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A histochemical and ultrastructural study of the ovary and vitellogenesis in the dogfish Squalus acanthias Reid, James Allen 1975

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A HISTOCHEMICAL AND ULTRASTRUCTURAL STUDY OF THE OVARY AND VITELLOGENESIS IN THE DOGFISH SQUALUS AGANTHIAS JAMES ALLEN REID B.Sc, U n i v e r s i t y of B r i t i s h Columbia, 1 9 7 0 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF 'MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s t hesis as conforming to the required standard THJ UNIVERSITY OF BRITISH COLUMBIA January, 1 9 7 5 In presenting th is thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada fi ABSTRACT Protein yolk formation i s being studied i n an ever increasing number of organisms and i n each the method of formation ( i . e . i n t r a -oocytic vs extraoocytic) and the organelles involved (Golgi, mito-chondria etc.) d i f f e r even though the f i n a l product i s markedly s i m i l a r . Previous emphasis on elasmobranchs i n t h i s laboratory and the a v a i l a b i l i t y of the specimen favoured the s e l e c t i o n of Squalus acanthias as the experimental animal as v e i l as the lack of any pertinent studies on i t s ovary or v i t e l l o g e n i c processes. While examining the problem of yolk formation with both histochemical and u l t r a s t r u c t u r a l techniques, several other c r i t i c a l areas of i n v e s t i g a t i o n were pursued. As a r e s u l t , the composition of the f o l l i c u l a r envelope and the formation of i t s associated membranes, the surface epithelium, the t u n i c a albuginea, the ovarian cortex and the lacunae border c e l l s were also examined. An examination of representative oocytes from 0.1 to k.O mm i n diameter revealed many important f a c t s . F i r s t , these oocytes are i n a prolonged diplotene with up to 100 n u c l e o l i present, each r i c h i n rRNA represented u l t r a s t r u c t u r a l l y by t h e i r granular nature. Secondly, a complex Bal b i a n i body r i c h i n p r o t e i n and RNA and composed of mito-chondria and SER i s present. Here, t h i s mitochondrial mass i s the center for mitochondrial m u l t i p l i c a t i o n . T h i r d l y , l i p i d yolk i s formed before proteinaceous yolk and i t a r i s e s de_ novo i n the ooplasm from extra-o o c y t i c a l l y derived materials. F i n a l l y , p r o t e i d yolk arises from two sources. I n i t i a l l y , multigranular (MGB) and m u l t i v e s i c u l a r (MVB) bodies form from Golgi, SER, micropinocytotic v e s i c l e s and v e s i c l e s derived from the nuclear membrane which fuse together forming granular-vesicular bodies (GVB). In oocytes l a r g e r than 0.k mm, distended Golgi lamellae, SER cisternae and loops of SER v e s i c l e s form l i m i t i n g membranes and take up MGBs, MVBs and small GVBs. The contents of these large GVBs then d i s s o l v e , reaggregate and c r y s t a l l i z e forming a mature yolk p l a t e l e t . Of the other areas examined, the basal lamina, the v i t e l l i n e membrane and the f o l l i c l e cells•were the most relevant to yolk formation. The f o l l i c l e c e l l s were implicated i n the formation of both the lamina and the v i t e l l i n e membrane. The former are predominantly acid and sulphated mucopolysaccharides while the l a t t e r i s a protein-polysaccharide complex. Both membranes provide support and chemical s e l e c t i v i t y . The evidence for s t e r o i d a c t i v i t y i n the f o l l i c l e c e l l s of these stages was inconclusive. i i i TABLE OF CONTENTS Page INTRODUCTION' . 1 MATERIALS AND METHODS Experimental Animals k Preparation f o r Histochemical Analysis k Histochemical Procedures - 5 Preparation f o r Electron Microscopy 7 RESULTS The Ovary 9 Histochemical Analysis 9 Protein 9 Mucoproteins and Neutral Mucopolysaccharides 1 1 •Ribonucleic .-Acid Ik A c i d i c and Sulphated Mucopolysaccharides 1 8 Phospholipids and L i p i d s E l ectron Microscopical Observations 30 The Ovarian Epithelium 30 The Tunica'Albuginea 3 7 The Cortex 3 7 The I n t e r c e l l u l a r Space k3 The Lacunae Border C e l l s k3 The Red Blood C e l l s ^ 6 The Thecal C e l l s ' ^ 8 The Basal Lamina-Lamella Complex 5 3 The F o l l i c l e C e l l s 6 2 The V i t e l l i n e Membrane 7 1 i v The Oocyte Nucleus 77 The Mitochondrial Mass 85 L i p i d Yolk 86 Protein Yolk 88 DISCUSSION Hi stocherai s t r y Oocyte l l h V i t e l l i n e Memhrane 115 F o l l i c l e C e l l s 115 Basement Lamina-Lamella Complex 117 Theca 119 Stroma .120 Tunica Albuginea 121 •Ultr.astructure 123 Ovarian Epithelium 123 Tunica Albuginea 125 Cortex 126 The I n t e r c e l l u l a r Space 127 Lacunae Border C e l l s 128 Red Blood C e l l s 129 Thecal C e l l s 129 Basement Lamina-Lamella Complex 130 F o l l i c l e C e l l s and the V i t e l l i n e Membrane 13^ Oocyte Nucleus 1^1 A. Nuc l e o l i 1*+1 B. Nuclear Pores 1U3 C. Nuclear Membrane V Mitochondrial Mass ikk L i p i d Yolk 1 U 7 Protein Yolk 1 5 0 SUMMARY 1 5 7 BIBLIOGRAPHY " l 6 l APPENDICES Histochemical Techniques Employed 1 7 ^ Staining 1 7 ^ Enzyme Treatment 1 7 5 S e l e c t i v e Blocking and Unblocking 1 7 6 VI LIST OF TABLES TABLE Page IA, B, C. Combined Histochemical R e s u l t s 29 v i i LIST OF ILLUSTRATIONS . FIGURE Page 1 to 2 DNFB st a i n i n g 1 0 ' 3 to k PAS-trichrome s t a i n i n g 1 2 5 PAS-trichrome s t a i n i n g 13 5 PAS-dichrome, c o n t r o l " 13 7 PAS-dichrome, diastase treated 13 8 to 9 Methyl Green-Pyronin s t a i n i n g 1 5 1 0 to 1 1 Toluidine blue for nucleic acids and a c i d 1 7 -mucopolysaccharides 1 2 Toluidine blue, RNase treated 17 1 3 A l c i a n blue s t a i n i n g 1 9 lk Hale's c o l l o i d a l i r o n r e a c t i o n 2 1 1 5 a , l 6 a Toluidine blue c o n t r o l f o r hyaluronidase. 2 2 Water mounted„ 1 5 b , l 6 b Toluinine blue hyaluronidase treated. Water mounted 2 2 , 1 7 to 1 8 Aldehyde Fuchsin s t a i n i n g 23 1 9 AF following Methylation-Demethylation (MDM) 23 , 2 0 Azure A s t a i n i n g 25 2 1 Azure A, MDM treatment 25 2 2 Azure A, hyaluronidase treated 2 5 2 3 Copper Phthalocyanin s t a i n i n g 2 6 2h to 25 Sudan black s t a i n i n g 28 2 6 Surface epithelium of the ovary 3 1 2 7 Surface epithelium, complete panorama 3 2 2 8 Apices of three surface e p i t h e l i a l c e l l s 3 3 2 9 to 30 . High magnification of several e p i t h e l i a l contact 3 ^ surfaces v i i i FIGURE Page 31 Surface v e s i c l e s ' o f the e p i t h e l i a l c e l l s 3 5 3 2 L a t e r a l e p i t h e l i a l contact surfaces 3 5 3 3 Tunica albuginea 3 8 3 ^ High magnification of an e l a s t i c f i b r e 3 8 3 5 to 3 6 Dark c e l l s i n the cortex hO 3 7 Intermediate dark c e l l U l 3 8 Light c e l l i n the cortex U l 3 9 to hi Light c e l l s i n the cortex h2 h2 to hh Lacunae border c e l l s . hh 1+5 to hj Nucleated red-blood c e l l s hi hQ Thecal c e l l s , 0 . 3 mm oocyte hg h9 Theca interna c e l l s , 2 . 0 mm oocyte h9 5 0 to .52 Theca interna c e l l s , „ 1 , . 0 mm .oocyte 5 0 5 3 to 5 5 Theca externa c e l l s , 1 . 0 mm oocyte 5 2 5 6 to 5 8 Various aspects of the f o l l i c l e , 0 . 1 mm oocyte 5 ^ 5 9 V i t e l l i n e membrane, 0 . 2 mm oocyte 5 5 6 0 F o l l i c l e l a y e r s , 0 . 3 mm oocyte 5 5 6 1 F o l l i c l e c e l l t r a n s i t i o n zone, 0 . 3 mm oocyte 5 6 6 2 to 6 3 Zona r a d i a t a of a 0 . 3 mm oocyte 5 6 6h to 6 6 Basal FC cytoplasm and formation of the basal 5 7 . lamina, O.h mm oocyte 6 7 to 6 8 F o l l i c l e layers with e a r l y VM formation, O.h mm 5 8 oocyte 6 9 to 7 1 Stages of VM formation i n one oocyte, O.h mm 5 9 7 2 to lh High magnification of VM formation, O.h mm oocyte 6 0 7 5 P s e u d o s t r a t i f i e d f o l l i c l e c e l l s , O.h mm oocyte 6 l 7 6 Extensive VM formation, O.H mm oocyte 6 l FIGURE Page 7 7 to 7 9 Various views of the FCs, 1 . 1 mm oocyte 6 5 8 0 t o , 8 2 V i t e l l i n e membrane and zona r a d i a t a , 1 . 0 mm 6 6 oocyte 8 3 Base of the zona r a d i a t a 6 7 8k to 8 5 Variations i n 1 . 1 mm oocyte f o l l i c l e c e l l s ' 6 7 8 6 to 8 7 Light micrograph of the f o l l i c l e l a y e r s , 1 to 6 8 2 mm oocyte 8 8 to 8 9 Basement lamina and lamella, 1 . 5 to 1 . 8 mm oocyte • 6 9 9 0 to 9 2 Basal to median l e v e l s of the p s e u d o s t r a t i f i e d 7 0 f o l l i c l e c e l l s , 1 . 5 to 1 . 8 mm oocyte 9 3 to 9k FC-VM i n t e r f a c e with pore canals i n VM, 1 . 5 to 7 5 1 . 8 mm oocyte 9 5 to 9 6 Zona r a d i a t a and c o r t i c a l l a y e r , 1 . 5 to 1 . 8 mm 7 6 oocyte 9 8 to 1 0 0 Nuclear region and nearby p r o t e i n yolk precursors ' 7 8 (PYP), 0 . 1 mm oocyte 1 0 1 Light micrograph, 0 . 1 mm oocyte 8 0 1 0 2 Nucleus, 0 . 1 mm oocyte 8 0 103 N u c l e o l a r - l i k e body i n the ooplasm, 0 . 1 mm oocyte 8 0 i; 10k Nucleolus, 0 . 1 mm oocyte 8 l 1 0 5 to 1 0 7 Mitochondrial mass and surrounding ooplasm, 0 . 1 8 3 mm oocyte 1 0 8 Nuclear region, 0 . 2 mm oocyte 8 U 1 0 9 Central ooplasm and organelles, 0 . 2 mm oocyte 8k 1 1 0 Nucleus, 0 . 3 mm oocyte 8 7 1 1 1 to 1 1 2 Light micrograph of a 0 . 3 . mm oocyte with l i p i d 8 7 yolk 113 to 1 1 5 Ooplasm with various mitochondria and PYP, 0 . 3 9 2 mm oocyte 1 1 6 to 1 1 7 Ooplasm with various PYP, 0 . 3 mm oocyte 9 3 FIGURE Page 118 L i p i d yolk and forming PYP, O.k mm oocyte 9^  119 L i p i d yolk with a developing granular-vesicular 9\ body 120 to 122 Various l i p i d yolk p a r t i c l e s and organelles, 95 O.k mm oocyte 123 L i p i d yolk with concentric rings of SER, 96 0.5 nun oocyte 12k Branching mitochondrion, 0.5 mm oocyte 96 125 L i p i d yolk associated with SER, 0.5 mm oocyte 96 126 Various stages i n PYP formation, 0.5 mm oocyte 97 127 Large l i p i d yolk bodies, 0.5 mm oocyte 97 128 Nuclear membrane, pores and SER i n the ooplasm, 98 0.8 mm oocyte 129 Small nucleolus, 0.8 mm oocyte 98 .,130 Mitochondrial mass., 0.8 mm oocyte . 9 8 131 Mitochondrial mass and SER, 0.8 mm oocyte 100 132 Polymorphic mitochondria, 0.8 mm oocyte 100 133 Ring-shaped mitochondrion and numerous loops of 100 SER, 0.8 mm oocyte 13h L i p i d yolk p a r t i c l e s , 0.8 mm oocyte 100 135 Light micrograph, nucleus with lampbrush 101 chromosomes and n u c l e o l i , 1.0 mm oocyte 136 Annulate l a m e l l a , 1.0 mm oocyte 101 137 Mitochondrial mass, 1.0.mm oocyte 101 138 Mitochondrial mass, 1.0 mm oocyte 102 ' 139 GVB with attached SER 102 lUO Organelles and PYP, 1.0 mm oocyte 102 lHl Golgi body and several SER configurations 10k lk2 A large mass of PYP i n various stages of develop- 10k ment x i FIGURE Pag lk3 A large G o l g i - l i k e body, 1 .0.mm oocyte 10k Ikh Two PYP systems, 1 . 0 mm oocyte 1 0 5 lh5 Extensive SER systems, 1 . 0 mm oocyte 1 0 5 l U 6 Pinocytotic v e s i c l e s and l i n i n g bodies, 1 . 0 mm oocyte 1 0 5 1 U 7 Convoluted plasma membrane, 1 . 0 mm oocyte 1 0 5 1 U 8 Nuclear membrane, 1 . 1 mm.oocyte 1 0 6 1 U 9 N u c l e o l i i n a 1 . 1 mm oocyte 1 0 6 1 5 0 L i p i d yolk and PYP systems, 1 . 1 mm oocyte 1 0 6 1 5 1 to 1 5 2 Large PYP, 1 . 1 mm oocyte 1 0 8 1 5 3 A developing GVB, 1 . 1 mm oocyte 1 0 8 1 5 U A large dense PYP, 1 . 1 mm oocyte 1 0 9 1 5 5 Several Golgi and other organelles, 1 . 1 mm oocyte 1 0 9 1 5 6 to 1 5 7 L i n i n g bodies and mitochondria with attached SER 1 0 9 1 5 8 Protein yolk p l a t e l e t s with a dense core, 2 . 0 mm oocyte 1 1 1 1 5 9 Protein yolk p l a t e l e t with a c r y s t a l l i z e d core, 3 . 0 mm oocyte 1 1 1 1 6 0 to 1 6 1 Protein yolk p l a t e l e t s with c r y s t a l l i z e d cores, U.O mm oocytes 1 1 2 ACKNOWLEDGEMENTS I wish to express my appreciation to my supervisor Dr. P. Ford f o r his support and h i s patience throughout the preparation of t h i s work. I am also g r a t e f u l to Dr. C V . Finnegan f o r the use of h i s laboratory and to Dr. A.B. Acton and Dr. P. Larkin f o r ' t h e i r advice'and support. My s p e c i a l thanks i s extended to Mrs. Maureen Douglas f o r her invaluable assistance and advice during the research, preparation and correction of t h i s manuscript. To Mr L.L. Veto I also owe a s p e c i a l debt of thanks f o r h i s assistance and encouragement. My thanks also goes to the captains and crews of the Investigator No. I and the A.P. Knight, the F i s h e r i e s Research vessels used to obtain my specimens. F i n a l l y , I would l i k e to dedicate t h i s work to my wife Shelley, for .her patience, prodding, encouragement and assistance. . INTRODUCTION Yolk synthesis and deposition have been the subject of numerous, investigations i n the past two decades.. Yolk, both p r o t e i n and l i p i d , has been described as a r i s i n g within various animal oocytes under the influence of the mitochondria, Golgi, endoplasmic reticulum, nuclear extrusions, or d i r e c t l y out of the cytoplasm and o c c a s i o n a l l y by a combination of two or more of these methods (Raven, 1 9 6 l ; Nc^rrevang,. 1 9 6 8 ; Beams & Kessel, 1 9 7 3 ) . In a d d i t i o n to these i n t r a o o c y t i c methods, yolk formation may occur e x t r a o o c y t i c a l l y , the precursor materials f o r yolk being synthesized elsewhere and entering the oocyte by micropino-cytos i s with subsequent incorporation into developing yolk bodies (Raven, 1 9 6 l ; Nffcrevang, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ; Kessel, 1 9 7 1 ; Massover, 1 9 7 1 ; . Boyer, 1 9 7 2 ) . Thus the primary d i r e c t i v e of t h i s study was to examine the .development of the oocytes .of the-.dogfish Squalus 'acarithias . u l t r a -s t r u c t u r a l l y and determine the method of v i t e l l o g e n e s i s . A previous study on the morphology of the yolk of the dogfish Mustelus l a e v i s Risso (Grodzinski, 1 9 5 8 ) i n d i c a t e d the presence of an i n t e r n a l p l a t e l e t i n the p r o t e i n yolk sphere with properties more reminiscent of an amphibian p l a t e l e t than a t e l e o s t p l a t e l e t . Thus the u l t r a s t r u c t u r e of the. yolk p l a t e l e t was also examined to determine i f these properties were r e f l e c t e d i n the structure of the various p l a t e l e t s . While considering the p o s s i b i l i t y that yolk material was synthesized elsewhere and transported to the ovary for uptake into the oocyte i t became necessary to examine the f o l l i c u l a r envelope of the oocyte to e s t a b l i s h i t s effectiveness as a b a r r i e r to the passage of yolk m a t e r i a l , as well as any other possible functions. This f o l l i c u l a r envelope or wall was examined i n Squalus acanthias by Lance & C a l l a r d ( 1 9 6 9 ) who found i t to consist of a zona r a d i a t a , a v i t e l l i n e membrane, a single layer of columnar e p i t h e l i u m — t h e granulosa, the theca i n t e r n a and the theca externa. TeWinkel (1972) examined the oocyte f o l l i c l e {% k.O mm) of the smooth dogfish Mustelus canis and described a zona r a d i a t a , a p s e u d o s t r a t i f i e d columnar granulosa, a basement membrane and a theca. Considering these discrepancies i n two dogfish species, the f i r s t point was to e s t a b l i s h the histochemical and u l t r a s t r u c t u r a l composition of t h i s f o l l i c u l a r envelope. The thecal layer has often been described as a connective t i s s u e layer (Lance & C a l l a r d , 196'9Dumont, 1972; Peel & B e l l a i r s , 1972; TeWinkel, 1972) i n the lower vertebrates p o s s i b l y with s t e r o i d a c t i v i t y i n the theca interna (Lance & C a l l a r d , 1969; TeWinkel, 1972) . With these seemingly contradictory f a c t s i n mind the theca was examined to determine i t s .properties and function. The basement membrane, co n s i s t i n g of the basal lamina and i t s underlying.collagenous basal lamella (Nadol ejb\al», 1969), poses an in t e r e s t i n g problem. F i r s t of a l l , the l i t e r a t u r e disagrees on the presence of t h i s complex beneath the f o l l i c u l a r epithelium of two dogfish species, including the species under i n v e s t i g a t i o n where i t was not observed (Lance & C a l l a r d , I969) . However, preliminary l i g h t microscopy f o r t h i s study i n d i c a t e d the presence of a well developed basement lamina and lamel l a . Further, i t was thought that only f i b r o b l a s t s were capable of forming a basement membrane complex but r e c e n t l y , basal laminae have been found beneath e p i t h e l i a l a c k i n g underlying f i b r o b l a s t s (Hay & Revel, 1969; B e r n f i e l d & Wessels, 1970) while other evidence (Hay & Revel, 1969; Beisswenger & Spiro, 1973) has d i r e c t l y implicated e p i t h e l i a l c e l l s i n basal lamina formation. So, in v e s t i g a t i o n s were c a r r i e d out on the basal lamina-lamella complex to determine i t s chemical composition and to determine from which portion of the f o l l i c u l a r envelope i t was formed. The granulosa or f o l l i c u l a r sheath i s a l a y e r of e p i t h e l i a l c e l l s found around animal oocytes that i n Squalus has been implicated i n s t e r o i d synthesis (Lance & C a l l a r d , 1 9 6 9 ) while generally f o l l i c l e c e l l s have "been shown to synthesize yolk materials (Raven, 1 9 6 l ; Davidson, 1 9 6 8 ) f o r the oocyte and to transport extraoocytic yolk precursors i n t o the oocyte (Raven, 1 9 6 l ; Davidson, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ) . The granulosa of Squalus  acanthias was examined to .clarify these possible functions. The v i t e l l i n e membrane was thought to be formed by the oocyte (Raven, . 1 9 6 l ) but i n many recent studies (N^rrevang, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ; Cummings, 1 9 7 2 ; Dumont, 1 9 7 2 ; Sahota, 1 9 7 3 ) the v i t e l l i n e membrane was observed to be formed by the f o l l i c l e c e l l s . In t e l e o s t s , however, there i s evidence f o r •both hypotheses (Chaudhry, 1 9 5 6 ; Yamamoto, 1 9 6 3 ) . The zona r a d i a t a - o f elasmobranchs were described by Balfour ( 1 8 8 5 ) and Wallace ( 1 9 0 3 ) who believed them to be separate membranes i n the f o l l i c u l a r envelope. Recent electron microscopical studies have demonstrated that the zona r a d i a t a i s formed by m i c r o v i l l i a r i s i n g from the egg surface (Schjeide, Wilkins et a l , 1 9 6 3 ; Yamamoto, 1 9 6 3 ; Press, I 9 6 I + ; B e l l a i r s , 1 9 6 5 , 1 9 7 1 ) . Thus, important areas of i n v e s t i g a t i o n are the examination of the v i t e l l i n e membrane to determine i t s chemical composition, i t s r e l a t i o n s h i p to the oocyte r e l a t i v e to the existence of a zona r a d i a t a , and i t s development. Ad d i t i o n a l observations are also reported on the ovarian surface epithelium, the tunica albuginea, the ovarian cortex, the lacunae border c e l l s , and the red blood c e l l s as l i t t l e i f any previous work had been done on these aspects of the spiny dogfish ovary. MATERIALS AND METHODS - EXPERIMENTAL ANIMALS Spiny dogfish, Squalus acanthias, were c o l l e c t e d i n the s t r a i t of Georgia during the months of March 1973, May 1973, August 1971, September 1971 & 1972 and November 1971. Ontogenetically, the ovaries a r i s e i n the d o r s o l a t e r a l l i n i n g of the peritoneum, one on each side of the dorsal mesentery i n the g e n i t a l ridges. The primordial germ c e l l s , generally assumed to a r i s e from yolk sac endoderm migrate into the peritoneum and the g e n i t a l ridges then d i f f e r e n t i a t e into ovarian t i s s u e . The paired.ovaries of immature (93.5 cm ± 5.5 cm; Ketchen, 1972) Squalus acanthias are s o f t , oval bodies each measuring h to 6 cm i n length, 3 to k cm i n width and 1 to 1.5 cm i n thickness. In the mature ovary the presence of 2 to 6 r i p e eggs measuring 2 . 5 to U cm i n diameter increases the o v e r a l l dimensions of the ovary. Morphologically, i t i s a compact ovary s i m i l a r to those found i n many t e l e o s t s as w e l l as i n r e p t i l e s , birds and mammals. PREPARATION FOR HISTOCHEMICAL ANALYSIS The ovaries were removed from the l i v i n g animal and f i x e d i n 10% neutral buffered formalin ( L i l l i e , 195*0 at ambient temperature, dehydrated through a graded ser i e s of ethanols, cleared i n benzene and embedded i n Paraplast (Fisher S c i e n t i f i c Co.). Sections of 7 Vm thickness were cut on an AO Spencer "820" Microtome and mounted on albuminized glass s l i d e s . For the preservation of l i p i d s , the material was embedded i n Cryoform ( i n t e r n a t i o n a l Equipment Co.) and frozen. Sections were cut at l 6 urn on an IEC CTF Microtome-Cryostat, picked up on glass c o v e r s l i p s and a i r d r i e d . HISTOCHEMICAL PROCEDURES Ten histochemical techniques were employed to demonstrate p r o t e i n s , l i p i d s , nucleic acids and carbohydrates. A modified dinitrofluorobenzene (DNFB) technique was used to demonstrate proteins according to the method of Tranzer and Pearse (196k). DNFB i s a general protein r e a c t i o n that complexes with N-terminal cx-amino groups, the phenolic hydroxyl group of tyrosine and the sulphydryl group of cysteine producing the coloured r e a c t i o n product (Sanger, 19^+5; Pearse, 1 9 6 8 ; Chayen et a l , I969). Trichrome p e r i o d i c a c i d - S c h i f f (PAS) s t a i n i n g was used to demonstrate 'mucoproteins, neutral mucopolysaccharides and a c i d o p h i l i c proteins (Hotchkiss, 1 9 ^ 8 ) . The re a c t i o n i s based on the presence of a 1 : 2 g l y c o l group o r . i t s equivalent amino or a l k y l amino d e r i v a t i v e which the p e riodic acid can oxid i z e into two aldehydes with subsequent attach-ment of the recoloured fuchsin of the S c h i f f ' s reagent. Methyl green-pyronin was used to detect RNA (Jordan & Baker, 1 9 5 5 ; C u l l i n g , 1 9 6 3 ) . This i s a competitive s t a i n i n g r e a c t i o n wherein a planar monovalent c a t i o n i c dye such as pyronin or t o l u i d i n e blue w i l l react with nucleic acids having f r e e l y a c c e s s i b l e purine and pyrimidine bases (single stranded RNA), whereas non-planar diva l e n t c a t i o n i c dyes such as methyl green w i l l s t a i n polymerized DNA s e l e c t i v e l y (Brachet, 1 9 5 ^ ; Scott, I 9 6 7 ) . I t should also be noted that p y r o n i n o p h i l i a may also be due to other polymeric acid substances such as a c i d i c muco-polysaccharides (Chayen et a l , 1 9 6 9 ) . Toluidine blue (l% aqueous) was used a f t e r RNase to detect RNA (Brachet, 1 9 H 0 a , 1 9 ^ 2 , 19hh; Pearse, 1 9 6 8 ) and as a c o n t r o l f or hyalur-onidase treatment. A l c i a n blue (AB) was employed to demonstrate a c i d mucopolysaccharides (Steedman, 1 9 5 0 ) as was the di a l y z e d i r o n technique (Hale, I 9 H 6 ) . A l c i a n blue i s a phthalocyanin d e r i v a t i v e that stains the uronic groups of a c i d i c and sulphated mucopolysaccharides at an a c i d pH. A l c i a n blue, a c a t i o n i c dye, i s thought to form r e v e r s i b l e e l e c t r o s t a t i c bonds between the c a t i o n i c dye molecules and the anionic s i t e s on the glycosamino-glycans (mucopolysaccharides). Hale's also stains a c i d i c mucopolysacch-arides at a low pH, but t h i s i s due to the a f f i n i t y of free a c i d i c groups for c o l l o i d a l F e + + + at a pH of 1 . 8 - 2 . 0 . Gomori's aldehyde fuchsin as modified by Halmi and Davies was used to 'stain'for sulphated mucopolysaccharides (Pearse, 1 9 6 8 ) . The c a t i o n i c t h i a z i n e dye Azure A (pH 1 . 5 ) (McConnachie & Ford, 1 9 6 6 ; Pearse, 1 9 6 8 ; Kvist & Finnegan, 1 9 & 9 ) was also used to demonstrate sulphated mucopolysaccharides. Cationic dye binding i s thought to be e l e c t r o s t a t i c i n nature and i t therefore depends upon the pH of the st a i n i n g medium and the pK value of the anionic groups involved. Lowering the pH (below 2 . 0 ) decreases the d i s s o c i a t i o n of carboxyl groups and therefore at pH 1 . 5 , only the sulphated groups are ion i z e d and can bind the dye (Szirmai, 1 9 6 3 ) . The copper phthalocyanin technique of Kluver & Barrera ( 1 9 5 3 ) was used to s t a i n phospholipids. The dye i s an amine s a l t of a sulphonated CuPC that may have 1-k substituted sulphonyl groups per molecule and one of a number of bases. The mechanism of s t a i n i n g i s thought.to. be an ion association r e a c t i o n that l i n k s the base, (CuPC)SOoH, to the free oxygen 7 atom of the phospholipid phosphate (Pearse, 1968). To demonstrate the presence of l i p i d i n cryostat sections, Sudan black B was used (McManus, 19^6) . The s t a i n i s used i n a 70% ethanolic so l u t i o n and the s p e c i f i c i t y a r i s e s from the increased s o l u b i l i t y of the colourant i n the t i s s u e l i p i d s . Several enzyme methods were employed to increase the s p e c i f i c i t y of some of the st a i n i n g reactions. Malt, diastase ( N u t r i t i o n a l Biochemicals : Corp.) was used to remove glycogen p r i o r to staining with PAS. Ribo-nuclease (Sigma Chemical Corp.) was used before the methyl green-pyronin and t o l u i d i n e blue procedures. T e s t i c u l a r hyaluronidase (Sigma Chemical Corp.) was used to remove hyaluronic acid, chondroitin sulphate A and chondroitin.sulphate C; t o l u i d i n e blue and Azure A staining.followed. Methylation and methylation-demethylation (saponification) blocking and unblocking reactions ( C u l l i n g , 1963) were used i n conjunction with AF and Azure A s t a i n i n g . Methylation blocks anionic groups by forming methyl esters on carboxyl groups and hydrolyzing sulphate esters. Demethylation unblocks carboxyl groups only by hydrolyzing the methyl esters ( C u l l i n g , 1963) . The preceding techniques are outlined i n Appendix A. . PREPARATION FOR ELECTRON MICROSCOPY Ovaries of the dogfish Squalus acanthias were bathed i n Karnovsky's (1965) glutaraldehyde-paraformaldehyde f i x a t i v e , dissected out and placed i n fresh f i x a t i v e . Individual oocytes or c l u s t e r s of small oocytes were selected and placed i n fresh f i x a t i v e f o r 1 -2 hours at ambient temperature. The f i x a t i v e was buffered to a pH of 7 .2 with 0 .2 M cacodylate b u f f e r (pH 7 .^0. The samples were washed twice i n buffer and stored. Post-f i x a t i o n i n OsO^ (l% i n 0 .2 M Cacodylate buffer,. pH 7 .2 ) v a r i e d from 1 to 2 hours depending on the th i c k n e s s of the specimen. Subsequently, the oocytes were washed, dehydrated, embedded i n Spurr's (1969) medium (P o l y s c i e n c e s , Inc.) and sectioned at 600 X ( s i l v e r - g r e y ) on a P o r t e r -Blum MT-2 ultramicrotome. For l i g h t microscopy, t h i c k ( l ym) s e c t i o n s were placed on gl a s s s l i d e s and s t a i n e d w i t h 1% t o l u i d i n e blue i n 1% sodium borate w h i l e f o r e l e c t r o n microscopy, t h i n s e c t i o n s c o l l e c t e d on carbon coated g r i d s were s t a i n e d f o r 1 hour i n methanolic u r a n y l acetate and 1 hour i n Reynolds l e a d c i t r a t e (1963). Thin s e c t i o n s were subsequently examined w i t h a H i t a c h i HU-7S e l e c t r o n microscope. RESULTS THE OVARY The ovary of Squalus acanthias can "be divided i n t o four main regions: the medulla, an inner stroma of connective t i s s u e with numerous blood vessels; the cortex, a broad p e r i p h e r a l l a y e r containing the ova; the tunica albuginea, a fibrous connective t i s s u e l a y e r surrounding the cortex; and the germinal epithelium, the outer columnar epithelium of the ovary (Figures 7 , 8 , 10 , 1 6 ) . An important feature to note, however, i s that developing ova are found only on the dorsal surface of the ovary. This coincides with the l o c a t i o n of the ostium tubae, the common opening to the oviducts which i s found on the dorsal aspect of-the fused oviducts. The developing ova are e n c i r c l e d by four d i s t i n c t l a y e r s . These 'layers are the-non-cellular v i t e l l i n e membrane (-VM); the granulosa or f o l l i c u l a r c e l l l a y e r ; the f o l l i c u l a r basement membrane (FBM); and the theca. The FBM i s subdivided i n t o an outer (basal lamella) and an inner, (basal lamina) component. HISTOCHEMICAL ANALYSIS  PROTEIN The DNFB re a c t i o n (Figures 1 , 2) shows the ooplasm to be proteinaceous while the clear v e s i c l e s i n the ooplasm represent l i p i d l o s t during preparation. The VM i s proteinaceous while the f o l l i c l e and t h e c a l c e l l s show only a small amount of pr o t e i n . The f o l l i c l e basement membrane (basal lamina) stains moderately. Within the cortex there would appear to be two types of c e l l , one containing dark red granules and s t a i n i n g more intensely than a second l i g h t e r c e l l type lacking granules. The DNFB sta i n i n g of a O.h mm oocyte. Large arrowheads indicate cytoplasmic bridges anchoring the oocyte (0) which i s surrounded by numerous lacunae (L). Small arrowheads i n d i c a t e the basement lamina while the arrow points to n u c l e o l i i n the oocyte nucleus (N). Other symbols: S = stroma. 100 ym bar. DNFB st a i n i n g of a 0 .8 mm oocyte. Arrowheads correspond to the previous f i g u r e . Note the reac t i o n of the v i t e l l i n e membrane (VM) and the mitochondrial mass (M). Numerous l i p i d droplets (Li) are present as c l e a r v e s i c l e s i n the ooplasm. 100 ym bar. VM ^ L I • m 2 cytoplasm of the RBC stains darkly red and contains a few c l e a r v e s i c l e s possibly l i p i d i n nature. The tunica albuginea also gives a strong p o s i t i v e r eaction, here demonstrating collagen, as a r e s u l t of the complete reduction provided by titanous c h l o r i d e rather than that of sodium hydrosulphite or stannous ch l o r i d e (Tranzer & Pearse, 1 9 6 U ) . MUCOPROTEINS & NEUTRAL MUCOPOLYSACCHARIDES Figures 3 , h and 5 are representative of the Trichrome-PAS technique, a method which w i l l give blue-black n u c l e i , yellow RBC's and a c i d o p h i l i c proteins i n addition to the deep red re a c t i o n of mucoproteins and neutral mucopolysaccharides. Once again the ooplasm displays i t s proteinaceous and l i p i d nature. The nucleus (Figure 5 ) contains several n u c l e o l i . The v i t e l l i n e membrane stains i n t e n s e l y p o s i t i v e demonstrating i t s muco--pro.tein .or neutral .mucopolysaccharide nature. The cytoplasm of the f o l l i c l e c e l l s varies i n i t s response f o r a c i d o p h i l i c proteins from negative to p o s i t i v e while the cytoplasm of the t h e c a l c e l l s does not respond to the Trichrome-PAS at a l l . The inner component of the FBM (the basement lamina) i s s l i g h t l y pink, p o s i t i v e to PAS, a' r e a c t i o n that may i n d i c a t e the presence of a s i a l i c acid ( C u l l i n g , 1 9 6 3 ; White, Handler & Smith, 1 9 6 8 ) . The cortex again displays two types of c e l l . The f i r s t type has a deeply indented nucleus and stains very i n t e n s e l y f o r a c i d o p h i l i c p r o t e i n s . The second type has a round nucleus and stains l e s s i n t e n s e l y f o r the presence of a c i d o p h i l i c proteins. The f u l l methol (Trichrome-PAS) i s a s t a i n that i s also s p e c i f i c f o r RBC's and they s t a i n i n t e n s e l y f o r a c i d o p h i l i c proteins. The tunica albuginea (Figure 7 ) gives a s l i g h t l y p o s i t i v e a c i d o p h i l i c r e a c t i o n . The tunica can also be seen to send projections into the cortex. Another i n t e r e s t i n g feature i s the presence of PAS PAS-trichrome s t a i n i n g of a 0.8 mm oocyte.. Note the rea c t i o n of the VM and the basement lamina (small arrowheads) f o r neutral MPS. The mitochondrial mass (M) responds strongly f o r a c i d o p h i l i c proteins. L i p i d (Li) droplets are s t i l l noted i n the ooplasm (o). 