 112-116 Embryo (s) , chick, culture of thyroid gland from. 11, 361-367
388
CUMULATIVE INDEX
development, variations in nuclear alkaline phosphates, 11, 274-275 differentiation, mitochondria in, 111,
295-301 fibrogenesis of, VIII, 234-236 human, culture of ovarian cortex from, 11, 356-360 intracellular protein sources, V, 316-
317 macromolecular uptake by, V, 310-317 press juices, chemical properties of, 11,
331-336 rat, culture of limb buds from, 11, 349-
352 role of phagocytosis in, V, 310-311 tissue, radiation effect on, VII, 29-32 95-sulfate studies on, VII, 162-166 sex hormones and, Vol. 87-89 transfer of macromolecules to, V, 311 ff. Emission spectrography, of trace elements, VIII, 347-350 Emperipolesis, VII, 245, 263 End piece, sperm, structural aspects of, VII, 229-230 Endocrine glands, histochemistry of, VI,
320-322 Endoderm, visceral histochemistry of, VI, 332-333 Endolymph, stria vascularis and, VIII,
543, 563 Endolymphatic appendage, otic labyrinth and, VII, 537-538 Endoplasmic reticulum, ergastoplasm and, VII, 438-440,455-
461 isolated nuclei and, VIII, 321-322 Endospores, bacterial formation of, IX,
32-35 Ensis, muscle of, VII, 297 ff., 322-327 Entropy, protein solutions and, VIII, 399403 Enzymes, see also individual compounds activity, effect of fixatives on, IV, 9899 integrated, in particulate nomitochondrial systems, 11, 207-210 cellular, IX, 129-186
-VOLUMES
I-IX
in cellular nuclei, 111, 199-223; VIII,
287-316, 336-337 cellular transport and, I, 70-89 distribution in gustatory and olfactory epithelia, 11, 304-319,324-330 effect of various substances on, 11,
300 sense of smell and, 11, 321-322 sense of taste and, 11, 319-321 in egg, effect of fertilization on, VI,
122-125 of external protoplast, I, 115-116 sugar absorption and, I, 107-108 histochemistry of, azo dye methods in, 111, 329-358 hydrolytic, intracellular, 111, 260-261 inhibition by diazonium salts, 111, 350351 intracellular, in egg and embryo, 111,279-291 in vitro and in vivo activity, 111, 284291 in isolated nuclei, VI, 390-404 localization in cell membrane, I, 66-68 lymphocytes and, VII, 264-265,273 lysis of bacterial walls by, V, 36-38 kinetics of computer studies on, IX, 175-176,179 nucleolar, IV, 288-289 oxidizing, intracellular, 111, 261-262 phosphate-splitting, number of, 11, 310313 phosphorylating, intracellular, 111, 262265 localization, I, 69-70 proteolytic, effect on bacterial cell walls, V, 36 reticulocytes and, VIII, 152-154 role in embryonic differentiation, 111, 277-279 synthesizing, intracellular, 111, 265-267 tissue, artificial redistribution, 111, 245-250 differential centrifugation, in study Of, 111, 225-275 heterogeneous distribution of, I11 248-249 in transport of red cells, V, 289-298 use in cytochemistry, IX, 109-113
CUMULATIVE I N D E X
use in demonstration of bacterial nucleus, IV, 123-124 use in polysaccharide histochemistry, VI, 234-238 Enzyme adaptation, V, 55-56 inducer action, V, 62-64 in microorganisms, V, 51-87 mass action theory, V, 64-66 extended, V, 80-84 organizer theory, V, 71-80 plasmagene theory, V, 66-70 source of adaptive enzyme, V, 56-57 nomenclature, V, 53-55 Epidermis, cells, contact of, VII, 394-395 Golgi apparatus of, I, 272-273 healing of, X, 321-322 human, culture of, X, 332-334 keratinization in, I, 275-278; X, 324326, 328-329, 341-348 lipids of, I, 274-275 mammalian, cytology of, I, 265-304 mitochondria of, I, 270-272 mitotic activity of, X, 318-320 transparent chamber studies on, 111, 372 Epithelium, cells, distribution of enzymes in gustatory and olfactory, 11, 304-319 gustatory, histology of, 11, 290-301 intercellular matter in, VII, 593-594 intestinal, brush border of, V, 478-482 lymphocytes and, VII, 253,262-263 motility of, IX, 206-207 olfactory, histology of, 11, 302-304 vitamin A excess and, VII, 103-106 Ergastoplasm, 11, 411; V, 497-498; VII, 425-485 ; VIII, 15-18 Ericymba buccata, background response of, VIII, 81 protective coloration and, VIII, 178 Eriocheir, VIII, 183, 472 chromatophorotropins in, VIII, 187 Erythrocytes, enzymatically controlled transport in, I, 75-79 halometry and, VIII, 415-416 internal structure, VIII, 412-413 ionic equilibria in, VIII, 462-463, 467468, 471, 477
-VOLUMES
389
I-IX
lymphocytes and, VII, 252-253 mammalian, isolation, VI, 506-507 metabolism of enzymes in, V, 280-289 osmotic pressure and, VIII, 393, 404405, 409, 418-422 permeability of, VI, 469-511; IX, 133134, 142-143 separation from reticulocytes, VIII, 145-146 Escherichia coli, fimbriae studies on, IX, 38-39 L-forms of, IX, 67-68 pleomorphism in, IX, 55, 57, 61, 67 T-phages of, VI, 159 morphology of, VI, 155-156 virus-infected, particle formation in, VI, 160-161 Esterases, biochemical differences between, I, 321325 histochemistry of, I, 323-335 isolated nuclei and, VIII, 303-304, 327, 330, 337 “neurosecretory” cells and, VII, 363364 Estradiol, VII, 87-88, 91, 94,% S3b-sulfate studies on, VII, 676 Estrone, VII, 91, 94,248 methylcholanthrene effect and, VII, 95-
96 Euchromatin, chromosomal, IX, 102-106 Extrusion, V, 353-355
F Fascia, heterotropic ossification VIII, 258-260 Fast green, use in cytochemistry, 110-111 Fat cells, mast cells and, 111, 424 transparent chamber studies on, 374-375 Fatty acids, metabolism in plants, IV, 179 oxidation, enzymatic, 11, 223-225 Fertilization, effects upon egg, VI, 107-126 specificity in, acrosome reaction V, 391-392
and,
IX,
111,
and,
390
CUMULATIVE INDEX
Fertilizin, IX, 202-203 Fetus, neurosecretory material in, VII,
375-376 Feulgen reaction, 11, 26-30 in animal material, 11, 233 in bacteria, IV, 124-126 inhibition of, 11, 237-238 nucleae, 11, 231-247;IV, 235-240;VI,
85
- VOLUMES I-IX oxidizing action, IV, 98 for tissue sections, V, 5 Flagella, bacterial, I, 102;IX, 35-38 formation of, IV, 108-109 Fluorescein, -carbamido proteins, properties of, V,
3-4
