STEROIDOGENESIS AND THE ROLE OF STEROIDS IN THE ENDOCRINE CONTROL OF OOGENESIS AND VITELLOGENESIS IN THE GOLDFISH, CARASSIUS AURATUS by KHAY HUAT KHOO B.Sc. (Hons) University of Malaya, 1968 M.Sc. University of Malaya, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA Ju l y , 1974 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r equ i r ements f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l owed w i thout my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada ABSTRACT In t h i s thesis I studied the r o l e of ovarian steroids i n oogenesis and ovarian development of g o l d f i s h (Carassius auratus L.) and examined the i n t e r r e l a t i o n s h i p of p i t u i t a r y gonadotropin and ovarian steroids i n the endocrine control of tel e o s t reproduction. There are four main parts to the i n v e s t i g a t i o n : ( i ) the c y t o l o g i c a l analysis of v i t e l l o g e n e -s i s , ( i i ) the demonstration of steroidogenic tissues i n the ovary and t h e i r endocrine c o n t r o l , ( i i i ) the development and functions of the cor-pus luteum, and (iv) the ro l e of ovarian steroids on oogenesis and v i t e l -logenesis. The h i s t o l o g i c a l and histochemical examination demonstrated that two types of yolk inclusions are formed during v i t e l l o g e n e s i s : yolk v e s i c l e s , comprised of mucopolysaccharides, and f i r s t formed, followed subsequently by yolk granules composed of proteins, phospholipids and neutral l i p i d s . Neither type of yolk develops i n the absence of the p i t u i t a r y ; estrogen regulates the formation of yolk v e s i c l e s while preg-nenolone was found to control deposition of yolk granules. The s i g n i f i -cance of these two kinds of yolk i s considered. This i s the f i r s t demon-s t r a t i o n of two yolk types i n g o l d f i s h with a separate endocrine control for the formation of each of them. The major s t e r o i d synthesizing tissues i n g o l d f i s h ovaries are the granulosa c e l l s of oocytes and the corpus luteum. The corpus luteum, whether pre- or post-ovulatory probably synthesizes estrogens. In t h i s study, no functional differences were established between pre- and post-i i i ovulatory corpora lutea. Treatment of post-ovulatory g o l d f i s h with t r i t i a t e d thymidine strongly suggests that the corpora l u t e a l c e l l s p r o l i f e r a t e to form another generation of oogonia. Estrogens are pro-bably active hormones i n t h i s reaction. The implications of these new findings are discussed. Administration of exogenous estrogen increases oogonia formation i n post-ovulatory g o l d f i s h whereas chronic administration of estrogens or testosterone into gravid f i s h induces extensive a t r e s i a . Treatment of gravid f i s h with progesterone or c o r t i c o s t e r o i d s induces ovulation. As a working hypothesis i t i s proposed that steroids are synthe-sized by the ovaries under gonadotropin stimulation. P i t u i t a r y gonado-tro p i n regulates the whole ovarian s t e r o i d synthetic process and not any s p e c i f i c reaction i n the s t e r o i d metabolic pathways. The synthesis of s p e c i f i c steroids during the reproductive cycle i s brought about by l o c a l i z e d i n h i b i t i o n of steroidogenic enzymes by s t e r o i d s , most l i k e l y estrogens. The p o t e n t i a l p r a c t i c a l applications of endocrine manipulations i n f i s h reproduction are discussed. i v TABLE OF CONTENTS Page Abs t r a c t i i L i s t of Tables ' . v i i i L i s t of Figures ..... i x Acknowl ed gemen t s x i i General I n t r o d u c t i o n 1 Se c t i o n I : The H i s t o l o g y and Histochemistry of Ovarian Development. 3 I n t r o d u c t i o n 4 M a t e r i a l s and Methods 6 ( i ) Maintenance of G o l d f i s h 6 (a) N a t u r a l G o l d f i s h 6 (b) Gravid G o l d f i s h 6 (c) R e f r a c t o r y g o l d f i s h 6 ( i i ) H i s t o l o g y and Histochemistry of Ovaries 7 Results 11 ( i ) H i s t o l o g y of Oocyte Growth and Development 11 (a) Oogonia H (b) F i r s t Growth Phase Oocytes 11 (c) Second Growth Phase Oocytes 13 ( i i ) H i stochemistry of V i t e l l o g e n e s i s 15 (a) D i s t r i b u t i o n of Polysaccharides 16 (b) D i s t r i b u t i o n of P r o t e i n s . . . . 20 (c) D i s t r i b u t i o n of L i p i d s 21 V Table of Contents (cont'd) Page Section I I : The Corpus Luteum and i t s Function.... 28 Introduction 29 Materials and Methods .. . 33 ( i ) Hypophysectomy 33 ( i i ) Histology of pre- and Post-ovulatory Corpus Luteum 35 ( i i i ) Histochemical Detection of Hydroxysteroid Dehydrogenases 35 3 (iv) Incorporation of Thymidine- H Into Ovaries 37 Results 39 ( i ) Histogenesis of a t r e t i c oocytes 39 (a) a-stage 39 (b) g-stage 39 (c) y-stage. 40 (d) 6-stage 40 (e) e-stage. ... 40 (ii).)Histogenesis of post-ovulatory Corpus Luteum 42 (a) Stage I....... 42 (b) Stage I I 42 (c) Stage III 43 ( i i i ) Hydroxysteroid dehydrogenases i n Normal Ovaries.. 43 (a) 3g-hydroxysteroid dehydrogenase 46 (b) 3a-hydroxysteroid dehydrogenase 46 (c) 17a-hydroxysteroid dehydrogenase 47 (d) 173-hydroxysteroid dehydrogenase.... 47 v i Table of Contents (cont'd) Page !• (iv) Hydroxysteroid Dehydrogenases i n Hypophy-sectomized F i s h . . . . . . . . 50 (v) Hydroxysteroid Dehydrogenases i n Hypophy-sectomized f i s h Treated with LH. 53 (vi) Hydroxysteroid Dehydrogenases i n Post-ovulatory Ovaries 55 ( v i i ) The Fate of Post-ovulatory Corpus Luteum as Determined by Autoradiography 57 Discussion 60 ( i ) Steroid Synthesis i n F i s h Ovaries 60 ( i i ) A t r e s i a of Second Growth Phase Oocytes... 61 ( i i i ) Post-ovulatory Corpora Lutea. 63 (iv) Localized Hormone Production 64 (v) Post-ovulatory P r o l i f e r a t i o n of Obgonia........ 65 (vi) Fate of Corpora Luteum C e l l s 66 (via.) The Reproductive Cycle Within the Ovary......... 67 Section I I I : The E f f e c t s of Ovarian Steroids on Oogenesis and V i t e l l o g e n e s i s 70 Introduction 71 Methods and Materials 73 (i ) E f f e c t s of Steroids on Oogonial Mitosis - 73 ( i i ) E f f e c t s of Steroids on V i t e l l o g e n e s i s 74 ( i i i ) E f f e c t s of Long Term Treatment of Steroids on Gravid F i s h : 75 v i i Table of Contents (cont'd) Page ( i v ) E f f e c t s of S t e r o i d s on O v u l a t i o n 76 R e s u l t s 78 ( i ) E f f e c t s of S t e r o i d s on Oogonial M i t o s i s 78 ( i i ) E f f e c t s of S t e r o i d s on V i t e l l o g e n e s i s 80 ( i i i ) E f f e c t s of Long Term A d m i n i s t r a t i o n of St e r o i d s on Gravid F i s h 86 ( i v ) E f f e c t s of St e r o i d s on O v u l a t i o n 91 D i s c u s s i o n 94 ( i ) E f f e c t s of S t e r o i d s on Oogonial M i t o s i s 94 ( i i ) E f f e c t of S t e r o i d s on V i t e l l o g e n e s i s . . . . . 95 ( i i i ) I n duction of Ovu l a t i o n by S t e r o i d s 98 ( i v ) The Mechanism of Endocrine C o n t r o l of Oogenesis 98 General D i s c u s s i o n 103 ( i ) P o s s i b l e P r a c t i c a l A p p l i c a t i o n s 105 References 107 v i i i LIST OF TABLES Page TABLE 1 L i s t of h i s t o l o g i c a l and histochemical techniques used with the appropriate preparative techniques 8 2 A summary of the histochemical properties of yolk i n c l u s i o n s 17 3 The degree of a t r e s i a i n various ovaries a f t e r hypophysectomy .... 39a, 4 Hydroxysteroid dehydrogenases detected i n the ovaries of normal v i t e l l o g e n i c ovaries. 45 5 Hydroxysteroid dehydrogenase detected i n the ovaries of hypophysectomized g o l d f i s h 51 6 Hydroxysteroid dehydrogenases i n the ovaries of hypophysectomized g o l d f i s h i n j e c t e d with LH 54 7 Hydroxysteroid dehydrogenases i n the ovaries of post-ovulatory f i s h . . . . ' 56 8 Summary of the e f f e c t s of s t e r o i d administration on oogonial mitosis 79 9 The e f f e c t s of steroids on v i t e l l o g e n e s i s i n hypo-physectomized g o l d f i s h 82 10 Comparison of the histochemical properties of preg-nenolone induced yolk granules with those of normal v i t e l l o g e n i c ovaries....... . 84 11 The long-term e f f e c t s of s t e r o i d administration on the ovaries of gravid g o l d f i s h 89 12 The e f f e c t s of steroids on ovulation i n gravid f i s h . . 92 i x LIST OF FIGURES FIGURE Page 1 An oogonia occuring s i n g l y i n the ovary.... 12 2 Oogonial cyst containing several oogonia....... 12 3 Chromatin nucleolar stage oocytes ......... 12 4 Perinucleolar stage oocytes.... 12 5 I n i t i a l yolk v e s i c l e stage oocyte.......... .. 14 6 Late yolk v e s i c l e stage oocyte 14 7 Early yolk granule stage oocyte ... 14 8 Late yolk granule stage oocyte 14 9 Ovarian section stained with p e r i o d i c a c i d - S c h i f f ' s r e a c t i o n . . . . 19 10 Oocytes stained with dinitroflurobenzene r e a c t i o n . . . . 19 11 Ovarian section stained with the t e t r a z o t i z e d o-d i a n i s i d i n e reaction f o r proteins 19 12 Ovarian section stained with the f e r r i c f e r r i c y a n i d e method for sulphydryl groups 19 13 Cryostat section of ovary stained with Sudan Black B. 19 14 Ovarian sections stained with acid haematin for phospholipids 19 15 A diagrammatic representation of oogenesis i n gold-f i s h ovary •.- 27 16 Diagrammatic presentation of the hydroxysteroid de-hydrogenase reaction... 2>&o~ 17 a-stage of f o l l i c u l a r a t r e s i a 41 18 (3-stage of f o l l i c u l a r a t r e s i a . . . . . . . . . . . 41 19 Y ~ s t a g e a t r e t i c f o l l i c l e 41 20 Higher magnification of the y-stage a t r e t i c f o l l i c l e . 41 X L i s t of Figures (cont'd) Page FIGURE 21 6-stage a t r e t i c f o l l i c l e . ... 41 22 e-stage a t r e t i c f o l l i c l e , showing the d i f f e r e n t i a -t i o n of oogonia ' 41 23 Stage I p o s t - o v u l a t o r y corpus luteum, w i t h evidence of the rupture of f o l l i c u l a r w a l l 44 24 Stage I p o s t - o v u l a t o r y corpus luteum without any s i g n of f o l l i c u l a r w a l l rupture 44 25 A h i g h m a g n i f i c a t i o n of Stage I post-ovulatory corpus luteum demonstrating hypertrophy of granu-l o s a c e l l s . . . . . . . . . . . . 44 26 Stage I I p o s t - o v u l a t o r y corpus luteum.... 44 27 A higher m a g n i f i c a t i o n of stage I I p o s t - o v u l a t o r y corpus luteum........ 44 28 Stage I I I p o s t - o v u l a t o r y corpus luteum 44 29 L o c a l i z a t i o n of 33-hydroxysteroid dehydrogenase i n the granulosa c e l l s of oocytes 48 30 Lack of hy d r o x y s t e r o i d dehydrogenase a c t i v i t y i n l a t e y o l k granule stage oocytes 48 31 Demonstration of 17 ( 3-hydroxysteroid dehydrogenase a c t i v i t y i n y s t a g e a t r e t i c oocytes of hypophy-sectomized g o l d f i s h 48 32 3f?-hydroxysteroid dehydrogenase i n the post-o v u l a t o r y f o l l i c l e s a f t e r o v u l a t i o n 48 33 Autoradiography of g o l d f i s h ovaries showing the l a b e l l i n g of f o l l i c u l a r c e l l s by thymidine-^H 59 34 Autoradiography of g o l d f i s h o v a r i e s showing the l a b e l l i n g of 6-stage corpus l u t e a c e l l s 59 35 Autoradiography showing the l a b e l l i n g of oogonia 30 days a f t e r thymidine-^H i n j e c t i o n 59 36 Autoradiograph of l a b e l l e d oocyte 30 days a f t e r the i n i t i a l thymidine- 3 H i n j e c t i o n 59 X X L i s t of Figures (cont'd) Page 37 A summary of corpus luteum formation i n g o l d f i s h . . . . . . 69 38 Yolk v e s i c l e s oocytes of hypophysectomized f i s h t r e a t e d w i t h estrogen 81 39 Ovary of hypophysectomized g o l d f i s h 81 40 Pregnenolone induced y o l k granule formation i n the oocyte of hypophysectomized g o l d f i s h 81 41 Histogram of the e f f e c t s of e s t r a d i o l , estrone, and e s t r i o l on y o l k v e s i c l e formation i n hypophysec-tomized f i s h . . . . . . . . 85 42 H i s t o l o g y of g r a v i d ovary t r e a t e d w i t h estrogen 88 43 H i s t o l o g y of g r a v i d ovary t r e a t e d w i t h d e o x y c o r t i -c o s t e r o l c o n t a i n i n g post-ovulatory corpora l u t e a . . . . . . 88 44 Ovaries of f i s h that are ready to ovulate 88 45 Ovaries of f i s h w i t h immature oocytes........ 88 46 Comparison of the e f f e c t s of estrone, e s t r a d i o l , e s t r i o l and t e s t o s t e r o n e on a t r e s i a of y o l k y oocytes i n g r a v i d f i s h 90 47 Diagram showing the i n t e r r e l a t i o n s h i p between p i t u i t a r y gonadotropin, steroidogenesis and oogenesis. 48 The s t e r o i d metabolic pathway i n t e l e o s t o v a r i e s 99 ACKNOWLEDGEMENTS x i i I t i s my great pleasure to acknowledge my indebtedness and gratitude to my research supervisor, Dr. W.S.Hoar, for his interest, advise and encouragements throughout the course of this study. I would l i k e to thank the members of my doctoral committee: Dr.E.M.Donaldson, Dr.H.D.Fisher, Dr.P.Ford, Dr.N.R.Li ley and Dr .A.M.Perks for t h e i r generous assistance and advise during the study and for reading the thesis. I extend my grat e f u l appreciation to Miss.D.Hards, for the prepara-tion of excellent h i s t o l o g i c a l sections, Mrs. Cathy Driedzic for her care-f u l preparation of the i l l u s t r a t i o n s , and Mrs, Pat Waldron, for typing the f i n a l copy of the thesis. I iam obligated to my fellow graduate students, Kenneth Chan, Ross Neuman, Jean-Guy Godin, B i l l Marshall and especially Norman Stacey for many stimulating discussions. My thanks to those that plunged into the i c y winter waters of Vancouver i n search of the elusive Coryphopterus ni c h o l s i • To everyone who have made my stay here i n Vancouver meaningful and enjoyable, I say thank you. Financial support for this research was received from the National Research Council of Canada through grants-in-aid to Dr.W.S.Hoar and a Canadian Commonwealth Scholarship to myself. I also wish to thank the U n i v e r s i t i Sains Malaysia for granting me a Fellowship i n thei r Academic Staff Training Scheme. GENERAL INTRODUCTION In t e l e o s t , the r e g u l a t i o n of gametogenetic a c t i v i t y by the p i t u i t a r y i s now w e l l e s t a b l i s h e d (see reviews by P i c k f o r d and A t z , 1957; Dodd, 1960; Hoar, 1965; 1969; L o f t s , 1968; Reinboth, 1972; Donaldson, 1973). Hypophysectomy r e s u l t s i n r e g r e s s i o n of ovaries and a t r e s i a of y o l k - l a d e n oocytes. A d m i n i s t r a t i o n of mammalian gonadotropin (Yamazaki and Donaldson, 1968; L i l e y and Donaldson, 1968; Sundararaj et a l . , 1972 a, b) m i t i g a t e s the e f f e c t s of hypophysectomy. In a d d i t i o n to o v a r i a n r e g r e s s i o n and a t r e s i a , hypophysectomy i n h i b i t s s teroidogenesis i n t e l e o s t o v a r i e s (Yamazaki and Donaldson, 1969; Wiebe, 1969) w h i l e a d m i n i s t r a t i o n of gonadotropin increase 3g-hydroxysteroid dehydrogenase a c t i v i t y i n hypophysectomized f i s h . However, i t i s s t i l l u n c e r t a i n whether gonadotropin acts d i r e c t l y on oogenesis and v i t e l l o g e n e s i s or whether i t operates through the production of a second hormone—possibly a s t e r o i d or whether both gonadotropins and s t e r o i d s are d i r e c t l y i n -volved. There i s only scanty i n f o r m a t i o n concerning endocrinology of ovum formation and development. Bullough (1942) observed a greater number of oogonia i n f i s h (Phoxinus phoxinus) tr e a t e d w i t h estrogens but d i d not d e s c r i b e the d e t a i l e d changes i n the ovary nor the i n t e r r e l a t i o n s h i p of gonadotropin and s t e r o i d s i n the c o n t r o l s . Again s t u d i e s of sex r e v e r s a l using s t e r o i d s suggest an e f f e c t of the s t e r o i d s on the p r i m o d i a l germ c e l l s but the c y t o l o g i c a l changes have not been w e l l d e s c r i b e d ; Yamamoto (1969) reviews the l i t e r a t u r e . F i n a l l y i n v e s t i g a t i o n s of a c t i o n s of 2 s t e r o i d s on y o l k formation have provided r a t h e r c o n t r a d i c t o r y evidence concerning t h e i r p o s s i b l e r o l e i n y o l k s y n t h e s i s and d e p o s i t i o n . A l -though estrogens s t i m u l a t e the l i v e r to synthesize y o l k precursors (Egami, 1955; B a i l e y , 1957; Ho and Vanstone, 1961; U r i s t and Schjeide, 1961; P l a c k et^ a l . , 1971; Aida e t a l . , 1973), Bullough (1942), Tavolga (1949) , Berkowitz (1951) , Egami (1955) observed that estrogen a d m i n i s t r a -t i o n i n h i b i t e d v i t e l l o g e n e s i s and caused f o l l i c u l a r a t r e s i a . The r o l e of s t e r o i d s i n inducing o v u l a t i o n i n t e l e o s t i s w e l l do-cumented s i n c e K i r s h e n b l a t (1952, 1959) f i r s t demonstrated that proges-terone and deoxycorticosterone induced o v u l a t i o n i n Misgurnus f o s s i l i s , whereas estrogens and androgens had no e f f e c t . S i m i l a r r e s u l t s were ob-t a i n e d w i t h Heterdpneustes f o s s i l i s (Ramaswami, 1962). F u r t h e r , Sundararaj and Goswami (1966) observed t h a t c o r t i c o s t e r o i d s induced o v u l a t i o n and i n some instances o v i p o s i t i o n i n both i n t a c t and hypophysectomized f i s h . In a d d i t i o n to the i n v i v o s t u d i e s , Goswami and Sundararaj (1971 a, b,) and Hirose (1972) demonstrated the i n d u c t i o n of i n v i t r o o v u l a t i o n by s t e r o i d s . This b r i e f summary i n d i c a t e s the u n s a t i s f a c t o r y s t a t e of knowledge concerning endocrine c o n t r o l of ovum formation and development. This i n v e s t i g a t i o n i s a study of the endocrine r e g u l a t i o n of oogenesis and o v a r i a n development i n g o l d f i s h , w i t h p a r t i c u l a r reference to the r o l e of s t e r o i d hormones. I t was convenient to d i v i d e the p r e s e n t a t i o n i n t o three s e c t i o n s , the h i s t o l o g i c a l and h i s t o c h e m i c a l a n a l y s i s of oogenesis and v i t e l l o g e n e s i s , the p i t u i t a r y c o n t r o l of steroidogenesis i n the ovary, e s p e c i a l l y the corpus luteum, and f i n a l l y the e f f e c t s of ovarian s t e r o i d s on oogenesis and v i t e l l o g e n e s i s . 3 SECTION I : THE HISTOLOGY AND HISTOCHEMISTRY OF OVARIAN DEVELOPMENT 4 INTRODUCTION The present i n v e s t i g a t i o n i s an analysis of c y t o l o g i c a l changes during the maturation of oocytes of g o l d f i s h , Carassius auratus. Such an analysis should provide a basi s f o r study of the endocrine control of oogenesis and v i t e l l o g e n e s i s i n tel e o s t ovaries. Although early stages of oocyte development have been w e l l documented, t-h& second growth phase has not previously been d e t a i l e d . In t h i s study a t t e n t i o n i s direct e d to the second growth p h a s e — e s p e c i a l l y the formation and chemical composition of yolk i n c l u s i o n s . There are numerous studies of ovarian histology during oocyte growth and development. Barr (1963) , L e h r i (1968) and Lambert (1970) have pro-vided good descriptions of tel e o s t oocyte growth. Yamazaki (1965) gave an excellent d e s c r i p t i o n of the h i s t o l o g i c a l changes i n the ovaries of g o l d f i s h , while e a r l i e r observations of ovarian histology i n g o l d f i s h were provided by Stomsten (1931) and Beach (1959). I t i s generally recognized that oocyte development occurs i n two phases. The f i r s t growth phase, which i s independent of p i t u i t a r y con-t r o l , involves the increase i n s i z e of oocytes with some nuclear changes (see review by Hoar, 1969). The second phase i s characterized by the deposition of yolk; there i s much confusion concerning t h i s phase espe-c i a l l y formation of the yolk in c l u s i o n s (Anderson, 1968; Guraya, 1965; Malone and Hisaoka, 1963). One of the reasons f o r the c o n f l i c t i n g opin-ions may be a t t r i b u t e d to a f a i l u r e to d i s t i n g u i s h the various yolk com-ponents. 5 I n i t i a l h i s t o c h e m i c a l s t u d i e s of v i t e l l o g e n e s i s were devoted to the d i s t r i b u t i o n of polysaccharides (Ihnumaxa and Tsukuda, 1952; Aketa, 1954; and Yamamoto, 1955; 1956; 1958) but few attempts were made to c o r r e l a t e the h i s t o c h e m i c a l d i s t r i b u t i o n of polysaccharides w i t h other chemical components. L a t e r , the emphasis s h i f t e d to the l i p i d composi-t i o n of oocytes and the o r i g i n of yo l k (Chopra, 1958; Yamamoto, 1958; Nath, 1960). These l a t e r workers have been concerned about the o r i g i n of y o l k and d i r e c t e d t h e i r a t t e n t i o n to the "yolk nucleus" now recog-n i z e d as a d i f f u s e d c o n c e n t r a t i o n of mitochondria. Some i n t e r e s t i n yo l k nucleus s t i l l remains (Guraya, 1963; 1968). Recently attempts have been made to sCtidy the h i s t o c h e m i c a l composition of y o l k i n c l u s i o n s i n t e l e o s t o v a r i e s . Malone and Hisaoka (1963), Guraya (1965) and Anderson (1968) s t u d i e d the h i s t o c h e m i c a l d i s t r i b u t i o n of p o l y s a c c h a r i d e s , l i p i d s , and p r o t e i n s i n t e l e o s t o v a r i e s . In s p i t e of these s t u d i e s , however, the chemical composition of the var i o u s y o l k i n c l u s i o n s i s s t i l l p o o r l y defined. 