100 urn bar. PAS-trichrome s t a i n i n g of a 0.5 mm oocyte. The r e a c t i o n of the basement lamina (BL) i s c l e a r l y i n d i c a t e d as i s that of the VM. Note l i g h t c e l l s (large arrowheads) and dark c e l l s (arrow) i n the stroma. Other symbols: FC = f o l l i c l e c e l l s , T = theca. 100 ym bar. 12A PAS-trichromestaining of a 0.8 to 1.0 mm oocyte. This i s a second technique used as a c o n t r o l f or the dichrome PAS method. Note n u c l e o l i (small arrowheads), nucleated red blood c e l l s (arrow) and a cytoplasmic bridge (large arrowhead). 100 ym bar. PAS-dichrome, diastase c o n t r o l . Not c l e a r l y indicated here i s a s l i g h t p o s i t i v e PAS r e a c t i o n i n the ooplasm and i n the cytoplasm of the stromal (S) and thecal. (T) c e l l s . The small arrowhead indicates the basement lamina while the arrow points to the nucleus of a lacuna border c e l l . 100 urn bar. PAS-dichrome diastase. The ooplasmic and cytoplasmic PAS reactions of the control are removed with diastase treatment. The r e a c t i v i t y of the VM and the BL i s ' undiminished. Note the surface epithelium (SE) of the ovary and i t s underlying tunica albuginea (TA). 100 ym bar. 13A p o s i t i v e material located i n t e r c e l l u l a r l y within the cortex. Frequently a " f l o c c u l e n t " material that i s s l i g h t l y PAS p o s i t i v e can be observed i n the lacunae external to the theca. The Dichrome technique, Figure 6, o f f e r s a few features obscured by . the f u l l Trichrome method. The ooplasm, the'cytoplasm of the stromal and thecal c e l l s of the co n t r o l a l l show a s l i g h t p o s i t i v e PAS r e a c t i o n . Figure 7 i l l u s t r a t e s the e f f e c t of malt diastase on Dichrome PAS s t a i n i n g . Here, the ooplasm and the stromal cytoplasm do not show a p o s i t i v e r e a c t i o n to PAS. The i n t e n s i t y of the s t a i n i g of the VM and the FBM i s only s l i g h t l y reduced. This i s an a r t i f a c t and i s not i n d i c a t i v e of the presence of glycogen i n these membranes. RIBONUCLEIC ACID :The methyl green-pyronin technique, Figures 8 and 9, was used to demonstrate the presence of RNA. The ooplasm i s pink (proteinaceous) and vacuolated i n d i c a t i n g the l i p i d l o s t during preparation. The n u c l e o l i s t a i n i ntensely red. The VM and the outer component of the FBM (basal lamina) stains b r i g h t l y orange. The n u c l e i of the. f o l l i c l e c e l l s and a l l other c e l l s s t a i n with methyl green i n d i c a t i n g the presence of DNA. The cytoplasm of the f o l l i c l e c e l l s e x h i b i t s a moderate degree of p y roninophilia. The cytoplasm of the stroma c e l l s would seem to respond d i f f e r e n t i a l l y with the dark c e l l s having the greater degree of pyroninophilia. The c e l l s of the theca are only s l i g h t l y p y r o n i n o p h i l i c . The tunica i s either negative or only s l i g h t l y p y r o n i n o p h i l i c The surface epithelium of the ovary i s covered by a coating of m a t e r i a l that i s also pyroninophilic (Figure 8 ) . Pyroninophilia by i t s e l f i s an i n d i c a t o r of the presence of RNA 15 8. Methyl Green-Pyronin s t a i n i n g of an oocyte at the surface epithelium (SE). There i s a p y r o n i n o p h i l i c response i n the f o l l i c l e c e l l s , the dark c e l l s of the stroma, and i n and on top of the surface e p i t h e l i a l c e l l s . There i s a p o s i t i v e r e a c t i o n i n the basement lamina (large arrowheads) and a negative r e a c t i o n . i n the v i t e l l i n e membrane. The arrows i n d i c a t e the i n t e r c e l l u l a r canals of the surface epithelium. 100 ym bar. 9 . Methyl Green-Pyronin s t a i n i n g of a 0 .8 mm oocyte demonstrating the p o s i t i v e r e a c t i o n of the n u c l e o l i and a f a i n t response i n the chromosomes (small arrow-heads). Other symbols: BL = basement lamina, FC = f o l l i c l e c e l l s . 100 urn bar. but only material s t a i n i g blue with t o l u i d i n e blue or red with pyronin and removable by treatment with ribonuclease i s RNA. Figures 1 0 and 1 1 demonstrate the t o l u i d i n e blue r e a c t i o n f or nucleic acids. The use of 1% aqueous t o l u i d i n e blue a f t e r RNase i s an a l t e r n a t i v e to Brachet's ( l 9 U 0 a , 1 9 ^ 2 , 1 9 U U ) technique with methyl green-pyronin. Toluidine blue w i l l s t a i n metachromatically when the a c i d i c substances are present as long polymeric molecules with t h e i r a c i d i c groups c l o s e l y packed as i n a c i d mucopolysaccharides (Sylven, 1 9 5*0 . In these f i g u r e s , the ooplasm i s s l i g h t l y orthochromatic although i n oocytes larger than 1 . 0 mm the reaction i s negative. The oocyte nucleus shows only a f a i n t trace of orthochromasia i n keeping with i t s s i z e and d i s t r i b u t i o n of nuclear m a t e r i a l . The n u c l e o l i , however, are deeply orthochromatic. The VM i s negative, e x h i b i t i n g neither orthochromasia nor metachromasia. The basement lamina-.exhibits ••-marked metachromasia while the basement lamella i s negative. The f o l l i c l e c e l l and thecal c e l l n u c l e i react orthochromatically as do the stromal c e l l n u c l e i and e p i t h e l i a l n u c l e i . However, the former with l e s s condensed chromatin do not s t a i n as darkly as the l a t t e r . The cytoplasm of the f o l l i c l e c e l l s reacts orthorchromatically and has the appearance of a f i n e homogeneous network of fibrous m a t e r i a l . The same can be s a i d of the cytoplasm of the thecal c e l l s . The cytoplasm of the stromal c e l l s i s also orthochromatic but the r e a c t i o n i s s l i g h t l y darker and i t s texture coarser. The t u n i c a albuginea (not v i s i b l e ) e x h i b i t s a very f a i n t metachromasia. This f a i n t metachromasia can also be seen at the border of the lacunae. The RBC n u c l e i are markedly orthochromatic while the cytoplasm of these c e l l s i s non-reactive to the s t a i n . Ribonuclease treatment followed by methyl green-pyronin almost 17 10. Toluidine blue for nucleic acids and a c i d i c muco-polysaccharides. This section c l e a r l y i n d i c a t e s the orthochromatic (blue) r e a c t i o n of the nucleic acids i n the n u c l e i of a l l c e l l s and i n the cytoplasm of the f o l l i c l e , stromal (S), and surface e p i t h e l i a l (SE) c e l l s . The metachromatic response (purple) of the basement lamina (BL) indicates a c i d mucopolysaccharides. 100 um bar. 11. Toluidine blue for nucleic acids and a c i d i c mucopoly-saccharides. This section more c l e a r l y i l l u s t r a t e s the r e a c t i o n of the f o l l i c l e , t h e c a l , and stromal c e l l s f o r nucleic acids. .100 .urn. .bar,. 12. Toluidine blue-RNase. A f t e r RNase treatment, a l l cytoplasmic (non-nuclear) orthochromasia i s removed. The basement lamina r e t a i n s i t s metachromasia and therefore lacks RNA. 100 um bar. 17A completely removes the p y r o n i n o p h i l i a of the n u c l e o l i . The ooplasm i s negative while the s t a i n i n g of the f o l l i c l e c e l l s i s reduced. The base-ment lamina has l o s t a considerable amount of p y r o n i n o p h i l i a while the stromal c e l l s are now negative. A l l other elements are unchanged. Hot t r i c h l o r o a c e t i c a c i d e x traction (Humanson, 1972) was also used. It produces e s s e n t i a l l y the same r e s u l t s (not shown) as RNase except that i t i s not as s p e c i f i c . TCA also reduces the methyl green s t a i n i n g of the n u c l e i . In contrast to these r e s u l t s , Figure 12 shows an RNase treated section stained with t o l u i d i n e blue. Here the n u c l e i and only the n u c l e i stand out against a blank background. A l l other cytoplasmic and ooplasmic stainin g has been removed. The f o l l i c u l a r basement lamina r e t a i n s i t s • metachromasia undiminished. Hot TCA extraction (not shown) as before 'is l e s s s p e c i f i c . Here, a l l 'the 'Elements "present 'exhibit a reduction i n s t a i n i n g . Most noticeable,however, i s the f i n e trace of cytoplasmic orthochromasia s t i l l remaining i n most of the stromal c e l l s . The n u c l e o l i of the oocyte also show a reduction i n s t a i n i n t e n s i t y . ACIDIC & SULPHATED MUCOPOLYSACCHARIDES The a l c i a n blue r e a c t i o n can be seen i n Figures 13a, b, c. The ooplasm i s negative for the presence of a c i d mucopolysaccharides (AMPS) having only a f a i n t b l u i s h background c o l o r . The VM i s also negative. The FBM i s p o s i t i v e for AMPS and stains b r i g h t blue-green. . Occasionally a p o s i t i v e reaction can also be seen between the c e l l s of the theca. The tunica albuginea i s the only other element to show a p o s i t i v e reaction to a l c i a n blue and i t i s s l i g h t l y l e s s intense than the r e a c t i o n of the FBM. The surface epithelium also appears to be covered i n a substance A l c i a n blue r e a c t i v i t y of several 0 .6 to 0 .8 mm oocytes. A metachromatic (blue-green) response demonstrates the presence of a c i d i c and sulphated mucopolysaccharides i n the basement lamina (arrows, Figures a and b ) , the tunica (TA, Figure c ) , and p o s s i b l y i n the theca (T, Figure a). Small arrowheads (Figure b) i n d i c a t e lampbrush chromosomes l y i n g between numerous n u c l e o l i i n the nucleus, (w). 100 ym bars. 19A that demonstrates a l c i a n o p h i l i a . The Hale's c o l l o i d a l i r o n r e a c t i o n i s i n d i c a t e d "by Figure 1 ^ . Here, the ooplasm (except for the non-staining l i p i d v e s i c l e s ) , the f o l l i c l e c e l l s , the t h e c a l c e l l s and the stromal c e l l s a l l react with t h i s t a i n . This, however, i s regarded as "background" s t a i n i n g . The most important features to note here are the negative VM and the FBM that gives a very intense r e a c t i o n . C l e a r l y the FBM has an inner and an outer component, the former being negative or only s l i g h t l y p o s i t i v e while the l a t t e r i s i n t e n s e l y p o s i t i v e . Figures 1 5 b and l 6 b i n d i c a t e the e f f e c t of t e s t i c u l a r hyaluronidase i n s a l i n e on the s t a i n i n g r e a c t i o n of t o l u i d i n e blue. There i s a s l i g h t reduction i n the orthochromasia of the stroma c e l l s that i s unexpected but i t i s obviously some a c i d i c molecule that i s hyaluronidaae l a b i l e . But, the most important aspect of t h i s method involves the FBM. Hyalur-onidase treatment almost completely removes the metachromasia of the basement lamina. According to Spicer and Meyer ( i 9 6 0 ) , aldehyde fuchsin, Figures IT & ' l 8 , stains sulphated mucins p r e f e r e n t i a l l y . The s t a i n i n g r e a c t i o n i a combination of the carboxyl groups with an intermediate metastable species of the a c t i v e dye molecule p a r a r o s a n i l i n e . The major r e a c t i o n product i s found i n the basal lamina. Secondary reactions are a l s o indicated i n the t u n i c a and i n the granules of the dark c e l l s . Following methylation, staining was t o t a l l y blocked i n a l l areas except for numerous short f i b r e s i n the t u n i c a and the basement lamina. It must be noted that the s t a i n i n g r e a c t i o n i n the basement lamina was markedly reduced.. Saponification or demethylation, Figure 1 9 , restored moderate Hale's C o l l o i d a l Iron r e a c t i o n i n a 1 . 0 to 2 . 0 mm oocyte. The v i t e l l i n e membrane (VM) and the basement lamina (large arrowheads) do not respond to t h i s r e a c t i o n while the basement lamella (small arrowheads) c l e a r l y does respond. 1 0 0 ym bar. 21A 22 1 5 a . Toluidine blue c o n t r o l f o r hyaluronidase. Water-mounted. l 6 a . Note the metachromatic (purple) response of the basement lamina (arrows). Other symbols: AC = abdominal c a v i t y , TA = tu n i c a albuginea. 1 0 0 ym bars. 1 5 b . Toluidine blue-hyaluronidase. Water-mounted. l 6 b . Hyaluronidase treatment c l e a r l y removes the metachromatic response (arrows) of the basement lamina as w e l l as diminishing the orthochromasia of a l l other components. Other symbols: L = lacuna, N = nucleus, 0 = oocyte, SE = surface epithelium. 1 0 0 ym bars. 22A 23 1 7 . Aldehyde fuchsin r e a c t i v i t y of a 1 . 0 to 2.0 mm oocyte. Strong response noted i n the basement lamina (large arrowhead) and the granules of dark c e l l s . Small arrow-heads i n d i c a t e n u c l e o l i i n the oocyte nucleus (N) while the arrow points to a lampbrush chromosome. 1 0 0 ym bar. 1 8 . Aldehyde fuchsin response of the t u n i c a albuginea. The e l a s t i c f i b r e s (E) s t a i n deeply with aldehyde fuchsin while the collagen (C) f i b r i l s react l e s s intensely. 100•um bar. 1 9 . Following methylation and demethylation (MDM), moderate aldehyde fuchsin s t a i n i n g i s restored to the basement lamina (arrow).and to the dark c e l l granules (not c l e a r l y shown). The e l a s t i c f i b r e s of the tunica (inset) also r e t a i n moderate s t a i n i n g . 1 0 0 ym bar. 23A staining to the basement lamina and the granules of the dark c e l l s . Azure A i s a c a t i o n i c t h i a z i n e dye s i m i l a r to t o l u i d i n e blue that i s used to demonstrate sulphated MPS when used at the pH of 1 .5 (Szirmai, . 1963; McConnachie & Ford, 1966; Kvist & Finnegah, 1970). Dye binding i s e l e c t r o s t a t i c and as the pH i s lowered below pH 2.0 only the sulphated groups are ionized and can bind dye (Szirmai, 19&3; K v i s t , 1968) . Azure A a c t i v i t y i s i n d i c a t e d by Figure 20. I t i s evident that at t h i s pH only the basement lamina i s metachromatic. The metachromatic a c t i v i t y of t h i s membrane also v a r i e s , being almost non-existent i n oocytes under 1 mm and increasing i n i n t e n s i t y as the oocyte enlarges. Methylation-demethylation, which blocks both carboxyl'and sulphate groups and subsequently unblocks only the COOH groups, a f f e c t s Azure A staini n g as shown by Figure 21. I t i s obvious that both the orthochromasi "•and "the metachromasia are only s l i g h t l y reduced. T e s t i c u l a r hyaluronidase when applied to Azure A s t a i n i n g , Figure 22, has v i r t u a l l y no e f f e c t on th metachromasia of the basement lamina while the orthochromasia of a l l other elements i s only s l i g h t l y reduced. PHOSPHOLIPIDS & LIPIDS The copper phthalocyanin (CuPC) method of Kluver & Barrera (1953) was used, Figures 23a, b, to demonstrate the presence of phospholipids. The reaction of the ooplasm w i l l vary from negative to only s l i g h t l y p o s i t i v e depending upon the length of d i f f e r e n t i a t i o n i n 0.05% aqueous l i t h i u m carbonate. This i n d i c a t e s that the amount of phospholipid present at t h i s stage i s small. The mitochondrial cloud, however, gives a strong p o s i t i v e reaction (not shown). The n u c l e i of a l l c e l l s s t a i n with neutral.red as do the n u c l e o l i of the germinal v e s i c l e . 2 0 . Azure A r e a c t i v i t y of a 0.8 mm oocyte c l e a r l y i n d i c a t e s the presence of sulphated mucopolysaccharides i n the . )( "basement jaamina (FBM). Arrow ind i c a t e s a m i t o t i c f i g u r e while the small arrowheads point to a l i g h t c e l l ( l e f t ) and a dark c e l l ( r i g h t ) . 1 0 0 ym bar. 2 1 . Azure A-MDM treatment in d i c a t e s the presence of sulphated MPS i n the basement lamina (arrow) by only f a i n t l y r e s t o r i n g the metachromasia. Several n u c l e o l i "can be'seen i n the nucleus (N). 1 0 0 ym bar. 2 2 . Azure A-Hyaluronidase treatment has l i t t l e e f f e c t on the metachromasia of the basement lamina (arrow). 1 0 0 ym bar. 26 23a, b. Copper phthalocyanin treatment f o r phospholipids reveals strong p o s i t i v e reactions i n the cytoplasm of the l i g h t (LC) and dark (D) c e l l s with a moderate response i n the thecal c e l l s and the ooplasm. Other symbols: FC = f o l l i c l e c e l l s , L = lacuna, 0 = oocyte, T = theca, VM = v i t e l l i n e membrane, arrow = basement lamina, large arrowhead = nucleated red blood c e l l . 100 ym bar. 26A The chromatin of the germinal v e s i c l e i s f i n e l y dispersed at t h i s stage and thus does not s t a i n with neutral red. The VM i s negative to CuPC and only very s l i g h t l y p o s i t i v e to neutral red. The basement lamina, • on the other hand, gives a p o s i t i v e neutral red rea c t i o n while the basal lamella i s negative to both CuPC and neutral red. The presence of phospholipid p o s i t i v e material i n the granulosa c e l l s i s d i f f i c u l t to ascertain. There seems to be a s l i g h t r e a c t i o n , but i t i s by no means as intense as the reaction o f the stroma c e l l s . S i m i l a r l y , the r e a c t i o n of the thecal c e l l s i s stronger than that of the f o l l i c l e c e l l s but not as strong as that of the stromal c e l l s . In the stroma, the cytoplasm of the dark c e l l s gives a strong p o s i t i v e r e a c t i o n f o r phospholipid. The l i g h t c e l l s , on the other hand, give a negative or only s l i g h t l y p o s i t i v e r e a c t i o n . The tun i c a albuginea also stains f o r phospholipid. The red blood c e l l s are the most in t e n s e l y r e a c t i v e -of a l l the elements found i n these sections. The haemoglobin gives an i n t e n s e l y p o s i t i v e r e a c t i o n fo r phospholipids while the n u c l e i s t a i n b r i l l i a n t l y red. Sudan black B, Figures 2k, 25 , has a high a f f i n i t y f o r phospholipids (Baker, 1 ° M , 1958; McManus, 19k6; Pearse, 1968) and i s used i n a 10% ethanolic s o l u t i o n . The l o c a l i z a t i o n of t h e ' s t a i n i n cryostat sections i s very i n t e r e s t i n g . The basement lamina i s homogeneously grey while the VM i s negative. The pertinent feature i s the extreme l o c a l i z a t i o n at the a p i c a l portion of the f o l l i c l e c e l l s and the appearance of small l i p i d droplets t r a v e r s i n g the v i t e l l i n e membrane. There appears to be a l o c a l i z a t i o n of l i p i d along the oocyte-VM border. The c e n t r a l region of the oocyte shows the dense accumulation of l i p i d at t h i s stage (approx. 3.0 mm i n diameter). Sudan Black B. Cryostat sections of a 3.0 to h.O mm oocyte. The v i t e l l i n e membrane i s a broad c l e a r band dotted with small l i p i d d roplets. The a p i c a l p o r t i o n of the f o l l i c l e c e l l s (large arrowhead) i s packed with l i p i d . The basement lamina i s i n d i c a t e d by a blunt arrow. Other symbols: 0 = oocyte, S = stroma. 100 ym bar. Sudan Black B. Cryostat sections of a 3.0 to h.O mm oocyte. The ooplasm indicates the presence of large quantities of l i p i d (black) d r o p l e t s . 100 ym bar. 29 Table IA, IB, IC: A Table of the histochemical r e s u l t s obtained i n t h i s study. Key to the Table: negative response +/- weak to negative response +/2 weak response + moderate response ++ good response +++ strong response ++++ intense response d droplets i n the VM e e l a s t i c f i b r e s m metachromasia o orthochromasia on orthochromatic nucleus onu orthochromatic nucleolus or orange r red y yellow (protein) TABLE I-A HISTOCHEMICAL RESULTS TUNICA DNFB PAS MG-P RNase TCA TOL BLUE RNase +++ ++y +/- +/- ++ + m ... TCA STROMA LIGHT DARK THECA + +++ ++ y +++ y ++ + /-• +/2 +/2 +/2 + + ++ o +/2 o +/2 o +/2 o FBM OUTER INNER ++ +/-++ +++ or +++ +++ m +++ m +++ m FC' +/- y ++ +/2 ++ o VM +++ ++++ +/- ++ OOCYTE +++ ++ y ++/+ + o RBC ++++ ++++ y ++ on NUCLEOLI ++ ++++ ++ o + o + O' MITO. CLOUD +++ +++ y TABLE I-B HISTOCHEMICAL RESULTS ALCIAN BLUE HALE'S HYALUR o ALDEHYDE CONTROL HYALUR. FUCHSIN METH. TUNICA STROMA LIGHT DARK THECA ++ +/-++ o ++ o ++ o + o + o + o +++ e ++ +++ + e ++ FBM OUTER INNER FC VM OOCYTE RBC NUCLEOLI MITO. CLOUD + ++++ ++++ +/- +++ m .++ o + o + m + o + o +/2 o + o + o +/2 o +++ ++++ ++ ++ METH. & SAP. + e ++ AZURE METH. A pH 1.5 & SAP. TUNICA - ... STROMA LIGHT DARK + onu +/2 o THECA + o +/2 o FBM OUTER INNER +/2 *-++ m + m FC + o +/2 o VM -OOCYTE RBC • • • • • • NUCLEOLI + o + o MITO. CLOUD TABLE I-C HISTOCHEMICAL RESULTS HYALUR. Cu PC + + +/2 o +++ +/2 o + + m ++ r +/2 o + + ++++ + o •++ r ++ SUDAN BLACK B • • • • • • ++ • • • + +++ -/++ a +++ ELECTRON MICROSCOPICAL OBSERVATIONS  THE OVARIAN EPITHELIUM The surface of the ovary i s p i t t e d by shallow invaginations that lead to the larger oocytes (Figures 8 , 16) as well as to the ovulated f o l l i c l e s and i s covered by a "germinal epithelium". This term p e r s i s t s although the evidence favours the extragonadal o r i g i n of the primordial germ c e l l s (Bloom & Fawcet.t, 1968; Hoar, 1970) . This epithelium, possibly cuboidal i n l i v i n g t i s s u e , i s columnar i n f i x e d material and i s devoid of c i l i a contrary to the observations of TeWinkel (Mustelus  canis, 1972) and Metten (Scylliorhynus c a n i c u l a , 19^1) . The plasma membrane i s thrown into shallow folds along the surface of each c e l l while numerous c l e a r and f i l l e d v e s i c l e s (180-250 ym X 360-UOO ym) are observed j u s t beneath the membrane (Figures 26 , 27 , 2 8 , 29 , 30, 31, 32). .^-Frequently, both types . of v e s i c l e can.be seen .fused with the plasma membrane exposing t h e i r contents to the abdominal c a v i t y . The material within the f i l l e d v e s i c l e s i s filamentous. The cytoplasm of these c e l l s i s f i l l e d with numerous small v e s i c l e s (iho ym), micro-filaments (36 to 1+5 A i n diameter), granular and agranular endoplasmic reticulum, often seen on the same cisternae, mitochondria and Golgi bodies. The most abundant organelle i s the free ribosome (Figures 26 to 32). The nu c l e i are c o n s t r i c t e d i n t o a varying number of lobes with only a narrow band of condensed chromatin present that i s applied to the inner l e a f l e t of the nuclear membrane. The nucleolus has a f i b r i l l a r core and an extensive granular cortex. The outer l e a f l e t of the nuclear membrane, which i s occasionally studded with ribosomes and blebs as i f gi v i n g o f f v e s i c l e s , i s separated from the inner membrane by a wide (100 to 150 %.) perinuclear c i s t e r n a . Nuclear pores are seen infre q u e n t l y Surface epithelium of the dogfish ovary. The dominant features of t h i s region are the large mucus f i l l e d channels (ICC) between the columnar c e l l s and the large n u c l e i of the e p i t h e l i a l c e l l s . The c e l l s are f i l l e d with microfilaments (large arrowhead), Golgi bodies ( G ) , mitochondria (M) and ribosomes. In t h i s f i g u r e , several of the membrane to membrane re l a t i o n s h i p s are i n d i c a t e d , p a r t i c u l a r l y the convoluted plasma membrane (concentric membranes) i n the upper right.. Other symbols: NU = nucleolus, P = plug of d e t r i t u s . 1 um bar. 31A A panorama of the surface epithelium of the dogfish ovary from the collagen (C) of the t u n i c a albuginea to the a p i c a l plasma membrane. The apices of four e p i t h e l i a l c e l l s can be seen separated by three membrane contact surfaces each of which ends i n a . t i g h t junction (Tj).and which may or may not. have desmosomes along i t s length. At the very apex of each c e l l , numerous v e s i c l e s can be seen (V, arrows) that often open into the abdominal c a v i t y . Also found i n the apex of each c e l l are multitudes of ribosomes (R), E.R. and Golgi bodies (G). The nucleus i s highly c o n s t r i c t e d and has a p e r i p h e r a l band of condensed chromatin. The basal aspects of these c e l l s r e s t on a c l e a r l y defined basement membrane or lamina (BL) with numerous p i n o c y t o t i c v e s i c l e s (PV) found i n the cytoplasm and opening into the space between the c e l l and the B.L. • The configuration of the i n t e r c e l l u l a r channel (ICS) i s shown as i s the c o n t i n u i t y of t h i s channel with the c l e a r zone between the B.L. and the c e l l . 1 um bar. 32A High magnification of the apex of three surface e p i t h e l i a l c e l l s and the i n t e r c e l l u l a r channel (ICC). Two t i g h t junctions (TJ) and several desmosomes (D) are indicated as are the numerous surface v e s i c l e s (V, arrows). Other symbols: AC = abdominal c a v i t y , CPM = convoluted plasma membrane, G = G o l g i , M = mitochondria, N = nucleus, P = pseudopodia, R = ribosomes. 1 pm bar. 3 3 A 34 2 9 . Pligher magnification of the apex of two surface e p i t h e l i a l c e l l s . Of p a r t i c u l a r i n t e r e s t are the t i g h t junctions (TJ) and desmosomes (D) found holding the two c e l l s together. Also evident are the many-surface v e s i c l e s (arrow) f i l l e d with a f l o c c u l e n t m a t e r i a l , the microfilaments (MF), mitochondria (M) and small, smooth v e s i c l e s (SV). Other symbols: AC = abdominal cav i t y , N = nucleus. 1 ym bar. 30. Higher magnification of the apex of two surface e p i t h e l i a l c e l l s . A w e l l defined t i g h t j unction (TJ) can be seen as w e l l as a region of convoluted plasma membrane (CPM) between the two c e l l s . . Numerous ribosomes (R) f i l l the cytoplasm. The arrow in d i c a t e s one of many large surface v e s i c l e s while the arrowhead points to a fibrous material on the exposed surface of the c e l l . 1 ym bar. 34A 31. An a p i c a l protuberance of a surface e p i t h e l i a l c e l l f i l l e d with surface v e s i c l e s some of which are opening (arrows) into the abdominal c a v i t y (AC). Other symbols: large arrowhead = microfilaments, F = folds of the plasma membrane. 1 urn bar. 32. The a p i c a l region of at l e a s t three c e l l s i n d i c a t i n g the various r e l a t i o n s h i p s of t h e i r l a t e r a l c e l l membranes, CPM = convoluted plasma membrane, D = desmosomes. Other symbols:, large arrowheads = microfilaments. 1 urn bar. 35A due to the angle of the section. In t y p i c a l fashion, these e p i t h e l i a l c e l l s r e s t on a basal lamina (basement membrane) a moderately dense, homogeneous band that varies i n thickness, depending on the angle of the section, from 250 to 500 & wide. The basal lamina follows the contour of the basal c e l l membrane but i s separated from i t by a l i g h t zone 500 R wide. The bulk of the present l i t e r a t u r e indicates that t h i s membrane i s composed of tropocollagen embedded i n an a c i d mucopolysaccharide matrix and that t h i s membrane i s a product of the e p i t h e l i a l c e l l s and not a condensation of the connective t i s s u e ground substance (Kurtz & Feldman, 1962; Pierce et a l , I96H). Supporting t h i s statement i s the presence of numerous v e s i c l e s i n the basal cytoplasm along with others undergoing "reverse pi n o c y t o s i s " at the c e l l membrane (Figure 27). The j u n c t i o n a l complexes of these e p i t h e l i a l c e l l s are represented by zonula occludentes (tight junctions) and macula adherentes (desmosomes). The former (Figures 26 to 30) are found nearest the abdominal c a v i t y and are characterized by the absence of an i n t e r c e l l u l a r space (I.C.S.) i n t h i s region. Beneath the zonula occludens, the I.C.S. widens (360 k) and then the membranes may exhibit one or more of three phenomena.. F i r s t , the l a t e r a l c e l l membranes may run unimpeded to the i n t e r c e l l u l a r channel (I.C.C.) (Figures 26, 27). Second, the membranes may be thrown into a series of concentric folds (Figures 26, 30, 32). F i n a l l y , there may be several desmosomes present (Figures 2 6 , 27, 29, 32). In any event, the I.C.S. abruptly widens into an i n t e r c e l l u l a r channel (Figures 26 j 27, 28) that p e r s i s t s to within- 100 nm of the basal lamina. Within t h i s channel i s a f l o c c u l e n t material that i s probably p r o t e i n or protein-polys.accharide i n nature as w e l l as a c e r t a i n amount of membranous d e t r i t u s . In Figure 26, a "plug" of mucoid debris can be seen trapped i n a f o l d of the surface epithelium. At the basal aspects of these c e l l s there are no j u n c t i o n a l complexes evident. In one case, Figure 27, the channel i s continuous with the l i g h t zone between the plasma membrane and the basal lamina. THE TUNICA ALBUGINEA The tunica albuginea which l i e s beneath the surface epithelium Is most commonly described as a fibrous layer of connective t i s s u e (Nelsen, 1953; Bloom & Fawcett, 1968; Mossman & Duke, 1973). TeWinkel (1972) describes t h i s layer i n Mustelus canis as being a collagenous connective t i s s u e layer with interwoven smooth muscle. In Squalus, the t u n i c a i s composed p r i m a r i l y of "collagen f i b r e s " , e l a s t i c f i b r e s and f i b r o b l a s t s (Figures 18, 27, 33, 3k). The "collagen f i b r e s " which are 1 to k um i n diameter .depending on t h e i r l o c a t i o n are composed of ..unit . f i b r i l s of collagen 500 to 800 R i n diameter and of indeterminate length. The unit f i b r i l s are c r o s s - s t r i a t e d , with transverse bands repeating every 520 R. The e l a s t i c f i b r e s also vary i n thickness from 0.5 to 2.0 um and they are composed of f i n e filaments 50 to 60 R i n diameter. The f i b r o b l a s t i n the tunica are t y p i c a l of quiescent c e l l s with e l l i p t i c a l n u c l e i , a few surface f o l d s , small Golgi v e s i c l e s and a few granular E.R. cisternae Also of i n t e r e s t are the v e s i c l e s that fuse with the plasma membrane surrounding a "collagen f i b r e " . However, contrary to TeWinkel's f i n d i n g s , there i s no evidence for the existence of smooth muscle i n the t u n i c a of Squalus acanthias. THE CORTEX The cortex of the ovary consists of a r i c h l y c e l l u l a r stroma The various elements of the t u n i c a albuginea, collagen (C), e l a s t i c f i b r e s (EF) and f i b r o b l a s t - l i k e c e l l s . 1 ym bar. High magnification of an e l a s t i c f i b r e (EF) demonstrati the 50 to 60 A filaments found within the f i b r e . The small arrowhead in d i c a t e s f i b r e s of s i m i l a r diameter found f r e e l y i n the tunica. 1 ym bar. 38A interspersed, with blood vessels and large lacunae. Within the cortex . there are three d i f f e r e n t types.of c e l l : "dark" c e l l s , " l i g h t " c e l l s and lacunae border c e l l s . Also within t h i s region are numerous c a p i l l a r i e s , some large blood vessels and a network of i n t e r c e l l u l a r collagen. The dark c e l l s (Figures 35, 36, 37) have polymorphic n u c l e i with a band of chromatin apposed to the inner nuclear membrane and small patches dispersed c e n t r a l l y i n the nucleoplasm. The outer nuclear membrane i s free of ribosomes and i s separated from the inner membrane by a space 550 to 650 R wide. The cytoplasm of these c e l l s i s f i l l e d with numerous small empty v e s i c l e s ( 6 5 O R) and many large v e s i c l e s (approx. 