-labeled globulin, VIII, 490-493 in plant material, 11, 233-234 Fluorescein isocyanate, plasmal, 11, 232-233 preparation, V, 3 procedure of, 11, 234-237 use for labeling of antibodies, V, 3 use in cytochemistry, IX, 110-111 Fluorescence microscopy, IX, 381 Fibrils, estimation of ascorbic acid by, 11, 108 smooth muscle and, VII, 306-309 in study of labeled antibodies, V, 5-6 transversely striated fibers and, VI, Flux, in enzyme kinetics, IX, 145-150 315-316 Folic acid, Fibrin, IX, 215, 386 effect on tissue cultures, 111, 51 Fibroblasts, lymphocytes and, VII, 265-266 cultures, carcinogen effect on, VII, 81- Formaldehyde, as fixative, IV, 80-82 82, 86 Fowl plague virus, VI, 171, 174 fibrogenesis and, VIII, 234-236 Fowl pox virus, VI, 172-173 heterotopic ossification and, VIII, 255, morphology of, VI, 177-178 261 Freezing and drying techniques, I, 35-63 lymphocytes and, VII, 251-252,263 Frog, refractive index and, VIII, 426-427 oocytes, lipids in, IX, 313-314 transformations of, VII, 402 skin, volume-osmotic pressure and, VIII, active transport of sodium ions 423, 425 through, IV, 383-387 Fibrous sheath, sperm, structure, VII, structure of, IV, 385-386 224-228 Fructose-6-phosphate, isolated nuclei and, Fick’s law, IX, 147-148,164 VIII, 304 Fimbriae, IX, 1, 3, 38-40 Fuchsin. see also Schiffs reagent Fish(es), Fucose, in connective tissue, VIII, 229 acrosome formation in, V, 407-410 Fumarase, chromatophorotropins in, VIII, 202-204 in isolated nuclei, VIII, 304, 322, 324, oocytes, lipids in, IX, 313-314 331 Fixation, see also Fixatives . reaction kinetics of, IX, 154 criteria for good, IV, 94-96 Fundulzu, effect on living protoplasm, IV, 87-96 egg volume of, VIII, 426 on properties of tissue and cell conmelanophores of, VIII, 202,203 stituents, IV, 96-99 Fungi, accumulatory mechanisms in, IV, histochemical, IX, 370-371 153 histological, comparison with freeze G drying, I, 53-57 Gambusia, VIII, 203 lymphocytes and, VII, 238-239,245 protective coloration and, VIII, 177 Fixatives, see also Fixation and individGastric mucosa, ual compounds secretion of H+ ions by, IV, 387-394 effect on enzyme activity, IV, 98-99 on tissue constituents, 111, 80-87 analogy with yeast, IV, 387-390
CUMULATIVE INDEX
Gastropods, acrosome reaction in, V, 377-379 Golgi apparatus of male germ cells, IV, 94-96 pulmonate, acrosome formation in, V, 414-421 Gastrulation, IX, 351-359, 363-364 Gelastorrhims bicolor, spermatid nucleus in, VII, 198, 200,201 Gelatin, pinocytosis and, VIII, 489 Gels, structure, IX, 215-216 protoplasmic contractility and, V, 199-227 use in cell adhesion studies, IX, 215216 Generative organs, radiation effects on, VII, 39-52 Genetic effects, radiation and, VII, 47-49 Germ cells, male, Golgi apparatus of, VI, 94-98 number of chromosomes in, 111, 178182 discrepancies in counts of, 111, 180181 radiation effects on, VII, 43-45 Gills, ion transport in, VIII, 472-473 y-Globulin, VII, 246, 281 Glomerulus, electron microscope studies on, 11, 410-411 Glossus, muscle of, VII, 300, 307 Glucose, cell utilization of, IX, 141-142 effect on bacterial growth, IX, 8-9 penetration into mammalian erythrocytes, VI, 473 ff. permeability of cells to, I, 70-85 ; IX, 131, 137-138 pinocytosis and, VIII, 494, 495, 498-500 Glucose phosphatases, isolated nuclei and, VIII, 304-305, 322, 337 Glucose-6-phosphate dehydrogenase, VIII, 331, 363-364 Glutamic dehydrogenase, VIII, 305, 322, 337 zinc and, VIII, 361-362 Glutathione, hydrolysis, isolated nuclei and, VIII, 306 in red cells, V, 287
-VOLUMES
I-IX
391
Glyceraldehyde dehydrogenase, isolated nuclei and, VIII, 306, 363-364 Glycerol, penetration into mammalian erythrocytes, VI, 473-474 transport, in red cells, V, 295-296 Glycerophosphate dehydrogenase, isolated nuclei and, VIII, 306, 363-364 Glycogen, of bacterial cells, IX, 45-47 in embryonic organs, VI, 304, 306, 333334 of epidermis, I, 281-282 in formation of teeth, VI, 314, 316 histochemistry of, VI, 310-311 intracellular, identification of, VI, 205207 lymphocytes and, VII, 264 in ossification, VI, 310-311 in placenta, VI, 330-331 in sebaceous glands, I, 2% synthesis, insulin and, VII, 113-114 Glycolysis, isolated nuclei and, VIII, 288, 306, 325 lymphocytes and, VII, 266 in plants, IV, 159-162 Glycoproteins, histochemistry of, VI, 216217 Goblet cells, IV, 299-334 Golgi apparatus, acroblast type, VI, 96-97 acrosome formation and, V, 422-429 electron microscopy of, 11, 413-416; X, 275-276 epidermal, I, 272-274 ergastoplasm and, VIII, 21-25 function of, VII, 470 lamelliform nature of, VI, 88-92 lymphocytes and, VII, 240-241 of male germ cells, VI, 94-98 of mast cells, 111,405 microscopy of, VI, 92-94 origin of, V, 447-448 role in cell function, V, 345-351 in sebaceous glands, I, 291-292 structure of, V, 471-478; VI, 85-106 Golgi bodies, see Gold apparatus Grafts, transparent chamber methods in studies on, 111, 376-393 autogenous, 111, 376 ff.
392
CUMULATIVE INDEX
homologous, 111, 386-387 preparation of, 111, 376 ff. tumor, 111, 387-392 Gram stain, IV, 119-120 Grana lamellae, molecular structure of, IV, 212-215 Granules, of mast cells, 111, 402-405 osmiophilic, of cytoplasm, V, 499 synaptic, V, 498-499 Ground substance, central nervous system and, VII, 601602 composition of, VII, 599 of cytoplasm, V, 499 definition of, VIII, 214 enzyme action, VII, 608-629 mucopolysaccharides and, VII, 599-601 Growth hormone, VII, 112-114 Gryllodes sigillatus, oocyte study on, IX, 305-309 Gryfdmw, muscle of, VII, 301, 307 Gullet, fine structure of, VIII, 122-125 gross structure of, VIII, 114-122 replication mechanisms and, VIII, 125128
H Hair, cells, structure of, VII, 571-575 Halometry, cell volume and, VIII, 415416 Haploid number in man, 111, 179-180 Haploidy, amphibian, I, 168-177 Hatching, IX, 345-348 Heart muscle, intercalated discs, V, 485 Heinz bodies, reticulocytes and, VIII, 137, 165 Hemiptera, acrosome formation in, V, 414 Hemoglobin, isolated nuclei and, VIII, 320-325, 329, ' 330 reticulocytes and, VIII, 149-151 Hemolysis, reticulocytes and, VIII, 138139, 148 Hensen cells, cochlea and, VII, 551 Heparin, histochemistry of, VI, 210-211 metachromasia of, VI, 247
- VOLUMES I-IX Hepatectomy, mitochondria1 changes after, IX, 248, 264-265 Hepatic cells, mitochondria of, IX, 237239, 242-245, 269, 274-277 Herbst processes, IX, 334-338 Herpes simplex viruses, growth of, VI, 178-179 Heterochromatin, chromosomal, IX, 84, 102-106 Heteroploidy, definition, 111, 188-189 somatic, differentiation and, 11, 192193 Heteroptera, acrosome formation in, V, 414 Hexokinase, in cells, IX, 138 ff. Hexose monophosphate shunt, in erythrocytes, V, 284-285 Hexoses, cell permeability to, IX, 132-133 penetration into mammalian erythrocytes, VI, 475-504 Hirosaki sarcoma, chromosomes of, VI, 31, 42-43 Histamine, mast cells and production of, 111, 424-425 Histidine, stain for, IX, 376-377 Histochemistry, of bacteria, IV, 115-142 of enzymes, azo dye methods in, 111, 329-358 of esterases, I, 323-335 of gustatory and olfactory epithelia, 11, 304-307 of phosphatases, 11, 249-260 of polysaccharides, VI, 193-263 quantitative, 11, 447-472 use of labeled antibodies in, V, 1-23 Historadiography, of nucleic acids, IV, 256-257 Holothuroids, acrosome reaction in, V, 372 Homogenizers, use in cell nuclei isolation, VIII, 280-284 Hormone(s), effect on tissue cultures, 111, 53-57 Human fetal lung, 3,4-benzopyrene and, VII, 82-83,86 tobacco condensate and, VII, 83
CUMULATIVE INDEX
