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) U n i v e r s i t y o f Malaya, 1968 M.Sc. U n i v e r s i t y o f Malaya, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Zoology We accept t h i s t h e s i s as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1974 In presenting this an a d v a n c e d degree the shall I Library further for scholarly by h i s of agree this thesis in p a r t i a l fulfilment of at University of Columbia, the make it that permission available for It financial is gain of The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada by the Columbia shall not requirements reference copying of I agree and copying or be a l l o w e d for that study. this thesis Head o f my D e p a r t m e n t understood that written permission. Department for for extensive p u r p o s e s may be g r a n t e d representatives. thesis freely British the or publication without my ABSTRACT I n t h i s t h e s i s I s t u d i e d t h e r o l e o f o v a r i a n s t e r o i d s i n oogenesis and o v a r i a n development o f g o l d f i s h ( C a r a s s i u s auratus L.) and examined the i n t e r r e l a t i o n s h i p o f p i t u i t a r y gonadotropin and o v a r i a n s t e r o i d s i n the e n d o c r i n e There a r e f o u r main control of teleost reproduction. p a r t s t o t h e 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 a n a l y s i s o f v i t e l l o g e n e sis, ( i i ) the demonstration t h e i r endocrine pus control, of s t e r o i d o g e n i c t i s s u e s i n the ovary and (iii) the development and f u n c t i o n s o f the c o r - luteum, and ( i v ) the r o l e o f o v a r i a n s t e r o i d s on oogenesis and v i t e l - logenesis. 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 examination two demonstrated t h a t types o f y o l k i n c l u s i o n s a r e formed d u r i n g v i t e l l o g e n e s i s : y o l k v e s i c l e s , comprised o f mucopolysaccharides, subsequently by y o l k g r a n u l e s neutral l i p i d s . and f i r s t formed, f o l l o w e d composed o f p r o t e i n s , p h o s p h o l i p i d s and N e i t h e r type o f y o l k develops i n the absence o f the p i t u i t a r y ; e s t r o g e n r e g u l a t e s the f o r m a t i o n o f y o l k v e s i c l e s w h i l e nenolone was found to c o n t r o l d e p o s i t i o n of yolk granules. cance o f these two k i n d s o f y o l k i s c o n s i d e r e d . preg- The s i g n i f i - T h i s i s the f i r s t demon- s t r a t i o n of two y o l k types i n g o l d f i s h w i t h a s e p a r a t e e n d o c r i n e c o n t r o l f o r t h e f o r m a t i o n o f each o f them. The major s t e r o i d s y n t h e s i z i n g t i s s u e s i n g o l d f i s h o v a r i e s are the g r a n u l o s a c e l l s o f oocytes and the corpus luteum. whether p r e - o r p o s t - o v u l a t o r y p r o b a b l y The corpus luteum, synthesizes estrogens. In t h i s study, no f u n c t i o n a l d i f f e r e n c e s were e s t a b l i s h e d between p r e - and p o s t - iii ovulatory corpora tritiated lutea. Treatment of p o s t - o v u l a t o r y thymidine s t r o n g l y suggests t h a t the c o r p o r a l u t e a l p r o l i f e r a t e to form another g e n e r a t i o n of oogonia. bably g o l d f i s h with a c t i v e hormones i n t h i s r e a c t i o n . f i n d i n g s are The Estrogens cells are pro- i m p l i c a t i o n s of these new discussed. A d m i n i s t r a t i o n of exogenous estrogen i n c r e a s e s oogonia formation i n p o s t - o v u l a t o r y g o l d f i s h whereas c h r o n i c a d m i n i s t r a t i o n o f or t e s t o s t e r o n e i n t o g r a v i d f i s h induces atresia. Treatment o f g r a v i d f i s h w i t h p r o g e s t e r o n e o r c o r t i c o s t e r o i d s induces ovulation. As a working h y p o t h e s i s s i z e d by extensive estrogens i t i s proposed t h a t s t e r o i d s are the o v a r i e s under gonadotropin stimulation. P i t u i t a r y gonado- t r o p i n r e g u l a t e s the whole o v a r i a n s t e r o i d s y n t h e t i c p r o c e s s s p e c i f i c r e a c t i o n i n the s t e r o i d m e t a b o l i c pathways. synthe- The and not any s y n t h e s i s of s p e c i f i c s t e r o i d s d u r i n g the r e p r o d u c t i v e c y c l e 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 s t e r o i d o g e n i c enzymes by s t e r o i d s , most likely estrogens. The p o t e n t i a l p r a c t i c a l a p p l i c a t i o n s of endocrine f i s h r e p r o d u c t i o n are discussed. manipulations in iv TABLE OF CONTENTS Page i i Abstract L i s t of Tables ' . L i s t of Figures ..... viii i x Acknowl ed gemen t s xii General I n t r o d u c t i o n 1 Section I : 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 Ovarian Development. 3 Introduction 4 M a t e r i a l s and Methods 6 ( i ) Maintenance o f 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) G r a v i d 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 H i s t o c h e m i s t r y of Ovaries Results 7 11 ( i ) H i s t o l o g y o f Oocyte Growth and Development (a) Oogonia 11 H (b) F i r s t Growth Phase Oocytes 11 (c) Second Growth Phase Oocytes 13 ( i i ) Histochemistry 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 P o l y s a c c h a r i d e s 16 (b) D i s t r i b u t i o n o f P r o t e i n s . . . . 20 (c) D i s t r i b u t i o n o f L i p i d s 21 V T a b l e o f Contents (cont'd) Page S e c t i o n I I : The Corpus Luteum and i t s F u n c t i o n . . . . 28 Introduction 29 M a t e r i a l s and Methods .. . ( i ) Hypophysectomy 33 33 ( i i ) H i s t o l o g y o f p r e - and P o s t - o v u l a t o r y Corpus Luteum 35 ( i i i ) Histochemical Detection of Hydroxysteroid Dehydrogenases 35 3 ( i v ) I n c o r p o r a t i o n o f Thymidine- H I n t o O v a r i e s 37 Results 39 ( i ) H i s t o g e n e s i s of a t r e t i c oocytes 39 (a) a - s t a g e 39 (b) g-stage 39 (c) y - s t a g e . 40 (d) 6-stage 40 (e) e-stage. ... ( i i ) . ) H i s t o g e n e s i s of p o s t - o v u l a t o r y Corpus Luteum 40 42 (a) Stage I . . . . . . . 42 (b) Stage I I 42 (c) Stage I I I 43 ( i i i ) H y d r o x y s t e r o i d dehydrogenases i n Normal O v a r i e s . . 43 (a) 3 g - h y d r o x y s t e r o i d dehydrogenase 46 (b) 3 a - h y d r o x y s t e r o i d dehydrogenase 46 (c) 1 7 a - h y d r o x y s t e r o i d (d) 1 7 3 - h y d r o x y s t e r o i d 47 47 dehydrogenase dehydrogenase.... vi T a b l e o f Contents (cont'd) Page !• ( i v ) H y d r o x y s t e r o i d Dehydrogenases i n Hypophysectomized Fish........ 50 (v) H y d r o x y s t e r o i d Dehydrogenases i n Hypophysectomized f i s h T r e a t e d w i t h LH. 53 ( v i ) H y d r o x y s t e r o i d Dehydrogenases i n P o s t ovulatory Ovaries 55 ( v i i ) The F a t e of P o s t - o v u l a t o r y 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 ) P o s t - o v u l a t o r y Corpora L u t e a . 63 ( i v ) L o c a l i z e d Hormone P r o d u c t i o n 64 (v) 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 Obgonia........ 65 ( v i ) Fate of Corpora Luteum C e l l s (via.) The R e p r o d u c t i v e Section I I I : 66 C y c l e W i t h i n the Ovary......... 67 The E f f e c t s of O v a r i a n 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 70 Introduction 71 Methods and M a t e r i a l s 73 ( i ) E f f e c t s of S t e r o i d s on O o g o n i a l M i t o s i s ( 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 - 73 74 ( i i i ) E f f e c t s of Long Term Treatment o f S t e r o i d s on Gravid F i s h : 75 vii T a b l e o f Contents (cont'd) Page ( i v ) E f f e c t s o f S t e r o i d s on O v u l a t i o n Results 76 78 ( i ) E f f e c t s o f S t e r o i d s on O o g o n i a l M i t o s i s ( 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 78 80 ( i i i ) E f f e c t s o f Long Term A d m i n i s t r a t i o n o f S t e r o i d s on G r a v i d F i s h ( i v ) E f f e c t s o f S t e r o i d s on O v u l a t i o n Discussion 86 91 94 ( i ) E f f e c t s of S t e r o i d s on O o g o n i a l M i t o s i s 94 ( i i ) E f f e c t o f 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 d u c t i o n o f O v u l a t i o n by S t e r o i d s 98 ( i v ) The Mechanism o f E n d o c r i n e C o n t r o l o f Oogenesis General Discussion (i) Possible Practical Applications References 98 103 105 107 viii LIST OF TABLES Page TABLE 1 2 3 4 5 6 7 8 9 L i s t o f 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 techniques used w i t h the a p p r o p r i a t e p r e p a r a t i v e techniques 8 A summary of the h i s t o c h e m i c a l p r o p e r t i e s o f y o l k inclusions 17 The degree of a t r e s i a i n v a r i o u s o v a r i e s a f t e r hypophysectomy .... 39a, H y d r o x y s t e r o i d dehydrogenases d e t e c t e d i n the o v a r i e s of normal v i t e l l o g e n i c o v a r i e s . 45 H y d r o x y s t e r o i d dehydrogenase d e t e c t e d i n the o v a r i e s of hypophysectomized g o l d f i s h 51 H y d r o x y s t e r o i d dehydrogenases i n the o v a r i e s of hypophysectomized g o l d f i s h i n j e c t e d w i t h LH 54 Hydroxysteroid post-ovulatory 56 dehydrogenases i n the o v a r i e s o f fish....' Summary of the 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 on oogonial mitosis 79 The e f f e c t s o f 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 82 10 Comparison of the h i s t o c h e m i c a l p r o p e r t i e s o f p r e g nenolone induced y o l k granules w i t h those of normal vitellogenic ovaries....... . 84 11 The long-term e f f e c t s o f s t e r o i d a d m i n i s t r a t i o n the o v a r i e s of g r a v i d g o l d f i s h 12 The on e f f e c t s o f s t e r o i d s on o v u l a t i o n i n g r a v i d f i s h . . 89 92 ix LIST OF FIGURES FIGURE Page 1 An oogonia o c c u r i n g s i n g l y i n the ovary.... 12 2 Oogonial cyst c o n t a i n i n g s e v e r a l oogonia....... 12 3 Chromatin 12 4 P e r i n u c l e o l a r stage o o c y t e s . . . . 5 I n i t i a l y o l k v e s i c l e stage o o c y t e . . . . . . . . . . 6 L a t e y o l k v e s i c l e stage o o c y t e 7 E a r l y y o l k g r a n u l e stage oocyte 8 L a t e y o l k g r a n u l e stage o o c y t e 9 Ovarian s e c t i o n stained with p e r i o d i c reaction.... n u c l e o l a r stage o o c y t e s ......... 12 14 14 ... 