800 nm). The large v e s i c l e s (dark bodies) can be e i t h e r empty, p a r t i a l l y f i l l e d with a f i n e granular m a t e r i a l , completely f i l l e d , or any of these with a varying number of fibrous or c r y s t a l l o i d i n c l u s i o n s . These dark bodies resemble the granules of mammalian e o s i n o p h i l i c leucocytes complete with t h e i r c r y s t a l l o i d i n c l u s i o n s (Fawcett, 1966). There are also a few mitochondria and free ribosomes present but no E.R. or Golgi bodies. The cytoplasm i s el e c t r o n dense and i s f i l l e d with a f i n e granular m a t e r i a l . The plasma membranes of adjoining c e l l s are separated by a space of 70 R. Where three or more c e l l s meet, the i n t e r -c e l l u l a r space can be f i l l e d with an electron dense substance. Figure 37 depicts what may be a dark c e l l except f o r the lack of a p e r i p h e r a l band of chromatin and the absence of dense c r y s t a l l o i d s i n the dark bodies. The presence of- four Golgi bodies enclosing forming dense bodies i s another feature that i s not t y p i c a l of a dark c e l l . The second type of c e l l found i n the cortex i s the l i g h t c e l l (Figures 38, 39, *+0, hi) which has a s p h e r i c a l or ovoid nucleus with a c e n t r a l l y located nucleolus and a chromatin pattern s i m i l a r to the A dark c e l l found i n the cortex of the ovary. Note i t s c h a r a c t e r i s t i c lohed nucleus (N) and the dark bodies i n the cytoplasm (FDB = forming dark body, LV = large v e s i c l e with a small amount of dark body m a t e r i a l ) . Also c h a r a c t e r i s t i c of a dark c e l l are the c r y s t a l l o i d s (large arrowheads) found i n the dark bodies. The cyto-plasm of a dark c e l l i s also f i l l e d with numerous small v e s i c l e s (V). 1 ym bar. A dark c e l l with i t s c h a r a c t e r i s t i c contents and lobed nucleus. The large arrowhead indicates two nuclear pores. Another i n t e r e s t i n g feature to note i s how loo s e l y these c e l l s seem to be anchored to one another. Other symbols: LC = l i g h t c e l l . 1 ym bar. 40A An intermediate dark c e l l with dark, bodies (DB) but no c r y s t a l l o i d s , a lobed nucleus (N) without a peripheral band of chromatin, and a c l u s t e r of Golgi bodies (G) an organelle not noted i n a dark c e l l . Other symbols: M = mitochondria. 1 ym bar. A l i g h t c e l l i n the ovarian cortex. This c e l l type i s characterized by an ovoid nucleus (N) with an outer nuclear membrane that i s covered with ribosomes and i s a c t i v e l y contributing (arrowheads) to the numerous large R.E.R. cisternae found i n the cytoplasm. Other symbols: M = mitochondria, SV = large membrane bound smooth v e s i c l e s seemingly i n t r a n s i t i o n to large f i l l e d v e s i c l e s (V). 1 ym bar. 41A Two l i g h t c e l l s p a r t i a l l y separated by a bundle of c o l l a g e n f i b r e s (c). The extent of the R.E.R. i n the cytoplasm i s evident as are s e v e r a l nuclear pores (arrow). Other symbols: small arrowheads = expanded outer nuclear membrane studded w i t h ribosomes, M = mitochondria, N = nucleus. 1 ym bar. A l i g h t c e l l w i t h a nucleolus (NU) and a c l u s t e r of d i s t o r t e d smooth v e s i c l e s (SV) and o u t l y i n g dense v e s i c l e s (DV), an arrangement s i m i l a r t o t h a t of a G o l g i body. 1 ym bar. A c l u s t e r of s e v e r a l l i g h t c e l l s separated by c o l l a g e n f i b r e s . Other symbols: RER = rough endoplasmic r e t i c u l u m . 1 ym bar. 42A 43 dark c e l l . The outer nuclear membrane i s studded with ribosomes and i s well removed from the inner membrane ( 3 6 0 R to 1+000 8.). The cytoplasm of these c e l l s i s f i l l e d with large v e s i c l e s of R.E.R. which encloses a l i g h t , f l o c c u l e n t m a t e r i a l . Figures 38 & 39 suggest that these large v e s i c l e s of R.E.R. are formed by pinching o f f folds of the outer nuclear membrane, a theory consistent with the b e l i e f that the E.R. and the nuclear membrane are c l o s e l y r e l a t e d i f not continuous (Fawcett, 1 9 6 6 ; Brown & Bertke, 1 9 6 9 ) . The cytoplasm contains a few mitochondria, polyribosomes, small v e s i c l e s and microtubules. A d d i t i o n a l l y , the cytoplasm of a l i g h t c e l l i s l e s s electron dense than that of a dark c e l l due to the absence of the f i n e granular matter found i n the dark c e l l s . Within the l i g h t c e l l , Golgi bodies are absent or highly d i s t o r t e d . However, a few medium-sized v e s i c l e s f i l l e d with a moderately e l e c t r o n dense granular substance-can be. seen l y i n g clo.se to .several d i s t o r t e d lamellae (Figures 3 8 , ho). Occasionally within a l i g h t c e l l one or more l i p i d drops can be found. THE INTERCELLULAR SPACE Frequently, the i n t e r c e l l u l a r space w i l l widen and the area w i l l be occupied by a v a r i a b l e number of collagen f i b r e s and an amorphous ground substance (Figures 3 9 , U l ) or large l i p i d droplets (Figures 1+2, hh). THE LACUNAE BORDER CELLS Within the cortex are numerous' lacunae or sinusoids each l i n e d by a s p e c i a l type of c e l l hereafter termed a lacunae border c e l l L.B.C. (Figures h2, 1+3, hh). S u p e r f i c i a l l y , the L.B.C. resembles the simple squamous endothelial c e l l of a c a p i l l a r y w a l l , thickened i n the region Three lacunae border c e l l s , a type found l i n i n g the numerous lacunae (L) of the cortex. These c e l l s are f i l l e d with free ribosomes, microfilaments (F), mito-chondria (M) and v e s i c l e s of various s i z e s . One type of v e s i c l e (small arrowhead) can be found i n the basal region where i t i s e i t h e r continuous with the plasma membrane or free i n the cytoplasm. These c e l l s r e s t on a basal lamina (BL). Other symbols: D = desmosome. 1 ym bar. Two lacunae border c e l l s and t h e i r basal lamina (BL) lying, "over" numerous collagen f i b r e s (C) and two dark c e l l s . The v e s i c u l a r content and the b a s a l pinocytosis (small arrowheads) of ..these c e l l s .is a lso evident. Rarely, v e s i c l e s can be seen opening into the lacuna (arrows). The surface f o l d (MF) i s a configuration implicated i n p i n o c y t o t i c a c t i v i t y . 1 ym bar. A lacuna border c e l l " o verlying" two l i g h t c e l l s (LC). Other symbols: C = collagen f i b r e s with p e r i o d i c i t y , F = filaments, L = lacuna, M = mitochondria. 1 ym bar. 44A of the nucleus and attenuated elsewhere. Unlike the c a p i l l a r y c e l l , the L.B.C. have few, i f any, Golgi bodies or c e n t r i o l e s (Bloom & Fawcett, 1968), but the cytoplasm i s packed with free ribosomes, microfilaments (30 to 50 R i n diameter), a few p r o f i l e s of granular endoplasmic reticulum and a few mitochondria. These organelles are scattered throughout the cyto plasm rather than concentrated near the nucleus as i n c a p i l l a r y c e l l s . Also quite numerous i n the cytoplasm of these c e l l s are v e s i c l e s of medium (210 nm) to large (270 nm) s i z e . The 210 nm v e s i c l e s are eit h e r clear or f i l l e d with a moderately e l e c t r o n dense f i n e l y granular material (Figures ^2, h3) while the larger v e s i c l e s are free of any e l e c t r o n dense matter. One of the most conspicuous and c h a r a c t e r i s t i c features of the lacunae border c e l l s i s the presence of many small v e s i c l e s 600 to 700 R i n diameter (Figures k2, h3) which seem to a r i s e from a large number of ..saccular .invaginations of the ..plasma .membrane that open onto the lacunar and basal surfaces of these endothelial c e l l s . These v e s i c l e s are most numerous at the basal surface where they open into the " l i g h t zone" that spearates the plasma membrane from the basal lamina. These v e s i c l e s i n d i c a t e that a great deal of micropinocytotic a c t i v i t y i s going on but none of these small v e s i c l e s have ever been observed fusing with any of the larger ones. The basal lamina (150 to 200 h t h i c k ) i s si m i l a r i n a l l respects to that of other.epithelia except that i t i s about one h a l f the thickness of the basal lamina of the surface e p i t h e l i a l c e l l s described e a r l i e r . As i s the case with the l a t t e r , the lamina i s probably composed of tropocollagen (Bloom & Fawcett, 1968). The lacunar plasma membrane r a r e l y shows v e s i c l e s opening onto the surface (Figure U3) but several v e s i c l e s of t h i s s i z e range (600 to 800 R) can be seen just beneath the plasma membrane (Figures U2, ^ 3). Additionally,, the surface membrane possesses a few broad but short pseudopodia as w e l l as the occasional narrow protuberance that resembles the surface f o l d generally implicated i n pinocytotic a c t i v i t y (Figure k3). At the junctions of these c e l l s the adjoining membranes come to within a few hundred angstroms of each other but for the most part remain d i s t i n c t l y separate. T y p i c a l l y , the a p i c a l region of the junction i s characterized by the presence of a desmosome. The only other form of j u n c t i o n a l complex between these c e l l s i s the occasional i n t e r d i g i t a t i o n of the plasma membranes (Figures h2, h3). Underlying the basal lamina i s a basement lamella of an amorphous ground substance and an ordered array of collagen f i b r i l s which seems to be t y p i c a l of a l l e p i t h e l i a l c e l l s (Bloom & Fawcett, 1968; Nadol et a l , 1969). ..THE RED-BLOOD .CELLS The nucleated red blood c e l l s of Squalus can be seen i n Figures ^5, U6, & H7. These e l l i p t i c a l c e l l s are about 2h ym long by 10 to 12 ym wide and 2.0 to 2.5 Via t h i c k . The nucleoplasm i s granular, homogeneous and electron dense. There i s no obvious pattern of condensed chromatin. The nuclear membrane has a wide perinuclear c i s t e r n a with the occasional nuclear pore that bridges the c i s t e r n a . The haemoglobin i s f i n e l y granular and homogeneous with the exception of a few i n c l u s i o n v e s i c l e s . Unlike human erythrocytes (Tanaka & Goodman, 1972) with r e l a t i v e l y few small c l e a r v e s i c l e s , the blood c e l l s of Squalus possess numerous large v e s i c u l a r inclusions (280 to 550 ym) that t y p i c a l l y contain whorls of membrane-like material (Figures 1+5, h6). This myelin-like configuration i s brought about by the p r e c i p i t a t i o n of phospholipids during f i x a t i o n (Matthews & Martin, 1971). In one of the red blood c e l l s (Figure H5) a Two red blood c e l l s and a nucleated c a p i l l a r y w a l l c e l l . There are myelin-like figures and "Golgi" bodies (G) present i n these c e l l s . 1 ym bar. Three red blood c e l l s , the lower with a p o r t i o n of i t s nucleus showing. There i s a marginal band of micro-tubules (arrow) present and several myelin-like v e s i c l e s (MV).. L u m b a r . High magnification of the marginal band of Figure k6 here c l e a r l y i n d i c a t i n g the microtubular (MT) nature of the band. 1 ym bar. 47A G o l g i - l i k e body can be seen. A marginal band of microtubules i s present e n c i r c l i n g the f l a t t e n e d e l l i p t i c a l c e l l at its,equator, l y i n g j u s t beneath the c e l l membrane. This band i s composed of 15 to 20 micro-tubules about 250 & i n diameter (Figures k6, U7). Figure H5 also shows a c a p i l l a r y w a l l , c e l l . I t i s s i m i l a r to a lacuna border c e l l except that i t lacks large f i l l e d v e s i c l e s , small v e s i c l e s that fuse with the basal plasma membrane and i t s basal lamina that i s thinner and l e s s conspicuous than that of the lacuna border c e l l . THE THECAL CELLS Around a 0 .3 mm oocyte, the t h e c a l c e l l s are elongate with f l a t t e n e d n u c l e i while the condensed chromatin i s applied to the inner nuclear membrane (Figure H8). The nucleoplasm i s e i t h e r l i g h t or dense depending on the density of the cytoplasm with the density of the innermost c e l l s obscuring any cytoplasmic d e t a i l while the more d i s t a l c e l l s possess numerous Golgi bodies, v e s i c l e s of a l l s i z e ranges, a few mitochondria, microtubules and microfilaments. There are only a few ribosomes present and no E.R. cisternae. The occasional l i p i d drop can also be found i n the thecal c e l l s at t h i s stage. The t h e c a l c e l l s appear to be grouped into " i s l a n d s " by the presence of an extremely dense, granular, i n t e r -c e l l u l a r matrix. Within that matrix are numerous p r o f i l e s of collagen. A s i m i l a r s i t u a t i o n can be seen within the ovaries of the hamster and g e r b i l except the matrix i s s t r u c t u r e l e s s (McReynolds et a l , 1973). When the oocyte has attained a diameter of 1.0 mm the c e l l s of the theca can be d i f f e r e n t i a t e d as either interna or externa. The c e l l s of the theca interna (Figures 50, 51, 52) have retained the f l a t t e n e d nuclei, and chromatin pattern of the e a r l i e r stages. The The thecal c e l l s of a 0.3 mm oocyte u n d i f f e r e n t i a t e d as to interna or externa. The n u c l e i are f l a t t e n e d and the cytoplasm contains numerous Golgi (G) and c l e a r v e s i c l e s . A granular matrix (GM) i s evident separating the thecal c e l l s from the f o l l i c l e and the cortex. Other symbols: BL = basement lamina, C = collagen, LC = l i g h t c e l l . 1 urn bar. The theca interna c e l l s of a 2.0 mm oocyte with numerous E.R. cisternae and mitochondria (M). 1 ym bar. 50 50. Theca interna c e l l s of a 1.0 mm oocyte with numerous free ribosomes, some on cisternae (arrows = RER) while • other areas are cl e a r (= SER). A well developed granular matrix (GM) i s present. 1 ym bar. 51. Theca interna c e l l s of a 1.0 mm oocyte with many free ribosomes but no R.E.R. Mitochondria (M) with granules and p l a t e - l i k e c r i s t a e are noted as are large v e s i c l e s containing granules (arrow) s i m i l a r to those of the granular matrix (GM). 1 ym bar. 52. A theca interna c e l l of a 1.1 mm oocyte. Note the granular matrix (GM) and i t s collagen f i b r e s external to the c e l l . Within the c e l l are numerous c l e a r . v e s i c l e s (LV, V) derived from e i t h e r the Golgi (G) or the nuclear membrane (small arrowheads). A p a i r of c e n t r i o l e s (CE) i s also evident i n t h i s c e l l . 1 ym bar. most conspicuous feature of the cytoplasm of these c e l l s i s the presence of numerous large, c l e a r v e s i c l e s ( 0 . 5 Mm). Figure 5 2 suggests that these v e s i c l e s may have originated, from the Golgi apparatus or from blebs of the nuclear membrane. V e s i c l e s of other sizes can also be seen, the smallest of which ( 0 . 0 9 ym) are c l o s e l y associated with the Golgi bodies. There i s very l i t t l e S.E.R. present and even l e s s R.E.R. The numerous polysomes and free ribosomes often give the impression of R.E.R. when they l i e close to some of the v e s i c l e s and the few S.E.R. tubules (Figures 5 0 , 5 l ) . Mitochondria are o c c a s i o n a l l y seen and at t h i s stage t h e i r c r i s t a e are p l a t e - l i k e . Microtubules of various lengths and the occasional droplet of l i p i d are also present i n these c e l l s . The dense granular matrix surrounding " i s l a n d s " of the c a l cell's i s s t i l l present and occa s i o n a l l y v e s i c l e s within the c e l l s can be seen f i l l e d with-a similar-'granular substance '(Figure 5 1 ) . The intimate association of t h i s i n t e r c e l l u l a r matrix and the collagen f i b r e s can be seen i n Figures 5 0 , 5 1 , and 5 2 . The theca externa c e l l s of a 1 . 0 mm oocyte (Figures 5 3 , 5*+, 5 5 ) have nuc l e i that are more sp h e r i c a l than those of the theca i n t e r n a . The chromatin pattern i s s i m i l a r but there i s a greater quantity of i t i n the core of the nucleus. These c e l l s lack the large c l e a r v e s i c l e s that are common to the c e l l s of the theca i n t e r n a . The v e s i c l e s that are present ( 0 . 1 to 0 . 3 ym) can be ei t h e r c l e a r , coated, or f i l l e d with an electron dense ma t e r i a l . In the l a t t e r case, t h i s m a t erial resembles the granular matrix.. The occasional v e s i c l e can also be seen emptying into the i n t e r c e l l u l a r space. Free ribosomes and polysomes are present as are frequent R.E.R. cisternae. Another i n t e r e s t i n g feature of these c e l l s i s the i n t r i c a t e r e l a t i o n s h i p s of t h e i r plasma membranes and the A theca externa c e l l of a 1.0 mm oocyte. Note the absence of the large c l e a r v e s i c l e s that characterize the theca interna. Several ribosomes and polysomes can be seen as w e l l as some R.E.R cis t e r n a e . Other symbols: small arrowhead = cytoplasmic v e s i c l e opening i n t o the i n t e r c e l l u l a r space, large arrowhead = SER continuous with the nuclear membrane. 1 um bar. Two theca externa c e l l s of a 1.0 mm oocyte with t h e i r i n t r i c a t e membrane r e l a t i o n s h i p s c l e a r l y i n d i c a t e d and a portion of a theca interna c e l l i n the lower l e f t on the other side of the granular matrix ('GM). Also evident i n the cytoplasm are microtubules, small v e s i c l e s and mitochondria (M). 1 u r a bar. The cytoplasm of several theca externa c e l l s of a 1.1 mm oocyte surrounded on the r i g h t by the granular matrix (GM) which separates the "thecal i s l a n d " from_ the c e l l s (LC) of the cortex. The i n t e r r e l a t i o n s h i p of the c e l l s of the externa i s a c h a r a c t e r i s t i c • missing i n the c e l l s of the theca i n t e r n a . Other symbols: R = ribosomes. 1 um bar. 52A large i n t e r c e l l u l a r spaces. At the 2 . 0 ram (Figure k9) stage, the c e l l s of the theca i n t e r n a have acquired more cisternae of E.R. than at the 1 . 0 mm stage. In most cases, these cisternae are only p a r t i a l l y studded with ribosomes. The number of mitochondria present has also increased s l i g h t l y . THE BASAL LAMINA-LAMELLA COMPLEX The basement lamina-lamella complex i s present around the smallest stage examined ( 0 . 1 mm). Thus any p r o t e i n or l i p i d m aterial en route, to the oocyte must cross them. At t h i s stage (Figures 5 6 , 5 7 , 5 8 ) , the basement lamina i s approximately O.h ym wide. I t i s a dense homogeneous band c l o s e l y applied to the plasma membrane of the f o l l i c l e c e l l . The • basal, lamella,however, i s about 2 . 1 ym wide and i t i s f i l l e d with numerous colflagen p r o f i l e s . 'There i-s a loose organization of the collagen f i b r i l s and scattered cross and l o n g i t u d i n a l section can be seen.. By 0 . 2 mm oocyte diameter, the lamina has attained an average width of 0 . 7 to 1 . 0 ym. Concomitant with t h i s increase i n width i s the appearnace of a l i n e a r f i b r i l l a r pattern within the membrane i t s e l f . This pattern i s most prominent i n the outermost 0 . 3 ym. The basal lamella has narrowed s l i g h t l y ( 1 . 7 5 Vim) and a granular component i s now present. Along the plasma membrane of the f o l l i c l e c e l l are small dense patches that appear to be l o c a l i z e d thickenings of the membrane. At 0 . 3 mm diameter (Figures 6 0 , 6 l ) the average thickness of the lamina i s approximately 1 . 0 ym. The f i b r i l s ( 2 0 0 R) are s l i g h t l y more prominent while an intimate r e l a t i o n -ship between the lamina and the collagen f i b r e s of the basal lamella seems to be developing. In the O.h to 0 . 5 mm oocytes observed, the basement lamina v a r i e d i n thickness from 0 . 3 5 to l.h ym. The f i b r i l l a r The f o l l i c l e of a 0.1 mm oocyte composed of a homogeneous basement lamina or membrane (BM) and i t s outlying basement lamella (BL), a f o l l i c l e c e l l l a y e r (FC) and a v i t e l l i n e membrane l y i n g between the f o l l i c l e c e l l s and the oocyte (0). Note the ribosomes on the outer nuclear membrane of the F.C. nucleus, the projections of the ooplasm into the F.C. (large arrow-heads) and the pin o c y t o t i c a c t i v i t y (arrow) at the base of the ooplasmic m i c r o v i l l i . 1 ym bar. A demonstration of the v a r i a t i o n i n f o l l i c l e thickness within a sing l e oocyte (re: previous f i g u r e ) . Note the number of v e s i c l e s within the F . C , the V.M. and the ..oocyte. BM • = 0-.h .ym, VM = ,0.,5 ym. 1 „ym bar.. A t y p i c a l f o l l i c l e c e l l of a 0.1 mm oocyte f i l l e d with numerous f r e e ribosomes, rough endoplasmic reticulum (ER), mitochondria (M) and Golgi ( G ) . The edge of a nucleus (N) with nuclear pores (arrow) i s in d i c a t e d as i s the basement lamina and the basal l a m e l l a (BL). The large arrowheads i n d i c a t e the p r o j e c t i o n of other f o l l i c l e c e l l s or the oocyte i n t o t h i s f o l l i c l e c e l l . Note the m i c r o v i l l o u s nature of the oocyte's (0) plasma membrane. BM = 0 . U ym, VM = 0.5 ym. 1 ym bar. The vitelline.membrane (VM) of a 0.2 mm oocyte showing i t s f i n e fibrous nature. The a p i c a l aspect of the f o l l i c l e c e l l (FC) i s f i l l e d with ribosomes and mitochondria and often projections of the F.C. i n t o the oocyte can be seen (arrow). Other symbols: large arrowheads = ooplasmic v i l l i . 1 ym bar. The f o l l i c l e of a 0.3 .mm oocyte. The basement lamina (EM) i s quite t h i c k and contains 200 A1 f i b r i l s (arrows). F o l l i c u l a r penetrations of the v i t e l l i n e membrane (arrow) are noted and when observed i n the oocyte (0) they u s u a l l y terminate with a de smosome (D). BM = 1.05 ym, VM = O.h to 0.7 ym. 1 ym bar. 5 5 A The f o l l i c l e of a 0 . 3 mm oocyte with f o l l i c l e c e l l s that have numerous free ribosomes, mitochondria, and microtubules (arrow). This area of the f o l l i c l e may be the region of t r a n s i t i o n from the animal to the vegetal pole. The occasional desmosome was noted along the f o l l i c u l a r v i l l i (large arrowhead). Other symbols: BM = basement lamina, 0 = oocyte. BM = 0 . 7 VM = 0.H to 0 . 8 ym. 1 ym bar. The zona r a d i a t a (PZ) of a 0.3 mm oocyte, a band of ooplasmic v e s i c l e s or the t i p s of the ooplasmic m i c r o v i l l i found within the V.M. pe r i p h e r a l to the oocyte (0). The large .arrowhead..indicates a dense v e s i c l e found within the oocyte. 1 ym bar. The zona r a d i a t a (PZ) of the same oocyte l 8 0 ° removed from the previous f i g u r e . 1 ym bar. The basal lamina (BM), lamella (BL) and basal cytoplasm of the f o l l i c l e c e l l s (FC) of a 0.1* mm oocyte. The . .small arrowhead i n d i c a t e s the banding pattern of the collagen of the lamella. The outer membrane of the nucleus can be seen giving o f f ribosome studded v e s i c l e s (arrows) which form part of the R.E.R. component found i n the basal cytoplasm. Also found i n the cytoplasm are numerous v e s i c l e s f i l l e d with a component s i m i l a r to the basement lamina (large arrowheads). BM = 1.06 ym. 1 ym bar. The basal f o l l i c l e c e l l cytoplasm f i l l e d with v e s i c l e s (large arrowheads) that are of s i m i l a r density and texture to the basal lamina (BM). Note too, the rough E.R. and the mitochondria. BM = 0.88 ym. 1 ym bar. The basal lamina around a O.h mm oocyte demonstrating i t s f i b r i l l a r pattern and interspersed granules. The basal lamella (BL) i s f i l l e d with interwoven collagen f i b r e s and here a f o o t - l i k e process from a t h e c a l (T) c e l l can be seen. BM = 1.1* ym. 1 ym bar. 5 7 A The f o l l i c l e of a O.U mm oocyte showing e a r l y stages i n the formation of the v i t e l l i n e membrane (arrows) with both ooplasmic and f o l l i c u l a r projections or v i l l i . The a p i c a l cytoplasm of the F.C. i s f i l l e d with ribosome Golgi (G) and mitochondria. • Other symbols: SER = smooth endoplasmic reticulum i n the c o r t i c a l ooplasm. BM = 0.6 to 0 .T ym, VM = 0.0 to 0.2 ym. 1 ym bar. The f o l l i c l e of a 0 .U mm oocyte i n the e a r l y stages of v i t e l l i n e membrane formation. Numerous ooplasmic and f o l l i c u l a r v i l l i are seen (arrows) with the l a t t e r often surrounded by a halo of microfilaments. The a p i c a l cytoplasm of the F.C. i s f i l l e d with Golgi (G), smooth v e s i c l e s and ribosomes. In the basal cytoplasm, v e s i c l e s (large arrowhead) that contribute to the formation of the basement lamina can be seen. BM = 0.7 Mm, VM = 0.0 to 0.2 ym. 1 ym bar. 59 69. The f o l l i c l e of a O.h mm oocyte (0) with no v i t e l l i n e membrane evident. The basement lamina (BM) i s completely f i b r i l l a r . These f o l l i c l e c e l l s are f i l l e d with Golgi (G), small v e s i c l e s (arrowhead) and some R.E.R. Other symbols: T = theca. BM = 0.3 ym. 1 ym bar. TO. A d i f f e r e n t region of the same oocyte with a well developed v i t e l l i n e membrane (VM) and the developing v i l l a r nature of the oocyte. Other symbols: arrowhead = stream of "material penetrating to the'S.;E.'R from the base of the v i l l i , M = mitochondria, N = nucleus, R = ribosomes. BM = 0.35 ym, VM = 0.28 ym. 1 ym bar. 71. Another region of the same O.h mm oocyte f o l l i c l e with extensive R.E.R. i n the F.C. cytoplasm and several c e l l u l a r or oocytic projections (arrowheads). Note the well developed S.E.R. band i n the oocyte and the l i p i d (L) yolk p a r t i c l e . BM = 0.37 ym, VM = 0.5 pm. 1 ym bar. 59A 60 7 2 . Development of the v i t e l l i n e membrane i n a O.h mm oocyte. In areas of f o l l i c l e c e l l - o o c y t e ( 0 ) contact, desmosomes can be seen (large arrowheads). The cytoplasm of the F.C. i s f i l l e d with ribosomes, v e s i c l e s , Golgi (G) and mitochondria (M) and some microfilaments (arrow). VM = 0.0 to 0 . 1 5 ym. 1 ym bar. 73. F i n e l y fibrous v i t e l l i n e membrane forming i n patches between oocyte (0) and f o l l i c l e c e l l v i l l i (large arrowhead). O.h mm oocyte. Note also the micro-f i l a m e n t s (arrows) associated w i t h the v i l l i . VM = 0.0 to 0.3 ym. 1 ym bar.-7^. Note the patchy formation of the v i t e l l i n e membrane and i t s fibrous nature (large arrowhead) i n a O.h mm oocyte. The dominant feature of these three f i g u r e s i s the abundance of organelles i n the F.C. near the V.M. and the lack of them i n the oocyte (0). VM = 0.0 to 0.2 ym. 1 ym bar. 60A A panorama of several p s e u d o - s t r a t i f i e d f o l l i c l e c e l l s (FC) from the basement lamina (BM) to the oocyte (0). O.H mm oocyte. The v i t e l l i n e membrane i s almost non-existent and desmosomes (D) can be seen i n areas of membrane to' membrane contact. Similar structures are noted between f o l l i c l e c e l l s . The cytoplasm of the F.C.s i s f i l l e d with ribosomes (R), polyribosomes, several v e s i c l e s , mitochondria and E.R. Oocytic v i l l i (arrow) can be seen and they are surrounded by micro-filaments. BM = 1.2 ym. 1 um bar. The t h i c k e s t v i t e l l i n e membrane (VM) observed around a O.h mm oocyte. The V.M. i s f i n e l y fibrous and i s pierced by pore canals (arrow) or v i l l i from e i t h e r the oocyte or the f o l l i c l e c e l l . Other symbols: G = G o l g i , N = nucleus, VI = ooplasmic v i l l i . VM = 2.5 to k.5 ym. 1 ym bar. pattern of the lamina (B.M.) now involves the e n t i r e membrane and at. times t h i s pattern i s augmented "by the appearance of granules between the layers (Figures 6k, 65, 66, 68, 69). The basal lamella (B.L.) i s about 0.6 to 0.8 um wide, i t s granular content has increased as has the complexity of the r e l a t i o n s h i p of the collagen f i b r e s both among themselves and with the basement lamina.(B.M.). At 0.6 mm oocyte diameter the basement lamina has an average thickness of 0.8 to 1.0 ym, there has been l i t t l e change. By 0.8 mm diameter, the lamina, has an average thickness of 1.1 um and the f i b r i l l a r pattern i s becoming l e s s evident. At a diameter of 1.5 to 1.8 mm (Figures 88, 89, 9 0 ) , the B.M. va r i e s i n thickness from k,J to 5.5 ym. The f i b r i l l a r pattern of the e a r l i e r stages i s absent and the membrane i s homogeneously granular. The B.L. i s about 6.0 um wide and i s l o o s e l y f i l l e d with an interwoven array of collagen .fibres and numerous .clusters of 0.025 .ym.,granules. ..There i s also a f i n e r component that seems to be derived from the B.M. i t s e l f . At the basement lamina-lamella i n t e r f a c e , the collagen f i b r e s are so arranged that some of them have t h e i r o r i g i n i n the substance of the basement lamina. The former orderly arrangement of these f i b r e s i s no longer evident, a "herringbone" pattern having taken i t s place. THE FOLLICLE CELLS The f o l l i c l e or granulosa c e l l s of the 0.1 mm oocyte (Figures 56, 57, 58) are low cuboidal and one layer t h i c k . This corresponds to the findings of Lance & C a l l a r d (1969) who noted that a l l elasmobranchs with a single c e l l - t y p e granulosa belong to the order S e l a c h i i while those with a two c e l l - t y p e granulosa belong to the order B a t o i d e i . T y p i c a l of e p i t h e l i a c e l l s , they r e s t on a basement membrane and face a. lumen (the oocyte). The n u c l e i are oval v i t h a f i n e l y dispersed chromatin pattern and s l i g h t condensations bordering the nuclear membrane. The cytoplasm contains numerous mitochondria with p l a t e - l i k e c r i s t a e , f r ee ribosomes and scattered v e s i c l e s (300 to 8 0 0 A). Rough E.R. i s present though not extensive and i s concentrated i n the mid to basal regions of the c e l l . A few S.E.R. tubules are also evident. In the basal region, several of the v e s i c l e s ( 8 0 0 it) are f i l l e d with material having the same ele c t r o n density and texture as the basement lamina. But, none of these v e s i c l e s can be observed fusing with the plasma membrane. Another i n t e r e s t i n g feature of these c e l l s i s the presence of several dense double membrane bound v e s i c l e s i n the a p i c a l region of the c e l l (Figures 5 6 , 5 8 ) . These " v e s i c l e s " , however, are projections of the ooplasmic membrane into the f o l l i c l e c e l l s . Similar phenomena have been reported by Yamamoto i n .•Oryzias • (.1-963).. In -i& . 0 . k-mm .oocyte .these ooplasmic projections are quite evident as are projections of the f o l l i c l e c e l l s i n t o the oocyte (Figures 6 0 , 6 7 , 6 8 , 7 3 ) . Often associated with the ooplasmic projections i s a "halo" of microfilaments (Figures 6 8 , 7 2 , 7 3 , lk, 7 5 ) . The Golgi bodies, v e s i c l e s and S.E.R. are s t i l l prominent i n the a p i c a l regions of these c e l l s (Figures 6 7 , 6 8 , 6 9 , 7 2 ) . Rough E.R. i s scattered throughout the cytoplasm but more highly concentrated i n the basal portion of the c e l l (Figures 6k, 6 5 , 7 1 , 7 *0 . In the basal region next to the lamina, ' electron dense v e s i c l e s ( 5 0 0 to l 6 0 0 it) are s t i l l present (Figures 6k, 6 5 , 6 6 , 6 8 ) . Free ribosomes and polysomes are also evident,as are small desmosomes (Figures 6 8 , 7 2 , 7 3 , 7 5 ) which are located between adjacent c e l l s and sometimes i n areas where the membranes of the oocyte and the f o l l i c l e c e l l meet. Another i n t e r e s t i n g feature of t h i s stage i s the varying thickness of the B.M. (lamina) ( 0 . 7 5 to 1 . 5 ym) from one side of the oocyte to the other. This corresponds r e s p e c t i v e l y to the vegetal-animal axis of the oocyte and to a decrease i n the density of the cytoplasmic and nuclear material of the f o l l i c l e c e l l s of the amimal hemisphere. The animal pole i s turned inward r e l a t i v e to the surface of the ovary (Wallace, 190k; Raven, 1 9 6 l ) . In a 1 . 