Human tissues, radiation and, VII, 3739, 43 Hyackthus orientalis, kinetochore structure in, VII, 125, 137, 138 Hyaline layer, IX, 333-343, 349-350 Hyaluronic acid, identification, VI, 209-210 metachromasia of, VI, 247-248 Hyaluronidase, activity of, VII, 609-610 as histochemical reagent, VI, 234-238 specificity of, VII, 608-610 Hybrids, amphibian, development of, I, 183-188 Hydrocortisone, VII, 269-270 Hydrogen ion concentrates, intracellular, V, 227-229 application of Donnan theory to, V, 262-265 changes in, V, 258-262 determination methods, V, 236-261 theories concerning, V, 230-236 Hydrogen ions, intercellular material and, VII, 636637 secretion, electron energy and, 11, 425426 Hydroxyproline, in connective tissue, VIII, 228, 231, 242-244 in wound healing, VIII, 237 Hyperpolarizatio.n, synaptic inhibition and, VIII, 86-87 Hypothalamo-hypophysial neurosecretion, VII, 337-389 anatomical consideration, VII, 340-353 cytological changes in, VII, 365-373 enzymes in, VII, 363-365 labeled cysteine and, VII, 362-363 “neurosecretory” cell and, VII, 354365
I Illumination, chromatophores and, VIII, 178-181 Induction, cell contact and, VII, 416-417 Infections, labeled antibodies in study of causative agents, . V. 17-19 mast cells and defense against, 111, 423
-VOLUMES I-IX
393
Influenza virus group, VI, 179-181 chemical disintegration of, VI, 173-174 growth of, VI, 180-181 localization of viral antigens in, V, 17 morphology of, VI, 170-171, 180 Infrared histospectroscopy, of nucleic acids, IV, 257-258 Infundibular process, VII, 345-353, 363 Inner border cells, organ of Corti and, VII, 544-545 Inner ear, V, 535-585 Inner hair cells, a-cytomembranes and, VII, 555 Inner pillar cells, organ of Corti and, VII, 545-546 Inner sulcus cells, organ of Corti and, VII, 544 Inorganic substances, used in tissue culture media, 111, 21-27 Inositol, effect on tissue cultures, 111, 52 Insects, acrosome formation in, V, 399-403 chromatophorins in, VIII, 181-182,201202 flagellate sperm of, V, 432-433 neurosecretory material in, VII, -373374 viruses of, electron microscopy of, VI, 148-155 Insulin, effect on cell permeability, IX, 131-132 effect on organ cultures, VII, 111-114 Integumentary system, embryonic, histochemistry of, VI, 303304 Intercellular material, VII, 590-639 Interferometry, of nucleic acids, IV, 258259 Intermedin, chemical nature of, VIII, 205 chromatophores and, VIII, 204 Interstitial cells, neurosecretion and, VII, 380-382 Intestine, enzymatically-controlled transport in, 111, 267-269 Invertase, in cells, I, 68 Iodine metabolism, thyroid structure and, VI, 278-285 Ion secretion, in -~lants. . 179-200 , 11 , role of mitochondria in, 11, 194
394
CUMULATIVE INDEX
Ion transport, localization of transport phenomena, 11,
193-194 through plant, 11, 1%-197 Irradiation, as cause of swollen mitochondria, IX, 246-247 Isocitric dehydrogenase, inhibitors of, VIII, 364 isolated nuclei and, VIII, 300,337 Isopods, chromatophorins in, VIII, 183-
185
J Joints, embryonic, histochemistry of, VI,
307
K Keratinization, in epidermis, I, 275-278 0-Ketoacids, activation, mechanism of, 11, 222-223 Kidney, enzymatically-controlled transport in, I, 79ff. glucose permeability in cells of, IX,
136-137
- VOLUMES I-IX Lamellibranch muscle, VII, 295-332 Lampbrush chromosome, 111, 154-161 structure of, 111, 157-161,174-175 Larvae, virus-infected, polyhedral bodies Of, VI, 148-149 Lead tetraacetate, use in histochemistry, VI, 227-230 Leader, VIII, 183,184,206 chromatophorins and, VIII, 194-1% Lesions, tractus hypopheseus and, VII,
367-371,376 Leucocytes, cation movement and, VIII, 410-411 mitochondria of, IX, 237,239,261 volume-osmotic pressure and, VIII,
423, 425 Leucophaea maderae, chromatophorotropins in, VIII, 182,202 Ligia oceanica, chromatophorins in, VIII,
183-184 L i m , muscle of, VII, 297 ff. Limacoitin sulfate, histochemistry of, VI,
213
heterotropic ossification and, VIII, 254-
258
Limb buds, culture from rat embryo, 11,
349-352
neck of, VII, 487-490 tubular cells of, structure, V, 513-516 Kinetochore, VII, 124-152 Kinetodesma, VIII, 100-102 Kinetosomes, centriole and, VIII, 26-28 kinetodesma and, VIII, 102,103 Klebsiella aerogenes, pleomorphism of, IX, 55-56,58-59,61,
66-67 polysaccharides of, IX, 42,45,47-48 volutin granules in, IX, 17-18,20-21 Klebsiella pneumoniae, capsules of, IX,
41-42 Krebs cycle, in plants, IV, 162-166 Kwashiorkor, mitochondria1 changes in, IX, 246, 248
L Labeling compounds, for antibodies, V, 2 Lactase, localization in cells, I, 68 Lactic dehydrogenase, in isolated nuclei, VIII,107, 330, 337, ,, zinc and, VIII, 362-363
Lipase, in epidermis, I, 282-283 in sebaceous glands, I, 298 Lipids, bacterial, IV, 118-119;V, 35 of cell membranes, IX, 218-220; X,
208 in cortex of sea urchin egg, VI, 108,
115-116,125 in embryonic organs, VI, 321-322,336 epidermal, I, 274-275 heterotropic ossification and, VIII,
263-264 histochemistry of, IX, 306-307 metabolism, in tissue cultures, 111, 44-
46 mitochondrial concentration, IX, 254,
257-258 nucleolar, IV, 288 reacting with PAS-technique, VI, 219-
223, 225 L i p o q t e i n structures, ciilular, V, 5&511 osmium staining of, V, 510-511
CUMULATIVE INDEX
Lithium, effect on developing egg, 111, 316-320; IX, 335-356 effect on larvae, IX, 350-351 Lithium chloride-induced pleomorphism, IX, 54, 61-62, 67 Liver, ergastoplasm and, VII, 441-442, 467 osmotic pressure and, VIII, 408-409 London forces, in cell adhesion, IX, 188, 194, 219 Lutruria, muscle of, VII, 300, 303, 319 Lymph, peripheral, cell counts in, VII, 258-259 Lymphatic vessels, growth of, 111, 371372 Lymphocytes, VII, 236-286 Lymphopoiesis, VII, 246-249 Lysophosphatides, role in activation of sea urchin egg, VI, 115-118, 125 Lysozyme, pinocytosis and, VIII, 492493
M Macromolecules, V, 303-322 Macrophages, formation of, VII, 402 lymphocytes and, VII, 250-251, 261,
286 Muctra, muscle of, VII, 300, 307, 319 Macula sacculi, innervation of, VII, 566567, 570 Macula utriculi, innervation of, VII, 565568, 570 Magnesium, enzyme activation and, VIII, 369-370, 373-375 Malaria, reticulocytes and, VIII, 164-165 Malic dehydrogenase, metals and, VIII, 363-364 Mammals, acrosome formation in, V, 421-426 embryos, histochemistry of, VI, 289341 microscopic studies with transparent chamber methods, 111, 359-398 Mammary tumors, of mice, VI, 166-167, 184 Man, haploid number in, 111, 179-180 Mast cells, 111, 399-474
-VOLUMES I-IX
395
Media, for tissue cultures, biological, 111, 5-14 synthetic, 111, 14-21 Median eminence, limits of, VII, 343344 Medulla terminalis, chromatophorotropins and, VIII, 182-183 Medusan eggs, IX, 326-328 Meiosis, kinetochore organization at, VII. 130-133 Membrane (s) , cellular. see Cytomembrane cytoplasmic, see Cytoplasmic membranes ergastoplasm and, VII, 449,460,474 ion permeability and, VIII, 451-462 pinocytosis and, VIII, 501-502 relation of redox pump to movements of free ions across, 11,431-435 synaptic transmission and, VIII, 92-93 Membrane potential, calculation of, VIII, 455-457 Membrane tectoria, Ssa studies on, VII, 679 Meningopneumonitis virus, VI, 176 specific infectivity of, VI, 167-168 Mersalyl acid, effect on cytokinesis and plasmagel structure of dividing eggs,
v, mff.