14 14 acid-Schiff's 19 10 Oocytes 11 O v a r i a n s e c t i o n s t a i n e d w i t h the t e t r a z o t i z e d dianisidine reaction for proteins 12 .. stained with d i n i t r o f l u r o b e n z e n e r e a c t i o n . . . . O v a r i a n s e c t i o n s t a i n e d w i t h the f e r r i c method f o r s u l p h y d r y l groups 19 o19 ferricyanide 19 13 C r y o s t a t s e c t i o n of ovary s t a i n e d w i t h Sudan B l a c k B. 19 14 O v a r i a n s e c t i o n s s t a i n e d w i t h a c i d haematin f o r phospholipids 19 A diagrammatic f i s h ovary 27 15 16 r e p r e s e n t a t i o n o f oogenesis i n g o l d •.- Diagrammatic p r e s e n t a t i o n o f the h y d r o x y s t e r o i d dehydrogenase r e a c t i o n . . . 17 a-stage 18 (3-stage o f f o l l i c u l a r 19 Y~ 20 Higher m a g n i f i c a t i o n of the y-stage a t r e t i c f o l l i c l e . s t a g e of f o l l i c u l a r atretic 2>&o~ atresia 41 atresia........... 41 follicle 41 41 X L i s t of F i g u r e s (cont'd) Page FIGURE 21 6-stage a t r e t i c f o l l i c l e 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 Stage I p o s t - o v u l a t o r y corpus l u t e u m , w i t h e v i d e n c e of the r u p t u r e o f f o l l i c u l a r w a l l 44 Stage I p o s t - o v u l a t o r y corpus luteum w i t h o u t any s i g n of f o l l i c u l a r w a l l rupture 44 A h i g h m a g n i f i c a t i o n o f Stage I p o s t - o v u l a t o r y corpus luteum d e m o n s t r a t i n g h y p e r t r o p h y o f granulosa 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 o f 3 3 - h y d r o x y s t e r o i d dehydrogenase i n the granulosa c e l l s of oocytes 48 Lack of h y d r o x y s t e r o i d dehydrogenase l a t e y o l k granule stage oocytes 48 23 24 25 30 31 32 33 34 35 36 . ... 41 activity in D e m o n s t r a t i o n o f 1 7 ( 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n y s t a g e a t r e t i c o o c y t e s o f hypophysectomized g o l d f i s h 48 3 f ? - h y d r o x y s t e r o i d dehydrogenase i n the p o s t ovulatory f o l l i c l e s after ovulation 48 A u t o r a d i o g r a p h y 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 f o l l i c u l a r c e l l s by thymidine-^H 59 A u t o r a d i o g r a p h y o f 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 o f 6-stage corpus l u t e a c e l l s 59 A u t o r a d i o g r a p h y showing the l a b e l l i n g o f o o g o n i a 30 days a f t e r t h y m i d i n e - ^ H i n j e c t i o n 59 A u t o r a d i o g r a p h o f l a b e l l e d o o c y t e 30 days a f t e r the i n i t i a l t h y m i d i n e - 3 i n j e c t i o n 59 H XX L i s t of Figures (cont'd) 37 A summary o f corpus luteum f o r m a t i o n i n g o l d f i s h . . . . . . 38 Y o l k v e s i c l e s oocytes of hypophysectomized treated w i t h estrogen Page 69 fish 81 39 Ovary o f hypophysectomized g o l d f i s h 81 40 Pregnenolone induced y o l k g r a n u l e f o r m a t i o n i n the o o c y t e o f hypophysectomized g o l d f i s h 81 41 H i s t o g r a m of the e f f e c t s o f e s t r a d i o l , e s t r o n e , and e s t r i o l on y o l k v e s i c l e f o r m a t i o n i n hypophysectomized f i s h . . . . . . . . 85 42 H i s t o l o g y o f g r a v i d ovary t r e a t e d w i t h e s t r o g e n 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 containing post-ovulatory corpora l u t e a . . . . . . 88 44 O v a r i e s o f f i s h t h a t are ready t o o v u l a t e 88 45 O v a r i e s o f f i s h w i t h immature o o c y t e s . . . . . . . . 88 46 Comparison of the e f f e c t s of e s t r o n e , 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 y o l k y o o c y t e s i n gravid 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 g o n a d o t r o p i n , s t e r o i d o g e n e s i s and o o g e n e s i s . 48 The s t e r o i d m e t a b o l i c pathway i n t e l e o s t o v a r i e s 99 xii ACKNOWLEDGEMENTS I t i s my great pleasure to acknowledge my indebtedness and g r a t i t u d e to my research s u p e r v i s o r , Dr. W.S.Hoar, f o r h i s i n t e r e s t , advise and encouragements throughout the course of t h i s study. I would l i k e to thank the members of my d o c t o r a l committee: Dr.E.M.Donaldson, Dr.H.D.Fisher, Dr.P.Ford, Dr.N.R.Li ley and Dr .A.M.Perks f o r t h e i r generous assistance and advise during the study and f o r reading the t h e s i s . I extend my g r a t e f u l a p p r e c i a t i o n to Miss.D.Hards, f o r the preparat i o n of e x c e l l e n t h i s t o l o g i c a l s e c t i o n s , Mrs. Cathy D r i e d z i c f o r her caref u l preparation of the i l l u s t r a t i o n s , and Mrs, Pat Waldron, f o r typing the f i n a l copy of the t h e s i s . I iam obligated to my fellow graduate students, Kenneth Chan, Ross Neuman, Jean-Guy Godin, B i l l Marshall and e s p e c i a l l y Norman Stacey for many s t i m u l a t i n g discussions. My thanks to those that plunged i n t o the i c y winter waters of Vancouver i n search of the e l u s i v e Coryphopterus n i c h o l s i • To everyone who have made my stay here i n Vancouver meaningful and enjoyable, I say thank you. F i n a n c i a l support f o r t h i s research was received from the National Research Council of Canada through g r a n t s - i n - a i d to Dr.W.S.Hoar and a Canadian Commonwealth Scholarship to myself. I a l s o wish to thank the U n i v e r s i t i Sains Malaysia f o r granting me a Fellowship i n t h e i r Academic S t a f f T r a i n i n g Scheme. GENERAL INTRODUCTION I n t e l e o s t , the r e g u l a t i o n o f gametogenetic a c t i v i t y by p i t u i t a r y i s now 1957; Dodd, 1960; w e l l e s t a b l i s h e d (see r e v i e w s by P i c k f o r d and Hoar, 1965; Donaldson, 1973). 1969; L o f t s , 1968; Reinboth, (Yamazaki and Donaldson, 1968; 1972; A d m i n i s t r a t i o n o f mammalian L i l e y and Donaldson, 1968; 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 i n t e l e o s t o v a r i e s (Yamazaki and Donaldson, 1969; a d m i n i s t r a t i o n of gonadotropin a c t i v i t y i n hypophysectomized f i s h . whether g o n a d o t r o p i n gonadotropin In addition steroidogenesis Wiebe, 1969) increase 3g-hydroxysteroid However, i t i s s t i l l and Sundararaj a, b) m i t i g a t e s the e f f e c t s o f hypophysectomy. whether i t o p e r a t e s Atz, Hypophysectomy r e s u l t s i n r e g r e s s i o n o f o v a r i e s a t r e s i a of y o l k - l a d e n o o c y t e s . e t a l . , 1972 the while dehydrogenase uncertain a c t s d i r e c t l y on o o g e n e s i s and v i t e l l o g e n e s i s o r t h r o u g h the p r o d u c t i o n o f a second h o r m o n e — p o s s i b l y a s t e r o i d o r whether b o t h g o n a d o t r o p i n s and s t e r o i d s a r e d i r e c t l y i n volved. There i s o n l y s c a n t y i n f o r m a t i o n c o n c e r n i n g f o r m a t i o n and development. o o g o n i a i n f i s h (Phoxinus Bullough endocrinology of ovum (1942) observed a g r e a t e r number o f phoxinus) t r 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 . A g a i n s t u d i e s o f sex r e v e r s a l u s i n g s t e r o i d s suggest an e f f e c t o f 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 t h e 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) r e v i e w s 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 f o r m a t i o n have p r o v i d e d rather c o n t r a d i c t o r y evidence c o n c e r n i n g 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 . though e s t r o g e n s s t i m u l a t e the l i v e r t o s y n t h e s i z e y o l k (Egami, 1955; 1961; B a i l e y , 1957; P l a c k et^ a l . , 1971; Ho and Vanstone, 1961; precursors U r i s t and A i d a e t a l . , 1973), B u l l o u g h The Schjeide, (1942), Tavolga (1949) , B e r k o w i t z (1951) , Egami (1955) observed t h a t e s t r o g e n 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 Al- administra- caused f o l l i c u l a r a t r e s i a . r o l e of s t e r o i d s i n i n d u c i n g o v u l a t i o n i n t e l e o s t i s w e l l cumented s i n c e K i r s h e n b l a t t e r o n e and (1952, 1959) deoxycorticosterone whereas e s t r o g e n s and do- f i r s t demonstrated t h a t p r o g e s - i n d u c e d o v u l a t i o n i n Misgurnus f o s s i l i s , androgens had no e f f e c t . tained w i t h Heterdpneustes f o s s i l i s S i m i l a r r e s u l t s were (Ramaswami, 1962). Further, ob- Sundararaj and Goswami (1966) observed t h a t c o r t i c o s t e r o i d s i n d u c e d o v u l a t i o n and in some i n s t a n c e s o v i p o s i t i o n i n b o t h 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 Hirose S u n d a r a r a j (1971 a, b,) and (1972) demonstrated the i n d u c t i o n o f 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 . T h i s 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 c o n c e r n i n g e n d o c r i n e c o n t r o l o f ovum f o r m a t i o n and development. This i n v e s t i g a t i o n i s a study of the e n d o c r i n e r e g u l a t i o n o f o o g e n e s i s 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 r e f e r e n c e of s t e r o i d hormones. I t was and t o the r o l e c o n v e n i e n t to d i v i d e the p r e s e n t a t i o n t h r e e 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 s t e r o i d o g e n e s i s e s p e c i a l l y the corpus l u t e u m , and on o o g e n e s i s and v i t e l l o g e n e s i s . into i n the o v a r y , f i n a l l y the e f f e c t s o f o v a r i a n steroids 3 SECTION I : THE HISTOLOGY AND HISTOCHEMISTRY OF OVARIAN DEVELOPMENT 4 INTRODUCTION The p r e s e n t i n v e s t i g a t i o n i s an a n a l y s i s o f c y t o l o g i c a l changes d u r i n g the m a t u r a t i o n o f o o c y t e s o f g o l d f i s h , C a r a s s i u s a u r a t u s . Such an a n a l y s i s s h o u l d p r o v i d e a b a s i s f o r study of the e n d o c r i n e c o n t r o l of o o g e n e s i s and v i t e l l o g e n e s i s i n t e l e o s t o v a r i e s . Although early stages o f o o c y t e development have been w e l l documented, phase has n o t p r e v i o u s l y been d e t a i l e d . t-h& second growth I n t h i s study a t t e n t i o n i s d i r e c t e d to the second growth p h a s e — e s p e c i a l l y the f o r m a t i o n and c h e m i c a l c o m p o s i t i o n of y o l k inclusions. There a r e numerous s t u d i e s o f o v a r i a n h i s t o l o g y d u r i n g o o c y t e growth and development. B a r r (1963) , L e h r i (1968) and Lambert v i d e d good d e s c r i p t i o n s o f t e l e o s t o o c y t e growth. an e x c e l l e n t d e s c r i p t i o n o f t h e h i s t o l o g i c a l (1970) have p r o - Yamazaki (1965) gave changes i n the o v a r i e s o f 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 p r o v i d e d by Stomsten (1931) and Beach (1959). It i s g e n e r a l l y r e c o g n i z e d t h a t oocyte development o c c u r s i n two phases. trol, (see The f i r s t growth phase, which i s independent o f p i t u i t a r y con- i n v o l v e s the i n c r e a s e i n s i z e o f oocytes w i t h some n u c l e a r changes r e v i e w by Hoar, 1969). The second phase i s c h a r a c t e r i z e d by t h e d e p o s i t i o n o f y o l k ; t h e r e i s much c o n f u s i o n c o n c e r n i n g t h i s phase especially f o r m a t i o n o f the y o l k i n c l u s i o n s Malone and H i s a o k a , 1963). (Anderson, 1968; Guraya, 1965; One o f t h e reasons f o r the c o n f l i c t i n g opin- i o n s may be a t t r i b u t e d t o a f a i l u r e to d i s t i n g u i s h t h e v a r i o u s y o l k components. 