1 mm oocyte, the cytoplasmic and ooplasmic projections are l e s s evident. The basal portions of the f o l l i c l e c e l l s s t i l l contain numerous dense v e s i c l e s ( 1 2 0 to 3 6 0 nm), mitochondria and R.E.R. cisternae (Figures 7 7 , 7 8 , 8k). None of these v e s i c l e s , however, are seen fusing with the plasma membrane. In the a p i c a l region (Figures 7 7 , 19, 8 5 ) , Golgi bodies are prominent as are occasional R.E.R. cistern a e . Also present i n the a p i c a l region at t h i s stage are l i p i d droplets (Figures 7 7 , 7 9 » 81+, 8 5 ) 0 . 6 to 0 . 7 um i n diameter. By 1 . 5 to 1 . 8 mm, the f o l l i c l e c e l l l a y e r has changed considerably The c e l l s are becoming columnar (see also Yamamoto,, 1 9 6 3 ; B e l l a i r s , 1 9 6 5 " , Cummings et a l , 1 9 7 1) or p s e u d o s t r a t i f i e d columnar, with the n u c l e i displaced towards the basement lamina (Figures 9 0 , 9 1 , 92). Tangential sections produce a multilayered f o l l i c u l a r epithelium. These c e l l s are i d e n t i c a l to the f o l l i c l e c e l l s of smaller oocytes i n a l l other aspects. The c e l l s c l o s e s t to the B.M. (Figure 9 0 ) are f i l l e d with numerous small v e s i c l e s of varying electron d e n s i t i e s . The most dense v e s i c l e s are closest to the basement lamina. Also within these c e l l s are several mitochondria, free ribosomes and v e s i c u l a r R.E.R. Microtubules (Figures 9 0 , 9 2 ) are p l e n t i f u l , forming a latticework throughout the c e l l . The i n t e r c e l l u l a r membranes are quite porous and even show the occasional i n t e r c e l l u l a r bridge (Figures 9 0 , 9 1 , 9 2 ) . At or above the l e v e l of the n u c l e i the f o l l i c l e c e l l cytoplasm now contains Golgi bodies and R.E.R. cisternae i n greater quantities than are found i n the basal regions The f o l l i c l e c e l l s of a 1.1 mm oocyte. The basal aspect (upper r i g h t ) i s f i l l e d with mitochondria (M), .dense v e s i c l e s and R.E.R. The a p i c a l region i s f i l l e d with numerous Golgi (G), c l e a r v e s i c l e s and some R.E.R. Other symbols: L = l i p i d , N = nucleus, VM = v i t e l l i n e membrane. BM = 3.0 to 3.5 ym, VM = 10 to 15 ym. 1 ym bar. The basal aspect of a f o l l i c l e c e l l (FC) of a 1.1 mm, oocyte. The layered nature of the basement lamina (BM) i s evident as are the dense v e s i c l e s (large arrowheads) i n the F.'C. '*-BM = 2.5 Pm. . 1 ym bar. The a p i c a l aspect of a f o l l i c l e c e l l of a 1.1 mm oocyte. Unlike the previous f i g u r e , the cytqplasm i s f i l l e d with mitochondria (M). and R.E.R. , The V.M. i s to the lower l e f t . 1 ym bar. 6 5 A 66 8 0 . The v i t e l l i n e membrane of a 1.1 mm oocyte ( 0 ) pierced by pore canals (large arrowhead)"for the passage of v i l l i . The zona r a d i a t a i s beginning to take shape at the oocyte's surface. 1 pm bar. 8 l . The zona r a d i a t a of a 1.0 mm oocyte (0). At the base..• of the zone are broad v i l l i (VI) with some pinocytotic". a c t i v i t y evident. The large arrowhead i n d i c a t e s ' a ' pore canal. 1 ym bar. 8 2 . An extensive zona r a d i a t a i n a 1.0 mm oocyte probably produced by the angle of sectioning. The broad v i l l i bases (OB) terminate i n smaller v e s i c l e - l i k e t i p s (0V) • buried i n the substance of the V.M. 1 ym bar. 6 6 A The broad v i l l i (OB) of the zona r a d i a t a (PZ) of a 1.0 mm oocyte also depicting the'pinocytotic a c t i v i t y at the oocyte's surface (arrows). 1 ym bar. Three f o l l i c l e c e l l s of a 1.1 mm oocyte each d i f f e r i n g i n background d e n s i t i e s . The large arrowhead in d i c a t e s a dense v e s i c l e of s i m i l a r composition to the lamina ,(BM),. Other symbols': . arrows*= jpore -eana'ls., L-- l i p i d , VM = v i t e l l i n e membrane. BM = 3.0 to 3.5 ym, VM = 10 to 15 ym. 1 ym bar. A f o l l i c l e c e l l of a 1.1 mm oocyte containing several dense l i p i d bodies, mitochondria (M), ribosomes (R), rough endoplasmic reticula.(RER) and numerous small v e s i c l e s . ( l a r g e arrowheads). 1 ym bar. 67A A low magnification, t h i c k section of the f o l l i c l e of a 1 to 2 mm oocyte (O). The angle of the section broadens the v i t e l l i n e membrane (VM) and gives a ps e u d o s t r a t i f i e d appearance to the t a l l , columnar f o l l i c l e c e l l s (FC) with t h e i r basal n u c l e i . The basement lamina (B) and- the lamella l i e between the F.C.s and the " i s l a n d s " of theca c e l l s . 370X. Higher magnification of the f o l l i c l e c e l l s , v i t e l l i n e membrane (VM), the oocyte (0) and the intervening zona ra d i a t a (PZ). 6 0 0 X. 68A The "basement membrane or lamina (BM) of a 1.5 to 1.8 mm oocyte. The texture of the membrane i s granular and i n the lower l e f t several F.C. v e s i c l e s of s i m i l a r composition can be seen. At the lamina-lamella i n t e r -face i t i s obvious that the collagen f i b r e s of the B.L. have t h e i r o r i g i n i n the basement lamina. BM = h.f ym. 1 ym bar. The basement lamella (BL) of a 1.5 to 1.8 mm oocyte. The collagenous nature of these f i b r e s i s c l e a r l y evident ( arrow) and intermingled within the he r r i n g -bone pattern are c l u s t e r s of granules (G) of unknown o r i g i n . The f i b r e s of the B.L. are also c l o s e l y associated with the theca interna c e l l s (T). 1 ym bar. 69A 70 90. The basement membrane (BM) and several f o l l i c l e c e l l s of a 1.5 to 1.8 mm oocyte. The numerous dense v e s i c l e s (arrow) of the F . C . are c l o s e l y associated with the basement lamina (BM) i n t e r f a c e . Of p a r t i c u l a r i n t e r e s t i s the large cytoplasmic c o n t i n u i t y between two f o l l i c l e c e l l s (large arrowhead). 1 um bar. 91. Numerous ps e u d o s t r a t i f i e d f o l l i c l e c e l l s (FC) at the l e v e l of t h e i r basal nuclei (N). The basement lamina of t h i s 1.5 to 1.8 mm oocyte i s to the upper l e f t . 1 inn "bar. 92. Two f o l l i c l e c e l l s above the l e v e l (closer to the oocyte) of the nuclei with several Golgi (G), short E.R. cisternae and numerous microtubules (arrows). The small arrowheads in d i c a t e breaks i n the l a t e r a l c e l l membranes. The V . M . i s to the lower r i g h t . 1 ym bar. 70A (Figure 9 2 ) . As the oocytes increase i n s i z e (greater than 2 . 0 mm), t h i s f o l l i c u l a r c e l l arrangement p e r s i s t s . In the a p i c a l regions there i s an increasing amount of l i p i d present and subsequent to t h i s i s the separation of the f o l l i c l e c e l l s and the v i t e l l i n e membrane. There are, however, cytoplasmic bridges that traverse t h i s gap and connect some f o l l i c l e c e l l s with the v i t e l l i n e membrane. THE VITELLINE MEMBRANE The relationship between the f o l l i c l e c e l l s , the c e l l membrane of the oocyte, the zona r a d i a t a and the V.M. i s intimate and complex (Figures 8 6 , 8 7 ) . Any consideration of the formation of the V.M. must include each of the aforementioned elements. In the smallest oocytes examined ( 0 . 1 mm) the v i t e l l i n e membrane (V.M.) i s we l l developed. I t i s a homogeneously dense structure of 0 . 5 to 0 . 7 ym thickness (Figures 5 6 , 5 7 , 5 8 ) . The oocyte plasma membrane i s highly folded and i t i s evident that some of these projections or v i l l i traverse the substance of the V.M. (Raven, 1 9 6 l ; B e l l a i r s , 1 9 6 5 ; N ? 5 r r e y a n g , 1 9 6 8 ) , approach and even penetrate the neighbouring f o l l i c l e c e l l s . At the base of these p r o j e c t i o n s , several small c l e a r v e s i c l e s (Figures 5 6 , 5 7 ) can be seen passing into the ooplasm. The material of the V.M. occupies f u l l y the space between the F.C. and the oocyte. This i s quite unlike chorion formation i n the t e l e o s t Oryzias (Yamamoto, 1 9 6 3 ) where the forming membrane i s deposited.in a wide i n t e r c e l l u l a r space By 0 . 2 mm, the V.M. has taken on a fibrous appearance (Figure 5 9 ) . The extent of i t s penetration of both the oocyte and the f o l l i c l e c e l l s i s evident, as i s the close r e l a t i o n s h i p of the f o l l i c l e c e l l s and the oocyte. The close proximity of the oocyte v i l l i to the f o l l i c l e c e l l s and the presence of f o l l i c l e c e l l cytoplasm beneath the V.M. at t e s t s to t h i s . The v i t e l l i n e membrane varies i n thickness from non-existent to 0 . 5 ym. At 0 . 3 mm the V.M. was observed at thicknesses ranging from 1 . 0 to 3 . 0 ym (Figures 6 0 , 6 l , 6 2 , 6 3 ) . The structure of the membrane i s unaltered and numerous f o l l i c u l a r penetrations are s t i l l evident. At the base and oc c a s i o n a l l y along the sides of these projections desmosomes or desmosome-like figures can be seen (Figures 6 0 , 6 l ) , Similar observations have been made i n a v a r i e t y of higher vertebrates (N ? 5 r r e v a n g , 1 9 6 8 ) and i n bi r d s p a r t i c u l a r l y ( B e l l a i r s , 1 9 6 5 ) . The zona r a d i a t a i s quite obviously that area created by the macro- and m i c r o v i l l i of the oocyte plasma membrane. The extent of t h i s zone va r i e s within a single oocyte and from oocyte to oocyte (Figures 6 0 , 6 2 , 6 3 ) . The .penetration of .clear and dense ^ v e sicles ri-nto 'the ' c o r t i c a l ooplasm also varies with the development of the V.M. and the Z.R. (Figures 6 0 , 6 l , 6 2 , 6 3 ) . Also present i n the c o r t i c a l ooplasm are a few smooth E.R. v e s i c l e s and cisternae. In O.h mm oocytes, there are c o n f l i c t i n g data as to the thickness and therefore the development of the v i t e l l i n e membrane. In one specimen the V.M. va r i e s i n thickness from non-existent to 3 . 0 to h.O ym (including the zona r a d i a t a ) . The specimen with the t h i c k e s t V.M. ( 3 . 0 to h.O ym) i s also fibrous and displays numerous pore canals (Figure 7 6 ) . Many m a c r o v i l l i are present on the ooplasmic border and interspersed among these v i l l i are v e s i c l e s of ooplasm that spread out in t o the v i t e l l i n e membrane. The spaces between the m a c r o v i l l i are devoid of fibrous V.M. material but are p a r t i a l l y f i l l e d with a f l o c c u l e n t substance. In a second specimen, the V.M. va r i e s from non-existent to 0 . 5 ym t h i c k (Figures 6 9 , TO, T l ) . Where the V.M. i s absent, the oocyte and the f o l l i c l e c e l l s are. separated by a space of approximately TO R. Local thickenings or electron dense patches are also evident along t h i s i n t e r - • • face. The boundary of t h i s i n t e r f a c e i s not smooth, as the two plasma membranes i n t e r d i g i t a t e extensively. Also of i n t e r e s t are the gaps i n the plasma membranes of neighbouring f o l l i c l e c e l l s (Figures 6 9 , TO). Presumably, as observations are made nearer to the opposite pole the t h i c k -ness of the V.M. increases. The i n t e r f a c e of the V.M. and the f o l l i c l e c e l l s i s remarkably smooth while the ooplasmic surface displays i t s c h a r a c t e r i s t i c v i l l i . Again, not a l l the spaces between the v i l l i are f i l l e d with the material of the V.M. C h a r a c t e r i s t i c p r o j e c t i o n s of f o l l i c u l a r m a t erial into the ooplasm are s t i l l present (Figures 6 9 , T l ) . Approximately 2 um beneath the ooplasmic membrane i s the beginning of «a band ,.of S.E.R. v e s i c l e s . This ..band .also -contains .a-few.mitochondria and Golgi bodies. Between t h i s band and the base of the ooplasmic v i l l i are "paths" of electron dense m a t e r i a l (Figures TO, T l ) . The most i n t e r e s t i n g specimens of t h i s stage are only beginning to form the v i t e l l i n e membrane (Figures 6 7 , 6 8 , 7 2 , 7 3 , lh, 7 5 ) . There are frequent desmosomes present along the apposed F.C. and oocyte plasma membranes. The f i n e fibrous nature of the V.M. i s noticeable but often i t i s d i f f i c u t t to discern. The membrane does not form evenly around the oocyte but forms i n clumps between the ooplasmic pr o j e c t i o n s (Figures 7 3 , 7 M . Often i n areas where these patches are located the F.C. plasma membrane can not be seen. I n t e r e s t i n g l y , the F.C. cytoplasm adjacent to the projections and V.M. patches contains many f i n e microfilaments (Figures 6 7 , 6 8 . 7 3 ) 2 5 R i n diameter. Above a l l , the most s t r i k i n g feature of t h i s and the other O.U mm specimens i s the abundance of c e l l u l a r organelles within the f o l l i c l e c e l l s close to the forming V.M. and the lack of them near the V.M. i n the oocyte. When the oocyte has reached a diameter of 1 . 0 to 1 . 1 mm the v i t e l l i n e membrane has changed. The V.M. i s now about 1 0 to. 1 5 ym t h i c k and i t occasionally appears to be granular (Figures 8 0 , 8 l , 8 2 ) . Pore canals are s t i l l present, but there are also holes or c l e a r v e s i c l e s i n the membrane that are the same s i z e as the pore canals (Figures 8 0 , 8 l , Qh). The i n t e r f a c e of the f o l l i c l e c e l l s and the V.M. i s r e l a t i v e l y even and only infrequently interrupted by i n t e r d i g i t a t i o n s . In the specimens observed, there are no desmosomes present along t h i s i n t e r f a c e . At the ooplasmic border the r e l a t i o n s h i p between the V.M. and the oocyte i s more complex than previously noted. Although some specimens s t i l l e x hibit simple m a c r o v i l l i , v i l l i and v e s i c l e s , others d i s p l a y an i n t r i c a t e •"array-of ooplasmic "buds", intermingled smaller ooplasmic v e s i c l e s and a band of ooplasmic v e s i c l e s (zona radiata) penetrating the V.M. (Figures 8 0 , 8 l , 8 2 , 8 3 ) . The spaces between the l a r g e r "buds" and the smaller v e s i c l e s i s only p a r t i a l l y f i l l e d with a material s i m i l a r to that of the v i t e l l i n e membrane. The l a r g e r "buds" contain v e s i c l e s of moderately electron dense material derived by micropinocytotic a c t i v i t y (Figure 8 3 ) . At a diameter of 1 . 5 to 1 . 8 mm the V.M. i s about 5 0 to 7 0 ym t h i c k . The e n t i r e membrane i s fibrous and i s extensively pierced by pore canals which i n turn are f i l l e d with a fibrous material (Figures 9 3 , 9 ^ , 9 5 ) . At the F.C. i n t e r f a c e , the f i b r e s of the V.M. are c l o s e l y associated with the F.C. plasma membrane and i n some instances may continue 5 0 0 to 9 0 0 ft past the plasma membrane (Figures 9 3 , 9 * 0 . The microfilaments within the pore canals extend into the f o l l i c l e c e l l s a distance of approximately 1 . 0 ym. In the f o l l i c l e c e l l s and c l o s e l y associated with the areas The v i t e l l i n e membrane-follicle c e l l (VM-FC) i n t e r f a c e of a 1.5 to 1.8 mm oocyte. The V.M. i s f i b r o u s and ofte n f i b r e s can be seen extending i n t o the f o l l i c l e c e l l . Note the pore canals (PC) which here are F.C. v i l l i . 1 pm bar. The f i b r o u s nature of the V.M. i s evident w i t h s i m i l a r f i b r e s found w i t h i n the pore canals (PC) and F.C. cytoplasm (arrows). V e s i c l e s (V) are a l s o noted i n the f o l l i c l e c e l l " c l o s e l y a s s o c i a t e d w i t h the pore canal and the membrane i n t e r f a c e . 1 um bar. The v i t e l l i n e membrane (VM) penetrated by pore canals (PC) of various o r i e n t a t i o n s (arrows). 1 pm bar. 7 5 A The zona r a d i a t a (PZ) of a 1 . 5 to 1 . 8 mm oocyte. I t i s composed of v e s i c l e s and l i e s within, the v i t e l l i n e membrane (VM) external to the oocyte and the broad oocyte v i l l i (OB). 1 um bar. The c o r t i c a l region of a 1 . 5 to 1 . 8 mm oocyte ( 0 ) showing pinocytotic v e s i c l e s (arrow) and v e s i c l e s i n s i d e of and outside of (double arrows) a l i n i n g body (LB). Note the "paths" (S) of e l e c t r o n dense m a t e r i a l that penetrate into the oocyte to a depth of 7 pm. Lining bodies are also i l l u s t r a t e d i n Figures l U 6 , l U 7 , 1 5 6 - , 1 5 7 . 1 ym bar. 76A where microfilaments penetrate the f o l l i c l e c e l l s are numerous v e s i c l e s 5 0 0 to 7 0 0 K i n diameter (Figure 9k). The pore canals (Figure 9 5 ) that penetrate the V.M. do not have any p a r t i c u l a r o r i e n t a t i o n , crossing each other at various angles. As the pore canals approach the oocyte (Figure 9 6 ) , they are no longer discernahle from the elements of the v e s i c u l a r band surrounding the oocyte. Depending on the angle of the secti o n , t h i s band i s from 2 . 0 to 5 . 0 um wide. The ooplasmic membrane i s s t i l l h i g h l y convoluted and the spaces between the m a c r o v i l l i only p a r t i a l l y f i l l e d with V.M. mat e r i a l . Within the c o r t i c a l region of the oocyte the "paths" of electron dense material f i r s t reported i n a O.h mm oocyte are quite extensive (Figure 9 7 ) . These "paths" anastomose f r e e l y and penetrate to a depth of 6 to 7 Vra beneath the oocyte membrane. Also seen throughout t h i s c o r t i c a l region and even deeper into the oocyte sare/numerous .double membrane bound'.structures. These structures .are f i l l e d with a fibrous material reminiscent of that within the pore canals.. Some of the l a r g e r structures also e x h i b i t v e s i c l e s 5 0 0 A i n diameter (Figure 9 7 ) s i m i l a r to those found near the F.C.-V.M. border. • When the oocytes have reached a diameter of 2 . 5 mm the v i t e l l i n e membrane has an average thickness of 6 0 to 9 0 ym. The membrane has a f i n e granular appearance and i t no longer has gaps between the oocyte v i l l i . The v i l l i are on the whole t a l l ( 2 . 2 ym) and regular with the appearance of a s t r i a t e d band. The v e s i c u l a r band (Z.R.) i s no longer evident although small (kkO K) c a n a l - l i k e structures are s t i l l v i s i b l e . THE OOCYTE NUCLEUS The nucleus of a 0 . 1 mm oocyte (Figure 1 0 1 ) has numerous n u c l e o l i and lampbrush chromosomes and i s therefore a primary oocyte i n mid-78 98. Nuclear region of a 0.1 mm oocyte. Large arrowheads in d i c a t e ribosomal p a r t i c l e s i n the nucleoplasm and ooplasm. Arrows i n d i c a t e E.R. connections with a G.V.B. Note the pores (P) and b l i s t e r s (B) of the nuclear membrane. 1 ym bar. 99. Nuclear region of a 0.1 mm oocyte with numerous yolk precursor bodies nearby. Notice also the proximity of the S.E.R. v e s i c l e s to the nuclear membrane. 1 ym bar. 100. Nuclear region of a 0.1 mm oocyte demonstrating the production of smooth membrane bound v e s i c l e s (B) from the nuclear membrane. 1 ym bar. 78A diplotene (Bloom & Fawcett, 1 9 6 8 ; Davidson, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ) . The nuclear membrane or envelope of the dogfish oocyte i s t y p i c a l , i n that i t i s composed of two membranes 5 0 to 9 0 ft t h i c k separated by a p e r i -nuclear c i s t e r n a 3 5 0 to 7 0 0 R wide (Figures 9 8 , 9 9 , 1 0 0 ) (Yamamoto, I 9 6 H ; Fawcett, 1 9 6 6 ) . The ooplasmic side of the membrane i s devoid of ribosome-like p a r t i c l e s thus resembling the smooth surfaced v e s i c l e s found i n the neighbouring ooplasm. The.outer nuclear membrane i s generally not p a r a l l e l to the inner membrane but d i s p l a y s numerous " b l i s t e r s " or "blebs" at i r r e g u l a r i n t e r v a l s along i t s length, the blebs being extremely close to or nearly continuous with the smooth surfaced v e s i c u l a r E.R. i n the ooplasm. The inner nuclear membrane follows a les s tortuous path and i n contrast i s twice as t h i c k as the outer nuclear membrane.. At t h i s stage, the nuclear envelope i s covered with .nuclear ...pores 5 0 0 t O ' 6 0 0 - A i n diameter,, i r r e g u l a r l y d i s t r i b u t e d -being clustered ( 1 6 / um ) i n c e r t a i n areas of the nuclear membrane and sparse elsewhere (h/ urir^). The n u c l e o l i of a 0 . 1 mm oocyte are granular i n nature and consist of 1 5 0 to 2 0 0 A p a r t i c l e s . The structure of the l a r g e s t ( 2 0 um diameter) observed nucleolus (Figures 1 0 2 , lOh) i s r e t i c u l a r with the outer h \im being l e s s densely packed, thus giving the impression of a c t i v e d i s i n t e g r a t i o n . In the nucleoplasm betwen the n u c l e o l i and the nuclear membrane there are numerous electron dense p a r t i c l e s 1 5 0 to 2 0 0 R i n diameter. P a r t i c l e s of the same size range can also be observed i n the ooplasm opposite the nuclear pores. Another i n t e r e s t i n g phenomenon observed (Figure 103) i s a large ( 8 0 0 nm) n u c l e o l a r - l i k e body i n the ooplasm. The nuclear morphology of a 0 . 2 mm oocyte i s s i m i l a r (Figure 1 0 8 ) with the exception of a s l i g h t increase i n the number of smooth v e s i c l e s 80 101. Cross-section of a 0.1 mm oocyte demonstrating i t s r e l a t i o n s h i p to the other elements of the ovary, note p a r t i c u l a r l y the surface epithelium (SE), the tun i c a (TA) and a nucleolus (NU) within the nucleus of the oocyte. 1*50 X, 10 um har. 102. Nucleus of a 0.1 mm oocyte (Figure 101) showing the granular nature of the nucleolus (NU), the ribosomal p a r t i c l e s (R) and the pores (P) of the nuclear membrane. 1 pm -bar. 103. Ooplasm of a 0.1 mm oocyte with a n u c l e o l a r - l i k e body (NU-B) present amidst several G.V.B.s. 1 um bar. 8 0 A 81 10k. Nucleolus (NU) of a 0.1 mm oocyte i n d i c a t i n g i t s granular and r e t i c u l a r nature, i t s fragmentation and d i s p e r s a l of 150 k p a r t i c l e s as well as i t s extreme s i z e . 1 um bar. 81A next to the outer nuclear membrane. The smooth, even nuclear membrane of 0 . 3 mm oocytes (Figure 1 1 0 ) r e f l e c t s an i n c r e a s e i n , n u c l e a r volume. Nuclear pores are abundant ( 2 5 to 30 / pm^) and the nucleoplasm s t i l l c ontains 1 5 0 to 2 0 0 & p a r t i c l e s . ' By 0 . 8 mm diameter (Figures 1 2 8 , 1 2 9 ) the most notable change i n the nuclear envelope i s the frequency (50 t o 6 0 / ' um ) of the nuclear pores. They are approximately 6 0 0 t o TOO 8. i n diameter and have a c e n t r e - t o -centre spacing of 1 2 0 t o 1 8 0 nm. In the nucleoplasm, there are dense patches of a f i n e l y g ranular m a t e r i a l adjacent t o the pores but c l e a r areas between the pores. As i n the e a r l i e r stages the nucleoplasm contains abundant e l e c t r o n dense p a r t i c l e s ( 1 5 0 t o 2 0 0 R) which occur s i n g l y and i n c l u s t e r s . C l u s t e r s of s i m i l a r p a r t i c l e s are a l s o found i n the ooplasm.. At t h i s stage there i s a p e r i n u c l e a r .band devoid of smooth E.R. v e s i c l e s and c i s t e r n a e . The granular n u c l e o l i have i n c r e a s e d i n number, and range i n s i z e from 2 . 6 to 2 5 ym. At 1 . 0 mm, l i g h t microscopy (Figure 135) r e v e a l s the smooth o u t l i n e of the nucleus and i t s l a r g e s i z e ( 3 5 0 t o hOO um). W i t h i n the nucleo-plasm, s e v e r a l lampbrush chromosomes can be seen, as w e l l as 1 2 to 1 3 (per s e c t i o n ) n u c l e o l i 6 t o 30 \im i n diameter. C l u s t e r e d around the nucleus are numerous mitochondria and l i p i d v e s i c l e s and a s o l i t a r y stack of annulate l a m e l l a e (Figure 1 3 6 ) . In a 1 . 1 mm oocyte the u l t r a s t r u c t u r e of the nucleus i s v i r t u a l l y the same as i t was i n a 0 . 1 mm oocyte, the only d i f f e r e n c e s being the o v e r a l l increase i n nuclear diameter, the number of n u c l e o l i and the i r r e g u l a r i t y of the nuclear membrane (Figure l U 8 ) . The frequency of nuclear pores i s at l e a s t e q uivalent to t h a t of an 0 . 8 mm oocyte w i t h 83 1 0 5 . Central ooplasm of a 0 . 1 mm oocyte with the outer f r i n g e of a 9 to 1 0 ym mitochondrial cloud (MM) shown. Note also the abundance of smooth membrane bound v e s i c l e s within the cloud and i n the ooplasm. The large G.V.B. also shows some d i s s o l u t i o n of i t s contents. 1 ym bar. 1 0 6 . A portion of the mitochondrial mass (.MM) of a 0 . 1 mm oocyte demonstrating the intimate a s s o c i a t i o n of the smooth endoplasmic reticulum of the cloud with the mitochondria as well as the out l y i n g granular and granular-vesicular bodies (GVB). 1 ym bar. 1 0 7 . Central ooplasm of a 0 . 1 mm oocyte with extensive S.E.R. near the mitochondrial mass. Small arrowhead in d i c a t e s a myelin-like f i g u r e within a G.V.B.; note the expanded Golgi (G) lamellae. 1 ym bar. 83A 84 108. Nuclear region of a 0.2 mm oocyte with several pores i n the membrane and numerous v e s i c l e s (V) at the ooplasmic side of the membrane. Numerous ribosomes (R) and S.E.R. . are also present i n the perinuclear ooplasm. The mito-chondria (M) dis p l a y stretched (dumbbell) or c o n s t r i c t e d configurations. 1 ym bar. 109. Central ooplasm of a 0.2 mm oocyte with extensive S.E.R. i n v e s i c u l a r and tubular configurations, Golgi bodies (G) with expanded lamellae and mitochondria (M). The p a r a l l e l alignment of c r i s t a e and the s l i g h t l y biconcave center may indic a t e the mitochondrion i s beginning to divi d e . 1 ym bar. 84A almost every fragment of a v a i l a b l e space occupied. At high magnification (Figure 1 H 9 ) the n u c l e o l i of t h i s stage appear to be b i p a r t i t e , having intermingled granular and amorphous regions. THE'MITOCHONDRIAL MASS In a 0.1 mm oocyte the d i s t r i b u t i o n of mitochondria through the ooplasm i s sparse with the exception of a densely packed mass of mito-chondria (Figures 105, 1 0 6 ) located midway between the c e n t r a l nucleus and the v i t e l l i n e membrane. This "cloud" of mitochondria i s 9 to 10 ym i n diameter and consists of both mitochondria and v e s i c u l a r smooth E.R. The mitochondria range from 0.3 to 0 . 8 um i n diameter and have p l a t e - l i k e or tubular c r i s t a e . In some instances, the c r i s t a e a l i g n themselves i n c l o s e l y apposed rows and frequently appear to fuse together ,Thsi "cloud" p e r s i s t s .in...all• .-sizes ^ examined (.pr.e-vitellogenic) with increasing dispersion within the ooplasm. V a r i a t i o n s i n mito-chondrial morphology are also noted. In a 0.2 mm oocyte, the incidence of mitochondria within the ooplasm has r i s e n . In ad d i t i o n to the "normal mitochondria, several unusual configurations, are seen. One form of mitochondrion displays i t s c r i s t a e aligned i n long p a r a l l e l rows (Figure 109). The second (Figure 1 0 8 ) displays several forms of c o n s t r i c t i o n , i n one case the mitochondrion i s stretched extremely t h i n i n the centre and presents a "dumb-bell" shape. Another type of c o n s t r i c t i o n i s reminiscent of binary f i s s i o n of u n i c e l l u l a r organisms. In a 0.3 mm oocyte the mitochondria of the ooplasm d i s p l a y few unusual features, with the exception of an occasional ring-shaped mitochondrion (Figures 113, l l U ) . In a 0.5 mm ooctye the numbers of ooplasmic mitochondria have increased considerably over the 0.1 mm oocyte (Figures 123, 1 2 6 ) and branched mitochondria Occur (Figure 12k). By 0.8 mm the ooplasmic mitochondria are quite numerous but t h e i r d i s t r i b u t i o n i s not uniform and does not follow any p a r t i c u l a r pattern., The mitochondrial mass of.a 0.8 mm oocyte i s quite l a r g e , at l e a s t 1 5 to 20 ym i n diameter (Figure 130), and consists of polymorphic mitochondria and extensive smooth E.R. systems both v e s i c u l a r and c i s t e r n a l (Figures 131, 132, 133). There are, however, three basic types of mitochondria within the "cloud": r i n g , dumb-bell and normal. In a 1.0 mm oocyte the number of mitochondria i n the ooplasm i s s i m i l a r to that of 0 . 5 to 0.8 mm oocytes, however, the mitochondrial mass (Figures 137, 138) i s not as densely packed and the amount of S.E.R. has decreased. Within the mitochondria, membranous whorls can now be seen i n the matrix and they may or may not be continuous with the c r i s t a e . S i m i l a r whorls are also found i n the ..ooplasmic .mitochondria (-Figure lU-l) . No further ultrastructural observations were made on the mitochondrial mass but i n l i g h t microscopic observations t h i s structure was s t i l l present i n oocytes up to U.O mm i n diameter. At t h i s stage the mass had axes of 0 . 5 mm and 0.2 mm. LIPID YOLK In the e a r l i e s t oocytes examined there -is l i t t l e l i p i d yolk present, however, i n 0.3 to O.H mm oocytes numerous l i p i d droplets can be found (Figures 111, 112, 1 1 8 , 120, 121, 122). These droplets are densely osmiophilic, ovoid and lack a l i m i t i n g membrane. Their average si z e i s 2 um x 3 um although numerous smaller droplets are found, the l a t t e r e ither r e f l e c t i n g the angle of sectioning through a l a r g e r droplet or demonstrating the formation of a l i p i d droplet. These droplets are 87 110. Nucleus df a 0.3 mm oocyte with a smooth even memhrane and abundant nuclear pores (P). Ribosome-like p a r t i c l e s are found on eit h e r side of the nuclear membrane. 1 ym bar. 111. Cross-section of a 0.3 mm oocyte i n d i c a t i n g the r e l a t i o n -ship of the oocyte (0) with i t s band of l i p i d yolk (LY) to the v i t e l l i n e membrane (VM, small arrowhead), the abasement lamina (large-arrowhead) and the interposed f o l l i c l e c e l l l a y e r . Other symbols: L i = l i p i d found i n t e r c e l l u l a r l y i n the stroma, LC = l i g h t c e l l s , DC = dark c e l l s . 380 X, 10 ym bar. 11 2 . Cross-section of a 0.3 mm oocyte. High magnification. The i n t e r d i g i t a t i o n of the V.M. and the oocyte i s seen . as i s a thecal layer (T) and the basal lamina (large arrowhead). 608 X, 10 ym bar. 87A d i s t r i b u t e d i n the ooplasm (Figures 1 1 1 , 1 1 2 ) midway between the nucleus and the v i t e l l i n e membrane. Frequently, only the v e s i c l e s of the S.E.R. are observed i n close a s s o c i a t i o n with the l i p i d d roplets, but o c c a s i o n a l l y , mitochondria and/or Golgi are also found nearby. In 0 . 5 mm oocytes (Figures 1 2 5 , 1 2 7 ) the a s s o c i a t i o n of the l i p i d yolk droplets with v e s i c l e s and cisternae of S.E.R. i s more pronounced than, that of O.H mm oocytes. Infrequently, a Golgi body can be found near a droplet and i n one instance (Figure 123) a droplet was found surrounded by concentric rings of S.E.R. cisternae and numerous mitochondria. In 0 . 8 mm oocytes with the increased development of the mitochondrial mass (Balbiani body), the l i p i d drops c l o s e s t to t h i s body (Figures 132, 133) become more in t i m a t e l y associated with the mitochondria, however, the.-chains of S.E.R. v e s i c l e s are . s t i l l .the ..organelles most often associated with the l i p i d yolk droplets (Figures 133, 13H). In 1 . 0 and 1 . 1 mm oocytes, t h i s a s s o c i a t i o n of the l i p i d droplets with ooplasmic organelles i s markedly reduced. However, where smaller droplets are observed, the smooth-E.R. v e s i c l e s can s t i l l be found close at hand (Figures 1399 1 5 0 ) . Further u l t r a s t r u c t u r a l observations were hampered by inadequate f i x a t i o n , however, normal l i p i d yolk drops were observed i n 3 . 0 mm oocytes. These droplets were quite l a r g e , 3 u m x H u m t o 5 u m x 7 ym, and appeared to be b i p a r t i t e having a l e s s dense core. PROTEIN YOLK Unlike l i p i d yolk, the precursors of p r o t e i n yolk can be found i n 0 . 1 mm oocytes as multigranular (M.G.B.), m u l t i v e s i c u l a r (M.V.B.) and granular-vesicular bodies (G.V.B.). The multigranular bodies are the l e a s t numerous and are the smallest of the three types. These membrane-bound bodies have a c l e a r background, vary i n s i z e from 1 2 0 nm to 2 5 0 nm and may contain any number of e l e c t r o n dense granules (yolk granules) 1 0 0 to 5 0 0 A1 i n diameter (Figures 9 9 , 1 0 5 , 1 0 6 ) . Similar bodies ( i n t r a c i s t e r n a l granules) have also been found i n crustacean (Kessel, 1 9 6 8 ) and trout oocytes (Beams & Kessel, 1 9 7 3 ) . M u l t i v e s i c u l a r bodies are also found i n 0 . 