Mesenchyme, cell contact and, VII, 407-408 embryonic, histochemistry of, VI, 304 ff. Mespila globulis, cytological studies on, IX, 335 ff. Metabolism, cellular, ascorbic acid and, 11, 122-126 embryonic determination and, VI, 346 ff. in amphibia, VI, 349-361 in chicken embryo, VI, 361-366 in sea urchin embryos, VI, 367-374 of erythrocytes, enzymes in, V, 280289 lymphocytes and, VII, 266-267 radiation and, VII, 16-17 Metachromasia, VI, 241-251 factors affecting, VI, 243-247
396
CUMULATIVE INDEX
relation between PAS staining polymerization and, VI, 250-251 of substances in histological preparations, VI, 247-250 Metachromatic granules, bacterial, IV, 130-131 Metalloenzymes, definition of, VIII, 350352 Metals, distribution, VIII, 378-380 enzyme complexes and, VIII, 367-375 subcellular fractions and, VIII, 375380 Metastasis, adhesion role in, VIII, 206211 Methemoglobin reductase, in erythrocytes, V, 286-287 Methyl green, IX, 110-111 staining of nucleic acids with, IV, 241245 20-Methylcholanthrene1 estrone and, VII, 95-96 mouse fibroblast cultures and, VII, 8182, 86 vitamin A and, VII, 106-107 Methylene blue extinction, VI, 251-253 Mice, brephotransplantation of gonads in, 11, 369-372 culture of ovarian cortex from, 11, 352-356 Michaelis-Menten enzyme, IX, 138-139, 153 Microbodies, mitochondria and, VII, 497498 Microfibrils, VIII, 40-58 Microorganism, enzyme adaptation in, V, 51-87 radiation and, VII, 26-29 Microscopic reversibility principle, IX, 169-175 Microsomes, VI, 453 ff. ergastoplasm and, VII, 436, 467-468, 471-476; VIII, 18 identification of, VII, 438-440 isolated nuclei and, VIII, 321-322 as mitochondria precursors, VIII, 266267
- VOLUMES
I-IX
of pancreas, V, 328-355 role in cellular secretion, V, 328-335 Microspectrophotometry, ultraviolet, ascorbic acid and, 11, 108-110 Microtomy, of freeze-dried tissues, I, 50-53 of virus-infected cells, VI, 135-139 Micrurgy, cytochemical, IV, 1-29 Millipedes, vesiculiform sperm of, V, 438439 Minerals, in epidermis, I, 283-284 nucleolar, IV, 289 Mitochondria, carbon tetrachloride and, VIII, 322324 changes in according to media, IX, 274278 composition of, IX, 252-260 destruction of, IX, 248-258 differentiation of nuclei and, in bacterial protoplast, 11, 142-152 distribution of, 111, 301-311 epidermal, I, 270-272 ergastoplasm and, VII, 433-434, 465 fate in flagellate sperm, V, 429-433 filamentous, X, 280-288, 305 function of, VI, 447-453 genesis of new in embryonic differentiation, 111, 295-301 heterogeneity, IX, 278-282 isolated nuclei and, VIII, 322-324 of mast cells, 111, 405 melanin granules and, 11, 413 membrane, IX, 241-242 metals and, VIII, 373,376-377 methods of study, IX, 272-273 microbodies and, VII, 497-498 in microorganisms, IV, 101, 103-134 mode of action in morphogenesis, 111, 314-316 morphology of, IX, 227-292 of muscle, V, 88164 -originated organelles, IX, 260-266 of pancreas, V, 335-345 pathological aspects of, VIII, 8-15 pinocytosis granules and, VIII, 496498 plant, IV, 144-187
CUMULATIVE INDEX
regeneration of, VIII, 5-8 reticulocytes and, VIII, 142-143 role in cell secretion, V, 335 in sebaceous glands, I, 297 of stained erythrocytes, X, 255-257 structure of, V, 336; VI, 445-447; VIII, 4-5; IX, 242-247; X, 277285 swelling of, IX, 246-247 thin segment and, VII, 509-511 ultrachondriome and, 11, 412-413 ultrastructure of, V, 461-470; IX, 229234 from electron microscope studies, 11, 411-412 Mitochondria1 helix, sperm, structural aspects of, VII, 222-223 Mitosis, I, 10 action of urethane on, 111, 113-130 in bacterial cell, 11, 165-170 behavior of alkaline phosphatase during, 11, 271-273 birefringence changes in the sea urchin egg during, I, 197-203 epidermal hormones and, VII, 112-113 evidence for release of chemical agents in, I, 203-209 kinetochore organization, VII, 130-133 nuclear DNA content during, V, 187191 radiation effects on, VII, 14-16 structural agents in, I, 195-210 vitamin C and, 11, 120 Modiolus, muscle of, VII, 299 Molluscs, acrosome reaction in, V, 374-379 amoebocytes in, IV, 33-43 role in digestion, IV, 53-59 role in shell repair, IV, 43-52 mantle, IV, 43-52 shell formation, amoebocyte activity and, IV, 49-50 Monocytes, VII, 250-251 Morphine, mutagenic properties of, X, 121 Motility, animal cells, radiation resistaye of, VII, 17-19 lymphocytes and, VII, 243-245
- VOLUMES I-IX
397
Motor end-plate, mammalian, IV, 337350 MTK-sarcomas, chromosome studies on VI, 27 ff. Mucin, ’histochemistry of, VI, 212 Mucoids, acid, VI, 214, 216 Mucoitin sulfuric acid, histochemistry of, VI, 211-212 Mucopolysaccharides, classification of, VI, 1%-198 in egg, VI, 294-296 ground substance, VII, 599-601 histochemistry of, VI, 208-213, 225 lymphocytes and, VII, 241 lysozyme and, VII, 241 “neurosecretory” cells and, VII, 369 secretion, mast cells and, 111, 425-426 “sulfomucase” and, VII, 86 Mucoproteins, histochemistry of, VI, 213216, 225 Multienzyme systems, integrated, 11, 202-207 in soluble extracts, 11, 201-230 Mumps, localization of viral antigens in, V, 17 Muragenesis, radiation and, VII, 3-4, 19-20, 47-49, 55 Muscle (s) , cation movement in, VIII, 410-411 cholinesterase activity of, IV, 357-361 cross-striated, ultrastructure of, I, 316318 cytochondria of cardiac and skeletal, V, 88-146 energy metabolism in, V, 140-142 enzymatically-controlled transport in, I, 71-73 heart, see Heart muscle heterotropic ossification and, VIII, 267268 ion permeability, VIII, 451,46849 mammalian, structure of cross-striated fibers, V, 518-521 membrane potential of, VIII, 457-458 potassium content of, VIII, 460-461 proteins, IX, 389-390 Mustards, mutagenic properties of, 116119 Mutagenesis, chemical, IX, 294-295
398
CUMULATIVE INDEX
Mya, muscle of, 299 ff. Mycobacteria, electron-scattering bodies in, 11, 143-148 Myelin sheath, lipoprotein structures of, V, 505-506 of nerve fibers, V, 506, 507 Myelocytes, mitochondrial origin, IX,
262 Myosin, histochemical identification, IX, 389 light scattering of, IX, 175 Mytilus, acrosome reaction in, V, 374-377, 384 muscle of, VII, 297 ff.