6 METHODS MAINTENANCE OF GOLDFISH . Gold f i s h (Carassius auratus L.) of the common comet v a r i e t y (10-15 cm standard length) were obtained from a commercial supplier (Hartz Mountain Pet Suppliers Limited, Richmond, B r i t i s h Columbia). They were kept i n f i b e r g l a s s tanks supplied with flowing dechlorinated fresh water which was areated with compressed a i r . G o l d f i s h were fed once a day with "Clark's New Age Trout Feed" (Moore-Clark Co., Salt Lake C i t y , Utah). This d i e t was supplemented, once a week, with frozen brine shrimp. "Natural" g o l d f i s h . The basic stock of f i s h was kept under natural photoperiod and temperature, except i n winter when the temperature was rais e d to about 10°C with immersion heaters. Gravid g o l d f i s h . Another group of g o l d f i s h was kept at 10-13°C with a d a i l y photoperiod of 16 hours. Fluorescent 40 watt lamps, con-t r o l l e d by time clocks, supplied the l i g h t . A f t e r about 3 months of such treatment most of the females become gravid. They remain gravid and w i l l not ovulate i f kept below 13°C (Yamamoto et a l . , 1966). These f i s h were used i n experiments requiring gravid g o l d f i s h . Refractory g o l d f i s h . A group of gravid g o l d f i s h was transferred to aquaria with dechlorinated water at 13°C. A f t e r t r a n s f e r , the water temperature was slowly raised to room temperature (22-25°C) and t h i s induced ovulation. The f i s h were allowed to ovulate at random. They were kept at room temperature and 16 hours d a i l y photoperiod f or 3-4 months when they became re f r a c t o r y and had ovaries s i m i l a r to those of 7 hypophysectomized g o l d f i s h . Under these c o n d i t i o n s , g o l d f i s h remain r e f r a c t o r y i f maintained at room temperature and cease to be r e f r a c t o r y only when placed i n water below 20°C. HISTOLOGY AND HISTOCHEMISTRY OF OVARIES The h i s t o l o g y and h i s t o c h e m i s t r y of g o l d f i s h o v a r i e s were studied at v a r i o u s stages of m a t u r i t y . F i s h from the " n a t u r a l " stock were sam-pl e d throughout the year to ensure that a l l stages of oogenesis were i n -cluded. The h i s t o l o g y of f r e s h l y ovulated eggs were a l s o examined. The ovaries of hypophysectomized f i s h were examined h i s t o l o g i c a l l y i n s t u d i e s on the enzymic a c t i v i t y of h y d r o x y s t e r o i d dehydrogenases. The o v a r i e s of p o s t - o v u l a t o r y f i s h were a l s o examined h i s t o l o g i c a l l y . The various techniques used are summarized i n Table 1. I n a l l cases, the f i s h were decapitated and t h e i r o v a r i e s placed i n v a r i o u s f i x a t i v e s i n c l u d i n g Bouin's f l u i d , Baker's n e u t r a l f o r m a l i n , Smith's dichromate and Carnoy's f l u i d (Gurr, 1962). A p o r t i o n of the o v a r i e s f o r h i s t o c h e m i c a l examination was frozen immediately w i t h an ace-tone-dry i c e mixture or "Cryowick" ( I n t e r n a t i o n a l Equipment Co., Massachu-s e t t s ) . These unfix e d but frozen o v a r i e s were sectioned at 10 y w i t h the c r y o s t a t and mounted on albuminized s l i d e s f o r h i s t o c h e m i c a l s t a i n i n g . Ovaries w i t h only e a r l y stage oocytes were embedded r o u t i n e l y i n p a r a f f i n . Yolk laden t i s s u e s are very b r i t t l e and d i f f i c u l t to s e c t i o n when embedded r o u t i n e l y i n p a r a f f i n . A technique which uses Smith's dichromate f i x a t i v e followed by double embedding of the ovaries was de-veloped to overcome t h i s problem. D e t a i l s are as f o l l o w s ; Table 1: L i s t of the va r i o u s h i s t o l o g i c a l and hi s t o c h e m i c a l techniques w i t h the appropriate preparative methods used. TECHNIQUES HISTOLOGY Haematoxylin and eosin (Gurr, 1962) Ma l l o r y ' s trichrome (Gurr, 1962) POLYSACCHARIDES Best's carmine (Pearse, 1968) P e r i o d i c a c i d - S c h i f f ' s (McManus, 1948) Di a l y s e d i r o n f o r a c i d mucopolysaccharides (Hale, 1946) A l c i a n Blue, pH 2.5 (Pearse, 1968) PROTEINS Din i t r o f l u r o b e n z e n e method (Burstone, 1955) Te t r a - a z o t i z e d o - d i a n i s i d i n e ( D a n i e l l i , 1953) Sakaguchi's r e a c t i o n (Baker, 1953) M i l l o n ' s r e a c t i o n (Baker, 1956) F e r r i c f e r r i c y a n i d e method f o r SH (Chevremont & F r e d e r i c k , 1943) DDD r e a c t i o n f o r SH (Barnett & Seligman, 1952) FIXATIVES B, S, NF B, S, NF B, S, NF B, S, NF B, S, NF S, NE, F S, NE, F S, NF, F S, NF, F B, S, NF SECTIONS P a r a f f i n P a r a f f i n P a r a f f i n P a r a f f i n P a r a f f i n P a r a f f i n P a r a f f i n , Frozen P a r a f f i n , Frozen P a r a f f i n , Frozen P a r a f f i n , Frozen P a r a f f i n Continued 0 0 TABLE 1 (Cont'd) TECHNIQUES FIXATIVES SECTIONS LIPIDS Sudan Black B (Chayen et a l . , 1969) O i l Red 0 ( L i l l i e , 1944) A c i d haematin (Baker, 1946) Copper phthalocyanin method (Kluver & B a r r e r a , 1953) NF, F NF, F B, S, NF, F NF, F Frozen Frozen P a r a f f i n , Frozen FFrozen Footnotes: - Key to a b b r e v i a t i o n of f i x a t i v e s , B, Bouin's f l u i d ; S, Smith's dichromate f i x a t i v e ; NF, Baker's n e u t r a l f o r m a l i n ; C, Carnoy's f l u i d ; F, u n f i x e d and frozen o v a r i e s . 2 P a r a f f i n s e c t i o n s (7 y) were sectioned on r o t a r y microtome. Frozen s e c t i o n s were cut on the cryostat at -15°C (10-12 u) . VO 10 The ov a r i e s were f i x e d i n Smith's f i x a t i v e f o r 12-24 hr and then washed i n running water f o r 6-12 hr. The washed ovaries were dehydrated through a s e r i e s of a l c o h o l s up to 95% a l c o h o l . Ovaries were not com-p l e t e l y dehydrated since some r e s i d u a l water gave b e t t e r h i s t o l o g i c a l r e s u l t s w i t h y o l k laden eggs (Rugh, 1968). The t i s s u e s were c l e a r e d i n methyl benzonate f o r up to 5 hr. A f t e r c l e a r i n g they were i n i t i a l l y i n -f i l t r a t e d w i t h 1% c e l l u l o i d i n i n methyl benzoate and then placed over-n i g h t i n chloroform before i n f i l t r a t i n g w i t h p a r a f f i n ( P a r a p l a s t , m.p. 56-58°C) f o r s e c t i o n i n g . A s m a l l piece of some of the ovaries was f i x e d overnight i n Baker's n e u t r a l f o r m a l i n . These t i s s u e s were washed i n running water f o r 12-24 h r , fr o z e n i n e i t h e r acetone-dry i c e mixture or w i t h Cryowick and mounted on specimen holders w i t h "O.C.T. compound" (Ames Co., Indiana) — a water s o l u b l e , embedding medium f o r frozen t i s s u e s . Sections of 10-12 y t h i c k -ness were cut on the c r y o s t a t f o r h i s t o l o g i c a l and h i s t o c h e m i c a l s t a i n i n g . The s p e c i f i c i t y of polysaccharide s t a i n s was confirmed by s t a i n i n g s e r i a l s e c t i o n s which were p r e t r e a t e d w i t h 0.5% d i a s t a s e (Chayen et a l . , 1969). The p e r i o d i c a c i d - S c h i f f ' s polysaccharide r e a c t i o n was f u r t h e r confirmed by a c e t y l a t i n g s e r i a l s e c t i o n s before s t a i n i n g ( L i l l i e , 1954). The s p e c i f i c i t y of the r e a c t i o n s f o r s u l p h y d r y l groups was confirmed by t r e a t i n g s e r i a l s e c t i o n s w i t h 0.1 M, N-ethyl-maleimide (Pearse, 1968). The occurrence of simple l i p i d s was confirmed by p y r i d i n e e x t r a c t i o n of s e r i a l s e c t i o n s (Baker, 1946) before s t a i n i n g i n e i t h e r Sudan Black B or O i l Red 0. 11 RESULTS HISTOLOGY OF OOCYTE GROWTH AND DEVELOPMENT Oogonia. These c e l l s occurred s i n g l y or i n small nests i n a l l ovaries examined (Fig. 1 & 2). They were most numerous i n post-ovula-tory f i s h and i n ovaries with numerous corpora lu t e a . Oogonial cysts were l e s s abundant i n maturing ovaries with yolky oocytes. Each oogon-ium was about 10-15 u i n diameter with a single nucleus and one large nucleolus which stained deeply with haematoxylin. In sections of ovaries stained with Mallory's trichrome the n u c l e o l i were deep orange. F i r s t Growth Phase. F i r s t growth phase oocytes were l a r g e r than oogonia. Primary oocytes were distinguished from oogonia by the presence of d i s t i n c t chromosomes i n various stages of meiotic prophase. This phase was sub-divided into two d e f i n i t e stages separated according to the fea-tures of the nucleus. These two stages were the chromatin nucleolar stage and the per i - n u c l e o l a r stage. The f i r s t stage a f t e r the development of oocytes i s the chromatin nucleolar stage. The oocytes i n t h i s stage had a nucleus with a single conspicuous nucleolus (Fig. 3). Chromatin threads were attached to the nucleolus. 06cyte at th i s stage were s l i g h t l y l a rger than oogonia and had diameters of 12-20 y. The chromatin nucleolar state i s followed by the peri- n u c l e o l a r stage. The per i n u c l e o l a r stage oocytes were e a s i l y i d e n t i f i e d by the peripheral arrangement of a large number of small n u c l e o l i on the inner side of the nuclear membrane ( F i g . 4). The nucleus was enlarged and the 12 Figure 1: An oogonium, as indicated by the arrow, occuring s i n g l y . Haematoxylin and eosin. Figure 2: Oogonial cyst (arrow) containing several oogonia with large n u c l e i deeply stained with haematoxylin. Figure 3: Chromatin nucleolar stage oocytes (arrows). Haematoxylin and eosin. Figure 4; Perinucleolar stage oocytes. The early p e r i n u c l e o l a r stage oocytes (Arrow 1) are smaller i n s i z e and stained deeply with haematoxylin as compared with l a t e p e r i n u c l e o l a r stage oocytes (Arrow 2). 13 chromosomes l o s t t h e i r d i s t i n c t nature. Together with the enlargement of the nucleus, the cytoplasm increased d r a s t i c a l l y i n volume. Oocytes, at the p e r i n u c l e o l a r stage, had diameters of 20-150 y. The cytoplasm of oocytes at t h i s stage stained deeply with haematoxylin. This was i n contrast to other stages where the cytoplasm was not stained by haema-t o x y l i n . Mallory's trichrome stained the cytoplasm deep red. The n u c l e o l i were very conspicuous with Mallory's trichrome and appeared deep orange ( F i g . 5). At the end of the p e r i n u c l e o l a r stage the cytoplasm l o s t i t s a f f i n i t y f o r haematoxylin. Second growth phase. The second growth phase oocytes were charac-t e r i z e d by the formation and accumulation of yolk. Two types of yolk i n c l u s i o n s were e a s i l y distinguished by t h e i r s i z e and s t a i n i n g properties. The larger yolk v e s i c l e (10-15 y i n diameter) stained blue with Mallory's trichrome; the smaller yolk granules (about 1 y i n diameter) stained deep red with Mallory's trichrome. These two types of yolk were formed sequen-t i a l l y with yolk v e s i c l e s deposited before yolk granules. Yolk v e s i c l e stage. The f i r s t i n d i c a t i o n of yolk formation was the presence of a r i n g of v e s i c l e s i n the periphery of the cytoplasm of oocytes (Fi g . 5). There was no pattern i n the l a t e r deposition of yolk v e s i c l e s . The v e s i c l e s were formed randomly. The oocyte at t h i s stage was about 150 y i n diameter. I n i t i a l l y , each v e s i c l e formed as a minute body but gradually increased i n s i z e u n t i l i t reached a diameter of 10-15 y. These yolk v e s i c l e s increased both i n s i z e and numbers u n t i l they occupied the whole cytoplasm of oocytes ( F i g . 6). In ovarian sections stained with 14 Figure 5: Oocyte i n the i n i t i a l yolk v e s i c l e stage with the c h a r a c t e r i s t i c r i n g of yolk v e s i c l e s (v). Mallory's trichrome. Figure 6: Late yolk v e s i c l e stage oocyte stained with Mallory's trichrome. The yolk v e s i c l e s (v) are more abundant. The zona rad i a t a (z) becomes thickened and i s stained red with Mallory's trichrome. Figure 7: Early yolk granule stage oocyte stained with Mallory's trichrome. The red yolk granules (g) accumulate between the blue yolk v e s i c l e s (v). Figure 8: Late yolk granule stage oocytes stained with Mallory's trichrome. The red yolk granules have accumulated toward the center of oocytes and d i s p l a c i n g the yolk v e s i c l e s to the periphery. 15 haematoxylin and e o s i n . the y o l k v e s i c l e s appeared to be vacuoles but t h i s proved to be an a r t i f a c t . With M a l l o r y ' s trichrome, these v e s i c l e s s t a i n e d l i g h t b lue. At t h i s stage of oocyte development the f o l l i c u l a r l a y e r s were f a i r l y w e l l developed. The t h e c a l and granulosa c e l l s were d i s t i n g u i s h -a ble. The zona r a d i a t a s t a r t e d to develop during the y o l k v e s i c l e stage. I n i t i a l l y the zona r a d i a t a was t h i n and s t a i n e d dark red w i t h M a l l o r y ' s trichrome. By the l a t e stages of y o l k v e s i c l e formation, however, the zona r a d i a t a was almost f u l l y developed and i t s r a d i a t e nature was apparent. Y o l k granule stages. The formation of y o l k granules c h a r a c t e r i z e d the f i n a l stage of v i t e l l o g e n e s i s and oocyte development. Yolk granules formed only i n oocytes w i t h f u l l y developed y o l k v e s i c l e s . The y o l k granules f i r s t appeared c l o s e to the zona r a d i a t a ( F i g . 7) and were e a s i l y d i s t i n g u i s h a b l e as red granules a f t e r s t a i n i n g w i t h M a l l o r y ' s trichrome; they were not very evident a f t e r s t a i n i n g w i t h haematoxylin and e o s i n . At a l a t e r stage y o l k granules were observed between the y o l k v e s i -c l e s . However, even at t h i s stage the y o l k granules were a l s o observed on the innner surface of the zona r a d i a t a ( F i g . 8). The oocyte at t h i s stage was about 350-500 u i n diameter w h i l e the i n d i v i d u a l granules were about 1 y or l e s s i n diameter. As more and more y o l k granules were formed, they migrated and accumulated towards the center of the oocytes. At the same time the y o l k v e s i c l e s were d i s p l a c e d towards the periphery of oocytes. HISTOCHEMISTRY OF VITELLOGENESIS Histochemical analyses focussed on the chemical composition of y o l k i n c l u s i o n s . Findings f o r the two types of y o l k i n c l u s i o n s are summarized 16 i n Table 2. D i s t r i b u t i o n of p o l y s a c c h a r i d e s . Polysaccharides were not detected i n the general cytoplasm of oogonia or primary oocytes, but only i n the y o l k v e s i c l e s which reacted w i t h a l l the polysaccharide s t a i n s . The v e s i c l e s s t a i n e d red w i t h Best's Carmine—an e m p i r i c a l method f o r d e t e c t -i n g glycogen which i s s t i l l w i dely used by histochemists (Pearse, 1968). C o n t r o l s e c t i o n s digested w i t h d i a s t a s e before s t a i n i n g gave a negative r e a c t i o n ; a b o l i t i o n of s t a i n i n g by d i a s t a s e suggested that the v e s i c l e s contained glycogen. I n c o n t r a s t to the y o l k v e s i c l e s , y o l k granules were not s t a i n e d by Best's carmine. The y o l k v e s i c l e s and zona r a d i a t a reacted w i t h p e r i o d i c a c i d -S c h i f f ' s reagent to form a dark red c o l o u r ( F i g . 9). This r e a c t i o n was abolished i n a c e t y l a t e d s e c t i o n s and t h i s confirmed the polysaccharide nature of y o l k v e s i c l e s and zona r a d i a t a . Since s e c t i o n s digested w i t h d i a s t a s e reacted p o s i t i v e l y w i t h p e r i o d i c a c i d - S c h i f f ' s reagents, the contents of y o l k v e s i c l e s are probably complex polysaccharides and not simple p o l y s a c c h a r i d e s . The y o l k granules and ooplasm of primary oocytes were not s t a i n e d by the p e r i o d i c a c i d - S c h i f f s r e a c t i o n . The y o l k v e s i c l e s a l s o s t a i n e d w i t h d i a l y s e d i r o n (Hale, 1948), and t h i s i n d i c a t e d that the y o l k v e s i c l e s contained a c i d mucopolysaccharides. The general cytoplasm of primary oocytes s t a i n e d greenish blue as com-pared to the blue s t a i n i n g of the y o l k v e s i c l e s . The y o l k granules were a l s o s t a i n e d greenish b l u e . Since t h i s greenish blue s t a i n was present i n both the cytoplasm and y o l k granules; i t was probably due to r e t e n t i o n of s t a i n i n g s o l u t i o n and not due to. a h i s t o c h e m i c a l r e a c t i o n . There was no change i n d i a l y s e d i r o n r e a c t i o n i n y o l k v e s i c l e s at v a r i o u s stages of Table 2: A summary of the histoc h e m i c a l p r o p e r t i e s of the two types of y o l k i n c l u s i o n s . TECHNIQUES POLYSACCHARIDES Best's Carmine (Pearse, 1968) Diastase - Best's Carmine P e r i o d i c a c i d - S c h i f f ' s (McManus, 1948) Dias t a s e - P e r i o d i c a c i d - S c h i f f ' s A c e t y l a t i o n - P e r i o d i c a c i d - S c h i f f ' s D i a l y s e d i r o n (Hale, 1946) A l c i a n B l u e , pH 2.5 (Pearse, 1968) YOLK VESICLES YOLK GRANULES + + PROTEINS Din i t r o f l u r o b e n z e n e (Burstone, 1955) ++ I I I I T e t r a - a z o t i z e d o - d i a n i s i d i n e ( D a n i e l l i , 1953) + +++ Sakaguchi's r e a c t i o n (Baker, 1947) —— I I M i l l o n ' s r e a c t i o n (Baker, 1956) - ++ F e r r i c f e r r i c y a n i d e r e a c t i o n (Chevremont & F r e d e r i c k , 1943) +++ + N-ethyl maleimide- F e r r i c f e r r i c y a n i d e -DDD r e a c t i o n (Barnett & Seligman, 1952) ++ N-ethyl maleimide - DDD r e a c t i o n -Continued Table 2 (Cont'd) TECHNIQUE LIPIDS Sudan Black B (Chayen et a l . , 1969) P y r i d i n e e x t r a c t i o n - Sudan Black B O i l Red 0 ( L i l l i e , 1944) P y r i d i n e e x t r a c t i o n - O i l Red 0 Ac i d haematin (Baker, 1946) P y r i d i n e e x t r a c t i o n - A c i d haematin Copper t h i o c y a n i n (Kluver & B a r r e r a , 1953) YOLK VESICLES YOLK GRANULES 4-f +4-++++ +44+ +4-t- 1 oo 19 Figure 9: Yolk v e s i c l e stage oocytes stained f o r polysaccharides with p e r i o d i c a c i d - S c h i f f ' s reaction. Yolk v e s i c l e s (v) showed a strong reaction. Figure 10: Oocytes stained with the dinitroflurobenzene method for proteins. The yolk granules (g) stained reddish whereas the yolk v e s i c l e s (v) stained yellowish. Figure 11: Oocytes stained with t e t r a z o t i s e d o - d i a n i s i d i n e reaction for proteins. The yolk granules (g) stained dark red while the yolk v e s i c l e s (v) stained brownish red, i n d i c a t i n g that the yolk v e s i c l e s contained less proteins than the yolk granules. Figure 12; Oocytes stained with the f e r r i c f e r r i c y a n i d e method for sulphydryl groups. Yolk v e s i c l e s (v) reacted p o s i t i v e l y though yolk granules (g) retained background s t a i n i n g only. Figure 13: Cryostat sections of ovary stained with Sudan Black B. Only the yolk granules (g) retained the s t a i n i n d i c a t i n g that they contained l i p i d s . Figure 14: Oocytes stained with acid haematin. The yolk granules (g) reacted p o s i t i v e l y i n d i c a t i n g phospholipids. 20 v i t e l l o g e n e s i s . A l c i a n blue (8GX) s t a i n e d the y o l k v e s i c l e s and zona r a d i a t a b l u i s h - g r e e n at pH 2.5. The y o l k granules were not s t a i n e d . The d i s -t r i b u t i o n s of the v e s i c l e s were s i m i l a r to those observed during p e r i o d i c a c i d - S c h i f f ' s s t a i n i n g ( F i g . 9 ) . I t i s concluded from polysaccharide s t a i n i n g that the y o l k v e s i c l e s c o n t a i n glycogen and complex a c i d i c p o l y s a c c h a r i d e s . By c o n t r a s t , y o l k granules do not c o n t a i n polysaccharides. D i s t r i b u t i o n of p r o t e i n s . O r i g i n a l l y introduced by Sanger (1945) as a reagent f o r N-terminal a-amino a c i d a n a l y s i s , d i n i t r o f l u r o b e n z e n e has been much employed as a reagent f o r d e t e c t i n g p r o t e i n end groups. For h i s t o c h e m i c a l d e t e c t i o n , the y e l l o w N l ^ - d i n i t r o p h e n y l compound i s reduced, d i a z o t i z e d and f i n a l l y coupled w i t h l-amino-8-naphthol-3, 6-disulphonic a c i d (H-acid) i n an a l k a l i n e s o l u t i o n . Both the y o l k granules and the zona r a d i a t a reacted w i t h d i n i t r o f l u r o b e n z e n e reagents to give a deep red colour ( F i g . 