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 o f v i t e l l o g e n e s i s were devoted t o the d i s t r i b u t i o n o f p o l y s a c c h a r i d e s 1954; (Ihnumaxa and Tsukuda, 1952; A k e t a , and Yamamoto, 1955; 1956; 1958) b u t few attempts were made t o c o r r e l a t e the histochemical d i s t r i b u t i o n o f polysaccharides w i t h chemical components. L a t e r , t h e emphasis s h i f t e d t o t h e l i p i d other composi- t i o n o f o o c y t e s and t h e o r i g i n o f y o l k (Chopra, 1958; Yamamoto, 1958; N a t h , 1960). These l a t e r w o r k e r s have been concerned about t h e 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 t o t h e " y o l k n u c l e u s " now r e c o g 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 o f m i t o c h o n d r i a . y o l k n u c l e u s s t i l l remains (Guraya, 1963; 1968). been made t o sCtidy t h e h i s t o c h e m i c a l c o m p o s i t i o n teleost ovaries. Some i n t e r e s t i n R e c e n t l y a t t e m p t s have of yolk inclusions i n Malone and H i s a o k a (1963), Guraya (1965) and Anderson (1968) s t u d i e d t h e 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 o f p o l y s a c c h a r i d e 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 . chemical defined. composition lipids, I n s p i t e o f t h e s e s t u d i e s , however, t h e of the v a r 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 poorly 6 METHODS MAINTENANCE OF GOLDFISH . G o l d f i s h ( C a r a s s i u s a u r a t u s L.) o f the common comet v a r i e t y (1015 cm s t a n d a r d l e n g t h ) were o b t a i n e d from a commercial s u p p l i e r (Hartz Mountain Pet S u p p l i e r s L i m i t e d , 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 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 f r e s h water which was a r e a t e d w i t h compressed air. G o l d f i s h were f e d once a day w i t h " C l a r k ' s New Age T r o u t Feed" (Moore-Clark Co., S a l t Lake C i t y , U t a h ) . T h i s d i e t was supplemented, once a week, w i t h f r o z e n b r i n e "Natural" goldfish. shrimp. The b a s i c s t o c k o f f i s h was kept under natural p h o t o p e r i o d and temperature, except i n w i n t e r when the temperature was r a i s e d to about 10°C w i t h immersion h e a t e r s . Gravid g o l d f i s h . Another group o f g o l d f i s h was kept a t 10-13°C with a d a i l y p h o t o p e r i o d o f 16 h o u r s . F l u o r e s c e n t 40 watt lamps, trolled by time c l o c k s , s u p p l i e d the l i g h t . A f t e r about 3 months o f such treatment most o f t h e females become g r a v i d . not Refractory goldfish. to They remain g r a v i d and w i l l o v u l a t e i f kept below 13°C (Yamamoto et a l . , 1966). used i n experiments r e q u i r i n g g r a v i d These f i s h were goldfish. A group of g r a v i d g o l d f i s h was a q u a r i a w i t h d e c h l o r i n a t e d water a t 13°C. transferred A f t e r t r a n s f e r , the water temperature was s l o w l y r a i s e d to room temperature induced o v u l a t i o n . con- (22-25°C) and t h i s The f i s h were allowed t o o v u l a t e a t random. They were kept a t room temperature and 16 hours d a i l y p h o t o p e r i o d f o r 3-4 months when they became r e f r a c t o r y and had o v a r i e s s i m i l a r t o those o f 7 hypophysectomized g o l d f i s h . r e f r a c t o r y i f maintained Under t h e s e c o n d i t i o n s , g o l d f i s h r e m a i n at room temperature and cease t o be r e f r a c t o r y o n l y when p l a c e d 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 o f g o l d f i s h o v a r i e s were s t u d i e d a t v a r i o u s s t a g e s of m a t u r i t y . F i s h from the " n a t u r a l " s t o c k were sam- p l e d throughout the y e a r t o ensure t h a t a l l stages o f o o g e n e s i s were i n cluded. The h i s t o l o g y of f r e s h l y o v u l a t e d eggs were a l s o examined. The o v a r i e s 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 o f h y d r o x y s t e r o i d dehydrogenases. o f 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 . techniques The The ovaries various used a r e summarized i n T a b l e 1. I n a l l c a s e s , the f i s h were d e c a p i t a t e d and t h e i r o v a r i e s p l a c e d 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 B o u i n ' 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 d i c h r o m a t e and Carnoy's f l u i d ( G u r r , 1962). 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 A p o r t i o n o f the f r o z e n immediately w i t h an ace- tone-dry i c e m i x t u r e o r " C r y o w i c k " ( I n t e r n a t i o n a l Equipment Co., setts) . These u n f i x e d but f r o z e n o v a r i e s were s e c t i o n e d a t 10 y w i t h c r y o s t a t and mounted on a l b u m i n i z e d Massachuthe slides for histochemical staining. O v a r i e s w i t h o n l y e a r l y stage oocytes were embedded r o u t i n e l y i n paraffin. Y o l k l a d e n t i s s u e s are v e r y b r i t t l e and d i f f i c u l t t o 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 t e c h n i q u e w h i c h uses Smith's d i c h r o m a t e f i x a t i v e f o l l o w e d by double embedding of the o v a r i e s was veloped t o overcome t h i s problem. D e t a i l s a r e as f o l l o w s ; de- T a b l e 1: L i s t o f t h e v a r i o u s 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 t e c h n i q u e s p r e p a r a t i v e methods used. TECHNIQUES w i t h the a p p r o p r i a t e FIXATIVES SECTIONS H a e m a t o x y l i n and e o s i n ( G u r r , 1962) B, S, NF Paraffin M a l l o r y ' s trichrome B, S, NF Paraffin HISTOLOGY ( G u r r , 1962) POLYSACCHARIDES Best's carmine ( P e a r s e , 1968) Paraffin P e r i o d i c a c i d - S c h i f f ' s (McManus, 1948) B, S, NF Paraffin D i a l y s e d i r o n f o r a c i d m u c o p o l y s a c c h a r i d e s ( H a l e , 1946) B, S, NF Paraffin A l c i a n B l u e , pH 2.5 ( P e a r s e , 1968) B, S, NF Paraffin D i n i t r o f l u r o b e n z e n e method ( B u r s t o n e , 1955) S, NE, F P a r a f f i n , Frozen 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) S, NE, F P a r a f f i n , Frozen S a k a g u c h i ' s r e a c t i o n (Baker, 1953) S, NF, F P a r a f f i n , Frozen M i l l o n ' s r e a c t i o n (Baker, 1956) S, NF, F P a r a f f i n , Frozen B, S, NF Paraffin PROTEINS 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 ( B a r n e t t & Seligman, 1952) Continued 00 TABLE 1 (Cont'd) TECHNIQUES FIXATIVES SECTIONS Sudan B l a c k B (Chayen et a l . , 1969) NF, F Frozen O i l Red NF, F Frozen LIPIDS 0 (Lillie, 1944) A c i d haematin ( B a k e r , Copper p h t h a l o c y a n i n Footnotes: - Key 1946) B, S, NF, method ( K l u v e r & B a r r e r a , 1953) NF, F P a r a f f i n , Frozen F FFrozen to a b b r e v i a t i o n o f f i x a t i v e s , B, B o u i n ' s f l u i d ; S, Smith's d i c h r o m a t e 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 ovaries. 2 P a r a f f i n s e c t i o n s (7 y) were s e c t i o n e d on r o t a r y microtome. were cut on the c r y o s t a t at -15°C Frozen sections (10-12 u) . VO 10 The o v 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 h r and t h e n washed i n r u n n i n g w a t e r f o r 6-12 h r . The washed o v a r i e s were d e h y d r a t e d t h r o u g h a s e r i e s o f a l c o h o l s up t o 95% a l c o h o l . O v a r i e s were n o t com- p l e t e l y d e h y d r a t e d s i n c e 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 l a d e n eggs (Rugh, 1968). m e t h y l benzonate f o r up t o 5 h r . The t i s s u e s were c l e a r e d i n A f t e r c l e a r i n g t h e y 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 m e t h y l benzoate and t h e n p l a c e d over- n i g h t i n c h l o r o f o r m b e f o r e 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 p i e c e of some o f neutral formalin. t h e o v a r i e s was f i x e d o v e r n i g h t i n Baker's These t i s s u e s were washed i n r u n n i n g w a t e r f o r 12-24 h r , f r o z e n i n e i t h e r acetone-dry i c e mixture o r w i t h Cryowick and mounted on specimen h o l d e r s w i t h "O.C.T. compound" (Ames Co., I n d i a n a ) — s o l u b l e , embedding medium f o r f r o z e n t i s s u e s . a water S e c t i o n s o f 10-12 y t h i c k - ness were c u t on t h e 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 o f p o l y s a c c h a r i d e s t a i n s was c o n f i r m e d by s t a i n i n g s e r i a l s e c t i o n s w h i c h 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 e t a l . , 1969). The p e r i o d i c a c i d - S c h i f f ' s p o l y s a c c h a r i d e confirmed r e a c t i o n was f u r t h e r 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 b e f o r e 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 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 - e t h y l - m a l e i m i d e ( P e a r s e , The o c c u r r e n c e of s i m p l e l i p i d s was confirmed by 1968). by p y r i d i n e e x t r a c t i o n o f s e r i a l s e c t i o n s ( B a k e r , 1946) b e f o r e s t a i n i n g i n e i t h e r Sudan B l a c k B o r O i l Red 0. 11 RESULTS HISTOLOGY OF OOCYTE GROWTH AND DEVELOPMENT Oogonia. These c e l l s o c c u r r e d s i n g l y o r i n s m a l l n e s t s i n a l l o v a r i e s examined ( F i g . 