N Nadi reaction, in sebaceous glands, I, 297 Natantians, chromatophorotropins in, VIII, 193-196 Nebenkern, ergastoplasm and, VII, 429 ff. mitochondrial, evolution of, V, 446-447 Nematodes, amoeboid sperm of, V, 439442 Nemerteans, cyto-embryology of, IX, 327 Nephron, thin segment, structural aspects, VII, 504-512 Nephrosis, mitochondria1 changes in, IX, 246, 252-253 Nerve (s), endings, ultrastructure of, VIII, 74-76 lamellibranch muscle and, VII, 328332 myelin sheaths of, ultrastructure of, I, 318 potassium permeability and, VIII, 466467 sodium permeability and, VIII, 466-467 transparent chamber studies on, 111, 373-374 Nerve cells, composition of, 111, 455-474 gravimetric determination of, 111, 466 mass determination, 111, 455-467 effect of development and differentiation on, 111, 469-472 ergastoplasm and, VIII, 449
-VOLUMES I-IX mitochondria of, V, 147-170; IX, 236237 early observations on, V, 148-151 quantitative determination of RNA in, 111, 467-469 Nervous system, embryonic, histochemistry of, VI, 301303 reticulocytes and, VIII, 157-159 Sa6-sulfate localization in, VII, 180181 Neurohypophy sis, innervation of, VII, 343-344 interstitial cells of, VII, 379-382 Neuromuscular junctions, degeneration and, VIII, 78-79 general features of, IV, 336-337 localization of cholinesterases at, IV, 335-375 ultrastructtlre of, VIII, 72-74 vertebral, IV, 337-355 Neurons, see Nerve cells Neuroproteofibrils, VIII, 72, 74 Neurosecretion, VII, 373-384 Neurosecretory material, VII, 342-373 Neutral red, lymphocytes and, VII, 240, 268 Nicotinic acid, effect on tissue cultures, 111, 51-52 Nissl bodies, ergastoplasm and, VII, 432, 449 Nonelectrolytes, permeability of mammalian erythrocytes to, VI, 469-511 Nuclear bodies, in bacteria, IX, 9-13 Nuclear membranes, structure, from electron microscope studies, 11, 408-409 Nuclear transplantation, IX, 325-326 Nuclei, see Nucleus Nucleic acids, I, 2-4 bacterial, IV, 121-122 in chromosomes, IX, 108-113 effect on tissue cultures, 111, 42 ff. formation cytoplasm and, VI, 406-407 histochemistry of, IV, 221-268 fiTation of tissue, IV, 229-230 physical methods, IV, 252-260
CUMULATIVE INDEX
quantitative, requirements for, IV, 222-229 staining methods, IV, 231-252 localization of, VI, 249-250 Nucleolonema, role in nucleolar origin, IV, 292-293 Nucleolus, chemistry of, IV, 280-289 composition, genetic evidence of, IV, 291-292 cycle of, IV, 271 DNA of, IV, 285-286 enzymes of, IV, 288-289 ergastoplasm and, VII, 462 function of, IV, 293-295 inclusions, IV, 272-273 interphase, morphology of, IV, 272-280 lipids of, IV, 288 lymphocytes and, VII, 238-239 morphology, changes in, IV, 275-276 experimental modification of, IV, 276-279 interphase, IV, 272-280 origin of, IV, 289-293 proteins of, IV, 286-288 ribonucleic acid content, IV, 281-285 role of nuclear sap, IV, 293 structure of, IV, 272-280; V, 505 Nucleoplasm, structure of, V, 505 Nucleoproteins, see also individual compounds quantitative aspects of nuclear, 11, 1-76 staining reactions for, 11, 17-31; IX, 382-383 study of, by photometric methods, 11, 3-17 Nucleoside, phosphorylase, isolated nuclei and, VIII, 307-308, 331 Nucleus, of Acetabularia, 11, 477-494 bacterial, IV, 104, 120-130 cytochemical demonstration of, IV, 122-130 cytological demonstration of, I, 9698 isolation of, IV, 136 structure of, 11, 165 cell debris and, VIII, 326-327 cell structure and, V, 351-355
-VOLUMES
I-IX
399
damage to, VIII; 331-335 deoxyribonucleic content of, V, 171-197 division, I, 10-12 endoplasmic reticulum and, VIII, 321322 enzyme studies on, 111, 199-223; VI, 390 ; VIII, 316-321, 336-337 ergastoplasm and, VII, 463-464 fragmentation of, VIII, 334-335 gel formation and, VIII, 333-334 homogenate and, VIII, 333-334 isolation, 111, 202; VI, 4, 383-411 contamination and, VIII, 316-327 methods of, VIII, 280-285 protein loss and, VIII, 327-331 isotope incorporation in, VIII, 285-316 of mast cells, 111, 402 membranes, V, 499-505 ; VI, 99 metabolic reactions in, VI, 404-418, 433, 444-445 mitochondria and, VIII, 322-324 osmotic behavior of, VIII, 426-427 protein synthesis and, VI, 393, 416-418 reproduction of, I, 9-26 ribonucleic acid and, VIII, 20-21 size of, I, 15-18 synthesis of RNA by, V, 433 ultrastructure of, I, 314-315, 316; V, 499-505 washing of, VIII, 329 Nucula, muscle of, VII, 299 ff. Nutrition, mast cells and, 111, 422 0 Oocytes, number of chromosomes in, 111, 178 Oogenesis, lipid histochemistry in, IX, 305-320 lipid synthesis in, IX, 317-318 Oogonia, human, chromosomes in, 111, 178-179 Ophiuroids, acrosome reaction in, V, 372374 Orcein, elastic fibers and, VIII, 215, 217218 Orconectes, VIII, 178 ff. chromatophorins in, VIII, 1%-198, 200 Organ cultures, use of, VII, 80-81 Organ of Corti, VII, 544-563
400
CUMULATIVE I N D E X
Organogenesis, histochemistry of, VI, 300-325 Orientation, contact guidance and, VII,
400-406 Orthoptera, acrosome formation in, V,
413-414 Osmium tetroxide, as fixative, IV, 83-84; VI, 136, 143 Osmoregulation, Golgi apparatus and, VIII, 24-25 Osmotic coefficient, VIII, 432-436 protein and, VIII, 393-398,403 Osmotic pressure, VIII, 393-427 Osmotic properties, of living cells, VIII,
387-448 Osmotic resistance, reticulocytes and, VIII, 146-148 Osmotic work, in cells, I, 135-164 Osteogenin, bone formation and, VIII,
264, 271 Ostrea, muscle of, 297 ff. Otic labyrinth, development of, VII, 537-
538 Otolith organs, physiologic aspects of, VII, 580-581 Otolithic membrane, S36-studies on, VII,
186-187 Outer pillar cells, organ of Corti and, VII, 546-550 Ovarian cortex, culture of, 11,352-360 Ovarian tissues, cultivated, 11, 380-383 Ovum, cleavage of, IX, 322-333 Oxidizing reactions, use in histochemistry, of carbohydrates, VI, 198-217 of other substances, VI, 217-223 of polysaccharides, VI, 223-241
P incorporation into isolated nuclei, VI, 404-407 Palaemonetes, chromatophores of, VIII, 178-180,185 chromatophorotropins in, VIII, 182P32,
183, 193-194 Pancreas, ergastoplasm and, VII, 442, 449, 467-
468
- VOLUMES I - I X exocrine cell, ingestion by, V, 326-328 structure of, in mouse, V, 516-518 sensory activity, V, 326-360 synthetic processes in, V, 328 Pandalus borealis, chromatophorotropin Of, VIII, 205-206 Panothenic acid, effect on tissue cultures, 111, 52 Papain, specificity of, VII, 613-614 Paphia, muscle of, VII, 301 ff. Papillae folliatae, 11, 291-295 Paramecia, antisera from, VI, 3-4 Paramecium, VIII, 97-128 Paramecium aurelia, antigen system, VI, 1-23 serotypes, VI, 3, 5, 6-17 Parm’lurus asotus, melanophores of, VIII, 203-204 Parasites, eggs of, transparent chamber study Of, 111, 392-393 Parasomal sac, VIII, 111, 124-125 Parasympathetic nerves, reticulocytes and, VIII, 158-159 Parathyroid gland, cultivated, grafting in man, 11, 372-377 Parathyroid hormone, bone and, VII,
110-111 Paraventricular nuclei, cytology of, VII, 341-342 labeled cysteine and, VII, 362 Particles, ergastoplasm and, VII, 452-454,
459-460,474 PAS reaction, see Periodic acid-Schiff reaction Pecten, muscle of, VII, 297 ff. Penicillin, effect on bacteria cell walls, IX, 57-58 effect on chromosomes, IX, 296 Peniculi, gullet and, VIII, 122-125 Pepsin, specificity of, VII, 612-613 use in histochemistry, IX, 111, 377 Peptidases, in isolated nuclei, VI, 394395 ; VIII, 308-309,337 Peptide synthesis, VI, 346 ff. Perforatonium, of mammalian sperm, V, 429
- VOLUMES
CUMULATIVE INDEX