10). The y o l k v e s i c l e s on the other hand s t a i n e d brownish-red w i t h the d i n i t r o f l u r o b e n z e n e method. Yolk granules a l s o s t a i n e d deep red w i t h the t e t r a z o t i z e d 0 — d i a n i s i d i n e r e a c t i o n , a general s t a i n f o r p r o t e i n s ( F i g . 11). The y o l k v e s i c l e s s t a i n e d l i g h t red. This r e a c t i o n i s s t i o c h i o m e t r i c (Chayen et a l . 1969). This suggested that the y o l k v e s i c l e s contained l e s s p r o t e i n than the y o l k granules. Sakaguchi reagents, which are s p e c i f i c f o r a r g i n i n e (Baker, 1947) gave a p o s i t i v e r e a c t i o n w i t h y o l k granules w h i l e the y o l k v e s i c l e s were h a r d l y s t a i n e d at a l l appearing l i g h t y e l l o w i n c o l o u r . 21 Yolk granules appeared dark red a f t e r t r e a t i n g with Millon's reagent which i s s p e c i f i c f o r the hydroxy-phenyl group. The only common amino acid containing hydroxy-phenyl group i s tyrosine. The yolk v e s i -c l e s , by contrast, stained yellow to orange with Millon's reagent. Yolk v e s i c l e s appeared blue i n colour a f t e r reacting with Chevremont-Frederick's method (Fig. 12). This method f or sulphydryl groups i s based on the reduction of fe r r i c y a n i d e to ferrocyanide. The r e s u l t i n g ferrocya-nide combined with f e r r i c ions to form an insoluble Prussian blue p r e c i -p i t a t e . This reaction i s not s p e c i f i c by i t s e l f , since many reducing substances reduce f e r r i c y a n i d e . Control sections immersed i n 0.1 M, N-ethyl maleimide before reacting with Chevremont-Frederick reagents ap-peared green and showed no Prussian blue p r e c i p i t a t e i n the yolk v e s i c l e s ; N-ethyl maleimide s p e c i f i c a l l y blocked sulphydryl groups. The green colour i s non-specific and due to retention of reagents by the yolk granules. Yolk v e s i c l e s stained blue with dihydroxy-dinaphthyldisulphide (DDD) method. The DDD method i s based on the s p e c i f i c oxidation of sulphydryls (Pearse, 1968). No blue colour was evident i n yolk v e s i c l e s of control sections where sulphydryl groups were blocked with N-ethyl maleimide. The yolk granules were not stained by dihydroxy-dinaphthyl-disulphide reagent. In summary, the various protein stains demonstrated that both the yolk v e s i c l e s and yolk granules contained proteins. Yolk v e s i c l e s contain less proteins than yolk granules; sulphydryl groups are present i n yolk v e s i c l e s but not i n granules. Histochemistry of l i p i d s . Yolk granules but not yolk v e s i c l e s stained with Sudan Black B (Fig. 13). Sudan Black B was removed from yolk granules 22 by pyridine extraction. The zona ra d i a t a was not stained by Sudan Black B. The d i s t r i b u t i o n of O i l Red 0 was s i m i l a r to Sudan Black B. Yolk granules but not yolk v e s i c l e s absorbed O i l Red 0. This O i l Red 0 s t a i n -ing of yolk granules was not evident i n control sections extracted with pyr i d i n e . Yolk granules and zona ra d i a t a stained blue-black to almost black with acid haematin ( F i g . 14). The d i s t r i b u t i o n of acid haematin s t a i n i n g was not changed by pyridine extraction; yolk v e s i c l e s stained l i g h t yellow, i n d i c a t i n g the lack of phospholipids. Luxol Fast Blue MBS stained yolk granules and zona r a d i a t a dark blue. This copper thiocyanin method i s s p e c i f i c f o r phospholipids. The blue colour of yolk granules was s t i l l present i n control sections extracted with pyridine. The yolk v e s i c l e s were not stained by the copper thiocyanin method. In conclusion, the l i p i d histochemistry of v i t e l l o g e n e s i s indicated that yolk v e s i c l e s did not contain any l i p i d while yolk granules had both neutral fats and phospholipids. The zona radiata contained phospholipid but not neutral f a t s . 23 DISCUSSION The h i s t o l o g i c a l and h i s t o c h e m i c a l f i n d i n g s demonstrated t h a t two types of y o l k i n c l u s i o n s (yolk v e s i c l e s and y o l k granules) are formed during v i t e l l o g e n e s i s i n g o l d f i s h . These two yo l k i n c l u s i o n s d i f f e r d i s -t i n c t i v e l y i n t h e i r morphology, t i n c t o r i a l p r o p e r t i e s , and chemical nature; they are deposited s e q u e n t i a l l y — f i r s t the y o l k v e s i c l e s and then the y o l k granules. The occurrence of two types of yo l k i n c l u s i o n s i n t e l e o s t oocytes was p r e v i o u s l y described by Yamamoto (1956, 1958). He found both y o l k v e s i c l e s and y o l k granules i n L i o p s e t t a obscura and Clupea p a l l a s i i , based on h i s t o l o g i c a l evidence. S i m i l a r l y , Malone and Hisoaka (1963) d i s -t i n g u i s h e d two types of y o l k , designated as e x t r a v e s i c u l a r and i n t r a v e s i -c u l a r , i n the z e b r a f i s h Brachydanio r e r i o . However, the occurrence of two types of y o l k i n c l u s i o n s i s not u n i -v e r s a l among t e l e o s t s . In some s p e c i e s , three types of y o l k have been ob-served; Yamamoto (1956) described y o l k v e s i c l e s , y o l k g l o b u l e s , and l i p i d g l o b u l e s , i n the smelt, Hypomesus japonicus w h i l e Guraya (1965) noted three types of y o l k i n c l u s i o n s i n Channa marulius. The f i r s t y o l k i n c l u s i o n s deposited i n g o l d f i s h oocytes are the y o l k v e s i c l e s which form as minute o v a l bodies that grow u n t i l they reached the s i z e of 15 y. These y o l k v e s i c l e s have been described by many i n v e s t i -gators using d i f f e r e n t names i n c l u d i n g c o r t i c a l a l v e o l i , i n t r a v e s i c u l a r y o l k , vacuoles, vacuome, y o l k g l o b u l e s , y o l k spheres and y o l k v e s i c l e s . In s p i t e of v a r y i n g nomenclature, y o l k v e s i c l e s always c o n s i s t of mucopoly-saccharides and react p o s i t i v e l y to the p e r i o d i c a c i d - S c h i f f ' s r e a c t i o n 24 (Aketa, 1954; Yamamoto, 1956 a, b, c; Malone & Hlsoaka, 1963; Guraya, 1965; Yamazaki, 1965). Yolk v e s i c l e s i n oocytes of Clupea p a l l a s i i and L i o p s e t t a obscura cont a i n simple mucopolysaccharides (Yamamoto, 1956 a, b) w h i l e the g o l d f i s h examined i n t h i s i n v e s t i g a t i o n have a c i d mucopoly-sacc h a r i d e s ; Hypomesus japonicus a l s o has y o l k v e s i c l e s w i t h a c i d i c mucopolysaccharides (Yamamoto, 1956 c ) . Aketa suggested that y o l k v e s i -c l e s of Oryzias l a t i p e s c o n s i s t e d of sulphated mucopolysaccharides. Towards the l a t t e r part of g o l d f i s h v i t e l l o g e n e s i s , the y o l k v e s i c l e s are d i s p l a c e d towards the periphery of oocytes and develop i n t o c o r t i c a l a l v e i o l i . The c o r t i c a l a l v e o l i play an important r o l e i n the c o r t i c a l r e a c t i o n at the time of f e r t i l i z a t i o n (Yamamoto, 1961; Manroy, 1965). The o r i g i n of y o l k v e s i c l e s had been a t t r i b u t e d to v a r i o u s c e l l components. Aketa (1954) suggested that y o l k v e s i c l e s o r i g i n a t e from the " p r e - c o r t e x " w h i l e Yamamoto (1961) suggested that y o l k v e s i c l e s o r i g i -nate from the ground cytoplasm. A c t u a l l y , there i s no r e a l d i f f e r e n c e between these two p o s t u l a t i o n s . By c o n t r a s t , Nath (1960) and Guraya (1965) proposed the "yolk nucleus" as the precursor of y o l k v e s i c l e s . The pre-cursors of y o l k v e s i c l e s , because of t h e i r small s i z e , are beyond the r e s o l v i n g power of the l i g h t microscope. Although no one has examined the y o l k nucleus i n t e l e o s t w i t h the e l e c t r o n microscope, B a l i n s k y and Davis (1963) demonstrated that the y o l k n u c l e i of amphibians were concen-t r a t i o n s of mitochondria which played no p a r t i n y o l k formation. At the e l e c t r o n microscope l e v e l , the best evidence of the o r i g i n of y o l k v e s i -c l e s was provided by Yamamoto and Onozato (1965), and Yamamoto and Oota (1967). Yamamoto and Onozato (1965) demonstrated that G o l g i complexes 25 gave r i s e to y o l k v e s i c l e s i n g o l d f i s h and these f i n d i n g s have been confirmed by Yamamoto and Oota (1967) i n Brachydanio r e r i o , and by Gupta and Yamamoto (1972) i n g o l d f i s h , Carassius auratus. The y o l k v e s i c l e s i n g o l d f i s h were d i s t i n g u i s h e d from y o l k granules by t h e i r l a r g e r s i z e and t i n c t o r i a l p r o p e r t i e s . Chemically the y o l k v e s i c l e s are polysaccharides and p r o t e i n s r i c h i n s u l p h y d r y l groups w h i l e the y o l k granules c o n s i s t of p r o t e i n s , phospholipids and n e u t r a l l i p i d s . The chemical composition of y o l k granules as determined by h i s t o c h e m i c a l s t a i n i n g i s very s i m i l a r to the composition of y o l k as determined by b i o -chemical s t u d i e s . Plack and Fraser (1970), i n a biochemical study, demon-s t r a t e d t h a t the y o l k of cod contains two major l i p o p r o t e i n s of s i m i l a r molecular weight (400,000) and chemical composition). E a c h h l i p o p r o t e i n i s about 79% p r o t e i n and 21% l i p i d w h i l e approximately 65% of the l i p i d f r a c -t i o n i s p h o s p h o l i p i d . The y o l k granules were deposited independently of the y o l k v e s i c l e s and only appeared i n oocytes a f t e r the y o l k v e s i c l e s were f u l l y i i d e v e l o p e d . There are s t i l l many c o n f l i c t i n g opinions about the o r i g i n of y o l k granules. D r o l l e r and Roth (1966) concluded from e l e c t r o n microscopic s t u d i e s of P o e c i l i a r e t i c u l a t a o v a r i e s that y o l k granules were formed by the endoplas-mic r e t i c u l u m and G o l g i bodies. These workers described m i c r o p i n o c y t o s i s i n the periphery of oocytes; the granules formed by p i n o c y t o s i s accumulated w i t h i n the endoplasmic r e t i c u l u m and G o l g i complex to form y o l k granules. Yamamoto and Oota (1967), and Gupta and Yamamoto (1972) disputed the suggest-ions of D r o l l e r and Roth; Yamamoto and Oota p o s t u l a t e d t h a t mitochondria were the precursors of y o l k granules. They argued that p i n o c y t o t i c bodies 26 were taken i n t o oocytes and resynthesized w i t h i n mitochondria to form y o l k granules. This suggestion seems u n l i k e l y i n view of the recognized f u n c t i o n s of mitochondria i n c e l l r e s p i r a t i o n . Further work, e s p e c i a l l y at the e l e c t r o n microscopic l e v e l , i s r e q u i r e d before these c o n f l i c t i n g opinions can be r e s o l v e d . I n s p i t e of these d i f f e r e n c e s i n o p i n i o n s , a l l i n v e s t i g a t o r s agreed that p a r t of y o l k granules were derived from an exogenous source and that y o l k precursor were deposited i n oocytes by p i n o c y t o s i s . Anderson (1968) and Guraya (1965) have a l s o provided evidence of p i n o c y t o s i s w i t h i n oocytes of Fundulus h e t e r o c l i t u s and Channa marulius, r e s p e c t i v e l y . The h i s t o g e n e s i s of y o l k granule formation supported the i d e a of an exogenous source of y o l k granules. I t i s w e l l known that i n many t e l e o s t the l i v e r accumulates l i p i d s and p r o t e i n s during the reproductive period ( B a i l e y , 1957* Ho and Vanstone, 1961; Cedard e_t al_. , 1961, P l a c k and F r a s e r , 1970). Very recent i n v e s t i g a -t i o n s by Aida e_t a l . (1973) a l s o support the e a r l i e r p o s t u l a t i o n s that t e l e o s t l i v e r i s the s i t e of y o l k p r o t e i n s y n t h e s i s . I t seems l i k e l y t hat the y o l k p r o t e i n s synthesized by the l i v e r are transported by blood plasma to the ovary and deposited w i t h i n oocytes by p i n o c y t o s i s . 27 Figure 15: A diagramatic representation of oogenesis i n g o l d f i s h ovaries. The stages i n oocyte development during oogenesis are -(I) oogonia within cysts; (II) chromatin nucleolar stage oocytes; (III) per i n u c l e o l a r stage oocyte; (IV) early yolk v e s i c l e stage oocyte; (V) intermediate yolk v e s i c l e stage oocyte; (VI) l a t e yolk v e s i c l e stage oocyte; (VII) i n i t i a l yolk granule stage oocyte; (VIII) l a t e yolk gran-ule stage oocyte; and (IX) mature oocyte. DIAGRAMATIC REPRESENTATION OF O O C Y T E G R O W T H SECTION IT : The Corpus Luteum and i t s Functions 29 INTRODUCTION Not a l l of the developing o v a r i a n f o l l i c l e s complete the formation of an ovum; many of them become a t r e t i c at some stage during t h e i r de-velopment. These a t r e t i c f o l l i c l e s have many s i m i l a r i t i e s to the post-o v u l a t o r y f o l l i c l e s and there has long been a l i v e l y i n t e r e s t i n t h e i r f u n c t i o n . At present n e i t h e r the physiology of the a t r e t i c f o l l i c l e s nor the p o s t - o v u l a t o r y corpus luteum i s f u l l y understood. A t r e t i c oocytes occur i n a wide v a r i e t y of t e l e o s t as evident by the reviews by P i c k f o r d and Atz (1957), B a l l (1960), Dodd (1960), Hoar (1965, 1969) and C h i e f f i (1970). They have been described f r e q u e n t l y s i n c e Bretschneider and Duyvene de Wit (1947) f i r s t discussed t h e i r h i s t o g e n e s i s i n Rhbdeus amarus. F o l l i c u l a r a t r e s i a has been described i n P o e c i l i a r e t i c u l a t a ( S t o l k , 1951; Lambert, 1970), Carassius auratus (Beach, 1959), Gasterosteus aculeatus (Tromp-Blom, 1959) , Scomber scomber (Bara, 1960), Mystus seenhgala (Sa'thyanesan (1961), Heteropneustes f o s s i l i s ( N a i r , 1963), Pleuronectes p l a t e s s a ( B a r r , 1963), Xenenotodon c a n c i l a ( R a s t o g i , 1966), Monopterus a l b a (Chan et a l . , 1967), and Sebastodes p a u c i s p i n i s (Moser, 1967) . The po s t - o v u l a t o r y f o l l i c u l a r s t r u c t u r e s which resemble mammalian corpora l u t e a , have been reported i n Scomber scomber (Bara, 1960), Mystus seenghala (Sathyanesan, 1962), Heteropneustes f o s s i l i s ( N a i r , 1963), E u c a l i a i n c o stans (Braekevelt and M c M i l l a n , 1967), and C l a r i u s batrachus ( L e h r i , 1968). Although Barr (1963), Rajalakshmi (1966), and Moser (1967) 30 reported an absence of p o s t - o v u l a t o r y corpora l u t e a i n t e l e o s t o v a r i e s , t h i s may have been due to random sampling or the ovaries were not sampled soon enough a f t e r o v u l a t i o n . The s i m i l a r i t y i n morphology and o r i g i n of these s t r u c t u r e s , whether pre- or p o s t - o v u l a t o r y , to mammalian corpora l u t e a l e d Bretschneider & Duyvene de Wit (1947) to consider them comparable to the mammalian corpus luteum and to suggest an endocrine f u n c t i o n based on the questionable " B i t t e r l i n g o v i p o s i t o r growth t e s t " . This work has s t i m u l a t e d many d i s -cussions of the f u n c t i o n of the pre- and p o s t - o v u l a t o r y o v a r i a n f o l l i c l e s but no d e f i n i t e conclusions have been reached due to the l a c k of c r i t i c a l evidence of t h e i r endocrine f u n c t i o n . Many authors, w h i l e r e c o g n i z i n g the l i m i t a t i o n s of the " B i t t e r l i n g o v i p o s i t o r growth t e s t " , s t i l l consider these s t r u c t u r e s comparable to the mammalian corpus luteum (see reviews by B a l l , 1960; Hoar, 1965). In a d d i -t i o n to the s i m i l a r i t y i n morphology and o r i g i n , pre- and p o s t - o v u l a t o r y bodies o f t e n have the appearance of true e p i t h e l i a n glands. Hoar (1965, 1969) suggested that t e l e o s t corpora l u t e a might produce estrogens r a t h e r than progesterone. The appropriateness of the term "corpuca luteum" i n t e l e o s t s i s not u n i v e r s a l l y accepted. The main argument against i t i s the l a c k of c r i t i c a l evidence of the endocrine f u n c t i o n of the a t r e t i c f o l l i c l e s . The two b a s i c endocrine fun c t i o n s of the corpus luteum are (a) a c t i v e s e c r e t i o n of the hormone progesterone and (b) the p i t u i t a r y c o n t r o l . In c o n t r a s t to the mammalian corpus luteum, s u c c e s s f u l gestations have been e s t a b l i s h e d i n the absence of these bodies i n ' t e l e o s t s (Lambert and Van Oordt, 1965; Lambert, 1970). Lambert (1970) suggested that i f the a t r e t i c f o l l i c l e s 31 were Important hormone producing glands they should be r e g u l a r l y present during g e s t a t i o n . However, the absence of a t r e t i c f o l l i c l e s d uring ges-t a t i o n does not r u l e out an endocrine r o l e of these s t r u c t u r e s but shows only that they p l a y no p h y s i o l o g i c a l r o l e i n g e s t a t i o n ; they may, however, produce hormones and p l a y some other reproductive r o l e . Some workers f e e l that the l a c k of p i t u i t a r y c o n t r o l i n the formation and maintenance of a t r e t i c f o l l i c l e s enhances doubts of an endocrine f u n c t i o n (Hisaw, 1963). Corpora l u t e a r e g u l a r l y form subsequent to hypo-physectomy (Bretschneider and Duyvenne de Wit, 1947; B a r r , 1963) and some authors who doubt the endocrine status of t e l e o s t corpora l u t e a , maintain that a t r e t i c oocytes are probably adaptive devices f o r d i s p o s i n g moribund ova (Dodd, 1960; P o l d e r , 1964). They consider the absence of progesterone s e c r e t i o n and the l a c k of a r o l e i n g e s t a t i o n strong arguments f o r using a d i f f e r e n t d e s c r i p t i v e term from the mammalian corpus luteum. However, the appropriateness of the term "corpora l u t e a " depends on i t s d e f i n i t i o n . The d e f i n i t i o n of corpus luteum, as an endocrine gland that secretes progester-one and maintains g e s t a t i o n , i s too r e s t r i c t i v e . I n v e r t e b r a t e s , e s p e c i a l l y t e l e o s t s , s t r u c t u r e s s i m i l a r to corpora l u t e a are not r e s t r i c t e d to v i v i -para (Hisaw, 1963; Brambell, 1960) and a narrow d e f i n i t i o n prevents compara-t i v e s t u d i e s of these s t r u c t u r e s i n oviparous s p e c i e s . From a comparative viewpoint i t may be more appropriate to consider the "corpus luteum" as an endocrine gland that secretes s t e r o i d hormones and p l a y a r o l e i n reproduc-t i o n but which need not n e c e s s a r i l y be concerned w i t h the maintenance of g e s t a t i o n . 32 The broader d e f i n i t i o n w i l l be adopted for t h i s study. The s t r u c -tures w i l l be termed corpora l u t e a and an attempt made to e s t a b l i s h the p h y s i o l o g i c a l function of both a t r e t i c oocytes and post-ovulatory f o l l i -c l e s i n the g o l d f i s h — a n oviparous t e l e o s t . This study w i l l i n v e s t i g a t e the corpus luteum as an organ capable of synthesizing steroids by examin-ing the histochemical l o c a t i o n of various hydroxysteroid dehydrogenases. Dorfman and Ungar (1965) established the r o l e of hydroxysteroid dehydro-genases i n s t e r o i d biosynthesis. The fate of the corpus luteum, either pre- or post-ovulatory, w i l l be followed past the 6-stage. 