1 & 2 ) . They were most numerous i n p o s t - o v u l a - t o r y f i s h and i n o v a r i e s w i t h numerous c o r p o r a l u t e a . Oogonial were l e s s abundant i n maturing o v a r i e s w i t h y o l k y o o c y t e s . ium was about Each oogon- 10-15 u i n diameter w i t h a s i n g l e n u c l e u s and one l a r g e n u c l e o l u s which s t a i n e d deeply w i t h haematoxylin. In sections of ovaries s t a i n e d w i t h M a l l o r y ' s t r i c h r o m e the n u c l e o l i were deep F i r s t Growth Phase. oogonia. cysts orange. F i r s t growth phase oocytes were l a r g e r than Primary oocytes were d i s t i n g u i s h e d from oogonia by the presence of d i s t i n c t chromosomes i n v a r i o u s s t a g e s o f m e i o t i c prophase. T h i s phase was s u b - d i v i d e d i n t o two d e f i n i t e stages s e p a r a t e d a c c o r d i n g to t h e f e a tures o f the nucleus. and These two s t a g e s were t h e c h r o m a t i n n u c l e o l a r stage the p e r i - n u c l e o l a r s t a g e . The first stage a f t e r the development o f o o c y t e s i s the chromatin n u c l e o l a r stage. The oocytes i n t h i s stage had a n u c l e u s w i t h a s i n g l e conspicuous n u c l e o l u s ( F i g . 3 ) . Chromatin nucleolus. threads were a t t a c h e d t o the 06cyte a t t h i s stage were s l i g h t l y l a r g e r than oogonia and had diameters o f 12-20 y. The stage. chromatin n u c l e o l a r s t a t e i s f o l l o w e d by the p e r i - n u c l e o l a r The p e r 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 p e r i p h e r a l arrangement o f a l a r g e number o f s m a l l n u c l e o l i on t h e i n n e r s i d e o f the n u c l e a r membrane ( F i g . 4 ) . The n u c l e u s was e n l a r g e d and t h e 12 Figure 1: An oogonium, as i n d i c a t e d by the arrow, singly. Figure 2: Haematoxylin Oogonial cyst and eosin. (arrow) c o n t a i n i n g several w i t h large n u c l e i deeply stained with Figure 3: Chromatin nucleolar Haematoxylin Figure 4; and stage oocytes oogonia haematoxylin. (arrows). eosin. P e r i n u c l e o l a r stage o o c y t e s . stage o o c y t e s occuring The e a r l y p e r i n u c l e o l a r (Arrow 1) are s m a l l e r i n size and s t a i n e d d e e p l y w i t h haematoxylin as compared w i t h 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 w i t h the enlargement of the n u c l e u s , the cytoplasm i n c r e a s e d d r a s t i c a l l y i n volume. at the p e r i n u c l e o l a r s t a g e , had diameters o f 20-150 y. of o o c y t e s a t t h i s stage s t a i n e d deeply w i t h haematoxylin. c o n t r a s t to o t h e r stages where the cytoplasm was toxylin. The Oocytes, cytoplasm T h i s was not s t a i n e d by haema- M a l l o r y ' s t r i c h r o m e s t a i n e d the cytoplasm deep r e d . The n u c l e o l i were v e r y conspicuous w i t h M a l l o r y ' s t r i c h r o m e and appeared orange ( F i g . 5 ) . A t the end o f the p e r i n u c l e o l a r stage the lost i t s affinity f o r haematoxylin. Second growth phase. The second cytoplasm Two types o f y o l k i n c l u s i o n s were e a s i l y d i s t i n g u i s h e d by t h e i r s i z e and s t a i n i n g (10-15 y i n diameter) with Mallory's trichrome. These two properties. stained blue with Mallory's t r i c h r o m e ; the s m a l l e r y o l k g r a n u l e s (about 1 y i n diameter) red deep growth phase oocytes were c h a r a c - t e r i z e d by the f o r m a t i o n and accumulation o f y o l k . The l a r g e r y o l k v e s i c l e in s t a i n e d deep types o f y o l k 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. presence (Fig. 5). The first the o f a r i n g o f v e s i c l e s i n the p e r i p h e r y o f the cytoplasm o f oocytes There was no p a t t e r n i n the l a t e r d e p o s i t i o n of y o l k v e s i c l e s . The v e s i c l e s were formed randomly. 150 i n d i c a t i o n o f y o l k f o r m a t i o n was y i n diameter. The oocyte at t h i s stage was about I n i t i a l l y , each v e s i c l e formed as a minute body but g r a d u a l l y i n c r e a s e d i n s i z e u n t i l i t reached a diameter o f 10-15 y. These y o l k v e s i c l e s i n c r e a s e d b o t h i n s i z e and numbers u n t i l they o c c u p i e d the whole cytoplasm o f o o c y t e s ( F i g . 6 ) . In o v a r i a n s e c t i o n s s t a i n e d w i t h 14 F i g u r e 5: Oocyte i n the i n i t i a l y o l k v e s i c l e stage w i t h characteristic ring of y o l k v e s i c l e s (v). the Mallory's trichrome. F i g u r e 6: L a t e y o l k v e s i c l e stage oocyte trichrome. The F i g u r e 7: The y o l k v e s i c l e s zona r a d i a t a stained with (v) are more abundant. (z) becomes t h i c k e n e d and red w i t h M a l l o r y ' s trichrome. E a r l y y o l k granule stage oocyte trichrome. The Mallory's i s stained stained with red y o l k g r a n u l e s Mallory's (g) accumulate between the b l u e y o l k v e s i c l e s ( v ) . F i g u r e 8: Late yolk granule trichrome. The stage oocytes Mallory's r e d y o l k g r a n u l e s have accumulated toward the c e n t e r o f oocytes t o the stained with periphery. and d i s p l a c i n g the y o l k v e s i c l e s 15 h a e m a t o x y l i n and e o s i n . t h e y o l k v e s i c l e s appeared t h i s proved t o be an a r t i f a c t . t o be v a c u o l e s b u t With M a l l o r y ' s t r i c h r o m e , these v e s i c l e s stained l i g h t blue. A t t h i s s t a g e o f o o c y t e development t h e f o l l i c u l a r f a i r l y w e l l developed. able. l a y e r s were The t h e c a l and g r a n u l o s a c e l l s were d i s t i n g u i s h - The zona r a d i a t a s t a r t e d t o develop d u r i n g t h e y o l k v e s i c l e s t a g e . I n i t i a l l y t h e zona r a d i a t a was t h i n and s t a i n e d d a r k r e d w i t h M a l l o r y ' s trichrome. By t h e l a t e s t a g e s of y o l k v e s i c l e f o r m a t i o n , however, t h e zona r a d i a t a was almost f u l l y d e v e l o p e d and i t s r a d i a t e n a t u r e was apparent. Yolk granule stages. The f o r m a t i o n o f y o l k g r a n u l e s c h a r a c t e r i z e d the f i n a l s t a g e of v i t e l l o g e n e s i s and o o c y t e development. formed o n l y i n o o c y t e s w i t h f u l l y d e v e l o p e d y o l k v e s i c l e s . g r a n u l e s f i r s t appeared Yolk granules The y o l k c l o s e t o t h e 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 r e d g r a n u l e s 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 t r i c h r o m e ; they were n o t v e r y e v i d e n t a f t e r s t a i n i n g w i t h h a e m a t o x y l i n and e o s i n . A t a l a t e r s t a g e y o l k g r a n u l e s were observed between t h e y o l k v e s i cles. However, even at t h i s s t a g e t h e y o l k g r a n u l e s were a l s o observed on the i n n n e r s u r f a c e o f the zona r a d i a t a ( F i g . 8 ) . The o o c y t e a t t h i s s t a g e was about 350-500 u i n d i a m e t e r w h i l e t h e i n d i v i d u a l g r a n u l e s were about 1 y o r l e s s i n d i a m e t e r . t h e y m i g r a t e d and accumulated As more and more y o l k g r a n u l e s were towards the center of the oocytes. formed, 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 t h e p e r i p h e r y o f o o c y t e s . HISTOCHEMISTRY OF VITELLOGENESIS H i s t o c h e m i c a l a n a l y s e s f o c u s s e d on t h e c h e m i c a l c o m p o s i t i o n o f y o l k inclusions. F i n d i n g s f o r t h e two types o f y o l k i n c l u s i o n s a r e 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 . i n the g e n e r a l c y t o p l a s m P o l y s a c c h a r i d e s were not detected o f o o g o n i a o r p r i m a r y o o c y t e s , but o n l y i n the y o l k v e s i c l e s w h i c h r e a c t e d w i t h a l l the p o l y s a c c h a r i d e s t a i n s . The v e s i c l e s s t a i n e d r e d w i t h B e s t ' s C a r m i n e — a n e m p i r i c a l method f o r d e t e c t i n g glycogen w h i c h i s s t i l l w i d e l y used by h i s t o c h e m i s t s ( P e a r s e , 1968). C o n t r o l s e c t i o n s d i g e s t e d w i t h d i a s t a s e b e f o r e s t a i n i n g gave a n e g a t i v e r e a c t i o n ; a b o l i t i o n o f s t a i n i n g by d i a s t a s e suggested contained glycogen. not s t a i n e d by B e s t ' s 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 g r a n u l e s were carmine. The y o l k v e s i c l e s and S c h i f f ' s reagent t h a t the v e s i c l e s zona r a d i a t a r e a c t e d w i t h p e r i o d i c a c i d - t o form a dark r e d c o l o u r ( F i g . 9 ) . a b o l i s h e d 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 c o n f i r m e d n a t u r e of y o l k v e s i c l e s and zona r a d i a t a . T h i s r e a c t i o n was the p o l y s a c c h a r i d e Since sections digested with diastase reacted p o s i t i v e l y with p e r i o d i c a c i d - S c h i f f ' s reagents, c o n t e n t s of y o l k v e s i c l e s are p r o b a b l y complex p o l y s a c c h a r i d e s and simple polysaccharides. The y o l k g r a n u l e s and ooplasm o f p r i m a r y the not 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 ( H a l e , 1 9 4 8 ) , and t h i s i n d i c a t e d t h a t the y o l k v e s i c l e s c o n t a i n e d a c i d The general cytoplasm o f p r i m a r y o o c y t e s s t a i n e d g r e e n i s h b l u e as com- pared t o the b l u e s t a i n i n g of the y o l k v e s i c l e s . also stained greenish blue. i n b o t h the c y t o p l a s m of mucopolysaccharides. The y o l k g r a n u l e s were S i n c e t h i s g r e e n i s h b l u e s t a i n was and y o l k g r a n u l e s ; i t was s t a i n i n g s o l u t i o n and not due p r o b a b l y due present to r e t e n t i o n to. a h i s t o c h e m i c a l r e a c t i o n . There 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 s t a g e s was of T a b l e 2: A summary of the h i s t o c h e m i c a l properties o f t h e two types o f y o l k TECHNIQUES YOLK VESICLES inclusions. YOLK GRANULES POLYSACCHARIDES B e s t ' s Carmine ( P e a r s e , 1968) D i a s t a s e - B e s t ' s Carmine P e r i o d i c a c i d - S c h i f f ' s (McManus, 1948) Diastase- Periodic acid- Schiff's Acetylation- Periodic acid- Schiff's Dialysed + + i r o n ( H a l e , 1946) A l c i a n B l u e , pH 2.5 ( P e a r s e , 1968) PROTEINS Dinitroflurobenzene Tetra-azotized ++ ( B u r s t o n e , 1955) o-dianisidine Sakaguchi's r e a c t i o n ( D a n i e l l i , 1953) + ( B a k e r , 1956) Ferric ferricyanide reaction (Chevremont & F r e d e r i c k , N - e t h y l 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 +++ (Baker, 1947) —— Millon's reaction I I II & Seligman, 1952) N - e t h y l maleimide - DDD r e a c t i o n 1943) I I - ++ +++ + ++ Continued T a b l e 2 (Cont'd) TECHNIQUE YOLK VESICLES YOLK GRANULES LIPIDS Sudan B l a c k B (Chayen et a l . , 1969) 4-f P y r i d i n e e x t r a c t i o n - Sudan B l a c k B O i l Red 0 ( L i l l i e , 1944) +4- P y r i d i n e e x t r a c t i o n - O i l Red 0 A c 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 +44+ Copper t h i o c y a n i n ( K l u v e r & B a r r e r a , 1953) +4- t- 1 oo 19 F i g u r e 9: Y o l k v e s i c l e stage oocytes s t a i n e d f o r p o l y s a c c h a r i d e s periodic acid-Schiff's reaction. a strong Figure 10: reaction. 