Periodic acid, as histochemical oxidant, VI, 223-227 Periodic acid-Schiff ( P A S ) reaction, basis of, VI, 198-200 substances reacting with, VI,225 Peroxidase, in sebaceous glands, I, 297 Phagocytosis, intracellular fibrils and, VII,417-418 mast cells and, 111, 423 mitochondria1 changes in, IX,250 role in embryo, V,310-311 Phase contrast microscopy, of bacterial cells, 11, 142 in bacterial histochemistry, IV, 129130 1,lO-Phenanthroline, alcohol dehydrogenase and, VIII,359-361 Pholas, muscle of, VII,301, 307 Phosphatase, acid, behaviof during organogenesis, VI, 301 ff. in bull prostate, VI,399-400 in cytoplasmic particles, VI,455-457 isolated and, VIII,290, 320, 322, 337 in placenta, VI, 330 Phbsphatase, alkaline behavior during organogenesis, VI, 300 ff. in chromosomes, 11, 271 during mitosis, 11, 271-273 nuclear, 11, 261-288; VIII, 294-295, 320, 327, 330, 336 in yolk, VI,332-333 Phosphatases, in epidermis, I, 282 in erythrocytes, V, 285 histochemistry of, 11, 249-260 localization in cells, I, 69 methods of investigation, 11,250-252 in sebaceous glands, I, 297 Phosphate (s) , ions, intercellular material and, VII, 635-636 transport in red cells, V,290-293 Phosphoglucomutase, IX, 160-161 Phosphopyridine nucleotidase, in erythrocytes, V, 286 Phosphorylation, cellular, IX,144, 160-161
I-IX
401
intracellular, 111, 262-265 of muscle mitochondria, V, 116-125 nuclei and, VIII,287-288 oxidative, 11, 226 in plants, IV,174-177 Photofluorometry, of nucleic acids, IV, 259-260 Photometric methods, in study of nuclear proteins, 11, 3-17 Photosynthesis, calcium transport in, IV,394-395 in plants, IV,177-179 vitamin C and, 11, 121-122 Phthalate, collagen and, VIII,245-246 Picric acid, as fixative, IV,85 Piekarskie-Robinow technique, for demonstration of bacterial nucleus, IV, 122-123 Pigments, epidermal, I, 284-287 Pigmentation, mast cells and, 111, 423 Pinctada, muscle of,VII,301 Pinna, muscle of, VII,299 ff. Pinocytosis, VIII,482-502 ; IX,250 Pituitary gland, chromatophores and, VIII,202-204 Placenta, cytoplasmic RNA in, VI,325 ff. histochemistry of, VI,325-331 Placuna, muscle of, VII,301 Plant (s) , adsorption of cations by, 11; 186 cells, wall organization of, VIII, 33-60 chloroplasts of higher, IV,201-203 ion secretion in, 11, 179-200 radiation and, VII,23-26,47 respiration, effect of neutral salts on, 11, 187-188 mitochondria and, IV,159-174 salt absorption, 11, 180-185 viruses, electron microscopy of, VI, 139-149 Plasma, cells, lymphocytes and, VII,241-242 membrane, structure of, V,478 ultrastructure, I, 315 Plasmagel, V, 199-227 Plasmoptysis, of bacteria, IX,51-52 Plastic-embedding media, IX,241-242 Pleomorphism, of bacteria, IX,50-68
,
402
CUMULATIVE INDEX
Podophyllin, effect on ascites tumor cells, VI, 68-69, 71 Poisons, mitotic, effect on nuclear phosphatase, 11, 276-278 Poliomyelitis virus, VI, 170, 182 particulate identification, VI, 166-167 Polyhedrons, of insects, VI, 148-155 Poly-f3-hydroxybutyrate,as lipid granule component, IX, 26-31 Polymetaphosphate, IX, 16, 21-24 Polyploidy, amphibian, I, 168-177 Polysaccharides, bacterial, IV, 117-118; IX, 40-48 classification of, VI, 195-198 connective tissue and, VIII, 229-230 elastic tissue and, VIII, 213-214, 216, 225-226 histochemistry of, VI, 193-263 in ossification, VI, 311-312 reaction with PAS-technique, VI, 225 Polyspermy, block to, I, 258-260 Polytrophic ovaries, IX, 309-310 Poromya, muscle of, VII, 301, 327 Postnuclear cap, of mammalian sperm, V, 427-428; VII, 212-213 Potassium, VIII, 149, 453-489 Potato virus X, identification, VI, 141142 Potato yellow dwarf virus, VI, 142 Pox group virus, VI, 172-173 Pro-acrosome, V, 443-444 Prolidase, activity of, VII, 617-618 Protamines, stains for, IX, 373 Protein (s) , animal, fate of injected, V, 11-14 denaturation of, IV, 88-89 egg, VI, 120-121 elastic fibers and, VIII, 225-226 entropy of mixing and, VIII, 399-403 folding of molecules of, in cells, I, 136141 iodinated, formation of, VI, 279-284 metabolism, in plants, IV, 180-182 “neurosecretory” cells and, VII, 360361 nuclear, 11, 62-67; VI, 416-418 nucleolar, IV, 286-288 osmotic properties of, VIII, 395-404
-VOLUMES I-IX pinocytosis and, VIII, 489, 500-501 reaction with fluorescein isocyanate, V, 3-4 stains for, IX, 307, 371-377 synthesis, VI, 345 ff.; VIII, 467-471, 476 turnover in bacterial cell, V, -57-62 Protein carbohydrate complexes, in embryo, VI, 311-312 Proteinases, in erythrocytes, V, 286 in pig kidney nuclei, VI, 393-394 Proteus, flagella in, IX, 35-36 Proteus mirabilis, L-forms of, IX, 67-68 Protms vulgaris, pleomorphism of, IX, 65 Protoplasm, contractility, gel structure and, V, 199227 effect of tixation on living, IV, 87-93 structure of, IV, 87-88 Protoplasmic bead, of mammalian sperm, V, 426-427 Protoplasmic gel structures, see Plasmagel Protozoa, mitochondria1 cristae of, IX, 237-238 Proximal convoluted tubule, proximal portion, VII, 490-500 terminal portion, structure of, VII, 501504 Psammobia, muscle of, VII, 301, 307 Psittacosis-lymphogranuloma-venerumvirus group, morphology, VI, 176-177 Purines, effect on tissue cultures, 111, 42 ff. Pyknosis, lymphocytes and, VII, 246-247, 253-254, 268-269 Pyridine nucleotide dehydrogenases, complex, 11, 212-217 Pyridinoprotein dehydrogenases, 11, 211212 Pyridoxine, effect on tissue cultures, 111, 50-51 lymphopoiesis and, VII, 249-272 Pyrimidines, effect on tissue cultures, 111, 42 ff. Pyronin, staining of nucleic acids with, IV, 245-248
CUMULATIVE INDEX
Q Quadrulus, gullet and,
VIII,122-125
R Radiation, VII, 1-60 Radiation chemistry, mode of action and, VII, 58-60 Radiation physics, VII, 54-56 Radiation sickness, VII, 8-9 Radiobiology, VII, 2-9 experimental, VII, 1-77 Radioisotopes, use in research, VII, 62-
64
'
Radiological protection, chemicals and, VII,60-61 permissible dose and, VII,55-56 Radiosensitivity, types of, VII,51-52 Rana pipiem sperm, radiation effects on, VII, 44-45 Rats, ascites tumors of, chromosome cytology of, VI, 25-84 limb bud culture of, 11, 349-352 Red cells, sodium and potassium movements in, VIII,449-480 Redox pump, evidence for, in active cation transport, IV,377-396 general principles, 11, 420-423 osmotic work and, 11, 419-444 relation to ion movement, 11, 431-435 Refractometry, cell volume and, VIII, 417-4 18 Relative biological efficiency, dose units and, VII, 56-58 Renal vessels, ligation, ossification and, VIII,254-258 Reproductive apparatus, embryonic, histochemistry of, VI, 324-325 Reproductive tract, VII,87-89 Reptile (s) acrosome formation in, V,406,407 oocytes, lipids of, IX,314-315 Respiratory apparatus, embryonic, histochemistry of, VI, 320 Resting potential, ion distribution and, VIII, 452-462 Reticular membrane, organ of Corti, Deiters' cells and, VII,550-551
-VOLUMES I-IX
403
Reticulin, VI,216-217; VII,592-598 Reticulocytes, VIII, 136-163 Reticulum, endoplasmic, structure of, V, 494-496; VI, 99-100 lymphocytes and, VII,246-247 Retinal rods, lipoprotein structures of, V, 506, 508, 509 ultrastructure of, V, 511-513 Rhodopspirillum rubrum, volutin granules in, IX,16 Riboflavin, effect on tissue cultures, 111,