33 METHODS AND MATERIALS HYPOPHYSECTOMY OF GOLDFISH Goldfish were hypophysectomized using a modification of Yamazaki's (1965) technique. The f i s h were f i r s t anaesthesized i n a cold (4-8 C) s o l u t i o n of 0.01 % t r i c a n e methane sulfonate ('MS222', Sandoz) u n t i l they l o s t t h e i r r e f l e x a c t i v i t y . The "operating t a b l e " f o r hypophysectomizing g o l d f i s h , consisted of a sponge with a groove i n i t . G o ldfish were placed i n t h i s groove, with the l e f t side uppermost and held i n place by e l a s t i c bands of 2.5 cm width. The operating table was kept cool with crushed i c e during the en t i r e operation. The l e f t operculum together with the f i r s t two g i l l arches was r e -tracted with hooked pins attached to e l a s t i c cords. An oblique cut, p a r a l l e l to the second g i l l arch, was made i n the dorsal buccal mucosa. The i n c i s i o n varied from 5-10 mm i n length; t h i s cut exposed the parasphen-oid bone, beneath which the p i t u i t a r y i s situated. A hole was then d r i l l e d through the parasphenoid bone with a round dental burr (No. 3) to expose the p i t u i t a r y . This hole was immediately i n front of the inner t i p of the pterygoid bone. Another landmark for the point of d r i l l i n g i s the o r i g i n of a p a i r of conspicuous nerves from the cranium. These nerves were i n -e v i t a b l y cut during hypophysectomy while i n the sham hypophysectomized f i s h they were i n t e n t i o n a l l y cut during the d r i l l i n g of the parasphenoid bone; i n sham hypophysectomized f i s h the p i t u i t a r y was exposed but not removed. To e f f e c t hypophysectomy, the exposed p i t u i t a r y was removed by suction with a curved p i p e t t e . Bleeding was much reduced with cold anesthetics 34 and ceased quickly i n most instances. The i n c i s i o n i n the roof of the mouth healed within a week. The exposed area of the s k u l l was replaced with connective t i s s u e s . During recovery hypophysectomized g o l d f i s h were placed i n cold d i -luted sea water (4-8°C) prepared by d i l u t i n g with three times i t s volume of dechlorinated water. A temperature shock a f t e r hypophysectomy r e s u l t s i n high mortality and consequently water temperatures were raised slowly to room temperature. High s u r v i v a l rates, averaging well over 80%, were obtained with the above techniques; most of the mortality was due to b r a i n damage. 35 HISTOLOGY OF PRE- AND POST-OVULATORY CORPUS LUTEUM Pre-ovulatory corpora l u t e a were induced i n gr a v i d g o l d f i s h by hypophysectomy. Four hypophysectomized f i s h were k i l l e d each week f o r e i g h t weeks. A p o r t i o n of each ovary was frozen f o r h i s t o c h e m i c a l ex-amination of hydroxysteroid dehydrogenases w h i l e another p o r t i o n was f i x e d i n Bouin's f l u i d to observe the h i s t o g e n e s i s of a t r e t i c oocyte. The f a t e of p o s t - o v u l a t o r y f o l l i c l e s was observed i n f i s h k i l l e d 1, 7 and 30 days a f t e r o v u l a t i o n . Likewise i n these experiments, one p o r t i o n of each ovary was frozen f o r the d e t e c t i o n of hy d r o x y s t e r o i d dehydrogenases and the other f i x e d i n Bouin's f l u i d . Ovaries f i x e d i n Bouin's f l u i d were embedded i n p a r a f f i n , sectioned s e r i a l l y at 7 y and s t a i n e d w i t h haema-t o x y l i n and e o s i n , or M a l l o r y ' s trichrome (Gurr, 1962). HISTOCHEMICAL DEMONSTRATION OF HYDROXYSTEROID DEHYDROGENASE Hydroxysteroid dehydrogenases i n ova r i e s of v i t e l l o g e n i c g o l d f i s h were examined h i s t o c h e m i c a l l y w i t h a m o d i f i c a t i o n of Warttenberg (1958) technique. Hydroxysteroid dehydrogenases i n o v a r i e s of hypophysectomized g o l d f i s h were examined at weekly i n t e r v a l s f o r e i g h t consecutive weeks a f t e r hypophysectomy and the f i n d i n g s were compared with those i n the ov a r i e s of ovulated g o l d f i s h t e s t e d 1, 7 and 30 days a f t e r o v u l a t i o n . D e t a i l s of the techniques are as f o l l o w s : G o l d f i s h were decapitated and a p o r t i o n of each ovary was frozen w i t h "Cryowick" (IEC Company), w h i l e a second p o r t i o n was f i x e d i n Bouin's f i x a t i v e f o r r o u t i n e h i s t o l o g y . Frozen o v a r i e s were sectioned w i t h a c r y o s t a t ( H a r r i s I n t e r n a t i o n a l ) at 36 10-16 y; the temperature of the c r y o s t a t , which was c r i t i c a l f o r proper s e c t i o n i n g , was maintained at -10°C to -15°C. Cryostat s e c t i o n s were 2 mounted on 22 m c o v e r s l i p s , d r i e d f o r about 10 min at room temperature and r i n s e d i n 0.1 M T r i s - H C l b u f f e r , pH 7.5 before being incubated i n the r e a c t i o n medium at 30°C. The r e a c t i o n mixture c o n s i s t e d of 1 mg t e t r a z o l i u m s a l t ( N i t r o BT or MTT), 2 mg NAD and 10 mg MgCl 2 d i s s o l v e d i n 1.6 ml of 0.1 M, T r i s - H C l b u f f e r , pH 7.5 and 0.4 ml dimethyl formamide c o n t r a i n i n g 50-100 yg s t e r o i d . MTT was r o u t i n e l y used as N i t r o BT gave a more intense diaphorase r e a c t i o n . S t e r o i d s used as substrates i n c l u d e d dehydroepiandrosterone (a spe-c i f i c s ubstrate f o r 3 3-hydroxysteroid dehydrogenases), androsterone (a s p e c i f i c substrate f o r 3 a-hydroxysteroid dehydrogenases) , 17 cx-hydro-progesterone (a' s p e c i f i c s ubstrate f o r 17 a-hydroxysteroid dehydrogenases), and t e s t o s t e r o n e (a s p e c i f i c s u b s t r a t e f o r 17 ^-hydroxysteroid dehydrogen-ases) . Each of these substrates has one hydroxyl group f o r the hydroxy-s t e r o i d dehydrogenase r e a c t i o n ( F i g . 16). Other o v a r i a n s t e r o i d s , 17 a-hydroxypregenolone, pregnenolone, and e s t r a d i o l were used as substrates f o r u n s p e c i f i c hydroxysteroid dehydrogenase r e a c t i o n s . To d i s t i n g u i s h h ydroxysteroid dehydrogenases from diaphorase which occurs i n most t i s s u e , s e c t i o n s were incubated i n a r e a c t i o n mixture w i t h -out any s t e r o i d s u b s t r a t e . At the end of the i n c u b a t i o n p e r i o d , s e c t i o n s were f i x e d i n 10% n e u t r a l f o r m a l i n (Baker, 1944) which i n h i b i t e d the en-zymes. Sections were then washed i n two changes of 25% dimethyl forma-mide to remove excess s t e r o i d s , and f i n a l l y i n d i s t i l l e d water. The 36a 3 - 1 6 : HYDROXYSTEROID DEHYDROGENASE REACTION BluePpt. 37 washed s e c t i o n s were mounted i n Ferrant's media and examined w i t h i n a day or two a f t e r s t a i n i n g . INCORPORATION OF THYMIDINE-3H INTO GOLDFISH OVARIES A group of grav i d g o l d f i s h was t r a n s f e r r e d t o experimental aquaria c o n t a i n i n g deoJalorinated water at 10°C. The water temperature s l o w l y r a i s e d to room temperature (22-25°C). With t h i s treatment the m a j o r i t y of these f i s h ovulated a f t e r two days. F i s h which d i d not ovulate were removed w h i l e the ovulated f i s h were spawned w i t h " a c t i v e " males. Two days a f t e r spawning the females were i n j e c t e d w i t h 1 yc thymidine-3 H (360 mc/mM, Amersham) per gram body weight. A f t e r the i n t r a p e r i t o n e a l 3 i n j e c t i o n of thymidine- H i n 0.1 ml of s a l i n e the f i s h were k i l l e d , i n 3 groups of f o u r , at 1, 2, 4, 8, 16, and 32 days a f t e r the thymidine- H i n j e c t i o n . A p o r t i o n of each ovary was f i x e d i n Bouin's f l u i d and embedded i n p a r a f f i n using r o u t i n e h i s t o l o g i c a l techniques. P a r a f f i n s e c t i o n s (7 y t h i c k ) were cut and mounted on albumen coated s l i d e s . These s e c t i o n s were dewaxed and hydrated through a s e r i e s of a l c o h o l . Hydrated s e c t i o n s were dip-coated w i t h nuclear t r a c k emulsions (Kodak NTB 2) i n the dark (Kopriwa and LeBland, 1962). The s l i d e s were a i r - d r i e d and then kept i n l i g h t proof s l i d e boxes i n the r e f r i g e r a t o r (0-5°C) f o r a month, a f t e r which the s l i d e s were processed w i t h photographic developer (Kodak D 19) and f i x e r (Kodak F 5 f i x i n g b a th). The autoradiographs were st a i n e d w i t h Mayer's haematoxylin and e o s i n (Beserga and Malamud, 1969), dehydrated i n a l c o h o l , c l e a r e d i n xylene, and mounted i n DPX ( K i l k p a t r i c k and Lendrum, 1941). 38 Autoradiographs of f i v e sections from each ovary were examined 3 for the incorporation of thymidine- H into the various c e l l types i n the ovaries. 39 RESULTS HISTOGENESIS OF ATRETIC OOCYTES Yolky oocytes became a t r e t i c a f t e r hypophysectomy. The p a t t e r n of a t r e s i a was the same i n a l l oocytes although the r a t e s at which v a r i o u s oocytes, w i t h i n an ovary, became a t r e t i c was v a r i a b l e (Table 3). Because of t h i s v a r i a b i l i t y i t was impossible to q u a n t i f y the r a t e of a t r e s i a f o l l o w i n g hypophysectomy. However, oocytes at the y o l k granule stage were c l e a r l y the f i r s t to become a t r e t i c and, i n g e n e r a l , oocytes w i t h more y o l k became a t r e t i c much f a s t e r than those w i t h l e s s y o l k . For d e s c r i p t i v e purposes, a t r e s i a i n g o l d f i s h can be sub-divided i n t o four consecutive stages according to the s u b d i v i s i o n s of Bretschneider and Duyvene de Wit (1947) and Beach (1959) w i t h an a d d i t i o n a l f i f t h stage des-c r i b e d here. tt-stage ( F i g . 17). The hypertrophy of the granulosa i n i t i a t e s a t r e s i a . The granulosa c e l l s change from a squamous to a columnar e p i t h e l i u m and, i n most i n s t a n c e s , y o l k v e s i c l e s rupture to form a continuous mass of c o l l o i d ; the e n t i r e oocyte shows gigns of degeneration. The puncture of the zona r a d i a t a by numerous pores marks the end of the <* -stage a t r e t i c oocyte. g-stage ( F i g . 18). Hypertrophied granulosa c e l l s invade the cytoplasm of the a t r e t i c oocyte through the pores i n the zona r a d i a t a . The invading granulosa c e l l s d i g e s t and resorb the y o l k i n c l u s i o n s ; the zona r a d i a t a d i s i n t e g r a t e s towards the end of the (3-stage. 3 W Table 3: The degree of at r e s i a i n various ovaries at various time int e r v a l s a f t e r hypophysectomy. Only second growth phase (yolky) oocytes were counted. Days a f t e r No. of yolky NO. OF OOCYTES IN EACH STAGE hypophysectomy oocytes counted Yolk vesic l e Yolk granule A t r e t i c 7 75 63 14 1 7 52 31 4 17 7 42 22 3 17 7 64 46 2 36 14 54 35 5 14 14 57 16 11 30 14 96 96 0 0 14 64 38 24 2 21 61 15 2 44 21 63 18 0 45 21 45 4 0 41 21 52 18 7 27 28 46 0 0 46 28 36 0 0 36 28 65 2 0 63 28 73 0 0 73 28(sham) 61 44 17 0 28 (sham) 69 43 26 0 28(sham) 34 6 22 6 28(sham) 81 66 15 0 : sham hypophysectomized g o l d f i s h , which serves as the control 40 y-stage ( F i g . 19). Hypertrophy of the granulosa c e l l s continues during t h i s stage. By the end of this stage a l l remnants of oocytes are completely resorbed and the hypertrophied granulosa c e l l s surround a small c a v i t y , the atrium; t h i s marks the space formerly occupied by the oocyte. The en t i r e structure i s surrounded by a membrane, one c e l l t h i c k , which i s s i m i l a r to the theca of the normal oocyte ( F i g . 20). This stage appears to have been overlooked by many in v e s t i g a t o r s , although they described a y s t a g e d i f f e r e n t from the ^-stage described here. 6-stage ( F i g . 21). During t h i s stage, the granulosa c e l l s collapse into the atrium to form an i r r e g u l a r c e l l u l a r mass and yellow l u t e i n pigments are observed among the c e l l s . This stage p e r s i s t s f o r long periods; Barr (1963) observed 6-stage preovulatory corpora lu t e a s i x months a f t e r hypo-physectomy. e-stage ( F i g . 22). In addition to these four stages of a t r e s i a a f i f t h and f i n a l stage was recognized. In the l a t e stages of a t r e s i a some c e l l s of the 6-stage corpus luteum appear to d i f f e r e n t i a t e i n t o oogonia. Various phases of d i f f e r e n t i a t i o n were observed and the best evidence for a 41 Figure 17: a-stage of a t r e t i c oocyte stained with haematoxylin and eosin. The granulosa c e l l s of the a t r e t i c oocytes (arrow 1) hypertrophied as compared with the normal granulosa c e l l s (arrow 2). Extensive degeneration of yolk v e s i c l e s was evident (homogeneous area opposite arrow 1). Figure 18: 3-stage of a t r e t i c oocyte stained with haematoxylin. Masses of granulosa and thecal c e l l s invade and digest the a t r e t i c oocytes. Figure 19: A section of the ovary of a hypophysectomized g o l d f i s h containing many y-stage a t r e t i c f o l l i c l e s (free arrow) with the c h a r a c t e r i s t i c empty ca v i t y within i t . Figure 20: A d e t a i l e d photomicrograph of the y s t a g e a t r e t i c f o l l i -c l e . The oocyte i s completely reabsorbed and the colum-nar granulosa c e l l s have the c h a r a c t e r i s t i c s of physiolo-g i c a l l y active c e l l s . Figure 21: 6-stage a t r e t i c f o l l i c l e (mass of c e l l s on right) stained with haematoxylin and eosin. The granulosa collapsed into the space formerly occupied by the egg cytoplasm to form an i r r e g u l a r mass of c e l l s . Figure 22: e-stage a t r e t i c f o l l i c l e s . This photomicrograph shows the d i f f e r e n t i a t i o n of one h a l f of the a t r e t i c f o l l i c l e s i n t o oogonia (arrow) while the other h a l f remain u n d i f f e r -entiated. 42 separate e-stage corpus luteum was the presence of the p a r t i a l l y d i f f e r e n t i a t e d cysts which show one h a l f com-posed of oogonia while the other h a l f looked l i k e a 6-stage corpus luteum with yellow l u t e i n pigments. HISTOGENESIS OF POST OVULATORY CORPUS LUTEUM At ovulation the f o l l i c u l a r membranes of f u l l y developed oocytes rupture and release the eggs. The f o l l i c u l a r membrane i s not released but retained within the ovary where i t hypertrophies. Three d i s t i n c t phases i n histogenesis can be distinguished. Stage I (Fig. 23, 24, 25). A f t e r ovulation both theca and granulosa c e l l s become hypertrophied with the more marked response i n the granulosa (Fig. 25). The morpho-l o g i c a l appearance of t h i s stage i s the same as the y-stage a t r e t i c f o l l i c l e . Sometimes the remains of the broken f o l l i c u l a r wall i s evident ( F i g . 23) and appears as a d i s t i n c t i v e feature but the majority of post-ovula-tory bodies look l i k e y-stage a t r e t i c oocytes with no i n d i c a t i o n of the release of eggs (Fig. 24); t h i s may be due to the way the ovaries were sectioned. Stage I I ( F i g . 26, 27). There were no differences be-tween the <5-stage corpus luteum and the stage II post-43 o v u l a t o r y corpus luteum. The granulosa c e l l s converged i n t o an i r r e g u l a r mass as i n the 6-stage a t r e t i c oocyte and l o s t t h e i r glandular nature; as the theca surrounds the compact mass of granulosa c e l l s . As i n the 6-stage a t r e t i c oocyte, y e l l o w l u t e i n pigments i n t e r m i n g l e w i t h granulosa c e l l s . Stage I I I ( F i g . 28). The i r r e g u l a r mass of c e l l s i n stage I I post-ovulatory f o l l i c l e s seem to d i f f e r e n t i a t e i n t o oogonial c y s t s as i n the 6-stage a t r e t i c oocyte. These oogonial c y s t s c o n s t i t u t e the stage I I I or e-stage corpus luteum. Again, p a r t i a l l y d i f f e r e n t i a t e d oogonial c y s t s were observed w i t h one h a l f composed of u n d i f f e r e n -t i a t e d s t a t e I I c e l l s c o n t a i n i n g y e l l o w l u t e a l pigments w h i l e the other h a l f c o n s i s t s of w e l l defined oogonia. This evidence prompted the p o s t u l a t i o n of the d i f f e r e n t i a -t i o n of stage I I c e l l s i n t o oogonial c y s t s . Histochemical l o c a l i z a t i o n of hydroxysteroid dehydrogenases. The r e s u l t s of the h i s t o c h e m i c a l l o c a l i z a t i o n of hy d r o x y s t e r o i d dehydrogenase are presented i n Table 4. Formazan deposits demonstrated a general d i s -t r i b u t i o n of diaphorase a c t i v i t y i n s e c t i o n s of ovaries incubated i n a medium c o n t a i n i n g Nitro-BT and NAD but l a c k i n g s t e r o i d s u b s t r a t e . The diaphorase a c t i v i t y was observed i n oocytes as w e l l as f o l l i c u l a r c e l l s ; w i t h i n oocytes, t h i s a c t i v i t y increased w i t h oocyte diameter. I t was Stage I post-ovulatory corpus luteum stained with Mallory's trichrome. This photograph documents f or the f i r s t time evidence of the rupture f o l l i c u l a r w a l l (arrow) following ovulation. Stage I post-ovulatory corpus luteum also stained with Mallory's trichrome. Here there i s no evidence of the rupture of the f o l l i c l e a f t e r ovulation. This i s mainly due to the sectioning of the ovarian t i s s u e . A d e t a i l e d photograph of the stage I post-ovulatory corpus luteum showing the hypertrophy of granulosa c e l l s (c) as compared to the f o l l i c u l a r c e l l s , both thecal and granu-l o s a of the normal ovary ( f ) . The radiate nature of the zona ra d i a t a (z) i s evident. Stage II post-ovulatory corpus luteum (arrows). The granu-lo s a and thecal c e l l s collapsed into an i r r e g u l a r mass of c e l l s . A d e t a i l e d photograph of the stage II corpus luteum. The c e l l s e s p e c i a l l y the n u c l e i are les s hyperthrophied than those of the stage I corpus luteum ( F i g . 25). The stage I II post-ovulatory corpus luteum stained with haematoxylin and eosin. This stage i s characterized by the d i f f e r e n t i a t i o n of some c e l l s into oogonia (arrow). Table 4: A summary of the histoc h e m i c a l r e a c t i o n of hydr o x y s t e r o i d dehydrogenases i n ova r i a n f o l l i c l e s . STEROID SUBSTRATE ENZYME 1st growth V e s i c l e STAGES IN OOGENESIS E a r l y y o l k V e s i c l e Late y o l k V e s i c l e Yolk granule DtUycU ueT. i •-C o n t r o l Dehydroepi-androsterone Androsterone 17a-hydroxy pregnenolone 17a-hydroxy-progesterone E s t r a d i o l Testosterone Diaphorase 33-HSD 3a-HSD 33-HSD or 17a-HSD 17a-HSD 173-HSD 176-HSD + ++7 + 4-4-44 4-4-4-4-I n t e n s i t y of reactions were graded (-) to ( M i l ) ; (-) denotes no demonstrable a c t i v i t y and ( M M ) a maximal a c t i v i t y . 1 T HSD i s hydroxysteroid dehydrogenase. 46 much reduced when the tetrazolium s a l t , MTT, was used as the f i n a l e l e ctron acceptor. No 33-hydroxysteroid dehydrogenase a c t i v i t y was evident i n oo-cytes at the l a t e yolk granule stages (Fig. 30) although some enzyme a c t i v i t y was discernable i n early yolk granule stage oocytes. 33-hydroxysteroid dehydrogenase. Ovaries reacted when incubated i n a medium used for the demonstration of 33-hydroxysteroid dehydro-genase with either NAD or NADP as a co-factor but the reaction was weaker with NADP as the co-factor. The substrate was dehydroepian-drosterone or pregnenolone. This r e a c t i o n was c l e a r l y l o c a l i z e d i n f o l l i c u l a r c e l l s of f i r s t growth phase oocytes (Fig. 29) but i t was impossible to determine whether the reaction was of thecal or granu-l o s a l o r i g i n since these tissues are not well d i f f e r e n t i a t e d . The smaller second growth phase oocytes, i n the yolk v e s i c l e stage, gave p o s i t i v e r e s u l t s with the reaction located i n the granulosa c e l l s ; o c c a s i o n a l l y 33-hydroxysteroid dehydrogenase was also detected i n the theca c e l l s during this stage. No 33-hydroxysteroid dehydrogenase a c t i v i t y was evident i n oocytes at the l a t e yolk granule stages ( F i g . 30) although some enzyme a c t i v i t y was d i s c e r n i b l e i n early yolk granule stage oocytes. 3ot-Hydroxysteroid dehydrogenase a c t i v i t y . The d i s t r i b u t i o n of 3a-hydroxysteroid dehydrogenase a c t i v i t y i n g o l d f i s h ovaries, was simi -l a r to that of 33-hydroxysteroid dehydrogenase. The best r e s u l t s were obtained with NAD as a co-factor; NADP gave a much weaker reaction. 47 This 3a-hydroxysteroid dehydrogenase a c t i v i t y was l o c a l i z e d i n the periphery of f i r s t growth phase oocyte as was the case f or 33-hydroxy-s t e r o i d dehydrogenase ( F i g . 29). Formazan deposits with androsterone as substrate were detected i n the granulosa of yolk v e s i c l e stage oo-cytes but the a c t i v i t y of 3a-hydroxysteroid dehydrogenase was much weaker than 33-hydroxysteroid dehydrogenase. As previously described f or 33-hydroxysteroid dehydrogenase, no 3a-hydroxysteroid dehydrogenase a c t i v i -ty was detected i n the f o l l i c u l a r c e l l s of yolk granule stage oocytes. 17a^-Hydroxysteroid dehydrogenase. 