11: method f o r The y o l k g r a n u l e s (g) s t a i n e d r e d d i s h whereas the y o l k v e s i c l e s (v) s t a i n e d Figure Y o l k v e s i c l e s (v) showed Oocytes s t a i n e d w i t h the d i n i t r o f l u r o b e n z e n e proteins. with yellowish. Oocytes s t a i n e d w i t h t e t r a z o t i s e d o - d i a n i s i d i n e r e a c t i o n for proteins. The y o l k g r a n u l e s (g) s t a i n e d dark r e d w h i l e the y o l k v e s i c l e s (v) s t a i n e d brownish r e d , i n d i c a t i n g t h a t the y o l k v e s i c l e s c o n t a i n e d l e s s p r o t e i n s than the y o l k granules. Figure 12; Oocytes s t a i n e d w i t h the f e r r i c s u l p h y d r y l groups. f e r r i c y a n i d e method f o r Y o l k v e s i c l e s (v) r e a c t e d p o s i t i v e l y though y o l k g r a n u l e s (g) r e t a i n e d background s t a i n i n g o n l y . Figure 13: Cryostat s e c t i o n s of ovary s t a i n e d w i t h Sudan B l a c k B. the y o l k g r a n u l e s they c o n t a i n e d Figure 14: Only (g) r e t a i n e d the s t a i n i n d i c a t i n g t h a t lipids. Oocytes s t a i n e d w i t h a c i d haematin. reacted p o s i t i v e l y i n d i c a t i n g The y o l k g r a n u l e s (g) phospholipids. 20 vitellogenesis. A l c i a n blue (8GX) b l u i s h - g r e e n a t pH 2.5. 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 The y o l k g r a n u l e s were not s t a i n e d . The dis- 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 t o those observed d u r i n g p e r i o d i c acid-Schiff's staining (Fig. 9). I t i s c o n c l u d e d from p o l y s a c c h a r i d e s t a i n i n g t h a t the y o l k v e s i c l e s c o n t a i n g l y c o g e n and complex a c i d i c p o l y s a c c h a r i d e s . granules do not c o n t a i n By c o n t r a s t , y o l k 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 i n t r o d u c e d by Sanger (1945) as a r e a g e n t f o r N - t e r m i n a l - a m i n o 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 a 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 r e d u c e d , d i a z o t i z e d and f i n a l l y coupled w i t h l - a m i n o - 8 - n a p h t h o l - 3 , a c i d ( H - a c i d ) i n an a l k a l i n e s o l u t i o n . 6-disulphonic Both the y o l k g r a n u l e s and the zona r a d i a t a r e a c t e d w i t h d i n i t r o f l u r o b e n z e n e r e a g e n t s t o g i v e a deep r e d ( F i g . 10). colour The y o l k v e s i c l e s on the o t h e r hand s t a i n e d b r o w n i s h - r e d 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 g e n e r a l s t a i n f o r p r o t e i n s ( F i g . 11). stained l i g h t red. 1947) yolk vesicles T h i s 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). T h i s suggested t h a t the y o l k v e s i c l e s c o n t a i n e d granules. The l e s s p r o t e i n t h a n the y o l k Sakaguchi r e a g e n t s , w h i c h are s p e c i f i c f o r a r g i n i n e (Baker, 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 g r a n u l e s 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 a p p e a r i n g l i g h t yellow i n colour. 21 Yolk granules reagent appeared dark r e d a f t e r t r e a t i n g w i t h M i l l o n ' s which i s s p e c i f i c f o r the hydroxy-phenyl group. amino a c i d c o n t a i n i n g hydroxy-phenyl group i s t y r o s i n e . c l e s , by c o n t r a s t , s t a i n e d y e l l o w to orange w i t h M i l l o n ' s The o n l y common The y o l k v e s i reagent. Y o l k v e s i c l e s appeared b l u e i n c o l o u r a f t e r r e a c t i n g w i t h Chevremont- F r e d e r i c k ' s method ( F i g . 12). T h i s method f o r s u l p h y d r y l groups i s based on the r e d u c t i o n o f f e r r i c y a n i d e t o f e r r o c y a n i d e . The r e s u l t i n g f e r r o c y a - n i d e combined w i t h f e r r i c i o n s to form an i n s o l u b l e P r u s s i a n b l u e pitate. T h i s r e a c t i o n i s n o t s p e c i f i c by i t s e l f , substances reduce f e r r i c y a n i d e . preci- s i n c e many r e d u c i n g C o n t r o l s e c t i o n s immersed i n 0.1 M, N - e t h y l maleimide b e f o r e r e a c t i n g w i t h Chevremont-Frederick reagents appeared green and showed no P r u s s i a n b l u e p r e c i p i t a t e i n the y o l k N - e t h y l maleimide s p e c i f i c a l l y b l o c k e d s u l p h y d r y l groups. i s n o n - s p e c i f i c and due to r e t e n t i o n o f reagents vesicles; The green c o l o u r by the y o l k granules. Yolk v e s i c l e s stained blue with dihydroxy-dinaphthyldisulphide method. (DDD) The DDD method i s based on the s p e c i f i c o x i d a t i o n o f s u l p h y d r y l s (Pearse, 1968). No b l u e c o l o u r was e v i d e n t i n y o l k v e s i c l e s o f c o n t r o l s e c t i o n s where s u l p h y d r y l groups were b l o c k e d w i t h N - e t h y l maleimide. y o l k g r a n u l e s were n o t s t a i n e d by d i h y d r o x y - d i n a p h t h y l - d i s u l p h i d e The reagent. In summary, the v a r i o u s p r o t e i n s t a i n s demonstrated t h a t both the y o l k v e s i c l e s and y o l k g r a n u l e s c o n t a i n e d p r o t e i n s . Yolk v e s i c l e s contain l e s s p r o t e i n s than y o l k g r a n u l e s ; s u l p h y d r y l groups a r e p r e s e n t i n yolk v e s i c l e s but n o t i n g r a n u l e s . Histochemistry of l i p i d s . Y o l k granules but not y o l k v e s i c l e s s t a i n e d w i t h Sudan B l a c k B ( F i g . 1 3 ) . Sudan B l a c k B was removed from y o l k granules 22 by p y r i d i n e e x t r a c t i o n . Black The zona r a d i a t a was not s t a i n e d by Sudan B. The d i s t r i b u t i o n o f O i l Red 0 was s i m i l a r t o Sudan B l a c k B. g r a n u l e s but n o t y o l k v e s i c l e s absorbed O i l Red 0. ing Yolk T h i s O i l Red 0 s t a i n - o f y o l k g r a n u l e s was not e v i d e n t i n c o n t r o l s e c t i o n s e x t r a c t e d with pyridine. Yolk granules and zona r a d i a t a s t a i n e d b l u e - b l a c k w i t h a c i d haematin ( F i g . 14). t o almost black The d i s t r i b u t i o n o f a c i d haematin s t a i n i n g was n o t changed by p y r i d i n e e x t r a c t i o n ; y o l k v e s i c l e s s t a i n e d l i g h t yellow, i n d i c a t i n g the l a c k o f p h o s p h o l i p i d s . L u x o l F a s t Blue MBS stained yolk granules and zona r a d i a t a dark b l u e . T h i s copper t h i o c y a n i n method i s s p e c i f i c f o r p h o s p h o l i p i d s . c o l o u r o f y o l k g r a n u l e s was s t i l l p r e s e n t with p y r i d i n e . The b l u e i n control sections extracted The y o l k v e s i c l e s were not s t a i n e d by the copper t h i o c y a n i n method. In c o n c l u s i o n , the l i p i d h i s t o c h e m i s t r y o f v i t e l l o g e n e s i s i n d i c a t e d t h a t y o l k v e s i c l e s d i d not c o n t a i n any l i p i d w h i l e y o l k g r a n u l e s n e u t r a l f a t s and p h o s p h o l i p i d s . but n o t n e u t r a l f a t s . The zona r a d i a t a c o n t a i n e d had b o t h phospholipid 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 o f y o l k i n c l u s i o n s ( y o l k v e s i c l e s and y o l k g r a n u l e s ) 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 . a r e formed These two y o l k i n c l u s i o n s d i f f e r 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 c h e m i c a l they are deposited disnature; s e q u e n t i a l l y — f i r s t t h e y o l k v e s i c l e s and then the y o l k granules. The o c c u r r e n c e of two t y p e s o f y o l k i n c l u s i o n s i n t e l e o s t o o c y t e s was p r e v i o u s l y d e s c r i b e d by Yamamoto (1956, 1958). He found b o t h y o l k v e s i c l e s and y o l k g r a n u l e s i n L i o p s e t t a o b s c u r a and C l u p e a p a l l a s i i , based on h i s t o l o g i c a l e v i d e n c e . S i m i l a r l y , Malone and H i s o a k a (1963) d i s - t i n g u i s h e d two types o f y o l k , d e s i g n a t e d 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, t h e o c c u r r e n c e o f two types o f y o l k i n c l u s i o n s i s n o t u n i v e r s a l among t e l e o s t s . I n some s p e c i e s , t h r e e types o f y o l k have been ob- s e r v e d ; Yamamoto (1956) d e s c r i b e d 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 t h e s m e l t , Hypomesus j a p o n i c u s w h i l e Guraya (1965) noted t h r e e types o f y o l k i n c l u s i o n s i n Channa m a r u l i u s . The f i r s t yolk 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 b o d i e s t h a t grow u n t i l they r e a c h e d the s i z e o f 15 y. These y o l k v e s i c l e s have been d e s c r i b e d by many i n v e s t i - g a t o r s u s i n g 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 , v a c u o l e s , 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 . I n s p i t e o f v a r y i n g n o m e n c l a t u r e , y o l k v e s i c l e s always c o n s i s t o f mucopolysaccharides and r e a c t p o s i t i v e l y t o t h e 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 ( A k e t a , 1954; 1965; Yamamoto, 1956 Y a m a z a k i , 1965). a, b, c; Malone & H l s o a k a , 1963; Guraya, 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 o b s c u r a c o n t a i n s i m p l e m u c o p o l y s a c c h a r i d e s (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 mucopolys a c c h a r i d e s ; Hypomesus j a p o n i c u s a l s o has y o l k v e s i c l e s w i t h m u c o p o l y s a c c h a r i d e s (Yamamoto, 1956 c). acidic A k e t a suggested t h a t y o l k v e s i - c l e s o f O r y z i a s l a t i p e s c o n s i s t e d of s u l p h a t e d mucopolysaccharides. Towards the l a t t e r p a r t o f 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 a r e d i s p l a c e d towards the p e r i p h e r y of o o c y t e s and alveioli. The c o r t i c a l a l v e o l i p l a y an i m p o r t a n t r e a c t i o n a t the time of f e r t i l i z a t i o n The develop i n t o c o r t i c a l r o l e i n the (Yamamoto, 1961; cortical Manroy, 1965). 