1 mm oocytes (Figure 9 9 ) . They are structures of moderate s i z e ( 0 . 7 5 to 1 . 0 ym i n diameter) bound by a membrane that may or may not completely e n c i r c l e the body. Within t h i s body are numerous smaller ( 5 0 to 1 5 0 nm) membrane bound v e s i c l e s and a f i n e l y granular product. The t h i r d and most numerous form of p r o t e i n yolk precursor found i n 0 . 1 mm oocytes i s the granular-vesi c u l ar body (G.V.B.) (Figures 9 9 , ' 1 0 5 , ~ ' 1 0 6 ) . These-are the l a r g e s t of Jthe three'-types ( l to 2 ym i n diameter) and contain the same structures found i n the M.V.B.s and M.G.B.s and i n addition, f r a c t i o n s of broken or d i s s o l v i n g v e s i c u l a r membranes that resemble myelin figures (Figures 9 8 , 9 9 > 1 0 7 ) . M y e l i n - l i k e f i g u r e s have also been noted i n the protein yolk precursors of Rana esculenta & Rana  temporaria (Kress & Spornitz, 1 9 7 2 ) , Ciona i n t e s t i n a l i s (Kessel, 1 9 6 6 ) and T r i t u r u s (Spornitz & Kress, 1 9 7 3 ) . The G.V.B.s are more ele c t r o n dense than either the M.G.B.s or the M.V.B.s'due to the d i s s o l u t i o n of the v e s i c l e s and granules within the body and t h e i r subsequent recondensation as a homogeneously granular matrix (Figures 9 8 , 9 9 , 1 0 5 , 1 0 6 ) . M u l t i -v e s i c u l a r bodies which correspond to the M.V.B.s and G.V.B.s described herein are also reported i n many other oocytes (Rana pipiens & Xenopus, Spornitz & Kress, 1 9 7 1 ; Oryzias, Yamamoto, I 9 6 H ; Ciona, Kessel, I 9 6 6 ; Norrevang, 1 9 6 8 ; Prostheceraeus f l o r i d a n u s , Boyer, 1 9 7 2 ; T r i t u r u s , Spornitz & Kress, 1 9 7 3 ) . The oocytes of t h i s s i z e ( 0 . 1 mm) possess extensive cisternae of ves i c u l a r smooth E.R., many of the v e s i c l e s being l a r g e r ( 2 5 0 nm) than the ones found within the G.V.B.s ( 1 0 0 nm). Also, there are many smaller S.E.R. v e s i c l e s associated with the various yolk precursor bodies (Figures 1 0 5 , 1 0 6 ) and others that contain yolk granules. Thus, one source of material f o r the G.V.B.s i s from the S.E.R. Further, there are other large S.E.R.-like bodies (300 to 6 0 0 nm) that are formed by repeated fusion with the smaller S.E.R.-M.G.B.s and therefore represent e a r l y stages i n the formation of a p r o t e i n yolk precursor body (Figures 9 9 » 1 0 6 ) . There are several other sources of small v e s i c l e s f o r the yolk precursor bodies, f i r s t l y from the nuclear membrane as previously described (pp. 7 9 and 8 2 & Figures 9 8 , 1 0 0 ) and secondly from the Golgi•apparatus. The -small Golgi v e s i c l e s ( 5 0 0 to 7 0 0 K) -are s i m i l a r i n s i z e to the v e s i c l e s found within the M.V.B.s and the G.V.B.s (Figures 1 0 6 , 1 0 7 ) and are frequently seen i n intimate a s s o c i a t i o n with a developing protein yolk precursor (Figure 1 0 6 ) . Occasionally, one or more of the cen t r a l lamellae of a dictyosome are expanded (Figure 1 0 7 ) which may indica t e a tang e n t i a l sectioning plane or the formation of a yolk precursor v e s i c l e . F i n a l l y , v e s i c l e s may be derived from pinocytosis which i s d i f f i c u l t to substantiate as p i n o c y t o t i c v e s i c l e s are i d e n t i c a l i n appearance to v e s i c l e s from other sources. In a 0 . 2 mm oocyte small v e s i c l e s associated with the nuclear membrane are p l e n t i f u l (Figure 1 0 8 ) . As i n the 0 . 1 mm oocytes, ribosomes are only t h i n l y dispersed i n the ooplasm. Smooth E.R. i s abundant and the Golgi bodies are s t i l l producing large and small v e s i c l e s (Figure 1 0 9 ) . At 0 . 3 mm most of the granular-vesicular bodies have become more electron dense and possess f i n e l y granular or dense cores (Figures 113, l l H , 115). Other G.V.B.s can he seen int i m a t e l y associated with v e s i c l e s and small granular bodies and often t h e i r l i m i t i n g membranes are broken or h ighly i r r e g u l a r (Figures l l H , 115, 1 1 6 ). The S.E.R. i s s t i l l abundant but o c c a s i o n a l l y long c h a i n - l i k e forms are found (Figures 113, 1 1 6 ) . The mitochondria from the mitochondrial cloud are beginning to spread throughout the ooplasm and Golgi bodies, when seen, are associated with mitochondria, a G.V.B. and the long tubules of S.E.R. (Figure l l 6 ) . This i s a pattern that i s s t i l l evident i n l a r g e r oocytes. In a d d i t i o n to the well developed G.V.B.s of the 0.3 mm dogfish oocyte there are also numerous new M.V.B.s and G.V.B.s forming (Figure 1 1 7 ) . . In some instances these developing bodies consist p r i m a r i l y of myelin figures while others are t y p i c a l M.G.B.s, M.V.B.s and G.V.B.s. By O.H.mm, l i p i d yolk bodies are-well•developed. Their a s s o c i a t i o n with the S.E.R., mitochondria and Golgi has been noted previously. Also, G.V.B.s i n various stages of development are often found i n close a s s o c i a t i o n with the l i p i d yolk (Figures 1 1 8 , 1 1 9 , 1 2 1 ) . Occasionally, a forming G.V.B. can be detected without a l i m i t i n g membrane or with a p a r t i a l one but i n such cases there i s u s u a l l y a c l u s t e r of S.E.R. v e s i c l e s w i t h i n 7 0 0 nm distance (Figures 1 1 9 , 1 2 1). The growth of the G.V.B. by fu s i o n of smaller v e s i c l e s with the l i m i t i n g membrane (Figure 1 1 8 ) and the incorporation of granular and membranous elements through G.V.B. membrane d i s c o n t i n u i t i e s (Figure 1 2 1 ) can s t i l l be demonstrated. In O.H mm oocytes, new configurations of S.E.R. (Figures 1 1 8 , 1 2 1 ) are seen: the S.E.R. appears as an elongated c i s t e r n a with expanded ends which are normally devoid of any contents and as such are reminiscent of ce r t a i n of the large v e s i c u l a r elements that contain the granules 92 113. Ooplasm of a 0.3 mm oocyte w i t h a w e l l developed g r a n u l a r - v e s i c u l a r body (GVB), s e v e r a l m itochondria, one ring-shaped mitochondrion (M) and s e v e r a l c h a i n -l i k e strands of S.E.R. 1 um bar. l l H . Ooplasm of a 0.3 mm oocyte demonstrating i n i t i a l coalescence of the G.V.B.s and the i r r e g u l a r or broken nature o f the l i m i t i n g membrane ( l a r g e arrowheads). A ring-shaped.mitochondrion . (M) i s a l s o shown. .1 ym bar. 115. Ooplasm of a 0.3 mm oocyte demonstrating v a r i o u s stages i n the formation of a G.V.B. Large arrowheads i n d i c a t e the p a r t i a l absence of l i m i t i n g membranes. 1 ym bar. 92A Ooplasm of a 0 .3 mm oocyte revealing developing G.V.B.s, mitochondria (M), a Golgi body (G) and S.E.R. tubules.-Arrow indicates a break i n a G.V.B. membrane for the uptake of more ma t e r i a l . Small arrowheads i n d i c a t e the presence of material within the S.E.R. of s i m i l a r s i z e to that found i n the G.V.B.s 1 ym bar. Ooplasm of a 0 . 3 mm oocyte i n a region of forming m u l t i v e s i c u l a r and granular-vesicular bodies. Note p a r t i c u l a r l y the frequency of myelin-like figures within the bodies. 1 ym bar. 93A 94 118. Ooplasm of a O.h mm oocyte with numerous l i p i d yolk (LY) droplets and c l o s e l y associated S.E.R., note the expanded cisternae of S.E.R., Also evident are numerous pro t e i n yolk precursor bodies often with incomplete l i m i t i n g membranes and fusing v e s i c l e s (large arrowhead). The arrow indicates the granular and v e s i c u l a r nature of a G.V.B. 1 ym bar. 119. Ooplasm of a O.h mm oocyte c l e a r l y demonstrating the lack of a l i m i t i n g membrane about the l i p i d yolk bodies. Also featured i s a developing G.V.B. with i t s v e s i c u l a r and granular (large arrowheads) composition. 1 ym bar. 94A 9 5 120. Ooplasm of a O.H mm oocyte with a small l i p i d droplet surrounded by v e s i c l e s of S.E.R. Also present are a few mitochondria and a G.V.B. 1 ym bar. 121. Ooplasm of a O.H mm oocyte showing a large l i p i d droplet with S.E.R. and a G.V.B. nearby. Note e s p e c i a l l y the incomplete membranes around the developing G.V.B.s ("large arrowheads') and the expanded ends df the S.E'.R. cisternae. 1 ym bar. 122. Ooplasm of a O.H mm oocyte i n a region of numerous l i p i d yolk droplets that surround a complex Golgi body, a few mitochondria and some S.E.R. v e s i c l e s . 1 ym bar. 95A A l i p i d yolk droplet i n a 0.5 mm oocyte that i s surrounded hy concentric rings of•S.E.R. and numerous mitochondria. 1 ym bar. A branching mitochondrion found i n a 0.5 mm oocyte, configuration taken to i n d i c a t e m u l t i p l i c a t i o n of mitochondrial numbers. 1 urn, bar. L i p i d yolk p a r t i c l e s i n a 0.5 mm oocyte demonstrating t h e i r a s s o c i a t i o n with S.E.R. v e s i c l e s . Note also the developing G.V.B.s and t h e i r incomplete membranes. 1 ym bar. 96A 97 126. Two large G.V.B.s i n a 0.5 mm oocyte c l e a r l y i n d i c a t i n g the membranous and granular elements of which they are composed. Also of i n t e r e s t are the v e s i c l e s apposed to the G.V.B. that contain s i n g l e granules. The smaller G.V.B.s are more densely packed and one of them has nearly completed the d i s s o l u t i o n and recondensation of i t s contents (large arrowhead). The arrows i n d i c a t e chains of S.E.R. v e s i c l e s . 1 ym bar. 127. Several large l i p i d yolk droplets i n a 0.5 mm oocyte c l e a r l y demonstrating the lack of a l i m i t i n g membrane as w e l l as the intimate r e l a t i o n s h i p with the S.E.R. 1 ym bar. 97A 98 128. The nuclear membrane of a 0.8 mm oocyte i n d i c a t i n g the increased frequency of nuclear pores as w e l l as the presence of 150 K p a r t i c l e s on both sides of the membrane. In the ooplasm i s a large S.E.R. v e s i c l e at the hub of several S.E.R. chains. Other symbols: N = nucleus, P = nuclear pores, R = 150 & ribosomal p a r t i c l e s . 1 ym bar. 129. A small nucleolus i n the nucleus of a 0.8 mm oocyte. Note that the electron dense p a r t i c l e s i n the nucleo-plasm are the same size as those found within the nucleolus. l'ynT'bar. 130. A segment of the mitochondrial mass of a 0.8 mm oocyte with S.E.R. cisternae intermingled with the poly-morphic mitochondria. 1 ym bar. 98A of a M.G.B. or a G.V.B. (Figure 1 1 8 ) . In a 0 . 5 mm oocyte the pro t e i n yolk precursors are much the same as i n a l l previous stages (Figures 1 2 5 , 1 2 6 ) . There are, however, very dense precursor bodies that have nearly completed d i s s o u l u t i o n and recondensation of t h e i r granular and membranous components as well as many large ( 3 . 2 ym) G.V.B.s. In a 0 . 8 mm oocyte small M.G.B.s ( . 2 0 0 to H-50 nm) are found scattered throughout the S.E.R. of the mitochondrial cloud (Figure 131). Developing G.V.B.s are found i n the immediate v i c i n i t y of most l i p i d droplets (Figures 1 3 3 , 13*0 with mitochondria nearby. The chains of S.E.R. v e s i c l e s that are associated with the l i p i d droplets are also associated with the developing G.V.B.s (Figure 1 3 3 ) . Near the nucleus (Figure 1 2 8 ) are several chains of S.E.R. with expanded v e s i c l e s s i m i l a r to those .described i n O-.h '•mm .oocytes. In 1 . 0 mm oocytes the formation or development of p r o t e i n yolk precursors (G.V.B.s) follows several pathways. F i r s t l y , the pattern of mitochondria, G o l g i , S.E.R. chains and G.V.B.s p e r s i s t s (Figures lko, lUl) with the Golgi providing v e s i c u l a r elements and the S.E.R. providing v e s i c u l a r and granular elements (Figures 1 3 9 , l U l ) . Secondly, there often e x i s t s large masses of v e s i c u l a r S.E.R., M.G.B.s, M.V.B.s and G.V.B.s (Figure lh2). Within these masses can be seen the addi t i o n of free v e s i c l e s , granules and small M.G.B.s to the G.V.B.s v i a discon-t i n u i t i e s i n the l i m i t i n g membrane and the fusion of moderately sized ( 5 0 0 to 6 0 0 nm) M.G.B.s to y i e l d a l a r g e r body and the incorporation of small v e s i c l e s ( 5 0 to 1 0 0 nm) into a large (l.U to 1 . 7 ym) G.V.B. by membrane to membrane contact. A t h i r d p o s s i b l e source of large v e s i c l e s ( 5 0 0 to 9 0 0 nm) f o r protein yolk precursors may be from a G o l g i - l i k e 100 131. A segment of the mitochondrial mass of a 0 . 8 mm oocyte with large quantities of S.E.R. present as well as numerous small multigranular todies (large arrowheads). 1 ym bar. 132. Polymorphic mitochondria i n the ooplasm of a 0 . 8 mm oocyte with numerous S.E.R. cisternae and a l i p i d yolk p a r t i c l e . Other symbols: L = l i p i d , D = dumbbell-shaped mitochondrion, R = ring-shaped mito-chondrion. 1 ym bar. 133. A ring-shaped mitochondrion i n the ooplasm of a 0 . 8 mm oocyte with numerous tubules of S.E.R. coursing about and c l o s e l y associated with both the l i p i d and the developing G.V.B.s (large arrowheads). 1 ym bar. 13k. Numerous l i p i d yolk p a r t i c l e s i n the ooplasm of a 0 . 8 mm oocyte with S.E.R. cisternae around the p a r t i c l e s . Note also the highly granular G.V.B. 1 ym bar. 100A 101 135. A lyum t h i c k cross-section of a 1.0 mm oocyte demonstrating the lampbrush chromosomes and numerous (NU) n u c l e o l i within the nucleus (N). 100 um bar. 136. A s o l i t a r y stack of annulate lamellae found within a 1.0 mm oocyte near the nucleus. 1 um bar. 137- A portion of the mitochondrial mass of a 1.0 mm oocyte. 1 um bar. 1 0 1 A 102 138. A segment of the mitochondrial mass of a 1.0 mm oocyte that now appears l e s s t i g h t l y packed, but now has numerous myelin-like figures within the mitochondria. 1 ym bar. 139. A granular-vesicular body i n the ooplasm of a 1.0 mm oocyte showing the accumulation of a f l u f f y component and the attachment of an E.R. c i s t e r n a containing several granules. S.E.R. .is s t i l l ,found .associated with l i p i d yolk. 1 ym bar. ikb. The ooplasm of a 1.0 mm oocyte showing the frequently-observed r e l a t i o n s h i p of mitochondria, G o l g i , G.V.B.s and S.E.R. (small arrowheads). 1 ym bar. 102A body (Figure 1 ^ 3 ) . Similar clea r v e s i c l e s with yolk granules have been found elsewhere i n smaller oocytes e.g. Figures l H l , lhk. A f i n a l p o s s i b l e source of the l i m i t i n g membrane f o r a p r o t e i n yolk precursor body are the chains o f S.E.R. v e s i c l e s . These chains or tubules with occasional expansions along t h e i r length course throughout the oocyte (Figure 1 H 5 ) often looping back on themselves. When t h i s occurs (Figure ihk) ,- the membranes delineate an area l e s s electron dense than the surrounding ooplasm but of s i m i l a r density to membrane bound bodies l y i n g nearby. The presence of yolk granules within these bodies and within the v e s i c l e s of the S.E.R. in d i c a t e that these v e s i c l e s may be one of several sources of yolk body precursors. Another source of v e s i c l e s and the material they are transporting to the yolk precursor bodies i s from the oocyte membrane v i a pinocytosis (Figures l U 6 , 1 ^ 7 ) . But these vesieles-sshow.no d i s t i n g u i s h i n g features and thus cannot be d i f f e r e n t i a t e d from v e s i c l e s a r i s i n g from the G o l g i , the nuclear membrane or the S.E.R. In t h i s region near the oocyte-V.M. border are numerous l i n i n g body complexes and streams of ele c t r o n dense matter that has d i f f u s e d through the oocyte membrane. In 1 . 0 mm oocytes, ribosomes are not numerous but are found more frequently than i n e a r l i e r oocytes and normally occur as polyribosomes (Figures lk2t ihk, 1 U 5 ) . In oocytes 1 . 1 mm i n diameter two methods of p r o t e i n yolk precursor formation are in d i c a t e d . F i r s t , there are numerous c l e a r v e s i c l e s p o s s i b l y derived from expanded S.E.R. cisternae (O.U mm oocytes) or from a G o l g i - l i k e body ( 1 . 0 mm oocytes) that possess e i t h e r granules (M.G.B.s) or granules and membranous elements (G.V.B.s) (Figures 1.51, 1 5 1 i , 1 5 2 , 1 5 3 ) . Secondly, there i s further evidence that the S.E.R. An unusual section of a Golgi body (G) i n a 1 . 0 mm oocyte, with a myelin-like f i g u r e i n a dumbbell mito-chondrion. Also present i s a multigranular body (GB) and several chains of S.E.R. tubules that are beginning to loop and also contain granules. 1 ym bar. A large mass of pro t e i n yolk precursor bodies demonstrating the addi t i o n of free v e s i c l e s , granules and small M.G.B.s to the G.V.B.s v i a membrane (arrowheads) . d i s c o n t i n u i t i e s . .The ...large ..central .G..V..-B. ..i*s ;<als.o undergoing d i s s o l u t i o n and recondensation of i t s contents. 1 ym bar. One of many G o l g i - l i k e bodies found i n 1 . 0 mm oocytes, note the expanded lamellae. 1 ym bar. 104A — 143 Two d i f f e r e n t p r o t e i n yolk precursor systems i n a 1.0 mm oocyte: i n one, multigranular bodies (GB) are formed within a membrane bound v e s i c l e that i s eit h e r translucent or opaque; i n the second, chains or tubules of S.E.R. loop on themselves d e l i n e a t i n g an area of s i m i l a r density to that of the opaque G.B., Note also the occurence of granules within the S.E.R. cisternae and a few polyribosomes. 1 ym bar. Extensive S.E.R.. membrane systems beneath the V.M. of a 1.0 mm oocyte that eminate or terminate i n expanded cisternae (EC) s i m i l a r to the v e s i c l e s of c e r t a i n p r o t e i n yolk precursor bodies. Other symbols: M = mitochondria, R = ribosomes. 1 ym bar. Numerous pin o c y t o t i c v e s i c l e s (PV) beneath the plasma membrane of a 1.0 mm oocyte. Note also the paths or streams of electron dense material amidst the P.V. and the l i n i n g bodies (LB). 1 ym bar. The highly convoluted plasma membrane of a 1.0 mm oocyte demonstrating the formation of pi n o c y t o t i c v e s i c l e s (PV and small arrowheads). Other symbols: LB = l i n i n g body. 1 ym bar. 105A 106 lkQ. The nuclear membrane of a 1 . 1 mm oocyte studded with numerous pores (P). The nucleus (N) i s f i l l e d with many polyribosome-like f i g u r e s . Other symbols: 0 = oocyte. 1 um bar. 1 ^ 9 . Two n u c l e o l i (NU) within the nucleus of a 1 . 1 mm oocyte., that are b i p a r t i t e with amorphous (A) and granular (G) regions. The arrow indicates the fragmentation of the nucleolus producing polyribosomal figures., (R). 1 um bar. 1 5 0 . Two l i p i d yolk (LY) p a r t i c l e s i n a 1 . 1 mm oocyte int i m a t e l y associated with the S.E.R. Note the large v e s i c l e (upper arrowhead) with a few granules i n i t and the c i r c l e of S.E.R. forming a v e s i c l e (lower arrowhead). 1 um bar. 106A 107 chains e n c i r c l e an area of ooplasm which then may become l e s s e l e c t r o n dense and evolve into a G.V.B. (Figures 1 5 0 , 1 5 2 , 1 5 3 ) . The S.E.R. v e s i c l e s that are to form the l i m i t i n g membrane may or may not contain granules to be added to the G.V.B. The f i r s t step i n the transformation i s the d i s s o l u t i o n of the ends of the v e s i c l e s followed by the end to end fusion of adjoining membranes which i s presumed due to the breaks i n the l i m i t i n g membrane of other e a r l y G.V.B.s (Figures 1 5 2 , 1 5 3 , 1 5 * + ) . This transformation, however, requires the l o s s of one h a l f of the ve s i c u l a r membrane eit h e r by incorporation i n t o the l i m i t i n g membrane, addition to the G.V.B. as one of the many membranous fragments, or complete d i s s o l u t i o n . Following the formation of the membrane, the body then begins to acquire the yolk granules and membranous elements c h a r a c t e r i s t i c of a G.V.B. with a f i n e l y granular matrix (Figures 1 5 3 , 1 5 *0 . Polyribosomes are s t i l l only sparsely d i s t r i b u t e d (Figures 1 5 0 . , 1 5 2 , 1 5 5 , 1 5 6 ) and when observed often resemble free yolk granules. Another i n t e r e s t i n g feature of oocytes i n t h i s s i z e range i s the r e l a t i o n s h i p of the S.E.R. chains with the mitochondria, Golgi and the l i n i n g bodies. The general proximity of the S.E.R. and the mitochondria notwithstanding, membrane contact between the two organelles i s often observed (Figures 1 5 6 , 1 5 7 ) . Further, the S.E.R. tubules have also been observed i n d i r e c t c o n t i n u i t y with the v e s i c l e s of a Golgi body (Figure 1 5 5 ) and with a granular product i n the cist e r n a e s i m i l a r to that found i n many of the smaller ( 5 0 0 to 7 0 0 K) Golgi v e s i c l e s . Membrane to membrane contact between the S.E.R. tubules and the l i n i n g bodies has also been observed (Figures 1 5 6 , 1 5 7 ) . The l i n i n g bodies as previously described are f o l l i c l e c e l l processes and one can r e a d i l y surmise the transfer of some f o l l i c l e c e l l product to the S.E.R. of the oocyte v i a 108 1 5 1 . Several l a r g e , translucent v e s i c l e s (GB, GVB) i n a 1 . 1 mm oocyte that contain granular and granular and v e s i c u l a r elements. 1 ym bar. 1 5 2 . A large G.V.B. i n a 1 . 1 mm oocyte l y i n g beside a forming v e s i c l e of s i m i l a r s i z e and density. The l a t t e r appears to be forming from a small granular body (GB) and a loop of -S.'E.R. that contains granules (large-arrowhead). 1 um bar. 1 5 3 . A forming granular-vesicular body (GVB) i n a 1 . 1 mm oocyte. Alongside i s a pooriy developed S.E.R. loop (arrowhead) that e n c i r c l e s several granules (GR) and an area of s i m i l a r density to that of the G.V.B. 1 um bar. 108A 109 1 5 1 * . A large protein yolk precursor (GVB) i n a 1 . 1 mm oocyte. Its outer l i m i t i n g membrane i s incomplete allowing incorporation of granular and v e s i c u l a r elements. S.E.R. v e s i c l e s are seen i n contact with the l i m i t i n g membrane. 1 ym bar. 1 5 5 . Two of several Golgi (G) bodies found i n a 1 . 1 mm oocyte with small dense v e s i c l e s (large arrowheads) approaching the forming face of the body. Of p a r t i c u l a r i n t e r e s t i s the r e l a t i o n s h i p of a strand of S.E.R. with the forming face of the body. Other symbols: M = ring-shaped mito-chondrion, R = polyribosomes. 1 ym bar. 1 5 6 . In the region of the oocyte plasma membrane of a 1 . 1 mm oocyte showing the fibrous v i t e l l i n e membrane (VM), several p i n o c y t o t i c v e s i c l e s (PV) and a few l i n i n g bodies (LB). Also evident i s the attachment of the S.E.R v e s i c l e s to a mitochondrion (large arrowhead) and to a l i n i n g body. 1 ym bar. 1 5 7 - The c o r t i c a l region of a 1 . 1 mm oocyte, with numerous l i n i n g bodies (LB), p i n o c y t o t i c v e s i c l e s (PV), smooth E.R. tubules and mitochondria. The r e l a t i o n s h i p of the S.E.R. with the mitochondria and l i n i n g bodies i s again evident (large arrow-heads). Also found between the oocyte plasma membrane and f o l l i c l e c e l l plasma membrane ( the l i n i n g body complex) are numerous h00 to 5 0 0 & granules. 1 ym bar. 1 0 9 A 110 the l i n i n g body. In Figure 1 5 7 , note the numerous v e s i c l e s c l u s t e r e d about the base of the l i n i n g body and the hOO to 5 0 0 & granules i n the i n t e r c e l l u l a r space of the l i n i n g body. Figures 1 5 6 & 1 5 7 are of the c o r t i c a l region of a 1 . 1 mm oocyte and i n them one can observe numerous p i n o c y t o t i c v e s i c l e s and the occasional coated v e s i c l e . The l a t t e r resemble the coated v e s i c l e s of cockroach (Anderson, 1 9 6 H ) and mosquito (Roth & Porter, I 9 6 H ) oocytes as they take up yolk v i a p i n o c y t o s i s . . By 2 . 0 mm diameter, some of the primary yolk precursors (G.V.B.s) have formed dense cores and resemble t y p i c a l p r o t e i n yolk p l a t e l e t s ( F i gure 1 5 8 ) . That i s , they have an outer l i m i t i n g membrane, a l i g h t matrix often with v e s i c l e s or granules, and a dense core (Kessel, 1 9 6 8 ; N^rrevang, 1 9 6 8 ; Massover, 1 9 7 1 ; Spornitz & Kress, 1 9 7 1 , 1 9 7 3 ; Dumont, 1 9 7 - 2 ; Kress ,& Spornitz, 1 9 7 2 ) . .A c r y s t a l l i n e . l a t t i c e pattern was not observed i n yolk p l a t e l e t s of 2 . 0 mm oocytes. In 3 . 0 and h.O mm oocytes yolk p l a t e l e t s with a c r y s t a l l i n e l a t t i c e pattern can be found (Figures 1 5 9 , l 6 0 , l 6 l ) . Thus the t r i p a r t i t e p l a t e l e t described f o r 2 . 0 mm oocytes has changed only i n that the dense core now shows a c r y s t a l l i n e pattern with a p e r i o d i c i t y of 9 0 to 1 0 0 R. I t i s the presence of t h i s c r y s t a l l i n e pattern that defines these organelles as yolk p l a t e l e t s (Massover, 1 9 7 1 ) . Further, the complete c r y s t a l l i z a t i o n of the core, i n amphibian oocytes, i s i n t e r p r e t e d as i n d i c a t i n g a mature yolk p l a t e l e t (Karasaki, 1 9 & 3 ; Spornitz & Kress, 1 9 7 1 , 1 9 7 3 ; King et a l , 1 9 7 2 ) . The centre to centre p e r i o d i c i t y of dogfish yolk p l a t e l e t s i s s i m i l a r to that of v e s i c u l a r yolk p l a t e l e t s of Rana catesbiana ( 8 5 A; Massover, 1 9 7 1 ) , Rana esculenta and R. temporaria ( 9 5 Kress & Spornitz, 1 9 7 2 ) and T r i t u r u s v u l g a r i s ( 1 1 0 A1; Spornitz & Kress, 1 9 7 3 ) . At no. time P r o t e i n y o l k p l a t e l e t (PYP) formation i n a 2.0 mm oocyte. By t h i s stage, the contents of a G.V.B. have d i s s o l v e d and reformed as a dense core (CO) bound by t h e i r outer l i m i t i n g membrane (LM). Between the L.M. and the core i s a l i g h t m atrix i n which granules and v e s i c l e s are s t i l l e v i dent. 1 um bar. A p r o t e i n y o l k p l a t e l e t i n a 3.0 mm oocyte w i t h i t s outer l i m i t i n g membrane and a 90 t o 100 8. c r y s t a l l i n e l a t t i c e p a t t e r n i n i t s core (arrows). 1 ym bar. 111A Two protein yolk p l a t e l e t s found i n a H.O mm oocyte. The outer l i m i t i n g membrane (LM) of the p l a t e l e t and i t s c r y s t a l l i n e core (arrow) are quite evident. 1 ym bar. 112A 113 ' were intramitochondrial yolk platelets similar to those of certain amphibia .observed (Massover, 1971). DISCUSSION HISTOCHEMISTRY  OOCYTE Fi x a t i o n and embedding d i f f i c u l t i e s r e s t r i c t e d t h i s study to oocytes l e s s than 5 mm i n diameter thus r e s t r i c t i n g any discussion of yolk formation i n Squalus acanthias to the e a r l i e s t stages (Raven, 1 9 6 1 ) . C y t o l o g i c a l l y (lampbrush chromosomes, numerous n u c l e o l i ) these oocytes were found to be i n mid- to l a t e diplotene of the f i r s t meiotic d i v i s i o n . The presence of n u c l e o l i i n the germinal v e s i c l e i n d i c a t e s rRNA synthesis (Davidson et a l , I 9 6 H ; Brown, 1 9 6 6 ; Van Gansen & Schram, 1 9 7 2 ) which was substantiated by RNase treatment. The r e a c t i o n of the n u c l e o l i with aldehyde fuchsin i n d i c a t i n g sulphated mucopolysaccharides i s unexpected, <but .•Stich ( 1 9 5 1 ) -found• -polysaccharides -in the •oocyte'•'nucleoli of A s c a r l s , Cyclops and Diaptomus while Yamamoto ( 1 9 5 6 b ) found them i n the e a r l y oocytes of Liopsetta„ The cytoplasm of the oocyte presents no unexpected r e s u l t s s t a i n i n g for p r o t e i n , RNA and phospholipids. The vacuoles present i n p a r a f f i n sections are l i p i d i n nature as demonstrated by the intense Sudan black r e a c t i o n . Mustelus canis (TeWinkel, 1 9 7 2 ) oocytes, 0 . 2 5 to 3 . 0 mm, also give a p o s i t i v e r e a c t i o n f or l i p i d s with O i l red 0 . The ooplasm of early Xenopus oocytes (Dumont, 1 9 7 2 ) also stains f o r protei n , RNA and l i p i d . A polysaccharide r e a c t i o n that does not occur, i n Squalus was noted i n Xenopus oocytes. The mitochondrial mass (yolk nucleus) which has been observed i n young oocytes of arachnids (van Bambeke, 1 8 9 8 ) through to t e l e o s t s ( L i v n i , 1 9 7 1 ) i s r i c h i n p r o t e i n , RNA, and phospholipids. This corresponds to the findings of both Raven ( 1 9 6 1 ) and Dumont ( 1 9 7 2 ) as well as Guraya ( 1 9 6 8 ) who reviewed the histochemistry of yolk n u c l e i . E l e c t r o n micrographs (Figures 1 0 6 , 130, 137) reveal that t h i s structure i s composed of densely packed mitochondria and i n keeping with the terminology of Ward ( 1 9 6 2 ) , Balinsky & Devis ( 1 9 6 3 ) and Dumont ( 1 9 7 2 ) i t i s r e f e r r e d to as a mitochondrial mass or cloud. -VITELLINE MEMBRANE The v i t e l l i n e membrane occupies an area between the ooplasm and the f o l l i c l e c e l l s and i t i s generally conceded that i t i s formed from the oocyte (Raven, 1 9 6 1 ) . This point, however, cannot be resolved with l i g h t optics and w i l l be dealt with l a t e r . Histochemically, the V.M. reacts only f o r the presence of pr o t e i n and polysaccharides or neutral muco-proteins. The aldehyde fuchsin r e a c t i o n can be regarded as non - s p e c i f i c . The presence of pr o t e i n and polysaccharide e i t h e r together or by themselves has :been demonstrated by Yamamoto , ( l 9 - 5 6 b ) -in .Liopset.ta; Arndt ( 1 9 6 0 b ) i n cyprinoids; Tandler ( 1 9 5 7 ) , Franchi ( i 9 6 0 ) and Wartenberg & Stegner ( i 9 6 0 ) i n the zona r a d i a t a of human eggs and Dumont ( 1 9 7 2 ) i n Xenopus. Dumont notes that the re a c t i o n i s f i r s t most intense f o r polysaccharides, later- giving way to protein with a s l i g h t polysaccharide component. B e l l a i r s et a l ( 1 9 6 3 ) studied the composition of the avian v i t e l l i n e membrane and found i t to be a glycoprotein containing hexoses, hexosamines and s i a l i c a c i d . The presence of s i l a i c a c i d i n large q u a n t i t i e s i n the V.M. of Squalus i s doubtful since i t d i d not give a p o s i t i v e r e a c t i o n with a l c i a n blue, Hale's or t o l u i d i n e blue. FOLLICLE CELLS The t r a n s f e r of a l i p o p r o t e i n precursor molecule from the blood to the f o l l i c l e c e l l to the oocyte has been well documented i n the domestic hen ( F l i c k i n g e r & Rounds, 1 9 5 6 ; Mclndoe, 1 9 5 9 ; Schjeide et a l , 1 9 6 3 & 1 9 7 0 ; Husbands, 1 9 7 0 ; C h r i s t i e & Moore, 1 9 7 2 ; Gormal & Kuksis, 1 9 7 3 ) , i n Xenopus (Wallace, Jared & Nelson, 1 9 7 0 ) and i n an ins e c t ( T e l f e r & Melius, 1 9 6 3 ) . . In view of t h i s , the l i p i d droplets seen t r a v e r s i n g the V.M. i n 'Squalus may he considered a p h y s i c a l manifestation of a s i m i l a r phenomenon. Supporting t h i s previous statement i s the presence of dense l i p i d droplets i n the a p i c a l region of the f o l l i c l e c e l l s , an area that had previously remained unstained, and free l i p i d droplets located i n and external to the theca. Bonhag ( 1 9 5 5 a , b, 1 9 5 6 , 1 9 5 8 ) also found that i n i n s e c t s l i p i d i s transferred to the oocyte from f o l l i c l e c e l l s and trophocytes. The presence of phospholipid i n the f o l l i c l e c e l l s i s in d i c a t e d as i s the presence of some protein and RNA. However, there i s no morphologically apparent t r a n s f e r of t h i s RNA to the oocyte as i n the housefly and Drosophila (Bier, 1 9 6 3 ) . F o l l i c l e c e l l s are ^generally r i c h 'in RNA (Raven., 1 9 6 1 ) -a .condition that would point to a synthetic a c t i v i t y . The demonstration of RNA i n the f o l l i c l e c e l l s of Squalus d e f i n i t e l y i n d i c a t e s some form of proteinaceous synthetic a c t i v i t y . The presence of sulphated mucopolysaccharides (Chondroitin sulphate A or C) i n the f o l l i c l e c e l l s may be explained as the reaction of precursor molecules for the synthesis of the f o l l i c u l a r basement membrane. One would then expect a metachromatic response from Azure A (pH 1 . 