17a-hydroxy progesterone was s u c c e s s f u l l y used as a substrate f or the hydroxysteroid dehydrogenase reactio n . This reaction indicates the presence of 17a-hydroxysteroid dehydrogenase enzymes i n g o l d f i s h ovaries. The d i s t r i b u t i o n of t h i s enzyme was s i m i l a r to that of 13a- and 33-hydroxysteroid dehydrogenases. In short, 17a-hydroxysteroid dehydro-genase occurred i n the periphery of f i r s t growth phase oocytes and i n the granulosa of yolk v e s i c l e stage oocytes but yolk granule stage oocytes showed no sign of 17a-hydroxysteroid dehydrogenase a c t i v i t y . The 17a-hydroxysteroid dehydrogenase a c t i v i t y i n the ovaries i s les s intense than 33-hydroxysteroid dehydrogenase and s i m i l a r i n i n t e n s i t y with 3a-hydroxy-t e r o i d dehydrogenase. 173-Hydroxysteroid dehydrogenase. E s t r a d i o l and testosterone were su c c e s s f u l l y used as substrates f o r the demonstration of 173-hydroxysteroid dehydrogenase a c t i v i t y i n the ovary. As i n the hydroxysteroid dehydrogen-ase reactions NAD gave a better reaction than NADP. 48 Figure 29: Histochemical demonstration of 33-hydroxysteroid dehydro-genase i n ov a r i e s of normal f i s h . The formazan de p o s i t s (arrow) are l o c a l i z e d i n the granulosa c e l l s of oocytes. The n u c l e i (n) are l a b e l l e d to provide e a s i e r i n t e r p r e t a -t i o n of the i l l u s t r a t i o n . F igure 30: Ovaries of g r a v i d f i s h s t a i n e d w i t h 3 ( 3-hydroxysteroid de-hydrogenase. There were no a c t i v i t i e s i n the f o l l i c u l a r c e l l s ( f r e e arrow) of the y o l k granulestage oocytes. Figure 31: Ovaries of a hypophysectomized g o l d f i s h (two weeks a f t e r hypophysectomy) s t a i n e d f o r 173-hydroxysteroid dehydrogen-ase. A c t i v i t y , as i n d i c a t e d by the d e p o s i t i o n of formazan (arrow) was l o c a l i z e d i n y-stage a t r e t i c f o l l i c l e s . No enzyme a c t i v i t y was present i n f o l l i c u l a r c e l l s of the f i r s t growth phase oocytes. Figure 32: Histochemical demonstration of 3 [ 3-hydroxysteroid dehydro-genase i n the pos t - o v u l a t o r y f o l l i c l e s of r e c e n t l y ovulated g o l d f i s h . High enzyme a c t i v i t y was detected i n the corpus luteum (arrow). 49 The distribution of 173-hydroxysteroid dehydrogenases in the ovaries was similar to other hydroxysteroid dehydrogenases. The reac-tion with either estradiol or testosterone was restricted to the periphery of first growth phase oocytes and the granulosa of yolk vesicle stage oocytes. It was impossible to determine the precise location of 173-hydroxysteroid dehydrogenase in the first growth phase oocytes as the follicular tissues were poorly differentiated. As in the other hydroxysteroid dehydrogenases examined, there was a gradual reduction of enzyme activity as the oocyte developed into the yolk granule stage which showed no evidence of 173-hydroxysteroid dehydro-genase activity. 50 HYDROXYSTEROID DEHYDROGENASES IN HYPOPHYSECTOMIZED FISH Table 5 summarizes the d i s t r i b u t i o n of the various hydroxysteroid dehydrogenases i n the ovaries of hypophysectomized g o l d f i s h . One week a f t e r hypophysectomy, only a-stage a t r e t i c oocytes were observed but no q u a n t i f i c a t i o n of the rate of a t r e s i a was attempted because of the great v a r i a b i l i t y as indicated e a r l i e r . Hydroxysteroid dehydrogenases were not detected i n c e l l s of e i t h e r a-stage a t r e t i c oocytes or i n f i r s t growth phase oocytes. The lack of enzyme a c t i v i t y i n the f o l l i c l e s of f i r s t growth phase oocytes characterized the ovaries of hypophysectomized g o l d f i s h at a l l times. Most yolky oocytes have become a t r e t i c by the end of the second or t h i r d weeks of hypophysectomy. A t r e s i a progressed as f a r as the Y-stage during this period. Although there were some a-stage a t r e t i c oocytes, they were mainly i n the 3- or y-stages and no s i g n i f i c a n t d i f -ferences were noted i n the ovaries of f i s h hypophysectomized f o r two or three weeks. Hydroxysteroid dehydrogenase a c t i v i t y was absent i n f o l l i c l e s of f i r s t growth phase oocytes as w e l l as « - and 3-stage a t r e t i c oocytes. In s t r i k i n g contrast, y-stage a t r e t i c oocytes had 173-hydroxysteroid dehydrogenase, a c t i v i t y i n the hypertrophied granulosa ( F i g . 31). This a c t i v i t y was obtained whether testosterone or e s t r a d i o l was used as the substrate. The other hydroxysteroid dehydrogenases, 3a-, 33-, and 17a-hydroxysteroid dehydrogenases, were not detected i n the y-stage a t r e t i c oocytes. Table 5. Histochemical Reaction of Hydroxysteroid Dehydrogenase i n Ovaries of Hypophysectomized G o l d f i s h . STEROID SUBSTRATE ENZYME 1st growth corpus luteum stages phase oocyte a 3 y 6 Con t r o l Dehydroepi-androsterone Androsterone 17a-hydroxyprogesterone E s t r a d i o l Testosterone I n t e n s i t y of hydroxysteroid dehydrogenase a c t i v i t y graded (-) to ( M i l ) ; (-) denotes no demonstrable-r e a c t i o n and ( M M ) maximal. ^HSD denotes hydroxysteroid dehydrogenase enzymes. 2 No enzyme a c t i v i t y was detected i n second growth phase oocytes when they were present. Ln Diaphorase 33-HSD1 3a-HSD 17a-HSD 173-HSD 173-HSD 52 No intact second growth phase oocytes were found in ovaries of fish k i l l e d four weeks after hypophysectomy. A l l yolk oocytes had become atretic and some of them reached the 6-stage while others were at the a-stage. These a-stage atretic oocytes were much smaller than those observed earlier; the majority of oocytes were at the 3- or y-stages. As i n the ovaries of fish hypophysectomized for two and three weeks hydroxysteroid dehydrogenases were absent from f i r s t growth phase oocytes and from the a-, 3- or 6-stages atretic f o l l i c l e s . Although 173-hydroxysteroid dehydrogenase activity was detected i n y-stage atre-t i c oocytes, 3a-, 33-> and 17a-hydroxysteroid dehydrogenases were not evident. The ovaries of fish k i l l e d in the f i f t h to eight week after hypo-physectomy had only f i r s t growth phase oocytes and 6-stage atretic f o l l i -cles. No hydroxysteroid dehydrogenases were found i n these ovaries. In summary, hypophysectomy inhibited a l l hydroxysteroid dehydrogen-ases except 17 3-hydroxy steroid dehydrogenase in the y-stage atretic f o l l i c l e s . 53 HYDROXYSTEROID DEHYDROGENASES IN HYPOPHYSECTOMIZED GOLDFISH INJECTED WITH PORCINE LUTEINIZING HORMONE F i s h hypophysectomized f o r one week were i n j e c t e d w i t h 100 ug/g porcine l u t e i n i z i n g hormone (NIH-LH-S8). Two days a f t e r the i n t r a p e r i -t o n e a l i n j e c t i o n s , the f i s h were k i l l e d and examined f o r h y d r o x y s t e r o i d dehydrogenases. T h e i r o v a r i e s gave strong r e a c t i o n s f o r a l l four enzymes (Table 6 ) . Apparently, the l u t e i n i z i n g hormone st i m u l a t e s formation of these enzymes s i n c e h y d r o x y s t e r o i d dehydrogenases were not detected i n untreated hypophysectomized g o l d f i s h . I n l u t e i n i z i n g hormone-treated f i s h the d i s t r i b u t i o n of the formazan deposits was normal except f o r the presence of a l l four h y d r o x y s t e r o i d dehydrogenases i n y-stage a t r e t i c oocytes. As i n the ovaries of hypophysectomized g o l d f i s h , no hydroxysteroid dehydrogenase enzymes were detected i n the a-, and 6-stage a t r e t i c oocytes of the hormoner:treated f i s h . By c o n t r a s t , the y-stage a t r e t i c oocytes had a c t i v i t i e s of 3a-, 3(3-, 17a-, and 173-hydroxysteroid dehydro-genases; of these, 173-hydroxysteroid dehydrogenase was the most a c t i v e . This i s d i f f e r e n t from hypophysectomized f i s h where only 173-hydroxy-s t e r o i d dehydrogenase was detected i n the y-stage a t r e t i c oocyte and again argues f o r s t i m u l a t i o n of enzyme formation by LH. Table 6. E f f e c t of LH Treatment on H i s t o c h e m i c a l l y Demonstrable Hydroxysteroid Dehydrogenases i n Ovaries of Hypophysectomized G o l d f i s h S t e r o i d Substrate Enzyme 1st growth phase oocytes Stages of a t r e s i a a g y * • 6 Co n t r o l Dehydroepi-androsterone Androsterone 17a-hydroxy-progesterone E s t r a d i o l Testosterone diaphorase 33-HSD 3a-HSD 17a-HSD 176-HSD 176-HSD + ++++ U l 55 HYDROXYSTEROID DEHYDROGENASES IN POST-OVULATORY CORPUS LUTEUM The histochemical detection of hydroxysteroid dehydrogenases was car r i e d out at three times a f t e r ovulation: on the day of ovulation and on the seventh and t h i r t i e t h days a f t e r ovulation. F i s h k i l l e d s h o r t l y a f t e r ovulation showed very active hydroxysteroid dehydrogenase reactions i n t h e i r ovaries. Blue formazan deposits developed i n the post-ovulatory f o l l i c l e s with a l l the substrates. The formazan deposits were mainly i n the granulosa c e l l s although o c c a s i o n a l l y the thecal c e l l s stained also. Except f o r th i s very active hydroxysteroid dehydrogenase reaction i n the post-ovulatory corpora l u t e a ( F i g . 32) the ovary looks l i k e a normal v i t e l l o g e n i c ovary. F i r s t growth phase oocytes developed formazan i n the f o l l i c u l a r c e l l s with a l l the s t e r o i d substrates. Likewise, oocytes i n the yolk v e s i c l e stages had a l l the hydroxysteroid dehydrogenases but these were c l e a r l y confined to the granulosa c e l l s . As i n normal ovaries, no hydroxysteroid dehydrogenase a c t i v i t y occured i n the f o l l i c u -l a r c e l l s during the yolk granules stages. The d i s t r i b u t i o n of the various hydroxysteroid dehydrogenases did not seem to change during the f i r s t seven days a f t e r ovulation. At seven days post-ovulation, there were very few stage I post-ovulatory corpora lutea but most of the post ovulatory f o l l i c l e s had reached stage I I . The stage I post ovulatory corpus luteum showed a l l four hydroxy-s t e r o i d dehydrogenases (Table 7) with formazan deposits i n the granulosa c e l l s . These deposits were observed with a l l steroids tested as sub-st r a t e s . Only s l i g h t a c t i v i t y was observed i n the stage II post ovulatory Table 7: Histochemical L o c a l i z a t i o n of Hydroxysteroid Dehydrogenases i n the Ovaries of Ovulated Goldfish Steroid Enzyme Stages i n Oogenesis Post Ovulatory F o l l i c l e s Substrate l g t Y o l k Y o l k g t a g e I g t a g e ^ growth v e s i c l e Granule Control Diaphorase + + + + 4-Dehydroepiand androsterone 33-HSD +++ 4+++ + 4-H- + Androsterone 3a-HSD +4- 444- + 4+ + 17a-hydroxyprogesterone 17a-HSD 4-4- 4+ +• 4- +• E s t r a d i o l 17B-HSD 4+4- +4-f + +4+ + ON 57 corpus luteum. The r e a c t i o n was not much greater than i n the c o n t r o l s e c t i o n s and suggested that the r e a c t i o n was due to diaphorase a c t i v i t y . The h i s t o c h e m i c a l d i s t r i b u t i o n of these enzymes i n the other o v a r i a n t i s s u e s i s s i m i l a r to the d i s t r i b u t i o n i n the normal v i t e l l o -genic ovary. The small oocytes i n the f i r s t growth phase had high h y d r o x y s t e r o i d dehydrogenase a c t i v i t y . This high a c t i v i t y was obtained w i t h a l l s t e r o i d s u b s t r a t e s . Yolk v e s i c l e stage oocytes showed 3a-, 36-, 17a- and 173-hydroxysteroid dehydrogenase a c t i v i t i e s i n the granu-l o s a ; r e a c t i o n s were comparable to those of the normal ovary. As i n the other o v a r i e s examined, there was no hydroxysteroid dehydrogenase a c t i v i -t y i n the f o l l i c l e s of y o l k granules stage oocytes. Only stage I I post-o v u l a t o r y corpora l u t e a are found t h i r t y days a f t e r o v u l a t i o n . These are m o r p h o l o g i c a l l y s i m i l a r to the 6-stage pre - o v u l a t o r y corpora l u t e a and h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was detected i n them. The d i s t r i b u -t i o n of h y d r o x y s t e r o i d dehydrogenase i n the other o v a r i a n t i s s u e s was s i m i l a r to t h a t of the e a r l i e r stages w i t h r e a c t i o n s i n the f o l l i c u l a r c e l l s of f i r s t growth phase oocytes as w e l l as the granulosa of y o l k v e s i -c l e stage oocytes. Again, no hydroxysteroid dehydrogenase a c t i v i t y oc-curred i n y o l k granule stage oocytes. I t i s concluded that the post-ovulatory corpus luteum i s capable of s t e r o i d hormone s y n t h e s i s . Very a c t i v e s t e r o i d s y n t h e s i s occurs i n the corpus luteum as w e l l as i n the granulosa c e l l s of p o s t - o v u l a t o r y o v a r i e s . The Fate of P o s t o v u l a t o r y Corpus Luteum as Determined by Autoradiography T r i t i a t e d thymidine l a b l e s were detected i n f o l l i c u l a r c e l l s of f i s h 3 k i l l e d one day a f t e r thymidine- H i n j e c t i o n ( F i g . 33). More of these c e l l s 58 were l a b e l l e d near the ovarian periphery than i n the center of o v a r i e s . F o l l i c u l a r c e l l s were l a b e l l e d i r r e s p e c t i v e of the stage of ovum develop-ment. None of the abundant stage I po s t - o v u l a t o r y corpora l u t e a were l a b e l l e d . 3 The p a t t e r n of thymidine- H i n c o r p o r a t i o n was s i m i l a r i n ov a r i e s of 3 f i s h k i l l e d two days a f t e r thymidine- H i n j e c t i o n w i t h f o l l i c u l a r c e l l s 3 l a b e l l e d randomly i n the oocytes. On the f o u r t h day a f t e r thymidine- H i n j e c t i o n , however, l a b e l s were observed i n the c e l l s of stage I I post-o v u l a t o r y corpora l u t e a ( F i g . 34) as w e l l as the f o l l i c u l a r c e l l s . Again, none of stage I corpora l u t a c e l l s were l a b e l l e d . The m a j o r i t y of the stage IT corpora l u t e a were l a b e l l e d i n ovaries on f i s h k i l l e d e ight days 3 a f t e r thymidine- H i n j e c t i o n . The increase i n number of stage I I corpora lutea. was accompanied by a red u c t i o n i n stage I corpora l u t e a . 3 The d i s t r i b u t i o n of thymidine- H l a b e l s i n ovaries of f i s h k i l l e d 16 days a f t e r t r i t i a t e d thymidine i n j e c t i o n was e s s e n t i a l l y s i m i l a r to that of f i s h kept f o r 8 days w i t h some l a b e l l e d c e l l s i n the m a j o r i t y of stage IT corpora l u t e a . The f o l l i c u l a r c e l l s were l a b e l l e d , i r r e s p e c t i v e of the stage of the oocyte. The only d i f f e r e n c e was the absence of stage I corpora l u t e a i n the ovaries of these f i s h . The f o l l i c u l a r c e l l s of oocytes as w e l l as c e l l s of stage IT corpora l u t e a were l a b e l l e d i n ovar-3 i e s of f i s h k i l l e d 32 days a f t e r thymidine- H i n j e c t i o n . In a d d i t i o n many oogonia ( F i g . 35) and some oocytes were l a b e l l e d ( F i g . 36). The conclusion of the experiments described i n t h i s s e c t i o n i s that both f o l l i c u l a r and stage I I corpora l u t e a c e l l s are a c t i v e l y m u l t i p l y i n g . One of these two c e l l types d i f f e r e n t i a t e s i n t o oogonia and u l t i m a t e l y oocytes. 59 Figure 33: Autoradiography of the ovary seven.days a f t e r the 3 i n j e c t i o n of thymidine- H. The f o l l i c u l a r c e l l s (arrow) were l a b e l l e d . Figure 34: Autoradiography of the ovary, seven days a f t e r the 3 i n j e c t i o n of thymidine- H. In a d d i t i o n to the l a b e l -l i n g of f o l l i c u l a r c e l l s ( F i g . 33) c e l l s of 5-stage corpus luteum were a l s o l a b e l l e d (arrows). Figure 35: Autoradiography of g o l d f i s h o v a r i e s 30 days a f t e r thy-3 midine- H i n j e c t i o n . Some oogonia w i t h i n oogonial c y s t s 3 were l a b e l l e d by thymidine- H (arrow). 3 Figure 36: Autoradiography of oocyte l a b e l l e d w i t h thymidine- H. t h i s oocyte was from the ovary of g o l d f i s h i n j e c t e d w i t h 3 thymidine- H f o r 30 days. 60 DISCUSSION This investigation indicated that the f o l l i c u l a r c e l l s , especial-ly the granulosa, serve many functions. They are the major endocrine tissue i n the ovary, either as f o l l i c u l a r cells of normal oocytes, or as corpora lutea. In addition to steroid hormone synthesis, the f o l l i -cular cells are responsible for the removal of atretic oocytes by pha-gocytosis . After atresia or ovulation the f o l l i c u l a r cells developed into corpora lutea which later differentiate into oogonia. Steroid Synthesis in Fish Ovaries In the present study, hydroxysteroid dehydrogenases have only been found in the ovarian granulosa. The activities of 3a-, 33-, 17a-, and 173-hydroxysteroid dehydrogenases increased with oocyte development or differentiation, but at the yolk granule stage, a l l four hydroxysteroid dehydrogenases decreased so dramatically that during the latter part of this stage, no hydroxysteroid dehydrogenases could be detected in the granulosa. In post-ovulatory ovaries hydroxysteroid dehydrogenases were evident in first growth phase oocytes and in corpora lutea, but were not detec-table in the granulosa of large unovulated oocytes. In summary, these histochemical findings suggested that the granulosa c e l l s of developing oocytes are capable of steroid synthesis although the actual steroid hor-mones synthesized are not known. The absence of hydroxysteroid dehydrogenase activity during the yolk granule stage was surprising. It indicated an inhibition of steroid 61 synthesizing enzyme activity when oocytes reached the yolk granule stage. The steroid enzymes were evidently re-activated after ovulation. Hydroxysteroid dehydrogenase activity seems to depend on the p i t u i -tary gland. Following hypophysectomy, hydroxysteroid dehydrogenases were not detected i n granulosa of either f i r s t growth phase or yolk vesicle stage oocytes. Restoration of activity with bovine luteinizing hormone supports this argument. Although there are no comparable studies of hypophysectomy on hydroxysteroid dehydrogenases in the ovaries of teleosts, Yamazaki and Donaldson (1968) demonstrated a decrease in the 3g-hydroxy-steroid dehydrogenase of i n t e r s t i t i a l cells of goldfish testis after hypophysectomy. In their experiments, 33-hydroxysteroid dehydrogenase activity was restored with salmon gonadotropin, again confirming the pituitary involvement. Similarly, Yaron (1966) observed that 3B-hydroxy-steroid dehydrogenase decreased in testis of Tilapia mossembica after hypophysectomy, while Wiebe (1969) found that methallibure administration decreased 33-hydroxysteroid dehydrogenase activity in i n t e r s t i t i a l and Suertoli cells of Cymatogaster aggregata; methallibure inhibited gonado-tropin function i n this teleost (Hoar et a l . , 1967). In conclusion, i t i s evident that steroidogenesis i n the ovaries and testes of teleosts is under the control of pituitary gonadotropin. In the absence of gonadotropin a l l hydroxysteroid dehydrogenase activity ceases. Atresia of Second Growth Phase Oocytes In this study, atresia was induced by hypophysectomy; i n nature, adverse environmental or physiological conditions probably induce atresia 62 i n y o l k y oocytes (Yaron, 1971). The h i s t o l o g y of a t r e t i c f o l l i c l e s i n g o l d f i s h i s s i m i l a r to that described f o r other t e l e o s t (Bretschneider and Duyvene de Wit, 1947; S t o l k , 1951; Beach, 1959; B a r r , 1963; L e h r i , 1968; Lambert, 1970). I t i s c h a r a c t e r i z e d by the hypertrophy of granu-l o s a c e l l s , or t h e c a l c e l l s , or both as demonstrated by Chan et a l . (1967). The e a r l y stages ( a - and 3-stages) i n v o l v e d the breakdown and r e s o r p t i o n of moribund oocytes. The phagocytic a c t i o n of f o l l i c u l a r c e l l s i n a t r e t i c oocytes was g e n e r a l l y recognized (see review by B a l l , 1960; Dodd, 1960; Hoar, 1965; C h i e f f i , 1970), t h i s f u n c t i o n of the a t r e -t i c f o l l i c l e has been observed i n a l l v e r t e b r a t e groups i n c l u d i n g mammals (Brambell, 1955). Hypertrophy of granulosa c e l l s i n g o l d f i s h continues even a f t e r the y o l k i s completely resorbed. The y-stage a t r e t i c f o l l i c l e s have one or two l a y e r s of hypertrophied c e l l s surrounding an inner c a v i t y , the atrium ( F i g . 19). Though many i n v e s t i g a t o r s recognized a y-rstage during a t r e s i a (Beach, 1959; B a r r , 1963; Lambert, 1970), t h e i r ^ d e s c r i p t i o n of y-stage was s i g n i f i c a n t l y d i f f e r e n t from ours and Involve only the i r r e g u l a r mass of c e l l s noted i n the 6-stage. These authors may have f a i l e d to observe an intermediate stage before the c o l l a p s e of the a t r e t i c f o l l i c l e s . The present study i n d i c a t e s that t h i s stage, c h a r a c t e r i z e d by gland u l a r mor-phology, i s very important to the p h y s i o l o g i c a l f u n c t i o n s of a t r e t i c f o l l i c l e s . These y-stage a t r e t i c f o l l i c l e s i n g o l d f i s h appear to be true e p i -t h e l i a l glands ( F i g . 