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 components. cell A k e t a (1954) suggested t h a t 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 t h a t y o l k v e s i c l e s o r i g i n a t e from the ground c y t o p l a s m . A c t u a l l y , t h e r e i s no r e a l d i f f e r e n c e between t h e s e 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 " y o l k n u c l e u s " as the p r e c u r s o r of y o l k v e s i c l e s . The pre- c u r s o r s of y o l k v e s i c l e s , because of t h e i r s m a l l s i z e , are beyond the r e s o l v i n g power of the l i g h t m i c r o s c o p e . A l t h o u g h no one has examined t h e y o l k n u c l e u s i n t e l e o s t w i t h the e l e c t r o n m i c r o s c o p e , B a l i n s k y and D a v i s (1963) demonstrated t h a t the y o l k n u c l e i of amphibians were concent r a t i o n s of m i t o c h o n d r i a w h i c h p l a y e d no p a r t i n y o l k f o r m a t i o n . At the e l e c t r o n m i c r o s c o p e l e v e l , the b e s t e v i d e n c e of the o r i g i n of y o l k v e s i c l e s was p r o v i d e d by Yamamoto and Onozato (1965), and Yamamoto and Oota (1967). Yamamoto and Onozato (1965) demonstrated t h a t G o l g i complexes 25 gave r i s e t o y o l k v e s i c l e s i n g o l d f i s h and t h e s e f i n d i n g s have been c o n f i r m e d 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 , C a r a s s i u s a u r a t u s . 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 g r a n u l e s 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 . C h e m i c a l l y the y o l k v e s i c l e s a r e p o l y s a c c h a r i d e s 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 g r a n u l e s c o n s i s t o f p r o t e i n s , p h o s p h o l i p i d s and n e u t r a l l i p i d s . The c h e m i c a l c o m p o s i t i o n o f y o l k g r a n u l e s 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 v e r y s i m i l a r t o the c o m p o s i t i o n o f y o l k as d e t e r m i n e d by b i o chemical s t u d i e s . P l a c k and F r a s e r (1970), i n a b i o c h e m i c a l s t u d y , demon- s t r a t e d t h a t t h e y o l k o f cod c o n t a i n s two major l i p o p r o t e i n s o f s i m i l a r m o l e c u l a r w e i g h t (400,000) and c h e m i c a l c o m p o s i t i o n ) . about 79% p r o t e i n and 21% l i p i d w h i l e a p p r o x i m a t e l y Eachhlipoprotein i s 65% o f t h e l i p i d frac- tion i s phospholipid. The y o l k g r a n u l e s were d e p o s i t e d i n d e p e n d e n t l y and o n l y appeared i n oocytes of the yolk vesicles a f t e r t h e 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 a r e s t i l l many c o n f l i c t i n g o p i n i o n s about t h e o r i g i n o f y o l k g r a n u l e s . D r o l l e r and Roth (1966) c o n c l u d e d from e l e c t r o n m i c r o s c o p i c s t u d i e s o f 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 t h a t y o l k g r a n u l e s were formed by t h e e n d o p l a s mic r e t i c u l u m and G o l g i b o d i e s . These w o r k e r s d e s c r i b e d m i c r o p i n o c y t o s i s i n the p e r i p h e r y o f o o c y t e s ; the g r a n u l e s formed by p i n o c y t o s i s accumulated w i t h i n t h e endoplasmic r e t i c u l u m and G o l g i complex t o form y o l k g r a n u l e s . Yamamoto and Oota (1967), and Gupta and Yamamoto (1972) d i s p u t e d t h e suggesti o n s o f D r o l l e r and R o t h ; Yamamoto and Oota p o s t u l a t e d t h a t were t h e p r e c u r s o r s o f y o l k g r a n u l e s . mitochondria They argued t h a t p i n o c y t o t i c b o d i e s 26 were t a k e n i n t o o o c y t e s and yolk granules. resynthesized w i t h i n mitochondria This suggestion functions of mitochondria recognized i n c e l l r e s p i r a t i o n . F u r t h e r work, e s p e c i a l l y at the e l e c t r o n m i c r o s c o p i c o p i n i o n s can be seems u n l i k e l y i n v i e w o f the t o form l e v e l , i s required before these c o n f l i c t i n g resolved. I n s p i t e of t h e s e 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 t h a t p a r t of y o l k g r a n u l e s were d e r i v e d from an exogenous s o u r c e t h a t y o l k p r e c u r s o r were d e p o s i t e d i n o o c y t e s by p i n o c y t o s i s . and Anderson (1968) and Guraya (1965) have a l s o p r o v i d e d e v i d e n c e o f p i n o c y t o s i s w i t h i n o o c y t e s o f Fundulus h e t e r o c l i t u s and Channa m a r u l i u s , r e s p e c t i v e l y . h i s t o g e n e s i s of y o l k g r a n u l e source of y o l k The f o r m a t i o n s u p p o r t e d the i d e a of an exogenous granules. I t i s w e l l known t h a t 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 d u r i n g the r e p r o d u c t i v e p e r i o d ( B a i l e y , 1957* Ho and 1961; Cedard e_t al_. , 1961, P l a c k and F r a s e r , 1970). Vanstone, Very r e c e n t i n v e s t i g a - t i o n s by A i d a e_t a l . (1973) a l s o s u p p o r t the e a r l i e r p o s t u l a t i o n s t h a t 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 the y o l k p r o t e i n s s y n t h e s i z e d by the l i v e r are t r a n s p o r t e d by b l o o d t o the o v a r y and d e p o s i t e d w i t h i n o o c y t e s by p i n o c y t o s i s . that plasma 27 F i g u r e 15: A d i a g r a m a t i c r e p r e s e n t a t i o n o f oogenesis i n g o l d f i s h ovaries. The stages i n oocyte development d u r i n g oogenesis are - (I) oogonia w i t h i n c y s t s ; ( I I ) chromatin n u c l e o l a r stage o o c y t e s ; ( I I I ) p e r i n u c l e o l a r stage oocyte; (IV) e a r l y y o l k v e s i c l e s t a g e o o c y t e ; (V) i n t e r m e d i a t e y o l k v e s i c l e stage o o c y t e ; (VI) l a t e y o l k v e s i c l e stage o o c y t e ; (VII) i n i t i a l y o l k g r a n u l e stage o o c y t e ; ( V I I I ) l a t e y o l k g r a n u l e stage o o c y t e ; and (IX) mature o o c y t e . DIAGRAMATIC REPRESENTATION OF O O C Y T E GROWTH SECTION I T : The Corpus Luteum and i t s Functions 29 INTRODUCTION Not a l l o f t h e d e v e l o p i n g o v a r i a n f o l l i c l e s complete t h e f o r m a t i o n of an ovum; many o f them become a t r e t i c a t some s t a g e d u r i n g 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 t o t h e p o s t - o v u l a t o r y f o l l i c l e s and t h e r e has l o n g been a l i v e l y i n t e r e s t i n t h e i r function. the A t present n e i t h e r the p h y s i o l o g y of the a t r e t i c f o l l i c l e s nor p o s t - o v u l a t o r y corpus luteum i s f u l l y u n d e r s t o o d . A t r e t i c o o c y t e s o c c u r i n a wide v a r i e t y o f t e l e o s t as e v i d e n t by t h e r e v i e w s by P i c k f o r d and A t z ( 1 9 5 7 ) , B a l l 1969) and C h i e f f i (1970). ( 1 9 6 0 ) , Dodd ( 1 9 6 0 ) , Hoar (1965, They have been d e s c r i b e d f r e q u e n t l y s i n c e B r e t s c h n e i d e r and Duyvene de W i t (1947) f i r s t d i s c u s s e d 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 d e s c r i b e d 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), C a r a s s i u s a u r a t u s (Beach, 1959), G a s t e r o s t e u s a c u l e a t u s (Tromp-Blom, 1959) , Scomber scomber ( B a r a , 1960), Mystus s e e n h g a l a (Sa'thyanesan ( 1 9 6 1 ) , H e t e r o p n e u s t e s f o s s i l i s P l e u r o n e c t e s p l a t e s s a ( B a r r , 1963), Xenenotodon Monopterus ( N a i r , 1963), c a n c i l a ( R a s t o g i , 1966), a l b a (Chan e t a l . , 1967), and Sebastodes p a u c i s p i n i s (Moser, 1967) . The p o 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 w h i c h resemble mammalian c o r p o r a l u t e a , have been r e p o r t e d i n Scomber scomber ( B a r a , 1960), Mystus s e e n g h a l a ( S a t h y a n e s a n , 1962), H e t e r o p n e u s t e s f o s s i l i s ( N a i r , 1963), E u c a l i a i n c o s t a n s ( B r a e k e v e l t and M c M i l l a n , 1967), and C l a r i u s b a t r a c h u s ( L e h r i , 1968). A l t h o u g h B a r r ( 1 9 6 3 ) , R a j a l a k s h m i ( 1 9 6 6 ) , and Moser (1967) 30 r e p o r t e d an absence of p o s t - o v u l a t o r y c o r p o r a 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 s a m p l i n g o r the o v a r i e s 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 o f t h e s e s t r u c t u r e s , whether pre- o r p o s t - o v u l a t o r y , t o mammalian c o r p o r a l u t e a l e d B r e t s c h n e i d e r & Duyvene de Wit (1947) t o c o n s i d e r them comparable t o the mammalian corpus luteum and t o suggest an e n d o c r i n e f u n c t i o n based on the q u e s t i o n a b l e " 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 " . T h i s work has s t i m u l a t e d many d i s - c u s s i o n s of the f u n c t i o n o f the p r e - 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 c o n c l u s i o n s have been reached due t o the l a c k of critical e v i d e n c e of t h e i r e n d o c r i n e f u n c t i o n . Many a u t h o r s , w h i l e r e c o g n i z i n g t h e l i m i t a t i o n s o f the "Bitterling o v i p o s i t o r growth t e s t " , s t i l l c o n s i d e r t h e s e s t r u c t u r e s comparable mammalian corpus luteum (see r e v i e w s by B a l l , 1960; Hoar, 1965). t o the In addi- 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 , p r e - and p o s t - o v u l a t o r y b o d i e s o f t e n have the appearance 1969) of t r u e e p i t h e l i a n g l a n d s . Hoar (1965, suggested t h a t t e l e o s t c o r p o r a l u t e a might produce e s t r o g e n s r a t h e r than p r o g e s t e r o n e . The a p p r o p r i a t e n e s s of the term "corpuca l u t e u m " 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 a g a i n s t i t i s the l a c k of c r i t i c a l e v i d e n c e o f the e n d o c r i n e 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 e n d o c r i n e f u n c t i o n s of t h e corpus luteum are (a) a c t i v e s e c r e t i o n o f the hormone p r o g e s t e r o n e and (b) the p i t u i t a r y c o n t r o l . I n c o n t r a s t t o the mammalian corpus luteum, s u c c e s s f u l g e s t a t i o n s have been e s t a b l i s h e d i n the absence of t h e s e b o d i e s i n ' t e l e o s t s (Lambert and Van O o r d t , Lambert, 1970). Lambert (1970) suggested t h a t i f t h e a t r e t i c 1965; follicles 31 were Important hormone p r o d u c i n g glands they s h o u l d be r e g u l a r l y p r e s e n t during