5 ) but t h i s does not occur. Instead there i s a s l i g h t orthochromatic response that i s only s l i g h t l y l a b i l e to methylation-sa p o n i f i c a t i o n and hyaluronidase i n d i c a t i n g that there are precursor molecules for the basement lamina present but the a c i d i c groups of the polymeric molecules are not c l o s e l y packed. This would account for the orthochromatic response (Sylven, 1 9 5 ^ ; Kvist & Finnegan, 1 9 7 0 ) . BASEMENT LAMINA-LAMELLA COMPLEX The f o l l i c u l a r basement membrane (F.B.M.) i s a unique str u c t u r e . TeWinkel ( 1 9 7 2 ) studying Mustelus canis mentions the basement membrane, while Lance and C a l l a r d ( 1 9 6 9 ) studying Squalus acanthias omit mentioning i t completely. I t i s present i n Squalus and one can observe two components to the membrane. Such a membrane i s associated with the basal surface of e p i t h e l i a and i t should be expected here as the f o l l i c l e c e l l s can be considered s p e c i a l i z e d e p i t h e l i a l c e l l s (Bloom & Fawcett, 1 9 6 8 ) . The inner component (closest to the oocyte) i s termed the basement lamina while the outer component i s r e f e r r e d to as an amorphous ground substance (Bloom & Fawcett, 1 9 6 8 ) or as the basement lamella (Nadol et a l , 1 9 6 9 ) . The basement lamella reacts negatively to a l l but Hale's and aldehyde fuchsin. The re a c t i o n with Hale's i s d i f f i c u l t to explain because i t should ,produce the same r e s u l t s ..as .* a l c i a n 'blue.. A d d i t i o n a l l y , the-aldehyde fuchsin r e a c t i o n i n d i c a t i n g sulphated mucopolysaccharides should be supported by Azure A (pH 1 . 5 ) metachromasia and i t i s not. In contrast to these r e s u l t s , the ground substance of e p i t h e l i a i s described as c o n s i s t i n g of unit f i b r i l s of collagen embedded i n an amorphous protein-polysaccharide matrix (Bloom & Fawcett, 1 9 6 8 ) . One would then expect the ground substance to s t a i n with DNFB and PAS. The basement lamella i s often i d e n t i f i e d as a product of the connective t i s s u e f i b r o b l a s t s (Bloom & Fawcett, 1 9 6 8 ) , here found i n the theca, and should then correspond to'the s t a i n i n g reactions of the theca. Confirmation of t h i s can be found i n the t a b l e s . The basal lamina i s considerably more i n t e r e s t i n g . I t s minor components are prote i n , l i p i d , n e u t r a l mucopolysaccharides, mucoproteins and/or s i a l i c a c i d . An e p i t h e l i a l basement lamina i s also.PAS p o s i t i v e (Bloom & Fawcett, 1 9 6 8 ) . I t s major components are a c i d and sulphated mucopolysaccharides (anionic and sulphated anionic glycosaminoglycans). The glycosaminoglycans that are present are hyaluronic a c i d , chondroitin sulphate A and chondroitin sulphate C, as indic a t e d by the hyaluronidase l a b i l e metachromasia of t o l u i d i n e blue. Hyaluronic a c i d i s a h i g h l y hydrated molecule and such a composition would make i t very viscous (Schubert & Hamerman, 1956). As such, i t could endow a s t r u c t u r a l function to the anionic glycosaminoglycans by providing r i g i d i t y to the lamina as well as the p h y s i o l o g i c a l function of c o n t r o l l i n g molecular d i f f u s i o n (Rogers, I 9 6 1 ) . The persistence of a s l i g h t amount of metachromasia following hyaluronidase treatment may in d i c a t e the presence of a s i a l i c a c i d f r a c t i o n ( C u l l i n g , 1 9 6 3 ; Kvist & Finnegan, 1 9 7 0 ) . Aldehyde fuchsin has a very high a f f i n i t y f o r the strongly a c i d i c sulphonate groups of anionic glycosaminoglycans (Spicer & Meyer, i 9 6 0 ) . .However, i t cannot b e . s p e c i f i c f o r .sulphonate..groups, as witnessed .by the persistence of st a i n i n g following methylation and a s l i g h t increase following demethylation. This s t a i n i n g of carboxyl groups following demethylation i s contrary to the findings of many other i n v e s t i g a t o r s (Scott.& Clayton, 1 9 5 3 ; Spicer & Meyer, I960; Spicer, 1 9 6 2 ) but finds support i n the work of Ortman et a l (1966) wherein aldehyde fuchsin solutions were found to be unstable and would give r i s e to intermediate compounds that react with carboxyl groups (Kvist & Finnegan, 1 9 7 0 ) . The persistence of st a i n i n g a f t e r methylation i s i n disagreement with a l l of the previously mentioned in v e s t i g a t o r s and ind i c a t e s that here the aldehyde fuchsin s o l u t i o n may also be st a i n i n g a s i a l i c a c i d f r a c t i o n or there may not be complete blockage of the sulphate or carboxyl groups. The incomplete blocking of Azure A s t a i n i n g a f t e r the methylation-demethylation sequence indicated that, at pH 1.5, not a l l the metachromasia and orthochromasia was due to the presence of sulphated mucopolysaccharides. Although i t had "been reported that most non-sulphated glycosaminoglycans are orthochromatic when stained with c a t i o n i c t h i a z i n e dyes (Sylven & Malmgren, 1 9 5 2 ; Sylven, 1 9 5 * + ; Walton & R i c k e t t s , 1 9 5 * + ) , h i g h l y polymerized or concentrated hyaluronic acid can give a metachromatic response (Meyer, 1 9 5 5 , 1 9 5 6 ; Schubert & Hamerman, 1 9 5 6 ) . The reduction i n Azure A meta-chromasia following hyaluronidase i n d i c a t e s that at l e a s t some of the metachromasia i s due to chondroitin sulphate A and/or chondroitin sulphate C and supports the r e s u l t s following methylation-demethylation. I t has also been recorded that f a i n t a z u r o p h i l i a p e r s i s t i n g a f t e r hyaluronidase digestion indicates the presence of chondroitin sulphate B (McConnachie & Ford, 1 9 6 6 ) . Thus, the basal lamina i s composed mainly of chondroitin sulphate A and/or chondroitin sulphate C with a moderate amount of hyaluronic -.acid...and ..po.s.sibly .some .-.sialic-acid..with .-some .chondroitin. sulphate ,B. THECA The theca interna, not di s t i n g u i s h a b l e from the externa i n these early stages, has been reported to consist mainly of connective t i s s u e c e l l s (Lance & C a l l a r d , 1 9 6 9 ) i n Squalus acanthias or of a narrow layer of c e l l s and blood c a p i l l a r i e s adjacent to the granulosa and the surrounding band of collagenous material i n Mustelus canis (TeWinkel, 1 9 7 2 ). F u n c t i o n a l l y , the theca has been implicated along with the granulosa i n steroid.biosynthesis (Lance & C a l l a r d , 1 9 6 9 ; L i v n i , 1 9 7 . 1 ) . The histochemical data presented indicate the presence of small quantities of glycogen, p r o t e i n , RNA, phospholipid and p o s s i b l y some a c i d i c and sulphated mucopolysaccharides. It would appear to have l i t t l e b iosynthetic a c t i v i t y (other than s t e r o i d , Lance & C a l l a r d , 1 9 6 9 ) although i t may contribute some a c i d i c and 120 sulphated mucopolysaccharides to the outer component of the basement membrane, the basal lamella. STROMA The d i s t i n c t i o n between l i g h t and dark c e l l s i n the stroma i s at best a tenuous one, l i g h t optics and section thickness combine to make di s t i n g u i s h i n g these two c e l l types d i f f i c u l t . Nevertheless, c e l l s with ovoid n u c l e i have been designated as l i g h t c e l l s , while dark c e l l s are those with pleomorphic n u c l e i . Generally, both types present the same p i c t u r e as the t h e c a l c e l l s , except that the dark c e l l s s t a i n more in t e n s e l y than either the l i g h t c e l l s or the t h e c a l c e l l s . . The stroma of Mustelus i s described as c o n s i s t i n g of an exceptionally loose network of connective t i s s u e membranes carrying blood vessels and presumably, lymphatic channels. -(.TeWinkel, . 1 9 7 2 ) , . .-Thus., r e s u l t s s i m i l a r to those ,of the. theca, vwhich i s also a connective t i s s u e l a y e r , would be expected. The response of a small percentage (approximately 2 to h %) of the dark c e l l s with aldehyde fuchsin i s extremely i n t e r e s t i n g . The observed r e a c t i o n i n d i c a t e s the presence of both sulphated mucopolysaccharides and non-sulphated mucopolysaccharides (hyaluronic or s i a l i c acid) i n the dark c e l l s . Although such a response may indi c a t e that there are two types of dark c e l l , the po s s i b l e metabolic or f u n c t i o n a l s i g n i f i c a n c e of such a r e a c t i o n i s obscure. These c e l l s are randomly scattered i n the stroma, thus d i r e c t c o n t r i b u t i o n to the formation of either the f o l l i c u l a r membranes (lamina and lamella) or the e l a s t i c f i b r e s i n the tunica can be r u l e d out. A d d i t i o n a l l y , with such a strong aldehyde fuchsin r e a c t i o n , one would then expect a supporting a l c i a n o p h i l i c and/or a metachromatic response ( t o l u i d i n e blue, Azure A pH 1 . 5 ) . Such i s not the case. Cook ( 1 9 7 2 ) describes aldehyde fuchsin as a " c a p r i c i o u s " s t a i n r e q u i r i n g the use of a f r e s h l y opened b o t t l e of paraldehyde (Moment, 1 9 6 9 ) to achieve the desired r e s u l t s . The high l e v e l of background s t a i n present has been a t t r i b u t e d to an o l d and deteriorated s t a i n i n g s o l u t i o n (Cook, 1 9 7 2 ) but as i t was f r e s h l y prepared, the only remaining v a r i a b l e i s the paraldehyde which was not f r e s h l y opened. TUNICA The tunica albuginea, a connective t i s s u e layer beneath the surface epithelium and surrounding the cortex (Nelsen, 1 9 5 3 ) i s ignored by Lance and C a l l a r d . ( 1 9 6 9 ) i n t h e i r report on Squalus acanthias, but i s described by TeWinkel. ( 1 9 7 2 ) i n Mustelus canis as a dense c o r t i c a l collagenous, connective t i s s u e layer with interwoven smooth muscle. The strong DNFB reaction indicates the presence of abundant p r o t e i n but equating t h i s •protein t o smooth muscle .will -have'to await the demonstration.,of the .l a t t e r with the electron microscope. The use of titanous c h l o r i d e as a reducing agent i n DNFB also provides f o r improved s t a i n i n g of such t i s s u e components as collagen and e l a s t i c f i b r e s (Pearse, 1 9 6 8 ) . The other carbohydrates present include sulphated and a c i d i c mucopolysaccharides, r e s u l t s consistent with a connective t i s s u e nature. Again the r e s u l t s of aldehyde fuchsin (AF) and Azure A staining are contradictory. The complete blocking of AF staining following methylation and demethylation i s i n d i c a t i v e of a very high a f f i n i t y for the strongly a c i d i c sulphonate groups of the sulphated mucopolysaccharides (Conklin, 1 9 6 3 ; Spicer & Meyer, i 9 6 0 ; Spicer, 1 9 6 2 ; Kvist & Finnegan, 1 9 7 0 ) . Upon t h i s premise, an intense Azure A metachromatic response i s expected. The lack of such a response would i n d i c a t e that there were no chondroitin sulphates present. The c y t o l o g i c a l appearance of the tunica, Verhoeff's r e a c t i o n (unreported data) f o r collagen as well as TeWinkel's ( 1 9 7 2 ) observation of collagen i n Mustelus, a l l i n d i c a t e that collagen i s present i n the t u n i c a . The a s s o c i a t i o n of the chondroitin sulphates with the collagen of c a r t i l a g e (Swanson, 1 9 6 5 ) , a good i n d i c a t i o n that i t should be present here, argues that the Azure A r e a c t i o n i s erroneous. The only possible explanation f o r t h i s phenomenon i s that the molecules present are bound or masked, or the groups are not present i n s u f f i c i e n t quantity to produce metachromasia (Sylven, 1 9 5 H ; Chayen et a l , 1 9 6 9 ) . The presence of short e l a s t i c f i b r e s i n the t u n i c a emphasizes that i n a l l p r o b a b i l i t y t h i s layer i s viscous and f l e x i b l e . These f i b r e s i n t e r -woven with the collagen of the tunica lend support to the components of the ovary as well as serving to bind the ovary together and thus preventing the numerous (h to 8 ) large oocytes present p r i o r to and at ovulation from fragmenting the ovary. The e l a s t i c f i b r e s have the capacity to s t r e t c h „and then snap back i n t o t h e i r o r i g i n a l s tate. Presumably, t h i s property would be most important a f t e r ovulation has occurred to restore the ovary to i t s o r i g i n a l shape and to close the gaps created by the ovulated eggs. 123 ULTRASTRUCTURE  OVARIAN EPITHELIUM The term "germinal epithelium" p e r s i s t s even though the extragonadal o r i g i n of the primordial germ c e l l s i s well substantiated (Bloom & Fawcett, 1 9 6 8 ; Hoar, 1 9 7 0 ; B e l l a i r s , 1 9 7 1 ) . Throughout my observations of the developing oocytes, there was never any evidence to suggest that t h i s epithelium gave r i s e to the developing oocytes. Considering t h i s and the basic s i m i l a r i t i e s of the early dogfish and avian embryos, I f e e l that the primordial germ c e l l s of Squalus can be found i n the yolk sac region. The term "germinal epithelium"is inappropriate and w i l l "not be used i n t h i s study. The absence of c i l i a on the epithelium i s of i n t e r e s t considering i t s c i l i a t i o n i n at l e a s t .two other dogfish species. Mustelus canis (TeWinkel, . 1 9 7 2 ) -has .a c i l i a t e d ovarian .epithelium '-while Scylliorhynus .canicula (Metten, 1 9 ^ 1 ) has a c i l i a t e d abdominal c a v i t y . In a l l p r o b a b i l i t y t h i s c i l i a t i o n i s responsible f o r the di r e c t e d movement of an egg towards the o s t i a . Squalus eggs, however, are said to move p a s s i v e l y toward the anterior end of the oviduct and become engulfed by the ostium tubae (Weichert, 1 9 6 1 ) . In accounting f o r t h i s , one must consider one c r i t i c a l f a c t o r , Squalus  acanthias has fu n c t i o n a l r i g h t and l e f t ovaries and oviducts whereas the other species mentioned have but a sing l e f u n c t i o n a l ovary and oviduct. From t h i s , one can conclude that c i l i a t i o n i s a r e f l e c t i o n of the atrophy of one ovary and oviduct and therefore i t increases the p r o b a b i l i t y that any ovulated egg w i l l reach the ostium. The c e l l s of the ovarian.epithelium are s i m i l a r to most other t y p i c a l e p i t h e l i a l c e l l s (Bloom & Fawcett, 1 9 . 6 8 ; Matthews & Martin, 1 9 7 1 ) . They are columnar with a basa l , lobed nucleus having a prominent nucleolus and a p i c a l l y l o c a t e d organelles. The numerous mitochondria, Golgi bodies and E.R. indicate active synthesis and t h i s i s manifest by the preponderance of microfilaments within the c e l l s and the mucin—like v e s i c l e s , derived from the Golgi, at the surface of the ce l l : . In a l l p r o b a b i l i t y , the f l o c c u l e n t component of these v e s i c l e s functions as a p r o t e c t i v e or mucoid coating f o r the ovary s i m i l a r t o that found coating the i n t e s t i n a l e p i t h e l i a of many mammals (Fawcett, 1 9 6 6 ) and corresponds to "the p y r o n i n o p h i l i a (RNA) and a l c i a n o p h i l i a ( a c i d i c and sulphated mucopolysaccharides) demonstrated histochemically. The microfilaments, which are f a r more numerous than i n other e p i t h e l i a l c e l l s (Bloom & Pawcett, 1 9 6 8 ; Matthews & Martin, 1 9 7 1 ) are e a s i l y accounted f o r when one r e c a l l s the s i z e and number of r i p e eggs found i n a mature female. The microfilaments, i n a l l l i k e l i h o o d , serve t o maintain the i n t e g r i t y of the c e l l s under the d i s r u p t i v e i n f l u e n c e of the development and ovulation o f several eggs 3 to h cm i n diameter. Quite unlike the surface epithelium of the domestic fowl (.Dahl, . 1 9 7 2 ) , there are few, i f any, narrow surface crypts but there are extensive j u n c t i o n a l complexes. The absence of d e f i n i t e crypts or e p i t h e l i a l i n -growths indicates that there i s no c o n t r i b u t i o n of e p i t h e l i a l elements to the c o r t i c a l region either f o r use as f o l l i c l e c e l l s or as s t e r o i d producing t h e c a l gland c e l l s (Harrison., 1 9 6 l ; Narbaitz & Adler, 1 9 6 6 ; Dahl, 1 9 7 2 ) . The v a r i e t y of j u n c t i o n a l complexes found beneath the zonula occludentes presents an i n t e r e s t i n g problem—What purpose do they serve? Obviously they are i n t e r c e l l u l a r attachment devices that contribute to the maintenance of c e l l shape and thus to the i n t e g r i t y of the surface epithelium as well (Fawcett, 1 9 6 6 ; Matthews & Martin, 1 9 7 1 ) . The numerous regions of concentric f o l d s are by no means unique t o Squalus, but they are considerably more complex than others observed between c e r t a i n mammalian e p i t h e l i a l c e l l s (Matthews & Martin, 1 9 7 1 ) . I t i s quite probable that they too serve as i n t e r c e l l u l a r attachment devices but i t i s also probable that the extra surface area they provide may be u t i l i z e d i f the c e l l s were stretched by a developing 2 to h cm oocyte. The extensive i n t e r c e l l u l a r channels found between indivudual c e l l s i s another feature unique to the dogfish ovary. As f a r as I have been able to discern, no such channels have been reported i n any other vertebrate ovary. Their purpose i s obscure but several possible explanations are offered. F i r s t , they may serve as a reserve mucoid source for coating the e p i t h e l i a l surface. Second, they may act as an area to receive membranous det r i t u s which i s then concentrated and eliminated i n t o the abdominal cavi t y . Both explanations have f a u l t s however. In the f i r s t instance, the . .^zonula .oc.cludentes would -act as .-a iSeal to the ;passage of materials and f l u i d s i n either d i r e c t i o n (Fawcett, 1 9 6 6 ) . In the second instance, although there i s some evidence for membranous material i n the canal and possibly an eliminated "plug" of matter at the surface, the e l i m i n a t i o n of waste"in t h i s manner i s i n e f f i c i e n t . The only p o s s i b l e channel out of the body i s v i a the oviducts, a path that i s f u n c t i o n a l l y inappropriate. The well developed basement lamina (membrane) that separates the epithelium from the tunica i n Squalus was not observed beneath the epithelium of the smooth dogfish Mustelus canis (TeWinkel, 1 9 7 2 ) . TUNICA ALBUGINEA The nature of the tunica i n Squalus i s s i m i l a r to that of Mustelus (TeWinkel, 1 9 7 2 ) d i f f e r i n g only i n the absence of smooth muscle. The greater thickness of t h i s layer r e l a t i v e to that of most adult mammals 126 (Mossman & Duke, 1 9 7 3 ) r e f l e c t s the need to maintain the i n t e g r i t y of the organ again under the d i s r u p t i v e influence of the large eggs found i n adult ovaries. The collagen present o f f e r s resistance to a p u l l i n g force whereas the e l a s t i c f i b r e s w i l l return the t u n i c a to i t s o r i g i n a l shape. Although the p e r i o d i c i t y of the collagen f i b r e s i s somewhat l e s s ( 5 2 0 A vs 6k0 ft) than that of "native" collagen i t i s f e l t that t h i s i s not a s i g n i f i c a n t d i f f e r e n c e . . E l a s t i c f i b r e s have been described as "lacking v i s i b l e f i b r i l l a r subunits" (Bloom & Fawcett, 1 9 6 8 ) or c o n s i s t i n g .of an "amorphous electron-lucent substance with some l o n g i t u d i n a l filaments buried within the amorphous e l a s t i h " (Matthews & Martin, 1 9 7 1 ) . To the contrary, the e l a s t i c f i b r e s of the tunica show d i s t i n c t 5 0 to 6 0 8. filaments oriented p a r a l l e l to the long axis of the f i b r e . In view of t h i s i t would seem safe to conclude that these subunits are the c o n t r a c t i l e elements of the f i b r e s . CORTEX T y p i c a l l y , the ovarian cortex or stroma of non-mammalian vertebrates i s described as c o n s i s t i n g of f i b r o b l a s t - l i k e c e l l s , connective t i s s u e f i b r e s and smooth muscle (hen, Peel & B e l l a i r s , 1 9 7 2 ) , an exceptionally loose net of connective t i s s u e membranes (Mustelus canis, TeWinkel, 1 9 7 2 ) , a connective t i s s u e stroma (Squalus acanthias, Lance & C a l l a r d , 1 9 6 9 ) . However, the observations within t h i s study are not consistent with such a viewpoint. What i s indicated i s a r i c h l y c e l l u l a r cortex that i s i n t e r - " spersed with blood vessels and large lacunae. This seems to p a r a l l e l the mammalian cortex which i s described as being "embryonic" i n that i t i s compact, c e l l u l a r and l a c k i n g i n f i b r e s (Mossman & Duke, 1 9 7 3 ) . Although t h i s ! I d e s c r i p t i o n matches our observations, one must h e s i t a t e before drawing any conclusions as the descriptions are made from l i g h t micrographs and l i t t l e . i f any electron microscopy has'been done on the c e l l s of the cortex. Although there i s a d i s t i n c t morphological d i v i s i o n of the c e l l s of the cortex into at l e a s t two types, the. f u n c t i o n a l s i g n i f i c a n c e of the c e l l s remains obscure. Histochemically, they resemble the the c a l c e l l s , a response not unexpected considering the thecal c e l l s are probably modified c o r t i c a l c e l l s (Mossman & Duke, 1 9 7 3 ) . The bodies of the dark c e l l s may, i n f a c t , be lysosomes (Fawcett, 1 9 6 6 ) but p r e c i s e l y why there are so many c e l l s with such large numbers of lysosomes i s unknown. The p o s s i b i l i t y that these bodies are not lysosomes also e x i s t s and without performing enzymatic histochemical t e s t s we cannot say that these bodies are or are not lysosomes. The l i g h t c e l l s are characterized by the extensive development of distended cisternae of granular E.R.: a phenomenon that i s -typ i c a l of .-glandular .cells-.elaborating a,.protein r i c h secretory product (Fawcett, 1 9 6 6 ) . However, i f these c e l l s are producing a proteinaceous substance f o r export, there i s no i n d i c a t i o n as to how i t i s transported from the c e l l or as to where i t i s transported. An obvious supposition would be that these c e l l s are secreting a product that i s f i n d i n g i t s way into the developing oocytes as a constituent of the p r o t e i d yolk. THE INTERCELLULAR SPACE The collagen f i b r e s that are found woven throughout the ovary may be a product of either or both of the l i g h t and dark c e l l s . Their presence lends credence to the aforementioned discussion of the connective t i s s u e nature of the cortex (Lance & C a l l a r d , 1 9 6 9 ; Peel & B e l l a i r s , 1 9 7 2 ; TeWinkel, 1 9 7 2 ) . LACUNAE BORDER CELLS Undoubtedly, the lacunae border c e l l s are a form of endothelium which more than s u p e r f i c i a l l y resembles that of a c a p i l l a r y w a l l . More appropriately, the lacunae border c e l l s resemble the "continuous sinusoids" found i n the l i v e r of " c e r t a i n " vertebrate species (Bloom & Fawcett, 1 9 6 8 ) . As such, these c e l l s provide a p r o t e c t i v e epithelium f o r the c e l l s of the cortex. A d d i t i o n a l l y , the numerous 6 0 0 to 7 0 0 A1 v e s i c l e s may function i n the micropinocytotic t r a n s f e r of small quantities of f l u i d and solutes across the c e l l discharging i t into the c l e a r zone. The use of e l e c t r o n opaque tra c e r s and enzymes l i k e peroxidase have shown that such t r a n s f e r does occur i n c a p i l l a r y w alls. However, the l a b e l l e d v e s i c l e s are r e l a t i v e l y few and many are i n doubt as to whether t h i s mechanism could account f o r either the s e l e c t i v i t y of the process or the volume of material ..transported per .unit .time (Fawcett,', . 1 9 6 6 ; Bloom .&. Fawcett,, 1 9 6 8 ) . . Whether or not one must worry about the volume t r a n s f e r r e d or the s e l e c t i v i t y i n t h i s instance has not been determined. The enormous surface area involved leads one to the conclusion that there i s t r a n s f e r of some substance or substances across t h i s b a r r i e r and i f any s e l e c t i v i t y i s involved i t would be imposed by the basement lamina. I f some substance i s t r a n s f e r r e d across t h i s c e l l u l a r b a r r i e r , i t i s not intended f o r either the l i g h t or the dark c e l l s s p e c i f i c a l l y as both can be found underlying the lacunae border c e l l s . The lacunae show no evidence of electron opaque materials and one therefore concludes that ir± vivo they are f l u i d f i l l e d channels. 129 RED BLOOD CELLS The red blood c e l l s of Squalus are t y p i c a l nucleated red blood c e l l s . The most unusual features noted are the presence of myelin — l i k e bodies and a G o l g i - l i k e body. The myelin-like f i g u r e s are l i k e l y to be p r e c i p i t a t e d phospholipids (Matthews & Martin, 1 9 7 1 ) while the G o l g i - l i k e body i s eit h e r an a r t i f a c t or a degenerating Golgi apparatus. The microtubules of the marginal band function as s t i f f e n i n g , c y t o s k e l e t a l elements that help to maintain the fl a t t e n e d form c h a r a c t e r i s t i c of nucleated red blood c e l l s . THECAL CELLS The thecal layer i s often described as a connective t i s s u e l a y e r (Dumont, 1 9 7 2 ; Peel & B e l l a i r s , 1 9 7 2 ; TeWinkel,. 1 9 7 2 ) i n the lower vertebrates. However, i n mammals and many of the lower vertebrates, s t e r o i d a c t i v i t y has been ascribed- to the theca inter n a (as well as the granulosa) (Lance & C a l l a r d , 1 9 6 9 ; TeWinkel, 1 9 7 2 ) . The theca inter n a of the hen, on the other hand, i s a connective t i s s u e layer interspersed with l u t e a l c l u s t e r s that constitute the thecal gland, a s t e r o i d secreting structure (Peel & B e l l a i r s , 1 9 7 2 ) . The theca interna of Squalus acanthias has been described as co n s i s t i n g mainly of connective t i s s u e c e l l s (Lance & C a l l a r d , 1 9 6 9 ) while that of Mustelus canis as co n s i s t i n g of a narrow layer of c e l l s and blood c a p i l l a r i e s adjacent to the granulosa and the surrounding band of collagenous t i s s u e (TeWinkel, 1 9 7 2 ) . The theca externa of Squalus has not been described previously, whereas, i n Mustelus i t i s composed of loose membranes and blood vessels p e r i p h e r a l to a network of tubules (TeWinkel, 1 9 7 2 ) . What I have observed i n Squalus ovaries d i f f e r s markedly from t h i s . I n i t i a l l y the thecal c e l l s are f i b r o b l a s t - l i k e , u n d i f f e r e n t i a t e d as to interna or externa. There i s , however, an abundance of Golgi bodies and smooth v e s i c l e s that foreshadows the condition found i n slightly-l a r g e r oocytes when the theca can be divided into i n t e r n a and externa. The presence of smooth membrane bound v e s i c l e s , Golgi bodies and f l a t t e n e d nuclei define the theca interna c e l l s and lend credence to the p o s s i b i l i t y that these c e l l s may i n time secrete s t e r o i d s . This i s not unequivocal proof that these c e l l s do or w i l l secrete s t e r o i d s . However, the lack of such smooth membrane-bound organelles would preclude the p o s s i b i l i t y of t'hese c e l l s ever producing steroids (Fawcett, 1 9 6 6 ; Mossman & Duke, 1 9 7 3 ) . The appearance of the c e l l s of the theca externa indicates that they too are involved i n the synthesis of a product. Based on the morphology of c e r t a i n of the v e s i c l e s one may conclude that these c e l l s are responsible, at l e a s t i n part, for the formation of the dense granular matrix and -.possibly .the collagen f i b r e s ..found t h e r e i n . There .is ..also evidence to indicate that the c e l l s of the c e l l s of the theca interna contribute to t h i s matrix. In a l l , the thecal c e l l s appear to be modified connective t i s s u e c e l l s with c y t o l o g i c a l c h a r a c t e r i s t i c s s i m i l a r to those of c e l l s involved i n protein and s t e r o i d synthesis. This dense matrix may act as a basement membrane separating the thecal c e l l s from t h e i r associated f o l l i c l e and the surrounding c o r t i c a l c e l l s . In t h i s manner, we may have the formation of a thecal glend analogous to that-found i n the hen (Peel & B e l l a i r s , 1 9 7 2 ) and numerous mammals (Mossman & Duke, 1 9 7 3 ) . BASEMENT LAMINA-LAMELLA COMPLEX The basement membrane or basal lamina present around developing dogfish oocytes i s uncommonly t h i c k , having a counterpart only i n Descemet's membrane of the cornea (Fawcett, 1 9 6 6 ) and the di a b e t i c glomerular basement membrane (S i p e r s t e i n et a l , I 9 6 H ) . The average basement lamina i s a f i n e l y filamentous sheath l e s s than 1 0 0 nm t h i c k (Fawcett, 1 9 6 6 ; Hay & Revel, 1 9 6 9 ) . I have already noted such t y p i c a l laminae associated with the surface e p i t h e l i a l c e l l s and the lacunae border c e l l s . ' I t i s obvious from the data presented that the basal lamina (F.B.M.) i s a product of the f o l l i c u l a r e p i t h e l i o i d c e l l s . P r e c i s e l y , i t i s formed through the combined e f f o r t s of the granular E.R. and the Golgi v e s i c l e s located i n the basal regions of the e p i t h e l i a l c e l l s involved. S i m i l a r conclusions in v o l v i n g e p i t h e l i a l c e l l s forming basal lamina as well as an e x t r a c e l l u l a r collagenous matrix have been advanced concerning avian corneal epithelium (Hay & Revel, 1 9 6 9 ) , human glomerulus (Beisswenger & Spiro, 1 9 7 3 ) , the c u t i c l e of the roundworms Lumbricus and A s c a r i s as well as the byssus threads of Mytilus edulis (Gross,, 1 9 6 3 ) . Basal laminae have been shown to e x i s t beneath e p i t h e l i a regardless of the absence of f i b r o b l a s t s (Hay & Revel, 1 9 6 9 ; B e r n f i e l d & Wessells, 1 9 7 0 ) , once the only c e l l type thought capable of secreting collagen or such a connective t i s s u e matrix (Gross, 1 9 6 3 ; Bloom & Fawcett, 1 9 6 8 ) . This gains importance when one considers that basal laminae (glomerular) are s i m i l a r i n composition to collagen (Vernier, I 9 6 U ; Dische et a l , 1 9 6 5 ; Hay & Revel, 1 9 6 9 ; Beisswenger & Spiro, 1 9 7 3 ) but with lh times more hydroxyproline and 6 times more hydroxylysine than native (glomerular) collagen (Hay & Revel, 1 9 6 9 ) . Thus one would expect the basement lamina to exhibit s t a i n i n g properties s i m i l a r to those of collagen and e l a s t i c t i s s u e . Such a r e l a t i o n s h i p has already been demonstrated histochemically (See Tables IA to IC & pp 2 9 ). A d d i t i o n a l l y , two carbohydrate components have been i d e n t i f i e d within glomerular basal laminae. One component i s a hydroxylysine-linked (collagen-linked) glucosylgalactose unit (Beisswenger & Spiro, 1 9 7 3 ) while the other i s a free s i a l o f u c o -hexosaminoglycan (Dische et a l , 1 9 & 5 ; Hay & Revel, 1 9 & 9 ; Nadol et a l , 1 9 6 9 ) . The histochemical data presented e a r l i e r record the occurrence of aci d mucopolysaccharides within the basal lamina and p o s s i b l y within the f o l l i c l e c e l l s . In the l a t t e r instance, however, the techniques employed were not s u f f i c i e n t l y s e n s i t i v e to unequivocally demonstrate the presence of acid mucopolysaccharides. Using the s i l v e r methenamine and f e r r i t i n -l a b e l l e d antibody techniques, Vernier ( 1 9 6 U ) demonstrated that the e p i t h e l i a l c e l l s of human glomeruli contained material s i m i l a r to a component of the basement lamina and concluded that these c e l l s were at l e a s t p a r t i a l l y responsible for the synthesis and storage of basement lamina m a t e r i a l . Farquhar ( 1 9 6 H ) observed E.R. cisternae of these same c e l l s with a.material that was u l t r a s t r u c t u r a l l y .similar to the lamina. .Based on these observations and conclusions, i t i s obvious that p a r a l l e l observations made regarding basement membrane formation i n Squalus acanthias should lead to p a r a l l e l c o n c l u s i i n s . The following conclusions by Dische (Hay & Revel, 1 9 6 9 ) sum up the observations recorded herein and mirror the current dogma: "Basement membrane i s equivalent to a collagenous protein not organized above the l e v e l of tropocollagen but p o s s i b l y possessing c e r t a i n degrees of o r i e n t a t i o n with an amorphous substance between the collagenous p r o t e i n molecules... The collagen molecules themselves are apparently l i n k e d to one or more (hexosamine-free) glycans... whereas i n the amorphous matrix would be located (an a c i d mucopolysaccharide and a s i a l o f u c o -hexosaminoglycan)...the lowest l e v e l of organization (of collagen) i s represented by basement membrane, (here) collagen does not show i t s c h a r a c t e r i s t i c s t r i a t i o n due to the basic organization of tropocollagen into f i b r i l s therefore the membranes do not show a f i b r i l l a r y structure even i n electron micrographs." The basement lamella i s the more d i s t a l p o r t i o n of the "basement membrane complex that t y p i c a l l y contains a h i g h l y ordered array of collagen f i b r e s crossing approximately at r i g h t angles (Nadol et a l , 1 9 6 9 ) . In t h e i r studies of the skin of the t e l e o s t Fundulus, Nadol et a l ( 1 9 6 9 ) report observations of the basal lamella s i m i l a r to those reported herein for the basal lamella of Squalus. To wit, the collagen f i b r e s of the lamella are oriented i n a l t e r n a t i n g l o n g i t u d i n a l and c i r c u l a r l a y e r s with the layers crossing at 1 0 5 to 1 1 0 ° , producing a "herringbone pattern", the same pattern observed i n 1 . 