20). Histochemical data (Table 5, F i g . 31) show considerable 173-hydroxysteroid dehydrogenase a c t i v i t y . This enzyme 63. c a t a l y s e s e i t h e r the conversion of t e s t o s t e r o n e to androsterone, or e s t r a d i o l to estrone ( T a l a l a y , 1965) and i t s presence i n y-stage corpora l u t e a a t r e t i c a supports Hoar's (1965) suggestion that the t e l e -ost corpus luteum i s r e s p o n s i b l e f o r the s y n t h e s i s of estrogens. In c o n t r a s t to 173-hydroxysteroid dehydrogenase, there was no evidence f o r 33-hydroxysteroid dehydrogenase or 3a- and 17a-hydroxysteroid dehydro-genases i n y-stage a t r e t i c f o l l i c l e s . These observations are i n p a r t i a l agreement w i t h those of Lambert (1970) and Yaron (1971) who reported an absence of 3g-hydroxysteroid dehydrogenase i n a t r e t i c f o l l i c l e s of P o e c i l i a r e t i c u l a t a and T i l a p i a mossembica r e s p e c t i v e l y but d i d not t e s t f o r 3 17 ^-i,-. or 173-hydroxysteroid dehydrogenases. Hypophysectomized g o l d f i s h i n j e c t e d w i t h LH developed a l l four hy-d r o x y s t e r o i d dehydrogenases i n y-stage corpora l u t e a . This s t i m u l a t i o n of h y d r o x y s t e r o i d dehydrogenases suggests that p i t u i t a r y gonadotropins are r e s p o n s i b l e f o r the a c t i v i t y of these hydroxysteroid dehydrogenase enzymes i n the a t r e t i c f o l l i c l e s . Hypophysectomy impaired p a r t of the s t e r o i d s y n t h e s i z i n g c a p a b i l i t y of a t r e t i c f o l l i c l e s . P o s t - o v u l a t o r y Corpora Lutea Stage I p o s t - o v u l a t o r y corpora l u t e a are s i m i l a r i n appearance to y-stage a t r e t i c f o l l i c l e s and c o n s i s t of hypertrophied granulosa and t h e c a l c e l l s surrounding an empty c a v i t y . The main d i f f e r e n c e noted was the presence of an opening i n some, but not a l l of the p o s t - o v u l a t o r y f o l l i c l e ( F i g . 23 and 24). P o s t - o v u l a t o r y corpora l u t e a w i t h t h i s open-in g which marks the p o i n t of o v u l a t i o n have not been p r e v i o u s l y described. 64 In f a c t , most investigators have paid only cursory attention to post-ovulatory f o l l i c l e s , reporting only t h e i r r a pid d i s i n t e g r a t i o n and disappearance (Bailey, 1943; Bara, 1960; Rastogi, 1966; Braekevelt & McMillan, 1967). Barr (1963), Lambert (1966), Rajalakshmi (1966), and Moser (1967) maintained that no post-ovulatory corpus luteum existed i n t e l e o s t . Their conclusions were based on random sampling which did not include ovaries soon a f t e r ovulation. A de t a i l e d analysis of gold-f i s h ovaries from ovulated f i s h showed that the post-ovulatory f o l l i c l e s always existed and had a d e f i n i t e pattern of histogenesis. Localized hormone production. The d i s t r i b u t i o n of hydroxysteroid dehydrogenases i n stage I post-ovulatory f o l l i c l e s was s i m i l a r to y -stage a t r e t i c f o l l i c l e s i n f i s h i n j e c t e d with LH. In short, the tests were p o s i t i v e f o r 173-hydroxysteroid dehydrogenase as w e l l as 3a-, 33-and 17a-hydroxysteroid dehydrogenases. However, these stage I post-ovulatory corpora l u t e a with complete s t e r o i d synthesizing c a p a c i t i e s were rather transient and la s t e d no longer than a week. Seven days a f t e r ovulation hydroxysteroid dehydrogenase a c t i v i t y had d e f i n i t e l y declined. Bara (1965) demonstrated weak 33-hydroxysteroid dehydrogenases i n the theca of post-ovulatory f o l l i c l e s of Scomber scomber and t h i s also sug-gested that stage I post-ovulatory f o l l i c l e s were capable of st e r o i d hormone synthesis. The main difference between the post-ovulatory bodies described here and the mammalian corpus luteum i s the sparse v a s c u l a r i t y of the tel e o s t corpus luteum. As i n stage 1 post-ovulatory f o l l i c l e s , the y stage a t r e t i c f o l l i c l e s were poorly vascularized. Again, t h i s poor 65 v a s c u l a r i z a t i o n suggested that the hormone synthesized, most l i k e l y estrogen, acted l o c a l l y and were probably not released i n l a r g e amounts i n t o the c i r c u l a t o r y system. This l o c a l i z a t i o n of s t e r o i d s would r e s u l t i n high c o n c e n t r a t i o n of s t e r o i d hormones i n the corpus luteum. High l e v e l s of estrogens w i t h i n the ovary might be re q u i r e d to induce oogonial d i f f e r e n t i a t i o n ; t h i s l o c a l i z a t i o n of estrogens at the poi n t of a c t i o n would provide a mechanism f o r d i r e c t a c t i o n of estrogen on the t a r g e t t i s s u e s without a s i m i l a r r i s e i n blood estrogen which might i n h i b i t the production of gonadotropin. L o c a l i z e d a c t i o n s of s t e r o i d hormone provide a p l a u s i b l e e x p l a n a t i o n of the asynchronus development of oocytes i n the ovary. In h i s s t u d i e s on the t e s t i c u l a r c y c l e of another amniote, the r e p t i l e Naja n a j a , L o f t s (1972) found that the S e r t o l i c e l l s , a homologue of the granulosa c e l l s , synthesized androgens which were not rel e a s e d i n t o the p e r i p h e r a l v a s c u l a r system but induced the propagation of germ c e l l s l o c a l l y . In a d d i t i o n the S e r t o l i c e l l s of r e p t i l e s played an im-portant r o l e i n the s y n c h r o n i z a t i o n and maintenance of the germ c e l l s . P o s t - o v u l a t o r y p r o l i f e r a t i o n of oogohia. Yamazaki (1965) observed numerous oogonia i n ovaries of g o l d f i s h w i t h corpora l u t e a . Though oogonia are detected i n the ov a r i e s of many d i f f e r e n t t e l e o s t s throughout the year, a sharp r i s e i n number of f r e q u e n t l y observed s h o r t l y a f t e r spawning (Craig-Bennett, 1930; H i c k l i n g , 1935; Matthews, 1938; Mendoza, 1943; Bullough, 1942; B a r r , 1963; Yamazaki, 1965). The sudden increase of oogonia a f t e r o v u l a t i o n and the occurrence of hydroxysteroid dehydro-genases i n po s t - o v u l a t o r y f o l l i c l e s suggests some r e l a t i o n s h i p between 66 the two processes and the possibility that the post-ovulatory f o l l i c l e s may secrete steroid.hormones to induce oogonial formation. Bullough (1942) and Egami (1954) demonstrated that estrogens i n -duced an increase in the number of oogonia i n ovaries of Phoxirius laevis and Oryzias latipes respectively. Yamamoto (1969) has reviewed the many different studies on sex differentiation i n teleost that demon-strated the potency of estrogens as inducers of oogonial formation. These findings seem to support Hoar's (1965) hypothesis that the teleost corpus luteum synthesizes estrogens rather than progesterone and that the estrogens may induce germ cells to form oogonia. The Fate of Corpus Luteum Cells 3 The incorporation of thymidine- H into relatively large numbers of stage II post-ovulatory corpora lutea cells indicated that these cells were actively multiplying rather than being inactive and dying as some authors have suggested (Polder, 1964; Lambert, 1970). The autoradiographic evidence suggests that oogonia originated from either the f o l l i c u l a r cells or corpora lutea c e l l s . The histological picture of intermediate stages between stage IT corpora lutea and oogonial cysts supports the suggestion that some cells in the stage IT corpora lutea differentiate into oogonia. This postulation that oogonia were derived from corpora lutea cells of recently ovulated or resorbed ova i s supported by the sudden increase in oogonia after ovulation or resorption of ova. Yamazaki (1965) ob-served large numbers of oogonia in ovaries with atretic oocytes. Other 67 workers have also noted a sharp rise in the number of oogonia shortly after spawning (Craig-Bennett, 1930; Hickling, 1930; Matthews, 1938; Mendoza, 1941; Bullough, 1942; Barr, 1963; Yamazaki, 1965). Goldfish are multiple breeders that spawn i n the spring (Yamazaki, 1968). Like other teleosts that spawn more than once, ovulated eggs are replaced i n a cyclical manner (Fig. 37) after each spawning. The reproductive cycle within the ovary. During the i n i t i a l ma-turation, primodial germ cells differentiate into oogonia which form yolk-ladened oocytes. After vitellogenesis, oocytes follow one of two developmental paths. Under adverse conditions the yolk laden oocytes become atretic and develop into a - stage corpora lutea atretica. Dur-ing this stage, f o l l i c u l a r cells hypertrophy and phagocytose the atretic oocyte and form the g-stage corpora lutea which terminates the resorbtion of the aberrant oocyte. The absorption of the oocyte marks the beginning of the ystage. Steroid (probably estrogen) synthesis occurs in y-stage corpora lutea. The steroid synthesizing capability in y-stage f o l l i c l e s were par-t i a l l y impaired by hypophysectomy. Under normal circumstances (with the adverse conditions removed and the pituitary intact) the f u l l steroid synthesizing apparatus would be functioning as suggested by the occurrence of a l l four hydroxysteroid dehydrogenases in corpora lutea of hypophysec-tomized fish injected with LH. Estrogen synthesized bv y-stage corpora lutea i s probably localized within the 6-stage corpora lutea concerned with the differentiation of 68 oogonia into e-stage corpora l u t e a . I t i s suggested that t h i s gave r i s e to new oogonial cysts to replace the germ c e l l s l o s t during a t r e s i a . The majority of f i s h attained t h e i r f u l l maturity and ovulated r e g u l a r l y . The ovulated f o l l i c l e s formed the stage I post-ovulatory corpora l u t e a , comparable to the y-stage corpora lutea a t r e t i c a . These f o l l i c l e s had f u l l s t e r o i d synthesizing a b i l i t y . They probably synthe-s i z e d estrogens which were stored l o c a l l y . By the time the s t e r o i d syn-thesis was completed the corpora l u t e a developed into stage IT corpora lut e a . Estrogens that were synthesized e a r l i e r , induced some stage II c e l l s to d i f f e r e n t i a t e into oogonia. The newly d i f f e r e n t i a t e d oogonia within the stage I I I corpora lutea replaced oocytes l o s t during ovulation. 69 Figure 37: Summary of the f a t e of pre- and post-ovulatory f o l l i c l e s i n g o l d f i s h ovaries. (1) Adverse conditions cause yolky oocytes to undergo a t r e s i a . F o l l i c u l a r c e l l s hypertrophy and became phagocytic; the hypertrophied granulosa at the a-and 3-stages resorb the a t r e t i c oocyte. Once the oocyte i s removed the a t r e t i c f o l l i c l e s develops into the Y ~ s t a S e corpus luteum. This has the appear-ance of an endocrine gland and most l i k e l y synthesizes estrogens. The estrogens synthesized probably induce some of the d-stage c e l l s to d i f f e r e n t i a t e into oogon-i a as observed i n the €-stage corpus luteum. (2) The yolky oocytes normally mature and are ovulated; the ovulated f o l l i c l e s (Stage I post-ovulatory corpora lutea) synthesize s t e r o i d s . I suspect that the end pro-duct of s t e r o i d metabolism i s estrogens, l o c a l i z e d with-i n the post-ovulatory corpora l u t e a . These steroids probably induce some of the stage II corpus luteum c e l l s to d i f f e r e n t i a t e i n t o oogonia. This d i f f e r e n t i a t i o n of some c e l l s into oogonia give r i s e to the stage I I I or e-stage corpus luteum. THE FATE OF PRE-AND POST-OVULATORY FOLLICLES IN GOLDFISH OVARY r P Stage Atresia SECTION I I I : THE EFFECTS OF OVARIAN STEROIDS ON OOGENESIS AND OVARIAN DEVELOPMENT 71 INTRODUCTION There are very few s t u d i e s on the e f f e c t s o f s t e r o i d s on oogenesis and o v a r i a n development i n s p i t e of the extensive s t u d i e s of the e f f e c t s of s t e r o i d s on sex d i f f e r e n t i a t i o n (see review by Yamamoto, 1969), the secondary sexual characters and sexual behaviour (see review by L i l e y , 1969). There i s up to now no adequate study on the e f f e c t s of s t e r o i d s on oogonial m i t o s i s , though Bullough (1942) and Egami (1954) observed an abundance of oogonia i n f i s h t r e a t e d w i t h estrogen and speculated t h a t estrogen induces i n c r e a s e oogonia formation. Estrogen i n h i b i t e d y o l k d e p o s i t i o n i n oocytes of Phoxinus phoxinus (Bullough, 1942), Xiphophorus maculatus (Tavolga, 1949), P o e c i l i a r e t i c u - l a t a (Berkowitz, 1951), and Oryzias l a t i p e s (Egami, 1955). The i n h i b i -t i o n of v i t e l l o g e n e s i s r e s u l t e d i n f o l l i c u l a r a t r e s i a and ova r i a n regres-s i o n . Recently, Yamazaki (1972) demonstrated that t e s t o s t e r o n e a l s o suppressed ovarian development l e a d i n g to f o l l i c u l a r a t r e s i a . In sharp c o n t r a s t , Egami (1955), I s h i l and Yamamoto (1970), P l a c k et^ a l (1971) and Aida ejt a l . (1973) demonstrated that estrogens s t i m u l a t e d the l i v e r t o s y nthesize y o l k p r e c u r s o r s . K i r s h e n b l a t (1952) f i r s t demonstrated t h a t progesterone and deoxy-c o r t i c o s t e r o n e induced o v u l a t i o n i n the t e l e o s t , Misgurnus f o s s i l i s , whereas estrogens and androgens gave a negative response. The i n d u c t i o n of o v u l a t i o n , by c o r t i c o s t e r o i d s have been confirmed by Ramaswami (1962) , Sundararaj and Goswami (1966), Goswami and Sundararaj (1971) and Hi r o s e (1972). In t h i s s e c t i o n , I have examined the e f f e c t s of s t e r o i d hormones on oogonial m i t o s i s , v i t e l l o g e n e s i s , maintainence o f second growth phase oocytes and o v u l a t i o n of mature eggs. 73 METHODS AND MATERIALS THE EFFECTS OF STEROIDS ON OOGONIAL MULTIPLICATION The e f f e c t s of e s t r a d i o l , progesterone and deoxycorticosterone on m i t o s i s were s t u d i e d i n g o l d f i s h o v a r i e s . Female f i s h were i n j e c t e d 3 two days a f t e r they had spawned, w i t h 1 yc thymidine- H per gram body weight. The i n j e c t e d f i s h were then d i v i d e d i n t o four groups of f i v e f i s h each. F i s h i n each of the f i r s t . t h r e e groups were i n j e c t e d three times per week f o r four weeks w i t h 10 yg/g of a m i c r o c r y s t a l l i n e sus-pension of e i t h e r e s t r a d i o l , progesterone or deoxycorticosterone. The f o u r t h group, the c o n t r o l s , were i n j e c t e d at the same time i n t e r v a l s w i t h s a l i n e c o n t a i n i n g Tween 80. The f i s h were k i l l e d two days a f t e r the l a s t i n j e c t i o n and t h e i r o v a r i e s removed and f i x e d i n 10% n e u t r a l f o r m a l i n f o r autoradiography. Ovaries were r o u t i n e l y embedded i n p a r a f f i n , sectioned (at 7 y) , mounted on albumen coated s l i d e s , dewaxed and hydrated through a s e r i e s of a l c o h o l s . Hydrated s e c t i o n s were dip-coated w i t h n u c l e a r t r a c k emulsion (Kodak NTB 2) i n the dark (Kopriwa & LeBland, 1962); s l i d e s were a i r -d r i e d and kept i n a l i g h t proof box i n the r e f r i g e r a t o r (0-5°C) f o r a month. A f t e r one month the s l i d e s were processed w i t h photographic de-veloper (Kodak D19) and f i x e r (Kodak F 5 f i x i n g bath) s t a i n e d w i t h Mayer's haematoxylin and eos i n (Beserga & Malamud, 1969), dehydrated i n a l c o h o l , c l e a r e d i n xylene, and mounted i n "DPX" ( K i r k p a t r i c k & Lendrum, 1941). 74 The e s t i m a t i o n of percentage of oogonia and corpora l u t e a c e l l s 3 l a b e l l e d by thymidine- H was based on 50 random hig h power (x 450) f i e l d s of view from each ovary. The m i t o t i c frequency o f oocytes was based on the t o t a l number of oocytes which showed m i t o t i c f i g u r e s i n f i v e cross s e c t i o n s of each ovary. The m i t o t i c frequency i n f o l l i c u l a r c e l l s was based on t o t a l number of f o l l i c u l a r c e l l s showing m i t o t i c f i g u r e s i n the ten l a r g e s t oocytes of each ovary. The average number of f o l l i c u l a r c e l l s counted per ovary was 505 + 207. I n a d d i t i o n 790 + 248 corpora l u t e a c e l l s , 53 + 35 oogonia, and 469 + 245 oocytes were counted per ovary. The data were analysed s t a t i s t i c a l l y by a n a l y s i s of v a r i a n c e . The mean m i t o t i c frequencies of the v a r i o u s experimental groups of f i s h were compared by the " l e a s t s i g n i f i c a n t d i f f e r e n c e " &_ p r i o r i t e s t (Sokal & R o h l f , 1969). EFFECTS OF STEROIDS ON VITELLOGENESIS Female g o l d f i s h were hypophysectomized and kept f o r 5-6 weeks u n t i l a l l y o l k y oocytes were reabsorbed. At the end of t h i s p e r i o d , they were d i v i d e d i n t o groups of s i x f i s h and i n j e c t e d i n t r a p e r i t o n e a l l y w i t h one of the gonadal s t e r o i d s ; an a d d i t i o n a l group was i n j e c t e d w i t h a mixture of gonadal s t e r o i d s . S t e r o i d s were i n j e c t e d as m i c r o c r y s t a l l i n e suspensions i n s a l i n e (0.7% NaCl) c o n t a i n i n g 2% Tween 80. Each s t e r o i d was t r i t u r a t e d to a f i n e powder i n a glass homogenizer; Tween 80 was then added and followed by 0.7% NaCl to form the m i c r o c r y s t a l l i n e suspen-s i o n . 75 As i n the previous experiments, each g o l d f i s h was i n j e c t e d w i t h 10 yg/g of s t e r o i d i n 0.1 ml of s o l v e n t . The s t e r o i d s s t u d i e d were c h o l e s t e r o l , pregnenolone, 17a-hydroxypregnenolone, progesterone, 17a-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, e s t r a d i o l , estrone, e s t r i o l and t e s t o s t e r o n e . A c o n t r o l group was i n j e c t e d w i t h the solvent o n l y . The i n j e c t i o n schedule was once every 3 days f o r a p e r i o d of one month. At the end of the momth, f i s h were s a c r i f i c e d and t h e i r o v a r i e s examined h i s t o l o g i c a l l y and h i s t o c h e m i c a l l y . Completeness of hypophy-sectomy was ensured by h i s t o l o g i c a l examination of the heads. The heads were f i x e d i n Bouin's f i x a t i v e , d e c a l c i f i e d (Wiebe, 1968) and embedded i n p a r a f f i n . S e r i a l s e c t i o n s of the heads through the p i t u i t a r y r e g i o n were examined f o r p i t u i t a r y t i s s u e . EFFECTS OF LONG TERM STEROID INJECTIONS ON GRAVID GOLDFISH In t h i s group of experiments, the e f f e c t s of c h r o n i c i n j e c t i o n s of the f o l l o w i n g s t e r o i d s were compared: pregnenolone, progesterone, dehy-droepiandrosterone, androstenedione, e s t r a d i o l , estrone, e s t r i o l , t e s t o s -terone, d eoxycorticosterone, c o r t i s o n e , c o r t i c o s t e r o n e , and s a l i n e con-t r o l s . C a l c u l a t i o n of the percentage of y o l k y oocytes that were a t r e t i c was based on the assumption that a l l a t r e t i c oocytes were derived from y o l k y oocytes. The data were tre a t e d s t a t i s t i c a l l y by an a n a l y s i s of v a r i a n c e . The mean percentage of a t r e t i c oocytes induced by estrone, e s t r a d i o l , e s t r i o l , and t e s t o s t e r o n e were compared us i n g Tukey's W-76 procedure (Sokal & R o h l f , 1969). This i s an a p o s t e r i o r i t e s t as the d e c i s i o n to compare the percentage of a t r e s i a i n these four groups of experimental f i s h were made a f t e r the r e s u l t s were known. EFFECTS OF STEROIDS ON OVULATION IN GRAVID GOLDFISH In t h i s s e r i e s of experiments, each group of f i s h was i n j e c t e d w i t h one of the f o l l o w i n g s t e r o i d s : c h o l e s t e r o l , pregnenolone, 17a-hydroxypregnenolone, progesterone, 17a-hydroxyprogesterone, dehydroepiandro. sterone, e s t r a d i o l , estrone, e s t r i o l , t e s t o s t e r o n e , deoxycorticosterone, c o r t i s o n e , and C o r t i s o l . As i n the previous study, groups of 6-8 gravid g o l d f i s h were t r a n s f e r r e d to 50 l i t e r experimental tanks s u p p l i e d w i t h f l o w i n g d e c h l o r i n a t e d water (12+1°C) and a d a i l y photoperiod o f 16 hours. Each f i s h was i n j e c t e d , i n t r a p e r i t o n e a l l y , w i t h 100 yg/g of the appro-p r i a t e s t e r o i d suspended i n 0.1 ml of 0.7% NaCl c o n t a i n i n g 2% Tween 80. A c o n t r o l group was i n j e c t e d w i t h 0.1 ml of 0.7% NaCl c o n t a i n i n g 2% Tween 80. The i n j e c t i o n s were c a r r i e d out at noon. The t r e a t e d g o l d f i s h were examined f o r o v u l a t i o n at 9:00 a.m. the next day. Each f i s h was held i n the l e f t hand so that the abdomen was not grasped. Ovulated eggs were s t r i p p e d out of the oviduct by ge n t l y moving the f i r s t two f i n g e r s of the r i g h t hand over the abdomen i n the d i r e c t i o n of the c l o a c a . Excessive for c e was avoided as i t w i l l rupture the o v a r i e s and express unovulated oocytes. I f nothing or only f l u i d i s s u e d from the c l o a c a the f i s h were replaced and t e s t e d again a f t e r 24 hours. This procedure was c a r r i e d out d a i l y f o r 5 days a f t e r the i n i t i a l s t e r o i d i n j e c t i o n . Ovulated eggs that had been s t r i p p e d i n t h i s manner were then placed i n p l a s t i c t r a y s and a r t i f i c i a l l y f e r t i l i z e d . At the 77 end of the experiments the f i s h were k i l l e d by decapitation and the ovaries removed f o r h i s t o l o g i c a l examination. The gonadosomatic index of each f i s h was determined. 