gestation. However, the absence o f a t r e t i c f o l l i c l e s d u r i n g ges- t a t i o n does not r u l e out an e n d o c r i n e r o l e o f t h e s e s t r u c t u r e s but shows o n l y t h a t 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 ; t h e y may, however, produce hormones and p l a y some o t h e r r e p r o d u c t i v e r o l e . Some workers f e e l t h a t 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 f o r m a t i o n and maintenance of a t r e t i c f o l l i c l e s enhances doubts o f an e n d o c r i n e 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 t o hypo- physectomy ( B r e t s c h n e i d e r and Duyvenne de W i t , 1947; B a r r , 1963) and some a u t h o r s who doubt the e n d o c r i n e s t a t u s of t e l e o s t c o r p o r a l u t e a , m a i n t a i n t h a t a t r e t i c o o c y t e s a r e p r o b a b l y a d a p t i v e d e v i c e s f o r d i s p o s i n g moribund ova (Dodd, 1960; P o l d e r , 1964). They c o n s i d e r the absence o f p r o g e s t e r o n e 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 s t r o n g arguments f o r u s i n g 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 a p p r o p r i a t e n e s s o f the term " c o r p o r a 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 o f corpus luteum, as an e n d o c r i n e g l a n d t h a t s e c r e t e s p r o g e s t e r one and m a i n t a i n s g e s t a t i o n , i s too r e s t r i c t i v e . In vertebrates, especially 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 t o c o r p o r a l u t e a are not r e s t r i c t e d t o v i v i p a r a (Hisaw, 1963; B r a m b e l l , 1960) and a narrow d e f i n i t i o n p r e v e n t s 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 v i e w p o i n t i t may be more a p p r o p r i a t e t o c o n s i d e r t h e "corpus luteum" as an e n d o c r i n e g l a n d t h a t s e c r e t e s s t e r o i d hormones and p l a y a r o l e i n r e p r o d u c t i o n but which need n o t n e c e s s a r i l y be concerned w i t h the maintenance o f gestation. 32 The broader t u r e s w i l l be d e f i n i t i o n w i l l be adopted f o r t h i s study. termed c o r p o r a l u t e a and The s t r u c - an attempt made to e s t a b l i s h p h y s i o l o g i c a l f u n c t i o n o f both a t r e t i c oocytes and p o s t - o v u l a t o r y c l e s i n the g o l d f i s h — a n o v i p a r o u s teleost. folli- T h i s study w i l l i n v e s t i g a t e the corpus luteum as an organ capable o f s y n t h e s i z i n g s t e r o i d s by ing the the h i s t o c h e m i c a l l o c a t i o n o f v a r i o u s h y d r o x y s t e r o i d examin- dehydrogenases. Dorfman and Ungar (1965) e s t a b l i s h e d the r o l e o f h y d r o x y s t e r o i d dehydrogenases i n s t e r o i d b i o s y n t h e s i s . p r e - or p o s t - o v u l a t o r y , w i l l be The f a t e of the corpus luteum, e i t h e r f o l l o w e d p a s t the 6-stage. 33 METHODS AND MATERIALS HYPOPHYSECTOMY OF GOLDFISH G o l d f i s h were hypophysectomized u s i n g a m o d i f i c a t i o n of Yamazaki's (1965) t e c h n i q u e . s o l u t i o n of 0.01 The f i s h were f i r s t anaesthesized i n a cold The "operating table" f o r g o l d f i s h , c o n s i s t e d of a sponge w i t h a groove i n i t . t h i s groove, w i t h the l e f t bands of 2.5 C) % t r i c a n e methane s u l f o n a t e ('MS222', Sandoz) u n t i l lost their reflex activity. in (4-8 cm w i d t h . The hypophysectomizing G o l d f i s h were p l a c e d s i d e uppermost and h e l d i n p l a c e by o p e r a t i n g t a b l e was they elastic kept c o o l w i t h crushed i c e d u r i n g the e n t i r e o p e r a t i o n . The left operculum t o g e t h e r w i t h the f i r s t two t r a c t e d w i t h hooked p i n s a t t a c h e d to e l a s t i c c o r d s . p a r a l l e l to the second gill a r c h , was i n c i s i o n v a r i e d from 5-10 oid bone, beneath which the p i t u i t a r y i s s i t u a t e d . the parasphenoid the p i t u i t a r y . p t e r y g o i d bone. of An o b l i q u e c u t , immediately A h o l e was d e n t a l b u r r (No. nerves then drilled 3) t o expose i n f r o n t of the i n n e r t i p o f the Another landmark f o r the p o i n t of d r i l l i n g a p a i r of conspicuous re- i n l e n g t h ; t h i s cut exposed the parasphen- bone w i t h a round T h i s h o l e was arches was made i n the d o r s a l b u c c a l mucosa. The through mm gill from the cranium. i s the origin These nerves were i n - e v i t a b l y cut d u r i n g hypophysectomy w h i l e i n the sham hypophysectomized they were i n t e n t i o n a l l y cut d u r i n g the d r i l l i n g of the parasphenoid in sham hypophysectomized f i s h the p i t u i t a r y was B l e e d i n g was bone; exposed but not removed. To e f f e c t hypophysectomy, the exposed p i t u i t a r y was w i t h a curved p i p e t t e . fish removed by s u c t i o n much reduced w i t h c o l d a n e s t h e t i c s 34 and ceased q u i c k l y i n most i n s t a n c e s . mouth h e a l e d w i t h i n a week. with connective The i n c i s i o n i n the r o o f of the The exposed area of the s k u l l was replaced tissues. D u r i n g r e c o v e r y hypophysectomized g o l d f i s h were p l a c e d i n c o l d d i l u t e d sea water (4-8°C) prepared by d i l u t i n g w i t h t h r e e times i t s volume o f d e c h l o r i n a t e d water. A temperature shock a f t e r hypophysectomy r e s u l t s i n h i g h m o r t a l i t y and consequently water temperatures to room temperature. were r a i s e d s l o w l y High s u r v i v a l r a t e s , a v e r a g i n g w e l l over 80%, were o b t a i n e d w i t h the above t e c h n i q u e s ; most o f the m o r t a l i t y was damage. due to b r a i n 35 HISTOLOGY OF PRE- AND POST-OVULATORY CORPUS LUTEUM P r e - o v u l a t o r y c o r p o r a l u t e a were induced hypophysectomy. e i g h t weeks. amination i n g r a v i d g o l d f i s h by Four hypophysectomized f i s h were k i l l e d each week f o r A p o r t i o n o f each ovary was f r o z e n f o r h i s t o c h e m i c a l e x - o f h y d r o x y s t e r o i d dehydrogenases w h i l e a n o t h e r p o r t i o n was f i x e d i n B o u i n ' s f l u i d t o observe t h e h i s t o g e n e s i s o f a t r e t i c o o c y t e . The f a t e o f 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 after ovulation. L i k e w i s e i n t h e s e experiments, one p o r t i o n of each ovary was f r o z e n f o r t h e d e t e c t i o n o f h y d r o x y s t e r o i d dehydrogenases and the o t h e r f i x e d i n Bouin's f l u i d . Ovaries embedded i n p a r a f f i n , s e c t i o n e d s e r i a l l y t o x y l i n and e o s i n , o r M a l l o r y ' s t r i c h r o m e f i x e d i n B o u i n ' s f l u i d were a t 7 y and s t a i n e d w i t h haema( G u r r , 1962). HISTOCHEMICAL DEMONSTRATION OF HYDROXYSTEROID DEHYDROGENASE Hydroxysteroid dehydrogenases i n o v a r i e s o f 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 o f W a r t t e n b e r g (1958) technique. H y d r o x y s t e r o i d dehydrogenases i n o v a r i e s o f hypophysectomized g o l d f i s h were examined a t w e e k l y i n t e r v a l s f o r e i g h t c o n s e c u t i v e weeks a f t e r hypophysectomy and the f i n d i n g s were compared w i t h those i n t h e ovaries of ovulated g o l d f i s h D e t a i l s of the techniques 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 . a r e as f o l l o w s : G o l d f i s h were d e c a p i t a t e d and a p o r t i o n o f each ovary was f r o z e n w i t h " C r y o w i c k " (IEC Company), w h i l e a second p o r t i o n was f i x e d i n B o u i n ' 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 . F r o z e n o v a r i e s were s e c t i o n e d 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 ) a t 36 10-16 y; the temperature o f the c r y o s t a t , w h i c h was s e c t i o n i n g , was m a i n t a i n e d a t -10°C t o -15°C. c r i t i c a l f o r proper C r y o s t a t 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 a t room t e m p e r a t u r e 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 the r e a c t i o n medium a t 30°C. The r e a c t i o n mixture tetrazolium salt ( N i t r o BT o r MTT), i n 1.6 M, T r i s - H C l b u f f e r , pH 7.5 ml o f 0.1 c o n t r a i n i n g 50-100 yg s t e r o i d . before being incubated i n MTT 2 mg NAD was c o n s i s t e d of 1 and 10 mg M g C l and 0.4 2 mg dissolved ml d i m e t h y l formamide r o u t i n e l y used as N i t r o BT gave a more i n t e n s e d i a p h o r a s e r e a c t i o n . S t e r o i d s used as s u b s t r a t e s i n c l u d e d d e h y d r o e p i a n d r o s t e r o n e c i f i c substrate f o r 3 3-hydroxysteroid dehydrogenases), androsterone s p e c i f i c substrate f o r 3 a-hydroxysteroid progesterone (a spe- dehydrogenases) , 17 (a' s p e c i f i c s u b s t r a t e f o r 17 a - h y d r o x y s t e r o i d (a cx-hydro- 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 ^ - h y d r o x y s t e r o i d dehydrogenases) . Each o f t h e s e s u b s t r a t e s has one h y d r o x y l group f o r t h e h y d r o x y - s t e r o i d dehydrogenase r e a c t i o n ( F i g . 1 6 ) . Other o v a r i a n s t e r o i d s , 17 a- h y d r o x y p r e g e n o l o n e , p r e g n e n o l o n e , and e s t r a d i o l were used as s u b s t r a t e s for u n s p e c i f i c h y d r o x y s t e r o i d dehydrogenase r e a c t i o n s . To d i s t i n g u i s h h y d r o x y s t e r o i d dehydrogenases from d i a p h o r a s e o c c u r s i n most t i s s u e , s e c t i o n s were i n c u b a t e d out any s t e r o i d s u b s t r a t e . i n a r e a c t i o n mixture w i t h - A t the end o f 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 ( B a k e r , 1944) zymes. which S e c t i o n s were then washed i n two mide t o remove excess s t e r o i d s , and which i n h i b i t e d the changes o f 25% d i m e t h y l finally i n d i s t i l l e d water. en- formaThe 36a 3-16: HYDROXYSTEROID DEHYDROGENASE REACTION BluePpt. 37 washed s e c t i o n s were mounted i n F e r r a n t ' s media and examined w i t h i n a day o r two a f t e r staining. INCORPORATION OF THYMIDINE- H INTO GOLDFISH OVARIES 3 A group o f g r a v i d g o l d f i s h was t r a n s f e r r e d t o e x p e r i m e n t a l a q u a r i a c o n t a i n i n g d e o J a l o r i n a t e d water a t 10°C. r a i s e d t o room temperature (22-25°C). o f t h e s e f i s h o v u l a t e d a f t e r two days. The water temperature slowly W i t h t h i s t r e a t m e n t the m a j o r i t y F i s h which d i d n o t o v u l a t e were removed w h i l e the o v u l a t e d 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 t h e females were i n j e c t e d w i t h 1 yc t h y m i d i n e - 3 H (360 mc/mM, Amersham) p e r gram body w e i g h t . 