SO Ribonuclease, use in cytochemistry, IX, 110-114 Ribonucleic acid, 11, 58-62 in bacteria, IX,14-15 cytoplasmic, VI, 292-335 ergastoplasm and, VII,426,472-476 in microsomes, VI, 458-460 nuclear, metabolism of, VI, 415-416, 418-419 nucleolar, IV, 281-285 reticulocytes and, VIII, 151-152 role in cell secretion, V,329 ff. role in peptide formation, VI,346 ff. of tobacco mosaic virus, VI,147-148 of turnip yellow virus, VI, 145-146 in volutin granules, IX,16,.22 Ribonucleoproteins, of chromosomes, IX, 118-119 Ribosomes, of bacterial cells, IX,13-16 Rodents, histochemistry of early development, VI, 297-300 Roots, ascorbic acid in, 11, 110-113 salt absorption by, 11, 194-196 Rous sarcoma, causative agent, VI, 183184 S Saccharomyces cerevisiae, volutin granules in, IX, 17-1$ Sakaguchi reaction, IX,374-375 Salivary chromosome, 111, 136-154 structure, 111, 137-154, 172-173 Salivary gland, mitochondria of, V,340 ff. Salmortella typhimurum, IX,8-39 Salt absorption in plants, 11, 180-196
404
CUMULATIVE INDEX
Salyrgan, see Mersalyl acid Sarcolemma, lamellibranch muscle and, VII, 302-303 Sarcoma, cell motility in, 209-210 trypsin treatment of, 212-213 Schwann-cell, as mitochondria precursor, IX, 267 Scleroproteins, enzyme action on, VII, 603-607 histochemical identification, IX, 386388 Scorpions, acrosome formation in, V, 398-399 flagellate sperm of, V, 432-433 Sea urchin, cyto-embryology of, IX, 323-326 egg, birefringence changes during mitosis, I, 197-203 cortex of, VI, 107-110 development, enzymes and, 111, 277279 embryonic determination of, VI, 366374 metamorphosis of, IX, 362-365 Sebaceous glands, I, 290-299 Sccale cereale, chromosome organization in, VII, 152-153 Seedlings, germination and growth of, ascorbic acid and, 11, 118-119 Selectivity, cell contact and, VII, 408-416 Sensory field, 11, 294 Sepia oficinalis, color mimicry in, VIII, 179 Serum, effects on cell adhesion, IX, 213215 Sesurmu, VIII, 183-185 chromatophorotropins in, VIII, 188 Sex hormones, embryonic development and, VII, 87-89 Shigello fiexncri, fimbriae studies on, IX, 38-39 Shope fibroma, electron microscopy of, VI, 184-185 Silver nitrate, bone induction and, VIII, 268-269 Silver-staining technique, bone induction and, VIII, 268-269
-VOLUMES I-IX for localization of ascorbic acid, 11, 81 modification of, 11, 83-87 standard methods, 11, 82-83 Sinus gland, chromatophorotropins and, VIII, 182-183 Skeleton, embryological formation, IX, 360-361 Skin, brephotransplantation in man, 11, 368369 ion transport and, VIII, 472-474 96-localization in, VII, 185-186 Slime, bacterial, IX, 40-48 Sodium, VIII, 149, 463-489 Sodium chloride, pleomorphism inductivity, IX, 54, 60-63, 68 Solen, muscle of, VII, 297 ff. Somatic cells, chromosome number of, 111, 190 ff. Somatic inconstancy, 111, 186-195 DNA content of nuclei and, 111, 188 origin of, 111, 189-190, 194 Spectrophotometry, estimation of ascorbic acid by, 11, 94108 effect of oxidation on, 11, 103-107 infrared, 11, 107-108 Spectroscopy, ultraviolet, of nucleic acids, IV, 253-256 Sperm, see atso Spermatozoa flagellate, V, 397-433 head cap, structure of, VII, 203, 204, 205, 207 middle piece, VII, 216-224 neck, structure of, VII, 214-216 nonflagellate, V, 433-442 position of acrosome in, V, 443 principal piece, VII, 224-229 Spermatids, chromonemata of, IX, 91 mitochondria1 cristae of, IX, 236-237, 239 Spermatocytes, human, chromosomes in, 111,178 morphology of, VI, 94-% “synaptinemal complex” of, IX, 92-93 Spermatogenesis, chromatid bodies in, V, 442-443 cytology of, v, 395-453
CUMULATIVE INDEX
Spermatogonia, human, chromosomes in, 111, 178-179 Spermatozoa, chemotaxis of, I, 260-273 interaction with egg, I, 257-263 perforatoria in, VII, 212 physiological maturity, acrosome reaction and, v, 381-382 structure, VII, 195 ultrastructure of, VIII, 131 vacuole in head of, VII, 202-203 S p k r i u m , muscle of, VII, 301, 305 Spiders, acrosome formation in, V, 410-411 lipids in oocytes of, IX, 311-312 Spindle proteins, histochemical identification of, IX, 383-384 Spondylus, muscle of, VII, 301, 304, 327 Spores, bacterial, nucleus of, IV, 109 Sporulation, IX, 32-35 Staining, effect of dye concentration, I, 236-237 of pH on, I, 215-223 of elastic tissue, VIII, 217-229 isoelectric point and, I, 230-233 kinetics of, I, 246-248 nature of dye-protein bond in, I, 224239 reversibility of, I, 248-250 Staining solutions, effect of temperature on, I, 245-246 ionic strength of, I, 233 pH of, effect on protein-dye interaction, I, 215-223 Starch, formation in chloroplasts, IV, 212 Statoconia, composition of, VII, 580 Steatococcus, chromosome study of, IX, 85-86 Stemline cells, of rat ascites tumors, VI, 25-84 Stentor niger, mitochondria of, IX, 234235 Streptobacillus moniliformis, pleomorphism of, IX, 65 Streptococcus faecalis, cell wall of, IX, 50 pleomorphism of, IX, 63 Streptococcus hemolyticus, removal of “m” protein from, IX, 212
-VOLUMES I-IX
405
Stress, lymphocytes and, VII, 242-243, 248, 268, 271 osmotic, “neurosecretory” cells and, VII, 366-367, 369, 373, 375-376 Stria vascularis, endolymph and, VII, 543, 563 Striations, of double-obliquely striated fiber, VII, 316-324 transversely striated fibers and, VII, 309-315 Stroma, erythrocytic, metabolic processes in, V, 287-289 Strongylocentrofus, VII, 162-190, 622, 636 Submaxillary glands, change following duct ligation, V, 342-343 Submicromanipulation, volumetric, IV, 911 Substratum, adhesiveness and, 399-400 Succinic dehydrogenase, in isolated nuclei, VIII, 310, 337 “neurosecretory” cells and, VII, 364365 Succinoxidase, in isolated nuclei, VIII, 310-311, 322, 324-325, 337 Sudan black, as lipid stain, IX, 26, 306307 , . Sugar (s 1, absorption, enzymes and, I, 107-118 in bacterial cell walls, V, 30-32,46 cell permeability to, IX, 134, 149-150 effect on gastrulation, IX, 355-356 in erythrocyte metabolism, V, 280-283 in plant cell walls, VIII, 44-45 transport in red cells, V, 293-295 uptake by cells, IX, 131-132 Sulfatase, in isolated nuclei, VIII, 311, 324, 325, 337 Sulfhydryl groups, alcohol dehydrogenase and, VIII, 358, 361 epidermal, I, 278-280 stain for, IX, 373 “Sulfomucase,” mucopolysaccharides and, VII, 611 Sulfur granules, in bacteria, IX, 31-32
406
CUMULATIVE INDEX
Supraoptic nuclei, cytology of, VII, 341-342 labeled cysteine and, VII, 362-363 Synapse (s), VIII, 61-93,498-499 Synaptic vesicles, VIII, 79-93 Synaptinemal complexes, of chromosomes, IX, 92-94
T Takeda sarcoma, VI, 27 chromosomes of, VI, 31, 43-45 inoculation with, followed by MTKsarcoma, 11, 64-65 Target action, radiation and, VII, 48-49 Target theory, radiation and, VII, 54-
55, 59-60 Taste buds, structure of, 11, 295-298 Tectorial membrane, S3s-sulfate uptake in, VII, 562 structural aspects of, VII, 562-563 Teeth, development, histochemistry of, VI,
313-319 S35-sulfate localization in, VII, 184-
185 Tellina, muscle of, VII, 301 Telotropic ovaries, IX, 310-311 Teredo, muscle of, VII, 299, 301 Testis, isolated polyploid cells in, 111, 181 Testosterone, VII, 89,248 prostate glands and, VII, 96-97 reproductive tract explants and, VII,
87-88 Tetrahymenu, cell division in, IX, 23-25 nutritional requirements of, I, 28-32 Theophylline, -induced chromosome breakage, 1 10, 120 Thermodynamics, osmotic pressure and, VIII, 392-398 Thermoregulation, chromatophores and, VIII, 178-179 Thiamine, effect on tissue cultures, 111,
49-50 Thin segment, mitochondria of, VII, 509-511 nephron and, VII, 504-512 Thoracic duct, output of, VII, 258-260 Threshold action, radiation and, VII,
48-49
-VOLUMES I-IX Thymocytes, lymphocytes and, VII, 237-238 X-radiation and, VIII, 274-275 Thymus, antibodies and, VII, 284 lymphopoiesis and, VII, 248 Thyroglobulin, VI, 213,270,279 Thyroid gland, culture from chick embryos, 11,365-367 Cytology of, VI, 265-288 electron microscopy of, VI, 266-270 embryonic, phosphatases in, VI, 320-321 follicle, V1, 274-279 function of, VI, 284-286 morphology of, VI, 265-270 parafollicular cells, function of, VI,