78 RESULTS THE EFFECTS OF STEROIDS ON MITOSIS Table 8 summarizes the r e s u l t s of s t e r o i d a d m i n i s t r a t i o n on m i t o s i s i n g o l d f i s h o v a r i e s . When e s t r a d i o l , progesterone, or deoxy-c o r t i c o s t e r o n e was i n j e c t e d i n t o r e c e n t l y ovulated f i s h together w i t h 3 thymidine- H ( l u c / g ) , a c t i v e l y d i v i d i n g c e l l s i n c o r p o r a t e d t h i s l a b e l i n the DNA. I t should be emphasized that the c e l l s l a b e l l e d by thymi-3 d i n e - H, i n f i s h o v a r i e s are d i v i d i n g by m i t o s i s ; the m e i o t i c d i v i s i o n does not occur u n t i l oocytes are f u l l y mature and ready to o v u l a t e . The c e l l s l a b e l l e d were f o l l i c u l a r c e l l s , corpus luteum c e l l s , oogonia and oocytes ( F i g . 36, 37, 38, 39). I t was not p o s s i b l e to d i f f e r e n t i a t e between t h e c a l and granulosa c e l l s of the f o l l i c u l a r l a y e r s i n autora-diography . About 10% of the f o l l i c u l a r c e l l s were l a b e l l e d i n a l l f i s h — w h e t h e r experimental or c o n t r o l . An a n a l y s i s of variance i n d i c a t e d t h a t estrogen, progesterone, and deoxycorticosterone had no s i g n i f i c a n t e f f e c t on the 3 percentage of f o l l i c u l a r c e l l s l a b e l l e d by thymidine- H. The s i t u a t i o n , however, was q u i t e d i f f e r e n t i n the corpora l u t e a , where s t e r o i d s induced s i g n i f i c a n t i n c r e a s e i n mitoses (p < 0.01). The highest l e v e l of mitoses i n corpora l u t e a (3.9 + 1.0%) was found a f t e r the twice weekly i n j e c t i o n s f o r four weeks. Comparison of mean m i t o t i c frequencies by the l e a s t s i g n i f i c a n t d i f f e r e n c e method i n d i c a t e d t h a t the m i t o t i c frequency o f estrogen t r e a t e d f i s h were s i g n i f i c a n t l y higher than those of progesterone or deoxycorticosterone t r e a t e d f i s h (p < 0.01) Table 8: The e f f e c t s of e s t r a d i o l , progesterone, and deoxycorticosterone on m i t o s i s i n g o l d f i s h o v a r i e s . Treatment No. gold- % f o l l i c u l a r % corpora l u t e a % oogonia % oocyte •(10 yg/g i n f i s h c e l l s l a b e l l e d c e l l s l a b e l l e d l a b e l l e d l a b e l l e d 0.1 ml. s a l i n e ) C o n t r o l ( S a l i n e ) 5 Estrogen 5 Progesterone 5 Deoxycorticosterone 5 9.0 +.1.3 10.5 + 1.7 9.6 + 2.0 9.7 + 1.0 1.3 + 0.3 3.9 + 1.0 2.2 + 0.4 2.5 + 0.7 15.5 + 6.8 26.1 + 6.1 11.0 + 4.4 7.9 + 3.0 0.6 + 0.2 0.4 + 0.3 0.7 + 0.1 0.7 + 0.3 Each f i s h was i n j e c t e d w i t h 1 uc/g thymidine- H two days a f t e r o v u l a t i o n . F i s h was then i n j e c t e d , i n t r a p e r i t o n e a l l y , w i t h 10 ug/g of the appropriate s t e r o i d twice a week, f o r four weeks. Autoradio-graphs of ovarian sections were examined to c a l c u l a t e the percentage of v a r i o u s ovarian c e l l types l a b e l l e d by thymidine^H. * S t a t i s t i c a l s i g n i f i c a n c e against c o n t r o l f i s h at 0.01 < p < 0.05. ** S t a t i s t i c a l s i g n i f i c a n c e at p < 0.01. 80 w i t h no d e t e c t a b l e d i f f e r e n c e between the l a t t e r two s t e r o i d s . Comparisons of m i t o t i c frequencies i n oogonia of t r e a t e d f i s h a l s o showed a s i g n i f i c a n t e f f e c t of estrogen. Estrogen t r e a t e d f i s h had 26.1 + 6.1% of l a b e l l e d as compared to 15.5 + 6.8% i n the c o n t r o l group of f i s h . Progesterone and deoxycorticosterone had no s i g n i f i c a n t e f f e c t on oogonial m i t o s i s w h i l e deoxycorticosterone may be s l i g h t l y i n h i b i t o r y . I n c o n t r a s t to the oogonia, oocytes showed no response to any of the s t e r o i d s t e s t e d . Only s m a l l percentage of oocytes were l a -3 b e l l e d by thymidine- H (0.6%) at any time. THE EFFECTS OF STEROID ON VITELLOGENESIS IN HYPOPHYSECTOMIZED FISH A l l the estrogens ( e s t r a d i o l , estrone, and e s t r i o l ) induced the formation of y o l k v e s i c l e s i n the ovaries of hypophysectomized g o l d f i s h (Table 9 ) . In some oocytes the y o l k v e s i c l e s appeared i d e n t i c a l to nor-mal v i t e l l o g e n e s i s ; the v e s i c l e s o f t e n developed randomly throughout the ooplasm ( F i g . 38). There was no i n d i c a t i o n of y o l k granule formation i n the o v a r i e s of estrogen t r e a t e d hypophysectomized g o l d f i s h . E s t r a d i o l was the most potent inducer of y o l k v e s i c l e formation. E s t r a d i o l was followed by estrone. The d i f f e r e n c e between the two es-trogens was evident i n a response of f i v e out of s i x f i s h w i t h e s t r a d i o l compared to three out of s i x f o r estrone and e s t r i o l . Moreover, a greater percentage of oocytes (6.9 +1.9%) i n any one ovary developed y o l k v e s i -c l e s w i t h e s t r a d i o l ( F i g . 41); f i s h t r e a t e d w i t h s i m i l a r dose of e s t r i o l had fewest oocytes w i t h y o l k v e s i c l e s (5.6 + 1.6%). Estrogen induced yolk v e s i c l e (v) formation i n oocytes of hypophysectomized g o l d f i s h . This section was stained with Mallory's trichrome. The ovary of hypophysectomized g o l d f i s h stained with Mallory's trichrome. The ovary contains only f i r s t growth phase oocytes. Pregnenolone induced yolk granule formation i n the oocytes of hypophysectomized g o l d f i s h . Only yolk granules (g) were formed a f t e r pregnenolone t r e a t -ment. The ovarian section was stained with Mallory's trichrome. Table 9: The e f f e c t s of v a r i o u s s t e r o i d s ( i . p . , 10 ug/g once every 3 days f o r a month) on v i t e l l o g e n e s i s i n hypophysectomized g o l d f i s h . S t e r o i d s Body wt. G.S. ,1. No. No. o v a r i e s w i t h No. o v a r i e s (g) f i s h y o l k v e s i c l e s w i t h y o l k granules S a l i n e ( C o n t r o l 20.6 + 7.8 1.9 + 0.8 6 0 0 C h o l e s t e r o l 24.2 + 6.9 2.0 + 0.4 6 0 0 Pregnenolone 21.0 + 7.3 1.6 + 0.6 5 0 4 Progesterone 28.1 + 4.4 1.9 + 0.5 6 0 0 ^ 17 a-hydroxypro ges t erone 27.4 + 6.3 1.6 + 0.8 6 0 0 Dehydroepiandrosterone 23.8 + 3.9 1.6 + 0.8 6 0 0 Androstenedione 21.7 + 4.2 2.2 + 0.4 5 0 0 E s t r a d i o l 23.6 + 4.3 1.5 + 0.6 6 5 0 Estrone 25.0 + 8.7 2.2 + 0.7 6 3 0 E s t r i o l 23.9 + 2.9 1.8 + 0.8 6 3 0 Mixture of above s t e r o i d s 23.5 + 3.2 2.1 + 0.6 6 0 0 oo 83 Pregnenolone induced the formation of yolk granules without the p r i o r formation of yolk v e s i c l e s and t h i s curious f i n d i n g i s i n sharp contrast to normal v i t e l l o g e n e s i s where the various yolk i n c l u s i o n s are formed i n sequence with the yolk v e s i c l e s always appearing before yolk granules. Yolk v e s i c l e s were never observed i n oocytes of hypo-physectomized g o l d f i s h treated with pregnenolone. The yolk granules induced by pregnenolone were found close to the outer membrane of oocytes (Fig. 40). Although they were poorly d i f f e r e n -t i a t e d i n heamatoxylin and eosin stained s l i d e s , they were well defined as dark red granules with Mallory's trichrome. Histochemical studies showed these pregnenolone induced yolk granules to have the same chemical properties as normal yolk granules (Table 10); i n short they contained proteins, phospholipids and t y p i c a l neutral l i p i d s . A further point of i n t e r e s t concerns the v i t e l l i n e membrane. Under normal conditions t h i s i s w e l l developed by the time the yolk granules appear. However, i n con-t r a s t with normal v i t e l l o g e n e s i s , the v i t e l l i n e membrane was not evident i n oocytes with yolk granules induced by pregnenolone. None of the other steroids tested appear to a f f e c t yolk formation. The ovaries remain t y p i c a l of those i n hypophysectomized f i s h ( F i g . 39); s i m i l a r l y the f i s h injected with a complete mixture of gonadal steroids showed no changes i n v i t e l l o g e n e s i s . Table 10. Comparison of the histochemical staining properties of pregnenolone induced yolk granules with those of normal v i t e l l o g e n i c ovaries Techniques Normal Yolk Granules Pregnenolone-induced Yolk Granules P e r i o d i c a c i d - S c h i f f ' s Dinitroflurobenzene method +++ +++ Tetrazotized o-dianisidine method +++ +++ Sudan Black B ++ ++ Acid haematin for phospholipids +++ +++ CO 85 Figure 41: Comparison of the e f f e c t s of e s t r a d i o l , estrone, and e s t r i o l on y o l k v e s i c l e formation i n hypophy-sectomized g o l d f i s h . The mean gonadosomatic index of the groups of f i s h from l e f t to r i g h t are: 1.5 +0.6, 2.2+0.7, and 1.8+0.8 A n a l y s i s of variance i n d i c a t e d that there were no s i g n i f i c a n t d i f f e r e n c e s between the d i f f e r e n t t r e a t -ments . 10 8 CO 0> co £ 6 to cu o O 4 cu o 4> u cu 0_ Estradiol Estrone Estriol 86 E f f e c t s of long term a d m i n i s t r a t i o n of s t e r o i d s i n t o g r a v i d f i s h . Table 11 summarized the r e s u l t s of the experiment to determine the long term e f f e c t s of s t e r o i d a d m i n i s t r a t i o n i n gravid g o l d f i s h , e s p e c i a l l y on the maintenance of second growth phase oocytes. Though estrogens induce the synth e s i s and m o b i l i z a t i o n of y o l k precursors ( B a i l e y , 1957; Ho & Vanstone, 1961; U r i s t & Sch j e i d e , 1961; Plack et a l . , 1971), i t has been reported that estrogens i n h i b i t v i t e l l o g e n e s i s and cause ov a r i a n r e g r e s s i o n . Estrone, e s t r a d i o l , e s t r i o l , and testoste r o n e administered a t 10 ug/g twice a week f o r four weeks, induced a t r e s i a i n o v a r i e s of about 80% of these f i s h . A n a l y s i s of varia n c e i n d i c a t e d that a l l three e s t r o -genic s t e r o i d s (estrone, e s t r a d i o l , and e s t r i o l ) were e q u a l l y potent but that testosterone was s i g n i f i c a n t l y l e s s a c t i v e than the estrogens (p < 0.01). About 54% of yo l k y oocytes were a t r e t i c i n testosterone t r e a t e d f i s h as compared to 80% w i t h the estrogens ( F i g . 46). The a t r e s i a ap-peared to be s i m i l a r to that observed i n hypophysectomized f i s h but de-t a i l e d h i s t o g e n e s i s was not s t u d i e d . Some yo l k y oocytes remained i n t a c t and appeared completely normal ( F i g . 42). By c o n t r a s t , no evidence of a t r e s i a was found a f t e r treatment w i t h pregnenolone, progesterone, dehydroepiandrosterone, androstenedione, de-ox y c o r t i c o s t e r o n e , c o r t i s o n e , and c o r t i c o s t e r o n e . However, some deoxy-c o r t i c o s t e r o n e , c o r t i s o n e , and c o r t i c o s t e r o n e t r e a t e d f i s h had l e s s than 5% of po s t - o v u l a t o r y corpora l u t e a i n t h e i r o v a r i e s (Table 11), but these corpora l u t e a were d i s t i n c t from the a t r e s i a induced by e s t r a d i o l , estrone, e s t r i o l or te s t o s t e r o n e . The po s t - o v u l a t o r y corpora l u t e a were made up 87 of i r r e g u l a r masses of c e l l s ( F i g . 43) whereas the a t r e t i c f o l l i c l e s , e s p e c i a l l y those at the early stages, contained p a r t i a l l y digested cytoplasmic i n c l u s i o n s ( F i g . 42). These findings i n d i c a t e that estrogens and testosterone induce a t r e s i a and ovarian regression probably by negative feedback on the p i t u i t a r y gonadotropin. This hypothesis w i l l be considered i n the discussion. 88 Figure 42: Se c t i o n of an ovary of a g o l d f i s h i n j e c t e d w i t h 10 ug/g of e s t r a d i o l , twice a week f o r four weeks. Note the extensive a t r e s i a (arrows) i n the ovary. Figure 43: Se c t i o n of an ovary i n j e c t e d w i t h 10 ug/g of de-ox y c o r t i c o s t e r o n e , twice a week f o r four weeks. A l l y o l k y oocytes were i n t a c t ; o c c a s i o n a l l y there were some pos t - o v u l a t o r y corpora l u t e a (arrow). Figure 44: Ovary of a g r a v i d g o l d f i s h that i s ready to ovulate. The ovary c o n s i s t s of w e l l developed oocytes i n the l a t e y o l k granule stage. Figure 45: Ovary of g o l d f i s h where the oocytes have reached only the l a t e y o l k v e s i c l e stage and hence were not ready f o r o v u l a t i o n . Table 11: E f f e c t s of s t e r o i d s administered to g o l d f i s h f o r one month. R e s u l t s were recorded as the presence or absence of a t r e s i a , more d e t a i l e d data are presented i n F i g . 46. Treatment Body Wt. .G.S.I. 1 No. f i s h No. f i s h w i t h (g) t e s t e d - a t r e t i c oocytes S a l i n e (Control) 42.9 + 7.1 13.9 + 6.4 5 0 Pregnenolone 41.3 + 6.9 17.3 + -5.4 5 0 Progesterone 42.6+6.5 14.0 + 4.3 5 0 Dehydroepiandrosterone 41.7 + 6.5 12.0 + 4.3 5 0 Androstenedione 39.0 + 6.0 14.3 +4.7 5 0 E s t r a d i o l 48.1 + 6.0 15.9 + 5.0 5 5 Estrone 41.1 + 7.4 13.5 + 3.3 5 5 E s t r i o l 45.2 + 4.1 12.5 + 6.3 5 5 Testosterone 41.5 + 5.2 10.4 + 2.4 5 5 Deoxycorticosterone 42.7 +4.5 17.9 + 4.6 5 3 2 Cortisone 44.1 + 5.8 12.8 +4.6 5 I 2 C o r t i c o s t e r o n e 40.1+7.5 13.3 + 7.1 5 2 2 "Gonadosomatic Index (G.S.I.) determined at the end of experiment 'These f i s h had stage I I or 6-stage corpora l u t e a . These were probably the r e s u l t of o v u l a t i o n . They were d i s t i n c t from estrogen induced a t r e s i a which consisted mainly of «- and g-stage corpora l u t e a . ,90 Figure 46: Comparison of the e f f e c t s of estrone, e s t r a d i o l , e s t r i o l and t e s t o s t e r o n e on a t r e s i a o f yolk y oocytes i n gr a v i d g o l d f i s h . The mean gonadosomatic index of the groups of f i s h from l e f t to r i g h t are: 13.5 + 3.3, 15.9 + 5.0, 12.5 + 6.3, and 10.4 + 2.4. A n a l y s i s of vari a n c e i n d i c a t e d that there were s i g n i f i c a n t d i f f e r e n c e s i n some of the treatments (p<0.01). A com-pa r i s o n of the mean percentage of a t r e t i c oocytes induced by various s t e r o i d s by Tukey's W-procedure i n d i c a t e d that testosterone induced s i g n i f i c a n t l y l e s s a t r e s i a than the estrogens (p < 0.01). There were no s i g n i f i c a n t d i f f e r e n c e s among the va r i o u s estrogen treatments. Estrone Estradiol Estriol Testosterone 91 THE EFFECTS OF STEROIDS ON OVULATION IN GOLDFISH The effectiveness of various s t e r o i d suspension i n inducing ovu-l a t i o n i n gravid g o l d f i s h , maintained at 12 + 1°C and long photoperiod, are presented i n Table 12. I t should be r e c a l l e d that g o l d f i s h do not ovulate under these experimental conditions. Progesterone, deoxycorticosterone, cortisone, and corticosterone were found to induce ovulation i n gravid f i s h . Cortisone was the most potent of these steroids with four out of the f i v e f i s h responding on the f i r s t day a f t e r i n j e c t i o n of 100 ug/g cortisone; w i t h i n one day, ovulated f i s h developed ovaries with post-ovulatory corpora l u t e a ( F i g . 43). The most advanced oocytes i n these f i s h were i n the early yolk granule stage. The ovulated eggs were f u l l y developed as evidence by the fact that when a r t i f i c i a l l y f e r t i l i z e d they developed and hatched into f r y w ithin f i v e days. Of the f i v e f i s h i n j e c t e d with 100 ug/g progesterone, one ovulated on the next day and another on the t h i r d day a f t e r the i n i t i a l s t e r o i d i n j e c t i o n . The other three did not ovulate. Two out of the three unovu-lat e d f i s h had ovaries with incomplete v i t e l l o g e n e s i s . The most advanced oocytes had j u s t reached the l a t e yolk v e s i c l e stage ( F i g l 45). Three of the deoxycorticosterone treated f i s h ovulated. One f i s h ovulated each of the f i r s t three days of the experiment. The extent of ovulation was comparable to that i n cortisone treated f i s h . These ovulated eggs were also s u c c e s s f u l l y f e r t i l i z e d and hatched. One of the two unovu-lat e d f i s h i n t h i s group had oocytes only up to the l a t e yolk v e s i c l e Table 12. The response of gra v i d g o l d f i s h to s t e r o i d s . Each f i s h was i n j e c t e d w i t h 100 ug/g of s t e r o i d suspension d a i l y . F i s h were examined f o r o v u l a t i o n before each i n j e c t i o n . The experiment was terminated a f t e r four days. Treatment No. f i s h Body Wt. (g) G.S.I. 1 No. f i s h ovulated No. f i s h w i t h immature ovar-i e s S a l i n e (Control) 5 39.6 +5.3 24.3 + 6.6 0 1 C h o l e s t e r o l 5 45.0 + 8.9 14.8 + 5.8 0 1 Pregnenolone 5 54.0 +7.2 20.0 + 4.3 0 0 Progesterone. 5 35.9 + 6.7 10.8 + 6.1 2 2 Dehydroepiandrosterone 5 48.1 +7.9 10.9 + 4.7 0 0 Androstenedione 5 43.5 +9.9 12.7 + 3.2 0 1 E s t r a d i o l 5 36.7 + 9.4 16.5 + 4.2 0 1 Estrone 5 57.1 +9.1 . 19.9 + 6.9 0 1 E s t r i o l 5 53.8 + 8.7 13.6 + 5.8 0 1 Testosterone 5 40.0 + 12.9 12.3 + 5.1 0 1 Deoxycorticosterone 5 48.8 + 7.4 12.4 + 4.3 3 1 Cortisone 5 40.4 + 7.6 7.6 + 4.2 47. 0 C o r t i c o s t e r o n e 5 32.0 + 6.9 12.9 + 5.9 2 1 "''G.S.I.: Gonadosomatic index, as determined at the end of the experiment. VO hp 93 stages; the other had oocytes i n the early yolk granule stage. The three f i s h that ovulated had post-ovulatory corpora l u t e a i n t h e i r ovaries ( F i g . 47). Only two of the f i s h i n j e c t e d with corticosterone ovulated. One ovulated on the f i r s t day a f t e r s t e r o i d i n j e c t i o n and the second on the following day. None of the other three showed any sign of ovulation during the experiment. One of the three unovulated f i s h had oocytes at the l a t e yolk v e s i c l e stage only; the other two had oocytes i n the early yolk granule stage with a few i n the l a t e yolk granule stage. Ovaries of the ovulated f i s h were t y p i c a l of ovulated f i s h and contained many post-ovulatory corpora l u t e a and oocytes i n a l l stages of oocyte develop-ment except the l a t e yolk granule stage. The maturity of oocytes i n unovulated f i s h ovaries as presented i n Table 12, ind i c a t e s that although one of the c h o l e s t e r o l treated f i s h had oocytes i n the l a t e yolk v e s i c l e stage, the other f i s h treated with c h o l e s t e r o l had many f u l l y developed oocytes ( F i g . 44) which were capable of being ovulated. S i m i l a r l y one of the androstenedion treated f i s h , one e s t r a d i o l treated f i s h , one e s t r i o l treated f i s h and one f i s h treated with testosterone had incompletely mature ovaries but the remainder of these experimental f i s h had ovaries with f u l l y developed oocytes at the l a t e yolk granule stage. In summary, progesterone, deoxycorticosterone, cortisone, and c o r t i -costerone induced i n vivo ovulation i n g o l d f i s h . This e f f e c t i f quite s p e c i f i c as c h o l e s t e r o l , pregnenolone, dehydroepiandrosterone, androstene-dione, e s t r a d i o l , estrone, e s t r i o l , and testosterone had no e f f e c t on ovulation. 