5 to 1 . 8 mm dogfish oocytes. Their most s i g n i f i c a n t conclusion i s that the collagen f i b r e s of the lamella have t h e i r o r i g i n i n the substance of the basement laminaoWhere there are designated-areas that d i r e c t the deposition of collagen i n but one o r i e n t a t i o n , doing so continuously i n d i r e c t i o n . i f not i n time. .The ,.impli cat-ions of t h i s , are two-fold. F i r s t , as the oocyte and basement lamina grow, so w i l l the l a m e l l a , because the number of i n i t i a t i o n s i t e s on the lamina w i l l increase. Secondly, the e a r l i e r conclusions concerning the collagenous nature of the lamina are r e i n f o r c e d . A l s o , one may consider the l i n e a r pattern observed within the basement lamina to be the ordering of the tropocollagen molecules into non-striated f i b r e s . Whether t h i s r e f l e c t s the i n vivo s i t u a t i o n or i s an a r t i f a c t of preparation cannot be ascertained at t h i s time. However, a 0 . 5 1 M NaCl s o l u t i o n has been shown to produce reconstituted collagen f i b r e s that lack p e r i o d i c i t y (Gross, 1 9 6 3 ) . The intimate r e l a t i o n s h i p between the collagen f i b r e s of the basement l a b e l l a and the theca must also be considered. The basic s i m i l a r i t y of t h i s arrangement and that of the collagen and matrix of the "thecal i s l a n d s " leads one to b e l i e v e that the thecal c e l l s may be c o n t r i b u t i n g to 134 the growth of l.i.ie basal l a m e l l a . S i g n i f i c a n t l y , Grobsteins's l a b e l l i n g studies on thi: Interface materials of epitheliomesenchymal i n t e r a c t i o n s (1968) have K.l.iMwn that separately both e p i t h e l i a l and mesenchymal t i s s u e s can synthesi/.i.: collagen. Epithelium, however, produces only small amounts of hydroxylatf'ii, protein (Grobstein, 1968). • But when an epitheliomesenchymal i n t e r f a c e i s -I..;:;i,ed (with label)., i t i s noted that collagen production has increased. l i e concludes hydroxylated m a t e r i a l i s t r a n s f e r r e d from the mesenchyme to -I,he epithelium or that i n the presence of mesenchyme the epithelium may be stimulated t o hydroxylation. In e i t h e r case, one component of the macromolecular material accumulating at the epitheliomesenchymal i n t e r f a c e prob;i.h]y comes from the mesenchyme. Although convincing morphological ±.-vj.dence for t h i s i s l a c k i n g , the c i r c u m s t a n t i a l evidence i s s u b s t a n t i a l . For instance, there are extensive collagenous deposits at the s i t e s of eidtheliomesenchymal i n t e r f a c e s i . e . : beneath the surface epithelium (the txinica albuginea) and beneath the f o l l i c l e c e l l s (the basal lamina=-lM.iiiella complex). The o r i g i n of the other c e l l s i n the ovary i s mesodermal (Balinsky, 1966) and one would expect collagen to be deposited here but i n l e s s e r amounts than where there i s an e p i t h e l i o -mesenchymal i n t e r f a c e . Therefore, although the majority of the evidence presented favours the production of the basement lamina-lamella complex from the e p i t h e l i a l c e l l s one cannot completely eliminate the underlying tissues (mesodermal) f o r being responsible for at l e a s t part of the membranous complex. FOLLICLE CELLS and the "VITELLINE MEMBRANE The a c t i v i t y of the f o l l i c l e c e l l s i n basement lamina formation has been established but the u l t r a s t r u c t u r a l evidence also points to s e v e r a l other possible formative functions and t h e i r r e l a t e d phenomena. Generally, the f o l l i c u l a r sheath resembles that of a l l vertebrates (Raven, 1 9 6 1 and N ? 5 r r e v a n g , I 9 6 8 for reviews; Yamamoto, 1 9 6 3 ; J o l l i e & J o l l i e , I 9 6 U;' B e l l a i r s , 1 9 6 5 ; . Mossman & Duke, 1 9 7 2 ; Dumont, 1 9 7 2 ) and many invertebrates (Raven, I 9 6 I ; NtfSrrevang, I 9 6 8 ; Anderson & Spielman, 1 9 7 1 ; Cummings, 1 9 7 2 ) . The smallest oocytes examined ( 1 0 0 um) showed a well developed low cuboidal granulosa layer while Peel and B e l l a i r s ( 1 9 7 2 ) noted a cuboidal epithelium around 0 . 5 to 1 . 0 ym hen oocytes. A s i m i l a r s i t u a t i o n may also be found around dogfish oocytes of that s i z e . The u l t r a s t r u c t u r a l evidence for s t e r o i d a c t i v i t y i n these e a r l y oocytes i s c i r c u m s t a n t i a l . However, histochemical demonstration of 3BHSD i n the granulosa of Squalus acanthias oocytes was made by Lance and C a l l a r d ( 1 9 6 9 ) : the presence of a s t e r o i d dehydrogenase gen e r a l l y being accepted as evidence that the t i s s u e i s capable of s t e r o i d synthesis (Lance & C a l l a r d , 1 9 6 9 ) . The evidence of p o t e n t i a l steroidogenic a c t i v i t y l i e s with the amount of smooth membrane present i n the c e l l ( N i c h o l l s & Maple, 1 9 7 2 ; Peel & B e l l a i r s , 1 9 7 2 ) and c e r t a i n l y the f o l l i c l e c e l l s of e a r l y dogfish oocytes do possess considerable amounts of smooth membrane v e s i c l e s and smooth endoplasmic reticulum.. While the presence of such features alone does not c o n s t i t u t e conclusive proof of s t e r o i d secretion t h e i r absence provides strong argument against such a c t i v i t y ( N i c h o l l s & Maple, 1 9 7 2 ) . On the other hand, any attempt to r e l a t e these u l t r a s t r u c t u r a l features to endocrine a c t i v i t y are confused by the well established function of t h i s l a y e r i n the t r a n s f e r of materials to the oocyte and i n the formation of the basement lamina and the v i t e l l i n e membrane. From these data, i t i s impossible to state conclusively that the f o l l i c l e c e l l s of these stages are or are not secreting s t e r o i d s . The formation of the v i t e l l i n e membrane (zona pellucida) by the f o l l i c l e c e l l s has been established i n a v a r i e t y of mammals (N? 5 r r e v a n g , 1 9 6 8 ; Mossman & Duke, 1 9 7 3 ) , birds (N? 5 r r e v a n g , 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ) , insects (Aedes, Anderson & Spielman, . 1 9 7 1 ; Ephestia, Cummings, 1 9 7 2 ; Douglas F i r beetle, Sahota, 1 9 7 3 ) , and amphibia (Xenopus, Dumont, 1 9 7 2 ) . The si t u a t i o n i n the teleosts i s not as v e i l defined with the zona pellucida being formed by the f o l l i c l e c e l l s i n Trichiurus and Tracanthus (Chaudhry, 1 9 5 6 ) and from the oocyte i n Oryzias l a t i p e s (Yamamoto, 1 9 6 3 ) . V i t e l l i n e membrane formation i n the elasmobranch Squalus acanthias resembles most of the higher vertebrates i n that i t i s a product of the f o l l i c l e c e l l s . In those f o l l i c l e c e l l s just i n i t i a t i n g V.M. formation there are extensive stacks of R.E.R., numerous mitochondria, f r e e ribosomes and Golgi bodies. A similar s i t u a t i o n has been jioted i n Ephestia (Cummings., 1 9 7 2 ) where the f o l l i c l e c e l l s have extensive cisternae of R.E.R. and numerous Golgi at the beginning of V.M. formation. The only difference i s that i n Ephestia the V.M. i s formed from secretion bodies assembled i n the Golgi from material produced i n the R.E.R. cisternae. Squalus, however, has microfibres that closely resemble those of the V.M. within the peripheral regions of the f o l l i c l e c e l l s and these present a l l appearances of being intimately related to or added to the substance of the v i t e l l i n e membrane. Lacking direct evidence of secretion bodies fusing with the V.M. (Cummings, 1 9 7 2 ) i t seems l o g i c a l to assume that some unpackaged, monomeric secretion product of the f o l l i c l e c e l l s i s being polymerized i n the region of the developing v i t e l l i n e membrane (Chiquoine, i 9 6 0 ) and i s transformed into the protein-polysaccharide fi b r e s of the v i t e l l i n e membrane. The muco-polysaccharide nature of the v i t e l l i n e membrane has also been reported by Harrison ( 1 9 6 2 ) who speculated that the membrane probably functioned as a transport highway for the appropriately selected molecules and as a barrier to others. A mucopolysaccharide coat over the oolemma of the cockroach and cricket (Anderson & Spielman, 1 9 7 1 ) i s also implicated i n chemical s e l e c t i v i t y . Thus, i t i s l i k e l y that the dual chemical composition (protein and mucopolysaccharide) of the v i t e l l i n e membrane . r e f l e c t s two major functions—support and chemical s e l e c t i v i t y . Adding further support to the theory that the f o l l i c l e c e l l s , not the oocyte, form the V.M. i s the lack of s u f f i c i e n t ribosomes, R.E.R. and Golgi bodies i n the c o r t i c a l ooplasm to form a membrane of such magnitude (Cummings, 1 9 7 2 ) and the absence of V.M. material i n the deep crypts formed by the ooplasmic m a c r o v i l l i . The zona radiata i s a portion of the f o l l i c u l a r envelope f i r s t noted i n elasmobranchs by Balfour ( 1 8 8 5 ) and Wallace ( 1 9 0 3 ) . To them,, the zona radiata or zonoid.layer-was composed of " r a d i a l f i b r e s supported ...above and below by t h i n surfaces" a concept that does not hold true under electron microscopical examination. In t h i s study and several others (Schjeide, Wilkins et a l , 1 9 6 3 ; Yamamoto, 1 9 6 3 ; Press, I 9 6 H ; B e l l a i r s , 1 9 6 5 & 1 9 7 1 ) i t i s c l e a r l y evident that the zona radiata i s a m i c r o v i l l a r s p e c i a l i z a t i o n of the oocyte's plasma membrane. The t y p i c a l morphology of the zona radiata i s that of f i n g e r - l i k e m i c r o v i l l i (See Raven, 1 9 6 l and N?Srrevang, 1 9 6 8 for reviews) similar to that found around the smallest oocytes ( 1 0 0 ym) examined i n t h i s report. The unusual feature of t h i s "layer" i n larger Squalus oocytes i s that i t i s composed of a band of membrane bound vesicles derived from the oocyte, the surface of which i s thrown into numerous large "buds" and m a c r o v i l l i . S t r u c t u r a l l y , the r a i s i n g of the oocyte membrane into m i c r o v i l l i produces an increase i n the surface area of the oocyte ( B e l l a i r s , 1 9 7 1 ) . Kemp ( 1 9 5 6 ) estimated that the zona radiata of the frog p r o d u c e d a 35% i n c r e a s e i n s u r f a c e a r e a (Raven, 1 9 6 l ) . C o n s i d e r i n g t h e e x t e n t o f t h e m o d i f i c a t i o n s i n S q u a l u s , an i n c r e a s e i n s u r f a c e a r e a o f s i m i l a r magnitude w o u l d seem i n o r d e r . F u n c t i o n a l l y , t h i s i n c r e a s e i n s u r f a c e a r e a i s t a k e n t o r e f l e c t t h e enhanced passage o f m a t e r i a l s t h r o u g h t h e o o c y t e membrane ( B e l l a i r s , 1 9 7 1 ) . The r a i s i n g o f t h e o o c y t e and o c c a s i o n a l l y t h e f o l l i c l e c e l l b o r d e r i n t o m i c r o v i l l i i s a f e a t u r e common t o a l l c l a s s e s o f v e r t e b r a t e s (Raven, 1 9 6 l ; Yamamoto, 1 9 6 3 ; J o l l i e & J o l l i e , 196k; B e l l a i r s , 1 9 6 5 & 1 9 7 1 ; W i l l i a m s , 1 9 6 5 ; D a v i d s o n , 1 9 6 8 ; N ( z S r r e v a n g , 1 9 6 8 ; Dumont, 1 9 7 2 ) . a n d some i n v e r t e b r a t e s ( K e s s e l , 1 9 6 8 , Cummings, 1 9 7 2 ) . G e n e r a l l y , t h e s e m i c r o v i l l i o n l y p e n e t r a t e t h e v i t e l l i n e membrane (zona p e l l u c i d a ) s t o p p i n g a t o r b e f o r e t h e o p p o s i n g p l a s m a membrane. However, t h e r e a r e r e p o r t s o f f o l l i c l e c e l l p r o c e s s e s ( m i c r o v i l l i ) p e n e t r a t i n g t h e c o r t i c a l l a y e r o f an o o c y t e ( B e l l a i r s , 1 9 6 5 ; N ? 5 r r e v a n g , 1 9 6 8 ) and one r e p o r t o f " p r o l o n g a t i o n s o f t h e ' f o l l i c l e c e l l t h r o u g h t h e egg membrane f o r m i n g d i r e c t p r o t o p l a s m i c c o n n e c t i o n s w i t h t h e ooplasm" i n t h e S e l a c h i i ( W a l l a c e , 190k). F o r t h e most p a r t , t h e s e p r o c e s s e s a r e c o n f i n e d t o l a r g e y o l k e d eggs ( b i r d s and r e p t i l e s , B e l l a i r s , 1 9 6 5 ) h u t t h e y have a l s o been n o t e d i n t h e s m a l l e r . a m p h i b i a n eggs ( N ^ r r e v a n g , 1 9 6 8 ) . T h e r e f o r e t h e f i n d i n g s o f t h i s r e p o r t a r e c o n s i s t e n t w i t h p r e v i o u s work on t h e S e l a c h i i and w i t h t h e o t h e r v e r t e b r a t e c l a s s e s h a v i n g l a r g e y o l k e d eggs. These f o l l i c l e c e l l p r o c e s s e s have been examined t h e most c a r e f u l l y i n b i r d s ( P r e s s , I 9 6 H ; B e l l a i r s , I 9 6 5 ) and t h e t e r m i n o l o g y most f r e q u e n t l y a p p l i e d t o t h e s e " c o n t a c t a r e a s " a r e " t r a n s o s o m e s " and " l i n i n g b o d i e s " r e s p e c t i v e l y . U l t r a s t r u c t u r a l l y , t h e s e p r o c e s s e s a r e t h o u g h t t o have a r i s e n from desmosomes w i t h one membrane b e i n g p e c u l i a r l y s p e c i a l i z e d ( B e l l a i r s , 1 9 6 5 ; N ? 5 r r e v a n g , 1 9 6 8 ) . But t h r o u g h o u t t h e l i t e r a t u r e t h e r e i s no g e n e r a l agreement on th e i r morphology or function. B e l l a i r s ( 1 9 6 5 ) notes the l i n i n g body resembles a desmosome but i t s membranes are not arranged i n mirror-image pairs and that i t i s also larger than a desmosome. In Squalus oocytes, i t i s highly l i k e l y that the f o l l i c l e c e l l processes of the small ( 0 . 1 mm) oocytes and the double membrane bound structures of the larger oocytes ( 1 . 5 mm) are manifestations of a l i n i n g body-like phenomenon. The relationship here of the developing processes with desmosome-like structures adds further credence to the theory that these " l i n i n g bodies" developed from regular desmosomes. The functions of t h i s phenomenon have been speculated upon and the two most l o g i c a l are that they serve either for securing the oocyte i n place or providing a means of transferring nourishment across the v i t e l l i n e membrane (Press, I 9 6 U ; B e l l a i r s , 1 9 ^ 5 ; N^rrevang, 1 9 6 8 ) . In the l a t t e r instance, several of the e a r l i e r studies hinted at an ooplasmic-follicular continuity v i a the f o l l i c l e c e l l processes (Kemp, 1 9 5 8 ) but l a t e r electron microscopic studies (Raven, 1 9 ^ 1 ; Norrevang, 1 9 6 8 ) and t h i s report show no such continuity. Thus, i t must be noted that Wallace's ( 1 9 0 U ) conclusions were i n error, there being no direct connection between the f o l l i c l e c e l l and the oocyte. Other authors (Chiquoine, i 9 6 0 ; Adams & Hertig, I 9 6 U ) contended that these processes may s t i l l be related to n u t r i t i o n a l transport of materials unable to diffuse through the v i t e l l i n e membrane with the material entering the oocyte v i a micropinocytosis. W^rrevang ( 1 9 6 8 ) , however, holds the opposite view that the presence of desmosomes i n an area of contact would not permit the transport of materials into the oocyte and that i t was more l i k e l y that the processes served as anchoring structures for the oocyte. A t h i r d p o s s i b i l i t y exists and that i s that these " l i n i n g bodies" serve both functions. This i s the view held i n t h i s report with regard to the processes found i n Squalus oocytes. The desmosome—like•structures and the variety of angles that the processes penetrate the oocyte and the depths at which they are found support the "anchoring" statement. On the other hand, the presence of small vesicles at the f o l l i c u l a r o r i g i n of the processes and within and without the " l i n i n g bodies" of the oocyte as well as the paths or streams found associated with ±he l i n i n g bodies i n the c o r t i c a l ooplasm indicate the tra n s f e r of material v i a the processes. The streams of material probably represent some form of yolk precursor matter that has been passed through the v i t e l l i n e membrane v i a -the pore canals or l i n i n g bodies. Slauterback ( 1 9 6 3 ) suggested that microtubules found i n secreting c e l l s may act as a transport system and ,if t h i s assumption i s extended to closely packed microfilaments one.can r e a d i l y envision some material being channeled into the f o l l i c u l a r opening of the pore canals, transferred through the v i t e l l i n e membrane and d i f f u s i n g through the twin plasma membranes of the ooplasmic l i n i n g bodies. In opposition to TfySrrevang ( 1 9 6 8 ) i t i s obvious that the lack of desmosomes around the entirety of the l i n i n g body i s going to allow material to diffus e across the membranes and into the oocyte. In addition to the f o l l i c l e c e l l processes found i n Squalus oocytes there are also ooplasmic m i c r o v i l l i that penetrate the f o l l i c l e c e l l cyto-plasm. This i s a phenomenon infrequently recorded, being, noted i n but two teleost species, Lebistes r e t i c u l a t u s ( J o l l i e & J o l l i e , I 9 6 U ) and Oryzias  l a t i p e s (Yamamoto, 1 9 6 3 ) . In the former instance, the "microprojections of the ooplasmic processes pass through the pore canals and into the recesses of the inner f o l l i c l e plasma membrane". Once again there i s oocyte and f o l l i c l e c e l l contiguity but never cytoplasmic continuity and thus only ind i r e c t pathways for the passage of nutrients into the egg ( J o l l i e & J o l l i e , I 9 6 H ) . In the l a t t e r instance, oocyte m i c r o v i l l i are said to pass through perforations i n the zona pellucida and extend into the f o l l i c l e c e l l s forming complicated anastomoses. I t i s d i f f i c u l t , however, to t e l l from Yamamoto's micrographs that the m i c r o v i l l i actually penetrate the substance of the f o l l i c l e c e l l s and do not merely occupy an e x t r a c e l l u l a r space. The existence of these ooplasmic m i c r o v i l l i i n dogfish oocytes i s transient, with none being observed i n oocytes larger than 1 . 0 mm i n diameter Speculating on t h e i r function, one may assume that they do allow passage of some materials, possibly i n a reverse flow, or they serve as additional anchors for a young oocyte u n t i l superceded by the more highly developed f o l l i c l e c e l l processes or l i n i n g bodies. One feature that i s unique about these penetrating m i c r o v i l l i i n Squalus oocytes i s that there i s no record of any other vertebrate or .invertebrate oocyte .having both ooplasmic and . . f o l l i c u l a r c e l l processes that penetrate the substance of each other. OOCYTE NUCLEUS In a l l stages examined ( 0 . 1 to h.O mm) the oocyte nuclei were at diplotene of the f i r s t meiotic d i v i s i o n . Even i n the most immature females examined ( 6 0 to 9 0 cm) such was the case. This lead to the conclusion that the e a r l i e s t phases of oogenesis are to be found i n l a t e embryos ( 2 0 months) or i n the newly born young. A. NUCLEOLI The granular nature of Squalus oocyte n u c l e o l i i s a not uncommon feature. Raven ( 1 9 6 1 ) notes that electron microscopy has often revealed a purely granular composition of oocyte n u c l e o l i . The granules of the nucleoli are similar to those randomly dist r i b u t e d i n the nucleoplasm averaging 1 5 0 R i n diameter and when the periphery of the nucleolus i s examined i t can be seen that the nucleolus i s extruding these granules. Further, the 1 5 0 R granules found i n the ooplasm opposite the nuclear pores are, i n a l l l i k e l i h o o d , these same granules that have passed through the nuclear membrane v i a the nuclear pores. I t has been established that the hundreds of n u c l e o l i present during the prolonged diplotene stage represent an a m p l i f i c a t i o n of the genome associated with the synthesis of large quantities of rRNA (Brown, 1 9 6 6 ; Davidson, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ; Kessel & Decker, 1 9 7 1 ; Dumont; 1 9 7 2 ) . Thus these 1 5 0 R p a r t i c l e s i n the nucleolus, nucleoplasm and i n the ooplasm must be ribonucleoprotein (ribosomes) or the l 8 S and 2 8 S ribosomal RNA molecules that are contained i n i n a c t i v e s i n g l e ribosomes (Scheer, 1 9 7 3 ) . Lampbrush chromosomes, as found i n the diplotene stages of t h i s study have been implicated i n rRNA synthesis as well as i n DNA-like RNA synthesis (Rana, Kessel & Decker, 1 9 7 1 ) . The passage of nucleolar material into the ooplasm has been described i n so many cases (sponges, hydroids, platyhelminthes, annelids, t u r b e l l a r i a n s , molluscs, arthropods and vertebrates), that i t can hardly be doubted that i t i s of general occurrence (Raven, 1 9 6 1 ) . There are numerous theories on how t h i s nucleolar material crosses the nuclear mmebrane (See Raven, 1 9 6 l f o r a review) but the most commonly accepted theory i s that these 1 5 0 S rRNA p a r t i c l e s pass through the nuclear pores (Rhodonis, Anderson & Beams, 1 9 5 6 : Kemp, 1 9 5 6 b ; Kessel & Beams, 1 9 6 3 b ; Yamamoto, I 9 6 H ; Williams, 1 9 6 5 ; _ Priapulus, N ( z 5 r r e v a n g , 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ; Prostheceraeus, Boyer, 1 9 7 2 ; Xenopus, Scheer, 1 9 7 3 ; t r o u t , Beams & Kessel, 1 9 7 3 ) . I t i s also of i n t e r e s t to note that there i s "abundant c y t o l o g i c a l evidence" (Raven, 1 9 6 l ) and electron microscopical reports that whole n u c l e o l i have been extruded into the ooplasm (rat and some r e p t i l e s , B e l l a i r s , 1 9 7 1 ; Thyone, NpVrevang, 1 9 6 5 ) . . Thus, the 8 0 0 nm body observed i n the ooplasm of a 0 . 1 mm oocyte i s an extruded nucleolar body. A body th i s size would not pass through a nuclear pore and therfore the nuclear membrane would either have to break and reform or pinch o f f with the nucleolus contained inside. The l a t t e r p o s s i b i l i t y i s less l i k e l y due to the lack of an en c i r c l i n g double membrane or the remnants of one. B. NUCLEAR PORES The complete or p a r t i a l perforation of the nuclear envelope by pores i s discussed i n a l l papers focussed on the structure of the nuclear envelope of oocytes (N ( z 5 r r e v a n g , 1 9 6 5 ) . Regardless of species, both membranes of the nuclear envelope fuse i n an area approximately 5 0 0 & i n diameter and may or may not have a constricting diaphragm (NzSrrevang, 1 9 6 8 ) . In t h i s regard, the nuclear pores of dogfish oocytes are quite t y p i c a l . The presence or absence of an annular diaphragm was not established. "In older oocytes—and always at the onset of v i t e l l o g e n e s i s — n u c l e a r pores cover the entire nuclear envelope..."- (N? 5 r r e v a n g , 1 9 6 8 ) . Thus the increase i n nuclear pore frequency r e f l e c t s not only the approach of vitellogenesis but also the increase i n the amount of rRNA synthesized i n the nucleoli and transported to the ooplasm (Scheer, 1 9 7 3 ) . C. NUCLEAR MEMBRANE Throughout the stages examined the nuclear membrane shows only two major changes i n morphology: the increase i n the number of nuclear pores and the "blebbing" of the outer nuclear membrane. The l a t t e r i s not an uncommon phenomenon, being reported i n Priapulus (N? 5 r r e v a n g , 1 9 6 5 ) , trout (Beams & Kessel, 1 9 7 3 ) , Xenopus (Dumont, 1 9 7 2 ) , Triturus vulgaris (Spornitz & Kress, 1 9 7 3 ) , and numerous other species (N?$rrevang, 1 9 6 8 ) . These 1 vesicles or blebs from the nuclear membrane have been demonstrated to be part of the endoplasmic reticulum (N?$rrevang, 1965 & 1968; Beams 8s Kessel, 1973; Spornitz & Kress, 1973), e i t h e r granular or agranular depending on the species. Further, the postmitotic nuclear membrane has been shown to reform by the coalescence of f l a t saccular elements of the endoplasmic reticulum (Bloom & Fawcett, 1968) and thus i s considered to be an i n t e g r a l part, of the endoplasmic reticulum. Although no extensive connections between the nuclear membrane and the v e s i c u l a r smooth E.R. of the ooplasm were found i n t h i s study, the morphology of the membrane i s i n d i c a t i v e of at l e a s t a temporary connection. Given a r a p i d l y developing oocyte and the abundance of smooth E.R. adjacent to the nucleus one can r e a d i l y surmise that t h i s i s an act i v e process not e a s i l y demonstrated. A second phenomenon associated with the blebbing of the oocyte nuclear membrane i s the formation of annulate lamellae (N^rrevang, 1968; Spornitz & Kress, 1973). The structure found i n a 1.0 mm oocyte matches the c l a s s i c a l d e s c r i p t i o n , wherein i t consists "of p a r a l l e l f l a t t e n e d v e s i c l e s , pierced by pores, very s i m i l a r to those seen i n the nuclear envelope" (N?5rrevang, 1968). Such structures u s u a l l y appear at the onset of v i t e l l o g e n e s i s and are thought to play a r o l e i n p r o t e i n yolk synthesis, a function that was neither confirmed nor denied i n t h i s report. It i s also of i n t e r e s t to note that the increasing i r r e g u l a r i t y of the nuclear membrane p a r a l l e l s the increase i n n u c l e o l i number and that t h i s phenomenon has been recorded previously (N?5rrevang, 1968; Dumont, 1972) and related- to the further d i f f e r e n t i a t i o n of the oocyte. MITOCHONDRIAL MASS Also known as a Balb i a n i body or a yolk nucleus, the mitochondrial mass has been described i n a v a r i e t y of oocytes and was thought to be 145 involved i n the production of yolk. But e l e c t r o n microscopy has revealed that these bodies show a v a r i e t y of s t r u c t u r a l u n i t s , being composed of either a single element or several elements i n a complex structure (Raven, 1 9 6 1; Yamamoto, I 9 6 U ; Williams, 1 9 6 5 ; N<z$rrevang, 1 9 6 8 ) . In trout (Beams & Kessel, 1 9 7 3 ) the body i s a prominent, granular mass, i n S p i s u l a and sea urchins (N? 5 r r e v a n g , 1 9 6 5 ) , endoplasmic reticulum i s most prominent, i n mammals, the Balbiani body consists mainly of Golgi v e s i c l e s , i n b i r d s , ( B e l l a i r s , 1 9 7 1 ) the body, which disappears p r i o r to yolk deposition, consists of smooth E.R., mitochondria and Golgi v e s i c l e s , i n Xenopus, Trit u r u s and Rana esculenta (N^rrevang, 1 9 6 8 ; Dumont, 1 9 7 2 ) and Pentastomida (N? 5 r r e v a n g , 1 9 7 2 ) the B a l b i a n i body i s an enormous juxtanuclear mass of mitochondria intermingled with a few smooth membrane v e s i c l e s and granules. Spornitz and Kress ( 1 9 7 3 ) , however, maintain that T r i t u r u s v u l g a r i s lacks a Balbiani body. The juxtanuclear structure found i n Squalus oocytes most c l o s e l y resembles that found i n many amphibians and w i l l be r e f e r r e d to as a mitochondrial mass, as i n Ward ( 1 9 6 2 ) , Balinsky & Devis ( 1 9 6 3 ) , Ander son & Beams ( 1 9 6 H ) , and Dumont ( 1 9 7 2 ) . In t h i s study as well as many others (Yamamoto, I 9 6 H ; N ? 