285-286 structure of, VI, 266-285 Thyroxine, embryonic bone and, VII, 108-110 melanophores and, VIII, 205 mitochondria and, VIII, 9 ; IX, 246,276 secretion, VI, 284-285 Ticks, tubiliform sperm of, V, 439 Tides, color rhythms and, VIII, 190-193 Tissue cultures, caryometric studies on, 111, 69-111 chromosome number in, 111, 183-186 effect of developmental stage on reaction to medium, 11, 377-383 grafting of, 11, 367-384 lymphocytes and, VII, 256-257 media for biological, 111, 5-14 oxygen requirements of, 111, 33-34 radiation and, VII, 32-34 techniques of, IV, 4-5 uptake of macromolecules in, V, 315-
316 Tissue disintegration, mechanical factors in, VII, 638-640 Tissue homogenates, components of supernatant fractions, VI, 460-461 Tissue sections, electron microscopy of, I, 305-322;11,
403-407 preparation for use with labeled antibodies, V, 4-5 ultrathin, electron microscopy of, V,
458-459
CUMULATIVE INDEX
fixation of, V,459-460 preparation of, V,457-458 Tissue workers conference, 1950, report Of,
11, 499-505
Tissue (s) , antigenic substances native to, V, 14-16 connective, aging and, VIII,237-239 composition of, VIII, 213-215, 229-
230, 376-377 embryonic, VI,305 ff. histology of, VIII,217-219 mast cells in, 111, 405-415 morphology of, VIII,222-226 transparent chamber studies on, 111,
374 differentiation, organ culture and, 11,
349-367 elastic, 111, 437-453 embryonic, macromolecular uptake by,
V, 310-317 explanted, 11, 331-348 fractionation, 111, 259-269 intracellular material in, VII,590-603 localization of ascorbic acid in, 11, 77 ff. mammalian, cytochemistry of, VI, 425-
467 normal, DNA content of, V, 177-182 staining, with acid and basic dyes, I,
211-255 Tobacco condensate, human fetal lung and, VII, 83 Tobacco mosaic virus, structure of, VI,
146-148 Tobacco ring spot virus, morphology of,
VI, 145 Trace elements, emission spectrography and, VIII, 347-350 Tradescantiu, chromosome studies of, IX,85-87,92 colchicine-radiation treatment and, VII,
-VOLUMES I-IX
microscopic studies in living mammals with, 11, 359-398 Transplants, heterotropic ossification and,
VIII, 258-262 Transport, active, see Active transport of cations, V,296-298 enzymatic, I, 70-89 systems, ions and, VIII,475-477 Trehalose, localization in cells, I, 68 Trichocyst, structure of, VIII,112-113 Tridacnu, muscle of, VII,299 Triiodothyronine, embryonic bone and,
VII, 109-110 Trillium species, chromosome organization in, VII, 153-155 Triturus, kinetochore structure of, VII,
139, 142 Tropic hormones, pituitary, VI,213-215 Trypsin, activity of, VII,614-629 phosphate ions and, VII,636 specificity of, VII, 616-629 use in cytochemistry, IX, 110-112,115,
212-213,377 Trypsinogen, in pancreatic nuclei,
360-364
VI,
395-3% Tryptophan, -rich chromosomal protein, IX,383 status for, IX,375-376 * Tubercle bacilli, transparent chamber methods in study of, 111, 393-394 Tumor (s) , cells, lymphocytes and, VII,238,245 coaption and, VII,412,420-422 ergastoplasm and, VII,470-471 malignant, ascorbic acid in, 11, 117-118 mast cells and, 111, 423-424 Turbo, striated muscle in, VII,297 Turnip yellow virus, components of, VI,
145-146
U
24 DNA content of kinetochore, VII,142 kinetochore structure in, VII,125 ff. microsporocytes of, IX,117 Transparent chamber, construction and installation of, 111,
407
Uca, VIII, 178-206 Ultrachondrione, mitochondria and, 11,
412-413 Ultramicrotomy, I, 308-313 Ultraviolet microspectrophotometry, see under Microspectrophotometry
408
CUMULATIVE INDEX
Ulva lactuca, active transport of sodium ions in, IV, 395 Unio, muscle of, VII, 301 ff. Uranyl ion, effect on cell permeability, IX, 130-131, 139, 141 Ureter, heterotropic ossification and, VIII, 254-255, 258 Urethan, 111, 113-130 Uricase, isolated nuclei and, VIII, 312313, 322, 325, 337 Urinary tract, heterotopic ossification and, VIII, 254-262 Usubuchi sarcoma, VI, 27,31,45
V Vaccinia virus, studies on, VI, 172-173, 178 Vacuoles, bacterial, IV, 107-108 reticulocytes and, VIII, 143 Valine, elastic fibers and, VIII, 242-243 Valonia, VIII, 35-58 Venus, muscle of, VII, 299 ff. Versene, intercellular material and, VII, 634-635, 639 Vesicles, of cytoplasm, V, 497 Vesicular stomatitis virus, morphology Of, VI, 171-172 Vestibular sensory epithelia, structure and innervation, VII, 563-581 Viruses, animal and human, electron microscopy of, VI, 164-185 identification of infectious particles, IV, 165-169 incomplete forms, VI, 170-171, 174 infectivity, specific, VI, 167-169 morphology of, VI, 169-172 bacterial, see also Bacteriophages bacteria as hosts to, 11, 152-153 detection by electron microscopy, VI, 129-191 growth of, VI, 178-179 identification of, VI, 137-143 "incomplete," VI, 170 insect, electron microscopy of, VI, 148-155 ' morphology of, VI, 153-155
-
-VOLUMES I-IX labeled antibodies in studies of, V, 1718 origin of, I, 119-120 particulate nature of, VI, 130 plant, identification of, VI, 138-145 morphology of purified, VI, 145-148 staining of, VI, 143-144 tumorigenic, identification, VI, 141 pox group, enzymatic disintegration, VI, 172-173 Vitamin A, effect on tissue culture, III,46-47 excess, bone and, VII, 98-103 epithelium and, VII, 103-106 20-methylcholanthrene and, VII, 106107 S36-sulfate metabolism and, VII, 187188 Vitamin B,, effect on bone, VII, 107-108 Vitamin B,,, effect on tissue culture, 111, 534 Vitamin C, see Ascorbic acid Vitamin D, effect on tissue culture, 111, 47 Vitamin E, effect on tissue culture, 111, 47-48 Vitamin K, effect on tissue culture, 111, 48 Vitamins, see also individual compounds effect on tissue cultures, III,46-53 lymphocytes and, VII, 265-266 Volume, cellular, measurement of, VIII, 413-418 osmotic pressure and, VIII, 404-427 Volutin granules, IX, 2, 7, 16-25 formation of, IX, 17-19 stain for, IX, 3
W Wall, organization in plant cells, VIII, 33-60 Watanabe ascites hepatoma, VI, 27 chromosomes, VI, 31, 46 Water, cell permeability and, VIII, 427-428 cell volume and, VIII, 417-418
CUMULATIVE INDEX
diffusion inrells, VIII, 438-442 reticulocytes and, VIII, 145,149 Water permeaility, diffusion and, VIII, 436-438 measurement of, VIII, 431-432 White cells, transparent chamber studies on, 111, 372-373 Wound healing, cell matching and, VI,
409-412 Wounds, fibrogenesis and, VIII, 236-237
X Xenopzls leevis, chromatophorotropins in, VIII, 204-205 X-protein, of tobacco mosaic virus, VI,
140, 146-147 X-radiation, antibodies and, VII, 279 lymphocytes and, VIII, 238, 268-269,
271-277 X-ray diffraction, 223-242 X-ray microradiography, 111, 455-467 X-rays, cellulose and, VIII, 36-39 heterotropic ossification and, Will, 269 Xylan, cellulose and, VIII, 40-42,48, 50
-VOLUMES
409
I-IX
Y Yeast(s), active transport of cations in, IV, 378-
384 glucose permeability in cells of, IX,
137-138 uptake of glucose by, I, 73-75 Yolk sac, histochemistry of, VI, 331-333 Yoshida sarcoma, VI, 27, 32,35-38 chromosome cytology of, VI, 30,35 ff. mitosis in, VI, 32
z Zea maize, mitochondria of, IX,
238-239
Zinc, alcohol dehydrogenase and, VIII, 355-
361, 380 coenzymes and, VIII, 364-365 extrinsic, VIII, 356-357 metalloenzymes and, VIII, 353-365 pathology and, VIII, 365 Zymogen granules, ergastoplasm and, VII, 429, 431, 432,
468 mitochondria1 origin, IX, 263-264
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