94 I I I DISCUSSION This study demonstrates t h a t , i n a d d i t i o n to t h e i r w e l l known e f f e c t s on sex d i f f e r e n t i a t i o n (see review by Yamamoto, 1969), sexual behaviour, and secondary sexual characters (see review by L i l e y , 1969), o v a r i a n s t e r o i d s a l s o s t i m u l a t e oogonial m i t o s i s , v i t e l l o g e n e s i s , and o v u l a t i o n . U n l i k e male f i s h where testosterone s t i m u l a t e s complete spermatogenesis i n hypophysectomized f i s h ( L o f t s e t a l . , 1966; Sundararaj and Nayyar, 1967; Pandey, 1969), the o v a r i a n s t e r o i d s are s p e c i f i c i n t h e i r e f f e c t s . Estrogens which induced oogonial m i t o s i s had no e f f e c t on o v u l a t i o n and i n s t e a d induced a t r e s i a of y o l k y oocytes. Correspon-d i n g l y , progesterone and c o r t i c o s t e r o i d s which induced o v u l a t i o n had no e f f e c t on oogonial m i t o s i s . Estrogens, which s t i m u l a t e d the s y n t h e s i s of y o l k precursors by the l i v e r (Plack et al_ 1971) and the formation of y o l k v e s i c l e s , had no e f f e c t on y o l k granule formation. Instead another s t e r o i d hormone, pregnenolone, induced the formation of y o l k granules i n oocytes. These new f i n d i n g s on the endocrinology of oogenesis w i l l be discussed i n r e l a t i o n to the reproductive b i o l o g y of the female f i s h . E f f e c t of s t e r o i d s on oogonial m i t o s i s I t has been shown f o r the f i r s t time that estrogens stimulated oogonial m i t o s i s i n post-ovulatory g o l d f i s h . This e f f e c t appears to be q u i t e s p e c i f i c s i n c e progesterone and deoxycorticosterone had no e f f e c t . The extent of t h i s s p e c i f i c i t y i s not known as the present study was l i m i t e d to these three s t e r o i d s . In a d d i t i o n to the s t i m u l a t i o n of oogon-i a l m i t o s i s , estrogens induced m i t o s i s i n 6-stage corpora l u t e a c e l l s 95 which have been found to d i f f e r e n t i a t e i n t o oogonia. Our f i n d i n g s i n d i c a t e t h a t m i t o s i s w i t h i n the corpus luteum r e s u l t s i n a correspond-i n g i n c r e a s e i n oogonia. I n support of t h i s c o n c l u s i o n i t may be noted that Bullough (1942) and Egami (1954) observed an abundance of oogonia i n f i s h t r e a t e d w i t h estrogens w h i l e Cedard e_t a l . (1961) found a s i x -f o l d i n c r e a s e i n plasma estrogen at the time of spawning; t h i s i n c r e a s e i n plasma estrogen may be r e l a t e d to the marked i n c r e a s e i n the number of oogonia which many workers have noted i n post-spawning f i s h ( C r a i g -Bennet, 1930; Matthews, 1938; Bullough, 1942; B a r r , 1963 and Yamazaki, 1965). As a working hypothesis, i t i s proposed that corpora l u t e a synthe-s i z e estrogens which induce the genesis of oogonia from corpora l u t e a to replace oocytes l o s t during spawning. This may be the p h y s i o l o g i c a l f u n c t i o n of corpora l u t e a i n t e l e o s t where oogonial m i t o s i s goes on throughout the l i f e c y c l e . E f f e c t of s t e r o i d s on v i t e l l o g e n e s i s . The study on the e f f e c t s of s t e r o i d s on v i t e l l o g e n e s i s i n hypophysectomized g o l d f i s h i n d i c a t e d that only c e r t a i n aspects of v i t e l l o g e n e s i s were under the i n f l u e n c e of s t e r o i d s . The two types of y o l k i n c l u s i o n s were induced by q u i t e d i f -f e r e n t s t e r o i d s ; estrogens induced the formation of y o l k v e s i c l e s w h i l e pregnenolone induced the formation of the second type of y o l k i n c l u s i o n s — the y o l k granules. The s e q u e n t i a l formation of the two types of yolk i n c l u s i o n , as i n normal v i t e l l o g e n e s i s , have not been experimentally achieved. I t seems strange that the presence of a l l the ovarian s t e r o i d s , as i n the experiment using a mixture of o v a r i a n s t e r o i d s , had no e f f e c t 96 on y o l k formation. However, the s e q u e n t i a l formation of y o l k i n c l u s i o n s may depend on the s e q u e n t i a l synthesis of s t e r o i d s or some p r e c i s e com-b i n a t i o n of s p e c i f i c s t e r o i d s . Previous s t u d i e s demonstrated that estrogen a d m i n i s t r a t i o n stimulated the synthesis of a complex p r o t e i n i n the l i v e r (Egami, 1955; I s h i i & Yamamoto, 1970; P l a c k et a l . , 1971; and Aida et a l . , 1973). This p r o t e i n i s normally absent i n males and immature females but appears i n t h e i r blood f o l l o w i n g estrogen a d m i n i s t r a t i o n . This complex p r o t e i n i n the blood i s the presumptive y o l k p r o t e i n (Plack e_t a l . , 1971) . The uptake of y o l k precursors from the plasma by oocytes have not been s t u d i e d i n t e l e o s t s . In comparable s t u d i e s of v i t e l l o g e n e s i s i n amphibians, F o l l e t t e t a l . (1968), Wallace and Jared (1968), and Redshaw (1972) reported that no a c t i v e uptake of y o l k precursors by oocytes oc-curred i n the presence of high l e v e l s of estrogen. This response was very d i f f e r e n t from normal v i t e l l o g e n e s i s where r a p i d uptake of y o l k precur-sors was observed. Gonadotropin treatment, u n l i k e estrogen a d m i n i s t r a t i o n , s t i m u l a t e d both the synthesis of yolk precursors by the l i v e r and the subsequent i n c o r p o r a t i o n of these y o l k precursors i n t o oocytes. In c o n t r a s t to the synthesis of y o l k precursors i n the l i v e r of immature f i s h , the a d m i n i s t r a t i o n of estrogen to g r a v i d f i s h induces a t r e s i a i n second growth phase oocytes. Many workers have l i k e w i s e ob-served the i n h i b i t i o n of v i t e l l o g e n e s i s l e a d i n g to f o l l i c u l a r a t r e s i a i n va r i o u s f i s h : Phoxinus phoxinus (Bullough, 1942), Xiphophorus maculatus (Travolga, 1949), P e o c i l i a r e t i c u l a t a (Berkowitz, 1951), and Oryzias l a t i p e s (Egami, 1954, 1955). In a d d i t i o n , Yamazaki (1972) found that 97 test o s t e r o n e induced a t r e s i a i n second growth phase oocytes of Oncorhynchus gorbuscha. Our f i n d i n g s i n d i c a t e d that the responses to estrogens and testosterone were s p e c i f i c and not d u p l i c a t e d by pregneno-l o n e , progesterone, dehydroepiandrosterone, androstenedione, d e o x y c o r t i -costerone, c o r t i s o n e , and c o r t i c o s t e r o n e . During v i t e l l o g e n e s i s , estrogens appeared to have d i v e r s e e f f e c t s . Estrogens s t i m u l a t e d the synthesis of y o l k precursors by the l i v e r and i t s accumulation i n the plasma ( B a i l e y , 1957; Ho & Vanstone, U r i s t & Sc h j e i d e , 1961). We observed that estrogen s t i m u l a t e d the formation of y o l k v e s i c l e s i n oocytes. The formation of y o l k v e s i c l e s i s not r e l a t e d to the s y n t h e s i s . o f y o l k precursor i n the l i v e r . Yolk v e s i c l e s comprised of mucopolysaccharides and not l i p o p r o t e i n which i s the major c o n s t i t u e n t of the y o l k precursor synthesized by the l i v e r . I t i s the second type of y o l k i n c l u s i o n , the y o l k granules, that shares the same chemical com-p o s i t i o n as y o l k precursors described by Plack et_ a l . (1971). We found that the d e p o s i t i o n of y o l k granules i n oocytes i s not mediated by e s t r o -gens but by another o v a r i a n hormone, pregnenolone. Yamazaki and Donaldson (1968) have induced complete v i t e l l o g e n e s i s i n hypophysectomized g o l d f i s h w i t h salmon gonadotropin. This f i n d i n g was confirmed i n P o e c i l i a r e t i c u l a t a ( L i l e y and Donaldson, 1969) and Heteropneustes f o s s i l i s (Sundararaj et a l . , 1972b). The demonstration that both gonadotropin and ov a r i a n s t e r o i d s induced v i t e l l o g e n e s i s i n t e l e o s t s suggests that the gonadotropin i n d u c t i o n of v i t e l l o g e n e s i s i s mediated by i t s r e g u l a t i o n of s t e r o i d o g e n e s i s . 98 The I n d u c t i o n of Ovulation by S t e r o i d s . The present f i n d i n g s i n d i c a t e d that progesterone, deoxycorticosterone, c o r t i s o n e , and c o r t i -costerone were e f f e c t i v e i n inducing i n v i v o o v u l a t i o n of g r a v i d g o l d -f i s h . These observations were s i m i l a r to those obtained by K i r s h e n b l a t (1952, 1959) Ramaswami (1962), and Sundararaj and Goswami (1966). In a d d i t i o n to i n v i v o o v u l a t i o n , Goswami and Sundararaj (1971) demonstrated i n v i t r o o v u l a t i o n of o v a r i a n fragments by c o r t i c o s t e r o i d s . They ob-served that deoxycorticosterone was the most potent inducer.on i n v i t r o o v u l a t i o n . Recently Colombo e_t a l . (1973) demonstrated c o r t i c o s t e r o i d s y n t h e s i s i n t e l e o s t ( G i l l i c h t h y s m i r a b i l i s ) o v a r i e s and suggested that the endocrine c o n t r o l of o v u l a t i o n acts by p i t u i t a r y gonadotropin s t i m u l a t i o n of the s y n t h e s i s of c o r t i c o s t e r o i d s i n the ovary and not the i n t e r e n a l s as pos-t u l a t e d by Sundararaj and Goswami (1966). There i s no d e f i n i t i v e proof i n favour of any of these two p o s t u l a t i o n s . The Mechanism of Endocrine C o n t r o l of Oogenesis i n Teleost. The i n t e r a c t i o n between p i t u i t a r y gonadotropin and s t e r o i d o g e n e s i s , as sug-gested by our f i n d i n g s , present a p l a u s i b l e mechanism whereby one gonado-t r o p i n , as i s w i d e l y accepted i n t e l e o s t (Burzawa-Gerard and Fontaine, 1972; Donaldson et a l . , 1972) or at most two gonadotropins, c o n t r o l the d i v e r s e temporal changes i n o v a r i a n f u n c t i o n s during the reproductive c y c l e . The r e g u l a t i o n of s p e c i f i c s t e r o i d synthesis through the complex s t e r o i d metabolic pathway ( F i g . 48) may be brought about by the i n h i b i -t i o n or s t i m u l a t i o n of s p e c i f i c s t e r o i d o g e n i c enzymes. 99 Figure 48: The major s t e r o i d metabolic pathways i n te l e o s t ovaries. F i g u r e 4 8 i The m a j o r s t e r o i d m e t a b o l i c p a t h w a y s i n t e l e o s t o v a r i e s . ft Cholasftrol Pwgwnolone Progasterona Oaoxycorticosferona Cortiewterone Dehydrocorticostarone fro-Hy4-oxypre8nenolon<> fforHydroxyprogasti ll-Deoxycortijol Cortisol Oahydrcepiondrosterone Androttenadiona Tastostercna ll^ -Hydroxytestosterona H 0 Estrona Cortisona Esfrodiol-17^ ll-KetotesteMerone Ealriot 100 At the i n i t i a l stages of t e l e o s t reproductive cycle gonadotropin induces the synthesis of estrogens which f i r s t induce the d i f f e r e n t i a -t i o n of germ c e l l s into oocytes and l a t e r the formation of yolk v e s i -c l e s within oocytes. Some of the ovarian estrogens are released into the c i r c u l a t o r y system where they induce the l i v e r to synthesize yolk precursors. Estrogens w i l l be synthesized u n t i l they reach a threshold l e v e l which i s maintained by negative feedback i n t e r a c t i o n with p i t u i t a r y gonadotropin. I f as i n mammalian and b a c t e r i a l enzyme systems (Talalay and Marcus, 1955; Goldman, 1965), e s t r a d i o l i n h i b i t s 3g-hydroxysteroid dehydrogenase a c t i v i t y i n f i s h ovaries, then an accumulation of pregneno-lone, 17 <* -hydroxypregnenolone, and dehydroepiandrosterone w i l l r e s u l t . The increased synthesis of pregnenolone at t h i s stage w i l l induce the deposition of the yolk precursors i n oocytes as yolk granules. The l o -c a l i z e d i n h i b i t i o n of hydroxysteroid dehydrogenase by estrogens i s sup-ported by our observations of the lack of hydroxysteroid dehydrogenase a c t i v i t i e s i n the granulosa of yolk granule stage oocytes. As the i n h i b i t i o n of 3g-hydroxysteroid dehydrogenase by estrogens also prevents the synthesis of a d d i t i o n a l estrogens. The i n h i b i t i o n of 36-hydroxysteroid dehydrogenase, as w e l l as the negative feedback e f f e c t s of estrogens on p i t u i t a r y gonadotropin w i l l be removed when the e x i s t i n g estrogens are catabolized. The removal of negative feedback e f f e c t s w i l l cause a surge of gonadotropin synthesis and secretion, stimulating increased steroidogenesis throughout the whole metabolic pathway. The high l e v e l s of pregnenolone, which accumulated during estrogen i n h i b i t i o n of 33-hydroxysteroid dehydrogenase, w i l l r e s u l t i n increased synthesis ' » J . "no snfi tcrtico&iicioids _ , s 3 u l t i ^ ^ pcLfle 101 doe* Kerf &fitsr< 102 of progesterone and c o r t i c o s t e r o i d s r e s u l t i n g i n ovulation of mature oocytes. A l l the steroidogenic enzymes are apparently active during ovula-t i o n . The post-ovulatory f o l l i c l e s synthesize estrogens i n addition to c o r t i c o s t e r o i d s . The estrogens synthesized by post-ovulatory corpora l u t e a induce the genesis of a new batch of oogonia and at the same time i n a c t i v a t e the steroidogenic enzymes. This estrogen i n h i b i t i o n of 33-hydroxysteroid dehydrogenase repeats the reproductive c y c l e . 103 GENERAL DISCUSSION The ' p i t u i t a r y i s the primary endocrine gland r e g u l a t i n g reproduc-t i o n s i n c e hypophysectomy e l i m i n a t e s both v i t e l l o g e n e s i s and s t e r o i d o -genesis. However, t h i s study provides f u r t h e r evidence t h a t , i n a d d i -t i o n to p i t u i t a r y gonadotropin, t e l e o s t reproduction i s s t r o n g l y r e g u l a t e d by s t e r o i d hormones. V i t e l l o g e n e s i s i n g o l d f i s h i n v o l v e s the s e q u e n t i a l formation and d e p o s i t i o n of two types of y o l k i n c l u s i o n s . Yolk v e s i c l e s are f i r s t d eposited, followed subsequently by y o l k granules. The h i s t o c h e m i c a l study demonstrates that y o l k v e s i c l e s are comprised of mucopolysaccharides whereas y o l k granules are more complex and c o n t a i n p r o t e i n s , phospholipids and n e u t r a l l i p i d s . This i s the f i r s t chemical c h a r a c t e r i z a t i o n of two y o l k types i n g o l d f i s h . I t i s suggested that the y o l k v e s i c l e s may be i n v o l v e d i n the c o r t i c a l r e a c t i o n at f e r t i l i z a t i o n as described by Yamamoto (1961) while the y o l k granules serve as n u t r i e n t s f o r the develop-i n g embryo. A f t e r hypophysectomy a l l oocytes c o n t a i n i n g y o l k become a t r e t i c and are resorbed. The f o l l i c u l a r c e l l s may be i n v o l v e d i n the r e s o r p t i o n of dead oocytes as has been suggested so o f t e n but t h i s study provided no convincing evidence. A f t e r the cytoplasmic i n c l u s i o n s of a t r e t i c oocytes are completely removed, the f o l l i c u l a r c e l l s develop i n t o the pre - o v u l a t o r y corpus luteum. The pre-ovulatory corpus luteum synthesizes s t e r o i d hormones, probably estrogens s i n c e only 173-hydroxysteroid dehy-drogenase was detected i n the y-stage. 104 This study provides the f i r s t d e f i n i t e evidence of the rupture of theteleost ovarian f o l l i c u l a r membrane during ovulation p r i o r to t h e i r formation into corpus luteum. Post-ovulatory corpora lutea have very active 3a-, 33-, 17a-, and 173-hydroxysteroid dehydrogenases suggesting a c t i v e s t e r o i d hormone synthesis. A f t e r a stage of active hormone synthesis, the post-ovulatory corpus luteum forms an i r r e g u l a r mass of c e l l s i n d i s t i n g u i s h a b l e from the 6-stage pre-ovulatory corpus luteum. The s t e r o i d hormones synthesized by eit h e r the pre- or the post-ovulatory corpus luteum probably induce some of the corpora l u t e a l c e l l s to form the next batch of oogonia. This formation of oogonia from corpus luteum c e l l s i s strongly supported by autoradiographic evidence and has not previously been described. This study also demonstrated the presence of 3a, 33-, 17a-, and 173-hydroxysteroid dehydrogenases i n the granulosa c e l l s of a l l oocytes except those at the yolk granule stages. The presence of these steroidogenic enzymes suggests that the granulosa i s involved i n the synthesis of s t e r -oid hormones. Hypophysectomy i n a c t i v a t e s a l l these enzymes, they are reactivated by l u t e i n i z i n g hormone therapy demonstrating that steroido-genesis i n these tissues i s regulated by p i t u i t a r y gonadotropin. The s t e r o i d synthesized by the f o l l i c u l a r c e l l s of normal oocytes or the corpora l u t e a , both pre- and post-ovulatory, a f f e c t many aspects of oogenesis i n f i s h . Administration of exogenous steroids indicates that estrogens increase oogonia formation i n post-ovulatory g o l d f i s h . Estrogens also induce the formation of yolk v e s i c l e s i n hypophysectomized f i s h . Further, yolk granule deposition i n ovaries of hypophysectomized f i s h i s stimulated by pregnenolone. A d i f f e r e n t s e r i e s of steroids (pro-105 gesterone and the c o r t i c o s t e r o i d s ) induced o v u l a t i o n i n g r a v i d f i s h . The v a r i o u s processes i n oogenesis are st i m u l a t e d by d i f f e r e n t s t e r o i d s suggesting that a c e r t a i n degree of chemical s p e c i f i c i t y i s re q u i r e d f o r b i o l o g i c a l a c t i o n . This c h e m i c a l l s p e c i f i c i t y of s t e r o i d s provides a p l a u s i b l e e x p l a nation f o r the endocrine c o n t r o l of reproduc-t i v e c y c l e s . I t i s proposed that the s t e r o i d hormones synthesized by ov a r i a n t i s s u e s act l o c a l l y . This proposal provides an explanation of the asynchronous development of oocytes i n f i s h o v a r i e s . This demon-s t r a t i o n of gonadal s t e r o i d s a c t i n g d i r e c t l y on the o v a r i e s i s nov e l but not unique; i n comparable s t u d i e s , L o f t s et a l . (1966), Sundararaj and Nayyar (1967) , Donaldson and Yamazaki (1969) and Pandey (1969) demon-s t r a t e d t h a t a d m i n i s t r a t i o n o f androgens i n male hypophysectomized f i s h r e s t o r e d spermatogenesis. P o s s i b l e P r a c t i c a l A p p l i c a t i o n s . This study of the endocrine con-t r o l of oogenesis and v i t e l l o g e n e s i s suggests two p o s s i b l e p r a c t i c a l a p p l i c a t i o n s . f o r the endocrine manipulations of f i s h r e p roduction i n aquaculture using s t e r o i d hormones. The i n d u c t i o n of o v u l a t i o n by pro-gesterone and c o r t i c o s t e r o i d s o f f e r s a p o t e n t i a l l y easy and perhaps eco-nomical method to supplement the current p r a c t i c e of i n j e c t i n g i n d i v i d u a l f i s h w i t h gonadotropin or p i t u i t a r y e x t r a c t s (see reviews by P i c k f o r d and A t z , 1957; Clements, 1968; Shehadeh, 1970; Donaldson, 1973). Pro-gesterone or c o r t i c o s t e r o i d s can be a p p l i e d i n the water or the food enabling a greater number of f i s h to be tr e a t e d simultaneously. The economics of t h i s type of a p p l i c a t i o n should be examined. F u r t h e r , s t e r -o i d s are chemically more s t a b l e than gonadotropins and t h i s may be an important f a c t o r when c o n s i d e r i n g the i n t r o d u c t i o n of these techniques 106 into r u r a l aquaculture. Fish kept i n c a p t i v i t y , e i t h e r i n ponds or aquaria, frequently undergo ovarian degeneration. Prowse (1966) reported that 30-60% of t h e i r brood stock of grass carp, Ctenopharyngodon i d e l l a , had a t r e t i c oocytes and were of no use as spawners. The present i n v e s t i g a t i o n s suggests that pregnenolone, and perhaps the c o r t i c o s t e r o i d s , can be used to induce yolk deposition and ovarian maturation. Our studies on the long-term e f f e c t s of steroids on gravid f i s h demonstrated that pregnenolone, progesterone, and the c o r t i c o s t e r o i d s did not induce a t r e s i a whereas estrogens and testosterone cause extensive a t r e s i a of yolky oocytes. This adverse e f f e c t of long term administration of es-trogens may explain the ovarian degeneration observed by Funk e_t a l . (1973) a f t e r the treatment of j u v e n i l e salmon with salmon gonadotropin and estrogen for an extended period. This study opens up a new area i n teleost reproductive endocrino-logy which may have several p r a c t i c a l a p p l i c a t i o n s . However, research on the economics i s required and the possible species d i f f e r e n c e should be thoroughly investigated before t h i s endocrine manipulation of repro-duction can be applied commercially. The possible e f f e c t s of these hormones on. metabolism and growth should also be investigated. 107 REFERENCES Ahsan, S.N., and Hoar, W. S. 1963. Some e f f e c t s of gonadotropic hormones on the three-spine s t i c k l e b a c k , Gasterosteus a c u l e a t u s . Can. J . Zool. 41: 1045-1053. A i d a , K., H i r o s e , K., Yokote, M., and H i b i y a , T. 1973. 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