5 r r e v a n g , 1 9 & 5 & 1 9 6 8 ; Dumont, 1 9 7 2 ) i t has been shown that yolk formation i s morphologically independent of the Balbiani body and i t was urged that t h i s name be dropped. Further, as these structures are not morphologically homologous i t has also been considered unwise to use a general name for them. They do, however, exhibit c e r t a i n common features; they appear i n the immediate v i c i n i t y of the nucleus, mitochondria are often c l o s e l y associated with them as are E.R. components, they contain protein and are often r i c h i n RNA and they usually disappear p r i o r to v i t e l l o g e n e s i s (Raven, 1 9 6 I : Yamamoto, I 9 6 U ; B e l l a i r s , 1 9 7 1 ; Dumont, 1 9 7 2 ) . Therefore, I feel.that a common term such as Balbiani body devoid of i t s implication i n yolk formation i s more readily acceptable, than a variety of descriptive terms, one of which I have used extensively. The increase i n the number of mitochondria noted i n early Squalus oocytes i s a normal feature of oogenesis i n a l l vertebrates and invertebrate In Pentastomida (W^rrevang, 1 9 7 2 ) the m u l t i p l i c a t i o n of mitochondria and the concomitant increase i n ergastoplasm are taken as the f i r s t signs that the oocyte has entered i t s growth phase. In Priapulus the number of mitochondria r i s e s during oogenesis from 5 to 8 i n the oogonium to about 1 + 0 , 0 0 0 i n the mature oocyte with similar increases noted i n other oocytes (N^rrevang, 1 9 6 8 ) . The obvious question of the mechanism of mitochondrial m u l t i p l i c a t i o n now arises. Based on the variety of mitochondrial config-urations found i n dogfish oocytes i t i s l o g i c a l to presume that the mito-chondria p r o l i f e r a t e either by budding or by elongation and d i v i s i o n of pre-existing mitochondria. This i s a conclusion previously reached by . many authors. The dumb-bell and branched mitochondria described i n t h i s report have also been noted i n Priapulus, Drosophila, Lacerta, the rabbit, and the rhesus monkey (N(zSrrevang, 1 9 6 8 ) . These variations are generally believed to be signs of mitochondrial m u l t i p l i c a t i o n with the former case r e f l e c t i n g multiple d i v i s i o n s and the l a t t e r a budding process. The p a r a l l e l arrays of cri s t a e are either a further i n d i c a t i o n of an imminent d i v i s i o n or an indication of reduced metabolic a c t i v i t y (Fawcett, 1 9 6 6 ) . Given that t h i s configuration occurs i n a metabolically active oocyte, the l a t t e r supposition can be ruled out. The ring-shaped mitochondria are unusual but they may be produced as a resul t of the sectioning plane passing through the crook of a "question-mark" or dumb-bell shaped mitochondrion. Having already noted that the B a l h i a n i body i s not involved i n yolk formation, many authors have speculated on the function of t h i s s t r u c t u r e . L i v n i ( l 9 T l ) found G-6-P dehydrogenase (a cytoplasmic enzyme), ct-glycero-phosphate dehydrogenase and succinate dehydrogenase (mitochondrial enzymes) i n the mitochondrial B a l b i a n i body of three t e l e o s t s . Many other authors (Yamamoto, 196k; Guraya, 1 9 ^ 5 ; Abraham et a l , 1 9 6 6 ; U l r i c h , 1 9 6 9 ; Beams & Kessel, 1 9 7 3 ) intimate that i t forms or c o n s t i t u t e s an e s s e n t i a l precursor substance (usually RNA) which i s necessary f o r oocyte growth and v i t e l l o g e n e s i s . Other authors refuse to speculate on i t s function ( B e l l a i r s , 1 9 7 1 ; Dumont, 1 9 7 2 ) but allow that i t i s near enough to the nucleus to intercept e s s e n t i a l materials moving into the ooplasm from the nucleus, thus h i n t i n g that the B a l b i a n i body may i n d i r e c t l y be involved i n v i t e l l o g e n e s i s . Regardless of the confusion t h i s body of organelles creates, i t s function i n Squalus acanthias and various amphibian oocytes i s that of a centre for the m u l t i p l i c a t i o n of mitochondria. These mito-chondria then disperse and indeed the whole structure disperses p r i o r to v i t e l l o g e n e s i s allowing the mitochondria to associate with the numerous v e s i c l e s and cisternae of S.E.R. f o r the formation of p r o t e i d yolk. At no time were mitochondria observed with i n t e g r a l yolk p l a t e l e t s as i n many amphibian oocytes (Ward, 1 9 6 2 ; Massover, 1 9 7 1; Kress & Spornitz, 1 9 7 2 ) Thus, the mitochondrial mass or B a l b i a n i body i s involved i n yolk formation but only i n d i r e c t l y , i n that i t contributes e s s e n t i a l p r e r e q u i s i t e s , e i t h e r enzymes or r i b o n u c l e i c a c i d . LIPID YOLK Of the. three types of yolk found i n most oocytes (Raven, 1 9 6 l ) 148 Squalus acanthias oocytes lack only carbohydrate (glycogen) yolk. The pertinent histochemical data are inconclusive but u l t r a s t r u c t u r a l l y no • glycogen rosettes were observed i n any of the stages examined. L i p i d or f a t t y yolk i s formed before protein yolk (Raven, 1 9 6 l ) and i n amphibians i t i s formed at the end of the "pre-vitellogenic" period (Kemp, 1 9 5 6 b ) . L i p i d yolk i s usually represented as a dense, smooth or ir r e g u l a r body without a l i m i t i n g membrane (Raven, 1 9 6 l ; Yamamoto, I 9 6 H ; N^rrevang, 1 9 6 8 ; Bondi & Facchini, 1 9 7 2 ) . These ch a r a c t e r i s t i c s describe the large dense bodies found i n early dogfish oocytes. The methods of formation of l i p i d yolk are as varied as the species that produce i t . Numerous species (Lumbricus, Limnaea, Asterias, Rana, Raven, 1 9 6 l ; mollusc, c r a y f i s h , ascidians, Priapulus, N^rrevang, 1 9 6 8 ; Oryzias, Yamamoto, I 9 6 H ; Branchiobdella, Bondi & Facchini, 1 9 7 2 ) report the .formation of l i p i d yolk de .novo ( i . e . not -associated with -any organelles). Other species (earthworms, bi r d s , man, Raven, 1 9 6 l ; Arabacia, sea urchin, rabbit, NgSrrevang, 1 9 6 8 ) show i t s formation occuring i n association with mitochondria or a juxtanuclear accumulation of mitochondria (Raven, 1 9 6 1 ) . Only two reports of direct transformation of mitochondria into l i p i d are known (Drosophila & dog, Raven, 1 9 6 l ) . Formation of l i p i d yolk by or i n association with Golgi i s also reported frequently (Raven, 1 9 6 l ; N^rrevang, 1 9 6 8 ) . From the available morphological data, i t i s d i f f i c u l t to ascribe the formation of l i p i d yolk i n dogfish oocytes to any pa r t i c u l a r organelle. The relationship of the formed l i p i d elements with the vesicles of the smooth E.R. hints at some metabolic association. However, there i s no direct evidence to support such a claim. Thus, i t i s probable that as i n so many other species (Raven, 1 9 6 l ; Yamamoto, I 9 6 H ; N^rrevang, 1 9 6 8 ; Bondi & Facchini, 1 9 7 2 ) the l i p i d yolk i n dogfish oocytes can be said to arise de_ novo i n the ooplasm. This i n turn can be further q u a l i f i e d i f one considers Raven's ( 1 9 6 1 ) conclusion that the time and place at which l i p i d yolk becomes v i s i b l e does not necessarily correspond with the time and place of fat synthesis but probably represents a process of conden-sation and pr e c i p i t a t i o n of soluble l i p i d s which reach t h e i r saturation point and precipitate out possibly using various c e l l components as "condensation nuclei". In t h i s instance, the l i p i d yolk may be using the S.E.R. as i t s condensation nuclei with further growth of the l i p i d droplet occurring Independently of the smooth endoplasmic reticulum. This may explain the proximity of the S.E.R. to the l i p i d yolk and also, why no direct contribution to the developing droplet has been seen.-Again, the morphological data i s i n s u f f i c i e n t to conclusively explain the o r i g i n of th i s "dissolved l i p i d " . It-can-come"from-but "one of-two sources, the f i r s t being from within the oocyte i t s e l f . However, t h i s study has been unable to provide any evidence to substantiate t h i s theory. Secondly, the l i p i d may be extra-oocytic i n o r i g i n , t r a v e l l i n g to the ovary from i t s point of o r i g i n v i a the blood (Raven, 1 9 6 l ; N?Srrevang, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ) ultimately penetrating the oocyte either by d i f f u s i o n or by pinocytotic processes which have already been discussed i n terms of the dogfish oocyte v i t e l l i n e membrane. This- i s substantiated by the free l i p i d drops found i n the cortex, theca and f o l l i c l e c e l l layer. A d d i t i o n a l l y , the location of the l i p i d yolk i n the mid to outer regions of the oocyte and the pronounced formation of l i p i d concomitant with development of ooplasmic m i c r o v i l l i are factors that also prompted Bondi & Facchini ( 1 9 7 2 ) to conclude that the materials for l i p i d yolk penetrated the oocyte by dif f u s i o n . The relationship of the mitochondria and the l i p i d droplets has previously been considered to be consistent with the interpretation that the mitochondria play an important r o l e i n l i p i d metabolism as the mitochondria have been shown to contain the enzymes involved i n t r i g l y c e r i d e metabolism (Fawcett, 1 9 6 6 ; White, Handler & Smith, 1 9 6 8 ) . This relationship then'provides a store of energy for immediate or l a t e r use and a potential source of short carbon chains for the synthesis of s t r u c t u r a l components which contain l i p i d s , such as membranes. The l a t t e r i s of importance during protein yolk formation for the outer l i m i t i n g membrane of the yolk p l a t e l e t and for possible contribution to the numerous membrane bound vesicles that constitute, part of the primary yolk p l a t e l e t s . PROTEIN YOLK Protein yolk i s the second type of yolk found i n dogfish oocytes and although i t s precursor bodies are formed concurrently w i t h . l i p i d yolk, the pl a t e l e t s themselves do not become v i s i b l e u n t i l w e l l after l i p i d yolk has been formed. The development of G.V.B.s and M.V.B.s as precursors to protein yolk pl a t e l e t s i s a well established pathway observed i n tunicates (Ciona, Kessel, 1 9 6 6 ) , anurans (Kress & Spornitz, 1 9 7 2 ) and urodeles (Spornitz & Kress, 1 9 7 3 ) . In Ciona the abundant Golgi supply the large numbers of vesicles required while i n Rana esculenta and R_. temporaria the vesicles are derived' from micropinocytosis, the S.E.R. and the Golgi and i n Triturus vulgaris the vesicles of primary yolk are formed.from the nuclear membrane or de_ novo i n the ooplasm Secondary yolk i n T. vulgaris i s formed from p i n o c y t o t i c a l l y derived v e s i c l e s . In each instance the ooplasm has abundant free ribosomes. In Squalus acanthias oocytes, v e s i c u l a r yolk i s formed by v e s i c l e s from each of these sources, a phenomenon unreported i n any other yolk producing organism. A possible explanation f o r t h i s phenomenon l i e s i n the fact that only the S.E.R. i s moderately w e l l developed and therefore membranous contributions from a l l sources are required to produce the massive amounts of p r o t e i n yolk found i n dogfish oocytes. Another factor that must be considered i n dogfish v i t e l l o g e n e s i s i s the source of the yolk proteins incorporated i n t o the p l a t e l e t s . I t i s generally accepted that i n vertebrates the raw materials of yolk are formed i n the l i v e r and pass from there i n the blood plasma to the ovary where they enter the oocyte by pinocytosis (Rudack & Wallace, 1 9 6 8 ; Wallace & Dumont, 1 9 6 8 ; B e l l a i r s , 1 9 7 1 ; Kress &" Spornitz, 1 9 7 2 ) . This i s supported by several l i n e s of evidence. F i r s t l y , the rate of deposition i n b i r d ' s eggs i s too great for the yolk to have been manufactured i n the oocyte or i n .the f o l l i c l e c e l l s . In a hen, t h i s would require each f o l l i c l e c e l l to secrete a quantity of yolk equal to i t s own volume every h a l f hour during the l a s t phase of deposition. Secondly, the blood serum and l i v e r of l a y i n g hens has been shown to contain large amounts of l i p i d s and proteins with properties c l o s e l y resembling those of s i m i l a r m a terial found i n yolk, while these substances cannot be detected i n male b i r d s and non-laying hens (Heald & McLachlan, 1 9 6 5 ; Husbands, 1 9 7 0 ; B e l l a i r s , 1 9 7 1 ; Beuving & Gruber, 1 9 7 1 ; C h r i s t i e & Moore, 1 9 7 2 ) . T h i r d l y , there i s no biochemical data to support the conception of an i n t r a o o c y t i c synthesis of yolk proteins p a r t i c u l a r l y i n the ranidae (Kress & Spornitz, 1 9 7 2 ) and i n urodeles (Spornitz & Kress, 1 9 7 3 ) . But there i s considerable morphological and autoradiographic data to i n d i c a t e that yolk i s i n part synthesized i n t r a -o o c y t i c a l l y (Lebistes r e t i c u l a t u s , D r o l l e r & Roth, 1 9 6 6; Brachydanio r e r i o , Korfmeiser, 1 9 6 6 ; anurans, Spornitz & Kress, 1 9 7 1 , Kress & Spornitz, 1 9 7 2 , Spornitz & Kress, 1 9 7 3 ) Thus t h i s question a r i s e s , does Squalus acanthias produce i t s yolk autosynthetically (within the oocyte) or h e t e r o s y n t h e t i c a l l y (outside the oocyte)? Like many problems the answer i s not c l e a r cut. In a l l l i k e l i h o o d Squalus follows the vertebrate plan ( B e l l a i r s , 1 9 7 1 ) and produces i t s yolk proteins i n the l i v e r and transports them to the oocyte. In support of t h i s hypothesis one must consider the massive s i z e of the dogfish l i v e r , the si z e and numbers of the mature oocytes ( s i m i l a r to the hen's) and the pino-c y t o t i c v e s i c l e s found at the oocyte's plasma membrane. But i n the smallest oocytes, pinocytosis i s not occurring at a r a t e s u f f i c i e n t to account for the yolk precursor bodies (G.V.B.s) that are present. Thus, i t i s c l e a r that the membranous elements which c o n s t i t u t e the framework of the yolk p l a t e l e t s are produced a u t o s y n t h e t i c a l l y . However., the contents of these membranous elements may be formed under the influence of the free ribosomes as i s the case i n the tunicate Ciona (Kessel, 1 9 6 6 ) , the c r a y f i s h (Beams & Kessel, 1 9 6 3 ) and the l o b s t e r Homarus (Kessel, 1 9 6 8 ) , a proposal that i s compatible with the r o l e of the ribosome i n p r o t e i n synthesis. A l t e r n a t i v e l y , the small molecular precursors of p r o t e i n yolk may pass by d i f f u s i o n across the oocyte membrane to appear i n the E.R., Golgi and possibly the mitochondria (Ward, 1 9 6 2 ; Beams & Kessel, 1 9 6 3 ; Kessel, 1 9 6 8 a , b,c; Anderson, 1 9 6 9 ; Kessel, 1 9 7 1 ; Massover, 1 9 7 1 ; Boyer, 1 9 7 2 ) u l t i m a t e l y fi n d i n g t h e i r way to the granular-vesicular bodies. I n d i c a t i v e of t h i s i n dogfish oocytes are the intimate associations of the S.E.R. with the l i n i n g bodies, Golgi and mitochondria. In any event, these i n t r a o o c y t i c membranous elements serve a packaging r o l e f o r the accumulation of yolk m a t e r i a l over a period of time. The formation of the l i m i t i n g membrane of yolk p l a t e l e t precursors from distended Golgi or S.E.R. cisternae has a counterpart i n v i t e l l o g e n e s i s i n c r a y f i s h (Beams & Kessel, 1 9 6 2 , 1 9 6 3 a , b) and i n Lebistes ( D r o l l e r & Roth, 1 9 6 6 ) . In c r a y f i s h , large (300 to 8 0 0 K) yolk granules are formed v i t h i n the cisternae of R.E.R. l a t e r t o pass to agranular parts of the E.R. where they aggregate i n t o l a r g e r masses u l t i m a t e l y changing i n t o f i n e l y granular yolk bodies. Further, i n trout oocytes a system of precursor formation i d e n t i c a l to dogfish oocytes has been noted. Here S.E.R. cisternae with i n t r a c i s t e r n a l granules anastomose, fuse and swell to permit the accumulation of ICG or precursor yolk (Beams & Kessel, 1 9 7 3 ) . The looping of S.E.R. tubules to form a l i m i t i n g membrane has a s i m i l a r counterpart i n Priapulus oocytes. There the R.E.R. v e s i c l e s surround a small, i r r e g u l a r condensation of dense m a t e r i a l , with the incomplete mantle they form creating a.gradient, drawing i n a d d i t i o n a l m a t e r i a l . The l i m i t i n g membrane i s l a t e to form but i t s r e l a t i o n s h i p to the R.E.R. i s taken as an i n d i c a t i o n that i t s formation i s induced by the ergastoplasmic membrane (N? 5 r r e v a n g , 1 9 6 8 ) . .As previously mentioned, granular-vesicular bodies d i s p l a y f i g u r e s i n d i c a t i v e of the d i s s o l u t i o n and r e a s s o c i a t i o n of t h e i r contents as a granular matrix. Similar phenomena have been reported i n a l l oocytes that have a v e s i c u l a r form of yolk formation (Beams & Kessel, 1 9 6 2 , 1 9 6 3 a , b; Yamamoto, I 9 6 U ; Kessel, 1 9 6 6 , 1 9 6 8 ; Boyer, 1 9 7 2 ; Dumont, 1 9 7 2 ; Kress & Spornitz, 1 9 7 2 ; Beams & Kessel, 1 9 7 3 ; Spornitz & Kress, 1 9 7 3 ) . However, l i t t l e notice has been given to t h i s transformation of proteinaceous and l i p i d (membrane) precursors into a homogeneous mass. The f a c t that t h i s transformation i s p o s s i b l y enzyme mediated has gone unmentioned and may explain the proximity of the mitochondria to the G.V.B.s. One feature of v i t e l l o g e n e s i s i n dogfish oocytes that poses another question i s when does the c r y s t a l l i z a t i o n of the p l a t e l e t occur? The f a c t that i t i s not d i r e c t l y r e l a t e d to the development of the G.V.B. i s demonstrated by the presence of well developed electron dense G.V.B.s i n 0 . 1 mm oocytes and a t o t a l absence of yolk p l a t e l e t s . However, the c r y s t a l l i z a t i o n of the p l a t e l e t at 3 . 0 to U.O mm may r e f l e c t the threshold l e v e l f o r a p i n o c y t o t i c a l l y derived precursor required f o r c r y s t a l l i z a t i o n . A s i m i l a r phenomenon has been recorded i n T r i t u r u s v u l g a r i s (Spornitz & Kress, 1 9 7 3 ) wherein c r y s t a l l i z a t i o n of primary and secondary yolk p l a t e l e t s does not occur u n t i l micropinocytosis has begun. The c r y s t a l l i n e pattern i n amphibian yolk p l a t e l e t s has been a t t r i b u t e d to the arrangement of l i p o p r o t e i n subunits. These u n i t s are p h o s v i t i n (MW U 0 , 0 0 0 ) and l i p o v i t e l l i n (MW 2 1 0 , 0 0 0 ) (Wallace, 1963; Rudack & Wallace, 1968.; Massover, 1971; Leonard et a l , 1972) c l a s s i c a l l y present ,in the r a t i o of two pho s v i t i n to one l i p o v i t e l l i n hexagonally arranged to form the pattern observed. Recently, however, a freeze-etching study has suggested that the c r y s t a l l i n e pattern i s formed by l i p o v i t e l l i n dimers with one • phosvitin molecule associated with each monomer of the l i p o v i t e l l i n dimer (Leonard et a l , 197 2 ) . Analysis of the dogfish yolk p l a t e l e t and comparison of i t with the amphibian system was beyond the scope of t h i s study. But considering the s i m i l a r i t i e s of amphibian and elasmobranch yolk p l a t e l e t s , expecting such a molecular r a t i o ( 2 : 1 or 1 : 1 ) i n elasmobranch yolk would not seem out of order. For a discussion of the properties of various vertebrate phosvitins see Clark ( 1 9 7 2 ) . The formation and c r y s t a l l i z a t i o n of p h o s v i t i n and l i p o v i t e l l i n i n Rana pipiens was thought to involve a terminal phosphorylation mediated by an oocytic protein kinase (Wallace, 1 9 6 3 ) . But i n a recent chromatographic study (Wallace et a l , 1 9 7 2 ) , the lack of any s p e c i f i c association o f . ^ P -l a b e l l e d material with either the l i p o v i t e l l i n or, p a r t i c u l a r l y , the phosvitin 32 after incubation i n NagHPO^- P was taken as precluding the previous suggestion. Further, incubation of v i t e l l o g e n i n , the p h o s v i t i n - l i p o v i t e l l i n precursor substance, with ATP (y- P) and p u r i f i e d oocyte protein kinase did not dissociate the phosphorylated product into l i p o v i t e l l i n and phosvitin. Thus, the processing mechanism for the conversion of v i t e l l o g e n i n into yolk proteins remains unknown (Wallace e t . a l , 1 9 7 2 ) . Considering.the s i m i l a r i t y of amphibian and dogfish yolk (see below) these conclusions may hold true for Squalus as wel l . The presence of a c r y s t a l l i n e zone i s regarded as being t y p i c a l for yolk platelets (Massover, 1 9 7 1 ; Spornitz & Kress, 1 9 7 3 ) and even as defining the organelle as a yolk p l a t e l e t (Massover, 1 9 7 1 ) . However, Spornitz & Kress ( 1 9 7 3 ) also consider that the presence of a c r y s t a l l i n e or p a r a c r y s t a l l i n e arrangement i n structures found i n oocytes cannot alone be regarded as suf f i c i e n t evidence that such structures are i d e n t i c a l to yolk. This statement refers only to intramitochondrial c r y s t a l l i n e i n c l u s i o n bodies i n the ranidae which have different center to center spacing of the c r y s t a l cylinders r e l a t i v e to the pattern of the common amphibian yolk (vesicular yolk) p l a t e l e t (Spornitz, 1 9 7 2 ) . Thus i t i s safe to conclude that the c r y s t a l l i n e structures found i n dogfish oocytes are yolk p l a t e l e t s and based on the center to center spacing of the pattern that these p l a t e l e t s are i d e n t i c a l to the vesicular yolk p l a t e l e t s of the amphibia. The term " i d e n t i c a l " i s used loosely and i n a l l l i k e l i h o o d the s i m i l a r c r y s t a l l i n e l a t t i c e patterns of amphibian and elasmobranch yolk p l a t e l e t s r e f l e c t s similar ratios of phosvitin to l i p o v i t e l l i n (Spornitz, 1 9 7 2 ) . I t i s also of interest to note that yolk p l a t e l e t s with a c r y s t a l l i n e core (amphibian type) belong to the p h y l o g e n e t i c a l l y older vertebrate groups. These groups include the amphibia, the holosteans and now the elasmobranchs. The yolk of the p h y l o g e n e t i c a l l y newer groups, the t e l e o s t s , r e p t i l e s , birds and mammals lack a c r y s t a l l i n e core and are r e f e r r e d to as the b i r d type ( K i l a r s k i & Grodzinski, 1969). Based upon, the data presented the following pathway for p r o t e i n yolk formation i n dogfish oocytes has been postulated. In the smallest oocytes (< O.h mm), small M.G.B.s and M.V.B.s are formed by and from Golgi v e s i c l e s , S.E.R. v e s i c l e s , nuclear membrane and micropinocytotic v e s i c l e s . These i n turn fuse with one another forming granular-vesicular bodies. In l a r g e r oocytes (O.h mm +) the outer l i m i t i n g membrane of a yolk p l a t e l e t precursor i s formed either from distended Golgi or S.E.R. v e s i c l e s or from loops of small S.E.R. v e s i c l e s . In the former case these two v e s i c u l a r bodies are empty and appear f i r s t as large M.G.B.s. Once these precursor membranes form or begin to form they take up M.G.B.s, M.V.B.s and small G.V.B.s. By either method granular-vesicular bodies are formed and these i n turn d i s p l a y the breakdown and d i s s o l u t i o n of t h e i r contents ( i . e . m y e l i n - l i k e f i g u r e s and homogeneous granular matrix). The subsequent recondensation of t h i s material produces an electron dense granular matrix or core within the G.V.B. The f i n a l phase i n protein yolk p l a t e l e t formation i s the c r y s t a l l i z a t i o n of t h i s core. SUMMARY TUNICA ALBUGINEA The protein reaction suspected to in d i c a t e the presence of smooth muscle as i n the tunica of the smooth dogfish Mustelus canis (TeWinkel, 1972) was not corroborated by the electron micrographs. The histochemical analysis indicates that collagen should be present and our E.M. work demonstrates i t con c l u s i v e l y . E l a s t i c f i b r e s and t h e i r u l t r a s t r u c t u r e were also discussed. STROMA Histochemically the l i g h t c e l l s with ovoid nuclei possess small quantities of pro t e i n , R.N.A., phospholipid and neutral mucopolysaccharides while electron microscopy indicates extensive cisternae of rough endo-plasmic r e t i c u l a . The l a t t e r i n d i c a t e s a glandular-type c e l l elaborating a p r o t e i n - r i c h product, a conclusion only p a r t i a l l y supported by the histochemical data. The dark c e l l s with pleomorphic n u c l e i possess numerous dark bodies with c r y s t a l l o i d s that may correspond to the pro t e i n and non-sulphated mucopolysaccharide reactions. The p o s s i b i l i t y that two types of dark c e l l may exi s t was indicated by the aldehyde fuchsin reaction i n a small percentage of these c e l l s . This i s a hypothesis that i s supported by the discovery of an "intermediate dark c e l l " (Figure 37)- The lacunae border c e l l s were found to be of an endo-t h e l i a l type s i m i l a r to those l i n i n g c a p i l l a r y walls. THECAL CELLS The ultrastructure of these c e l l s i ndicates they are involved i n 158 protein and s t e r o i d synthesis, however, our histochemical observations only corroborate protein synthesis while the s t e r o i d a c t i v i t y of these c e l l s was noted by Lance and C a l l a r d (1969). U l t r a s t r u c t u r a l l y , the thecal c e l l s of Squalus are thought to contribute to the formation of the surrounding granular and collagenous matrix as well as the basal l a m e l l a . The presence of a c i d i c and sulphated mucopolysaccharides, which are often associated with collagen (Swanson, 196"5; Pearse, 1968), i n the thecal c e l l s supports t h i s hypothesis. BASEMENT LAMINA The basement lamina consists predominantly of ac i d and sulphated mucopolysaccharides and i s formed by the f o l l i c l e c e l l s . U l t r a s t r u c t u r a l l y , the v e s i c l e s i n the basal region of the f o l l i c l e c e l l s represent the muco-ipolysaccharide iprecursors that-..form the basement lamina. Hyaluronic a c i d , one of the glycosaminoglycans present i n the basement lamina i s a highly hydrated molecule and i s very viscous (Schubert & Hamerman, 1956). This v i s c o s i t y i s r e f l e c t e d i n the ele c t r o n density of the membrane and inturn- provides s t r u c t u r a l r i g i d i t y to the oocyte as we l l as a means for c o n t r o l l i n g molecular d i f f u s i o n . VITELLINE MEMBRANE Histochemically, the v i t e l l i n e membrane i s a protein-polysaccharide complex and the R.N.A. a c t i v i t y of the a p i c a l regions of the f o l l i c l e c e l l s r e f l e c t e d i n the ribosomes and R.E.R. of t h i s region i s r e l a t e d to the development of the membrane. The dual chemical composition of the membrane i s responsible for i t s two major f u n c t i o n s — s u p p o r t and chemical s e l e c t i v i t y . FOLLICLE CELLS Beyond the a c t i v i t y of the f o l l i c l e c e l l s i n basement lamina and v i t e l l i n e membrane formation l i t t l e else can be stated conclusively. The u l t r a s t r u c t u r a l evidence for steroid a c t i v i t y as noted by Lance and Callard ( 1 9 6 9 ) i n the f o l l i c l e c e l l s i s purely circumstantial and i s based only on the presence of quantities of smooth membrane bound vesicles and cisternae (Nicholls & Maple, 1 9 7 2 ; Peel & B e l l a i r s , 1 9 7 2 ) . OOPLASM • The major constituents of the ooplasm are protein and l i p i d . The M.G.B.s, M.V.B.s, and G.V.B.s comprise the proteinaceous portion while the l i p i d yolk droplets account for the l i p i d portion of the ooplasm. NUCLEOLI The numerous n u c l e o l i present are producing rRNA and u l t r a s t r u c t u r a l l y t h i s i s represented by t h e i r granular nature and the fragmentation of the peripheral regions of the body. MITOCHONDRIAL MASS Histochemically, t h i s organelle system i s r i c h i n protein, RNA and phospholipids. U l t r a s t r u c t u r a l l y , the cloud i s constructed of mitochondria, smooth endoplasmic reticulum and ribosomes. Primarily the cloud i s the centre for' mitochondrial m u l t i p l i c a t i o n and possibly some packaging of yolk material. Such juxtanuclear oocytic bodies are often mistakenly referred to as yolk n u c l e i . LIPID YOLK . The presence of l i p i d yolk i n e a r l y dogfish oocytes i s in d i c a t e d by the histochemical analysis and u l t r a s t r u c t u r a l l y i t was noted that' l i p i d yolk arose de novo i n the ooplasm from material of extra-oocytic o r i g i n . PROTEIN YOLK The amount of pr o t e i n present i n the ooplasm of ea r l y oocytes i s r e f l e c t e d i n the v e s i c l e s and granules found i n the electron micrographs. In the smallest oocytes (< O.h mm), multigranular (M.G.B.) and m u l t i -v e s i c u l a r (M.V.B.) bodies form from G o l g i , S.E.R., micropinocytotic v e s i c l e s and v e s i c l e s derived from the nuclear membrane. These fuse together forming granular-vesicular (G.V.B.) bodies. In la r g e r oocytes (O.h mm +), distended Golgi lamellae, S.E.R. cisternae and loops of S.E.R. v e s i c l e s form l i m i t i n g membranes and take up M.G.B.s, M.V.B.s and small G.V.B.s. 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Oocyte f o l l i c l e c e l l r e l a t i o n s h i p and f o r m a t i o n of c h o r i o n i n O r y z i a s la t i p e s . . J . Fac. S c i . , Univ. Tokyo 10:123-126. Yamamoto, M. 1964. E l e c t r o n microscopy of f i s h development. I l l . Changes i n the u l t r a s t r u c t u r e of the nucleus and cytoplasm of the oocyte d u r i n g i t s development i n O r y z i a s  l a t i p e s . J . Fac. S c i . , Univ. Tokyo. 10:335-346. *Clark, R.C. 1972. Sephadex fractionation of phosvitins from duck, turkey and ost r i c h egg yolk. Comp. Biochem. Physiol. 1+1:891-903. APPENDIX A HISTOCHEMICAL TECHNIQUES EMPLOYED  STAINING 1. DNFB (Tranzer & Pearse, 196k) Incubate 10 hrs. at 22°C i n 1% alcoholic DNFB-alkalinized with 0.2 ml IN NaOH, wash i n 90% EtOH followed by DHgO and reduce for 30 minutes i n titanous chloride. Wash i n c i t r a t e buffer, DHgO, and diazotize 5 minutes i n fresh nitrous acid. Wash i n DH2O and couple with H-acid for 5 minutes at U°C, wash, dehydrate, clear and mount. 2. PAS (Hotchkiss, 19^8) Oxidize 5 minutes i n alcoholic periodic a c i d , rinse i n 10% EtOH, immerse i n reducing bath for 1 minute, rinse i n 70% EtOH, treat with Schiff's (Barger & DeLamater) for 20 minutes. Wash, stain i n c e l e s t i n blue 2 to 3 minutes, Mayer's haemalum 2 .to 3 minutes, d i f f e r e n t i a t e with 1% acid alcohol, wash, counterstain with Orange G, wash, dehydrate, clear and mount. 3. METHYL GREEN-PYRONIN (Jordan & Baker, 1955) Stain 5 to 10 minutes i n methyl green-pyronin i n acetate buffer (pH U.8), rinse rapidly i n DH20, blot dry, dehydrate quickly i n acetone, clear and mount. h. TOLUIDINE BLUE (Pearse, 1968) Stain for 10 to 20 minutes i n 0.5 to 1.0% aqueous Toluidine blue (G.T. Gurr). Rinse, dehydrate, clear and mount i n permount. 5. ALCIAN BLUE (Steedman, 1950) Stain 20 minutes i n 1% a l c i a n blue ( A l l i e d Chemical) i n 3% acetic acid (pH 2.5). Wash, dehydrate, clear and mount. 6 . DIALYSED IRON (Hale, I 9 U 6 ) Flood with dialysed i r o n f or 10 minutes, wash, f l o o d with acid ferrocyanide for 10 minutes, wash, dehydrate, c l e a r and mount. 7. ALDEHYDE FUCHSIN (Halmi & Davies, 1953) Bring to 70% EtOH, s t a i n 20 minutes with a c i d i c AF, r i n s e i n 10%> EtOH, dehydrate, clear and mount. 8. AZURE A (Szirmai, 1963; McConnachie & Ford, 1966; K v i s t & Finnegan, I969) Stain f or 30 minutes i n 0.2% Azure A (Fisher S c i e n t i f i c Co.) i n phosphate buffer with the pH adjusted to 1.5 with 0.1N HC1. Dehydrate i n EtOH and c l e a r or dehydrate i n a i r and mount i n permount. 9 . COPPER PHTHALOCYANIN (Kluver & Barrera, 1953) Bring to absolute a l c o h o l , s t a i n i n Luxol Fast Blue MBS (Hartman-Leddon Co.) for 12 hours at 6o°C. Rinse i n 70$ EtOH and d i f f e r e n t i a t e "in 0; 05% aqueous l i t h i u m carbonate .'for -2 hours, r i n s e i n water. Counterstain for 20 minutes i n 1% aqueous neutral red, r i n s e , dehydrate, clear and mount. 10. SUDAN BLACK B (McManus, 19k 6) Cryostat.. Stain 30 minutes with saturated Sudan black B (E. Gurr Ltd.) i n 10% EtOH. Rinse quickly i n 10% EtOH, wash i n running water, mount i n Farrant's medium. ENZYME TREATMENT 1. DIASTASE (Humanson,- 1972) Digest sections i n 1.0% diastase of malt ( N u t r i t i o n a l Biochemicals Corp.) i n disodium phosphate b u f f e r for 1 hour at 37°C (pH 6.0) to remove glycogen. Control s l i d e s were incubated i n DH20 at 37°C for 1 hour. 2. PANCREATIC RIBONUCLEASE (Pearse, 1972) Incubate sections for 1 hour at 37°C i n 0.1% pancreatic Ribonuclease-A (Sigma Chemical Corp.) i n DB^O. Sections were then treated with 1.0% aqueous toluidine blue or methyl green-pyronin to demonstrate the loss of ba s o p h i l l i c material which i s considered to be RNA. Control slides were incubated i n DHgO at 37°C. 3. TRICHLOROACETIC ACID (Humanson, 1972) Sections were incubated i n 3% TCA (Fisher S c i e n t i f i c Co.) for 30 minutes at 60°C to remove RNA. Control s l i d e s were incubated i n DH20 at 60°C. k. TESTICULAR HYALURONIDASE (Pearse, 1972) Incubate sections for 3 hours at 37°C i n 0.1% t e s t i c u l a r hyaluronidase (Sigma Chemical Corp.) i n 0.85% s a l i n e . Control s l i d e s were treated at 37°C i n 0.85% 'saline only. Acidic mucopolysaccharides staining i n the controls but not i n enzyme treated slides may be either hyaluronic acid, chondroitin sulphate A and/or chondroitin sulphate C.. SELECTIVE BLOCKING AND'UNBLOCKING 1. METHYLATION-DEMETHYLATION (Cull i n g , 1963) Dehydrated sections were treated for h hours at room temperature i n 1% HC1 i n methanol, rinsed i n absolute EtOH, hydrated and stained. To saponify, methylated sections were rinsed i n EtOH and treated with 0.1N K0H i n DH20 for ^ 5 minutes at room temperature, washed i n water and stained. 

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