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 t h y m i d i n e - H i n 0.1 ml o f s a l i n e t h e f i s h were k i l l e d , i n 3 groups o f f o u r , a t 1, 2, 4, 8, 16, and 32 days a f t e r t h e t h y m i d i n e - H injection. A p o r t i o n o f each o v a r y 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 routine 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 c u t and mounted on albumen coated s l i d e s . dewaxed and h y d r a t e d through a s e r i e s o f a l c o h o l . These s e c t i o n s were Hydrated s e c t i o n s were d i p - c o a t e d w i t h n u c l e a r t r a c k e m u l s i o n s (Kodak NTB 2) i n the dark (Kopriwa and L e B l a n d , 1962). The s l i d e s were a i r - d r i e d and t h e n kept i n l i g h t p r o o f 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 w h i c h t h e s l i d e s were p r o c e s s e d w i t h p h o t o g r a p h i c d e v e l o p e r (Kodak D 19) and f i x e r (Kodak F 5 f i x i n g b a t h ) . The a u t o r a d i o g r a p h s were s t a i n e d w i t h Mayer's h a e m a t o x y l i n and e o s i n (Beserga and Malamud, 1969), d e h y d r a t e d i n a l c o h o l , c l e a r e d i n x y l e n e , and mounted i n DPX ( K i l k p a t r i c k and Lendrum, 1941). 38 A u t o r a d i o g r a p h s of f i v e s e c t i o n s from each ovary were examined 3 f o r the i n c o r p o r a t i o n of t h y m i d i n e - H i n t o the v a r i o u s the o v a r i e s . c e l l types i n 39 RESULTS HISTOGENESIS OF ATRETIC OOCYTES Y o l k y o o c y t e s became a t r e t i c a f t e r hypophysectomy. a t r e s i a was The p a t t e r n o f the same i n a l l o o c y t e s a l t h o u g h the r a t e s a t which v a r i o u s o o c y t e s , w i t h i n an o v a r y , became a t r e t i c was v a r i a b l e ( T a b l e 3 ) . of t h i s v a r i a b i l i t y i t was f o l l o w i n g hypophysectomy. Because i m p o s s i b l e t o q u a n t i f y the r a t e o f a t r e s i a However, o o c y t e s a t t h e y o l k g r a n u l e s t a g e were c l e a r l y t h e f i r s t t o become a t r e t i c and, i n g e n e r a l , o o c y t e s w i t h more y o l k became a t r e t i c much f a s t e r t h a n t h o s e w i t h l e s s y o l k . For d e s c r i p t i v e p u r p o s e s , a t r e s i a i n g o l d f i s h can be s u b - d i v i d e d i n t o f o u r c o n s e c u t i v e s t a g e s a c c o r d i n g t o the s u b d i v i s i o n s o f B r e t s c h n e i d e r 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 s t a g e desc r i b e d here. -stage tt atresia. (Fig. 17). The h y p e r t r o p h y o f the g r a n u l o s a i n i t i a t e s The g r a n u l o s a c e l l s change from a squamous t o 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 r u p t u r e t o form a c o n t i n u o u s mass of c o l l o i d ; the e n t i r e o o c y t e shows gigns of degeneration. The p u n c t u r e o f t h e zona r a d i a t a by numerous p o r e s marks the end o f t h e <* - s t a g e a t r e t i c g-stage ( F i g . 18). oocyte. Hypertrophied granulosa c e l l s invade the c y t o p l a s m o f the a t r e t i c o o c y t e t h r o u g h the p o r e s i n the zona radiata. The i n v a d i n g g r a n u l o s a c e l l s d i g e s t and r e s o r b 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. 3W Table 3: The degree of a t r e s i a i n various ovaries a t various time i n t 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 y o l k y hypophysectomy oocytes counted NO. OF OOCYTES IN EACH STAGE Yolk v e s i c l e Yolk granule Atretic 7 7 75 52 63 14 1 31 4 7 17 42 22 7 64 17 46 3 2 14 54 14 57 96 35 16 5 11 96 0 24 2 44 0 45 36 14 30 14 14 21 64 38 61 21 63 15 18 21 21 45 52 4 18 28 0 7 46 41 27 0 28 0 36 46 28 0 65 0 2 36 28 63 73 61 0 0 17 26 22 28(sham) 28 (sham) 28(sham) 28(sham) 69 34 81 0 44 43 6 66 0 2 73 15 : sham hypophysectomized g o l d f i s h , which serves as the c o n t r o l 0 0 6 0 40 y-stage ( F i g . 19). Hypertrophy continues during t h i s stage. all o f the g r a n u l o s a c e l l s By the end o f this s t a g e remnants of oocytes are c o m p l e t e l y r e s o r b e d and h y p e r t r o p h i e d g r a n u l o s a c e l l s surround the a t r i u m ; t h i s marks the space the o o c y t e . membrane, one The entire a small cavity, formerly occupied structure i s surrounded the by by a c e l l t h i c k , which i s s i m i l a r to the t h e c a of the normal oocyte ( F i g . 20). T h i s stage appears have been o v e r l o o k e d by many i n v e s t i g a t o r s , described a y s t a g e to although they d i f f e r e n t from the ^ - s t a g e d e s c r i b e d here. 6-stage ( F i g . 21). collapse During t h i s s t a g e , the g r a n u l o s a c e l l s i n t o the a t r i u m to form an i r r e g u l a r c e l l u l a r mass and y e l l o w l u t e i n pigments are observed T h i s stage p e r s i s t s among the f o r long p e r i o d s ; Barr 6-stage p r e o v u l a t o r y c o r p o r a l u t e a cells. (1963) observed s i x months a f t e r hypo- physectomy. e-stage ( F i g . 22). atresia a fifth and In a d d i t i o n t o these f o u r s t a g e s o f f i n a l stage was recognized. In the l a t e s t a g e s o f a t r e s i a some c e l l s of the 6-stage corpus appear t o d i f f e r e n t i a t e i n t o oogonia. d i f f e r e n t i a t i o n were observed luteum V a r i o u s phases o f and the b e s t evidence for a 41 F i g u r e 17: a-stage o f a t r e t i c oocyte s t a i n e d w i t h haematoxylin and e o s i n . The g r a n u l o s a c e l l s o f the a t r e t i c (arrow 1) h y p e r t r o p h i e d as compared w i t h the granulosa c e l l s (arrow 2 ) . y o l k v e s i c l e s was oocytes normal Extensive degeneration of e v i d e n t (homogeneous a r e a o p p o s i t e arrow 1 ) . F i g u r e 18: 3-stage o f a t r e t i c o o c y t e s t a i n e d w i t h haematoxylin. Masses o f g r a n u l o s a and t h e c a l c e l l s invade and digest the a t r e t i c o o c y t e s . F i g u r e 19: A s e c t i o n o f the ovary o f a hypophysectomized c o n t a i n i n g many y - s t a g e a t r e t i c f o l l i c l e s goldfish ( f r e e arrow) w i t h the c h a r a c t e r i s t i c empty c a v i t y w i t h i n i t . F i g u r e 20: A d e t a i l e d photomicrograph cle. of the y s t a g e a t r e t i c folli- The oocyte i s c o m p l e t e l y reabsorbed and the colum- nar g r a n u l o s a c e l l s have the c h a r a c t e r i s t i c s of p h y s i o l o g i c a l l y active F i g u r e 21: cells. 6-stage a t r e t i c f o l l i c l e (mass o f c e l l s on r i g h t ) w i t h haematoxylin and e o s i n . stained The g r a n u l o s a c o l l a p s e d i n t o the space f o r m e r l y o c c u p i e d by the egg cytoplasm t o form an i r r e g u l a r mass of F i g u r e 22: e-stage a t r e t i c f o l l i c l e s . cells. T h i s photomicrograph the d i f f e r e n t i a t i o n o f one h a l f o f the a t r e t i c shows follicles i n t o oogonia (arrow) w h i l e the o t h e r h a l f remain entiated. undiffer- 42 s e p a r a t e e-stage corpus luteum was the presence o f t h e partially differentiated posed c y s t s which show one h a l f com- of oogonia w h i l e the o t h e r h a l f looked l i k e a 6- stage corpus luteum w i t h y e l l o w l u t e i n pigments. HISTOGENESIS OF POST OVULATORY CORPUS LUTEUM At o v u l a t i o n the f o l l i c u l a r membranes o f f u l l y developed r u p t u r e and r e l e a s e the eggs. The f o l l i c u l a r membrane i s not r e l e a s e d but r e t a i n e d w i t h i n the ovary where i t h y p e r t r o p h i e s . Three phases i n h i s t o g e n e s i s can be d i s t i n g u i s h e d . Stage I ( F i g . 23, 24, 25). A f t e r o v u l a t i o n both t h e c a and g r a n u l o s a c e l l s become h y p e r t r o p h i e d w i t h the more marked response i n the g r a n u l o s a ( F i g . 25). The morpho- l o g i c a l appearance of t h i s s t a g e i s the same as the ystage a t r e t i c f o l l i c l e . broken Sometimes the remains o f the f o l l i c u l a r w a l l i s e v i d e n t ( F i g . 23) and appears as a d i s t i n c t i v e f e a t u r e but the m a j o r i t y o f p o s t - o v u l a - t o r y b o d i e s l o o k l i k e y-stage a t r e t i c oocytes w i t h no indication due of the r e l e a s e o f eggs ( F i g . 24); t h i s may be to the way the o v a r i e s were s e c t i o n e d . Stage I I ( F i g . 26, 27). oocytes There were no d i f f e r e n c e s be- tween the <5-stage corpus luteum and the stage I I p o s t - distinct 43 o v u l a t o r y corpus luteum. The g r a n u l o s a c e l l s converged i n t o an i r r e g u l a r mass as i n t h e 6-stage a t r e t i c oocyte and l o s t t h e i r g l a n d u l a r n a t u r e ; as the t h e c a s u r r o u n d s the compact mass o f g r a n u l o s a c e l l s . As i n t h e 6-stage a t r e t i c o o c y t e , 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 cells. Stage I I I ( F i g . 2 8 ) . The i r r e g u l a r mass o f c e l l s i n s t a g e I I p o s t - o v u l a t o r y f o l l i c l e s seem t o d i f f e r e n t i a t e i n t o o o g o n i a l c y s t s as i n t h e 6-stage a t r e t i c oocyte. These o o g o n i a l c y s t s c o n s t i t u t e t h e s t a g e I I I o r e - s t a g e 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 o b s e r v e d w i t h one h a l f composed o f 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 This evidence oogonia. prompted t h e p o s t u l a t i o n o f t h e 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 . 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 o f h y d r o x y s t e r o i d dehydrogenases. The r e s u l t s o f 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 o f h y d r o x y s t e r o i d dehydrogenase are presented i n T a b l e 4. t r i b u t i o n o f diaphorase Formazan d e p o s i t s demonstrated a g e n e r a l a c t i v i t y i n sections of ovaries incubated i n a medium c o n t a i n i n g N i t r o - B T and NAD b u t l a c k i n g s t e r o i d s u b s t r a t e . diaphorase a c t i v i t y was o b s e r v e d i n oocytes w i t h i n oocytes, dis- as w e l l as f o l l i c u l a r t h i s a c t i v i t y increased w i t h oocyte diameter. The cells; I t was Stage I p o s t - o v u l a t o r y corpus luteum s t a i n e d w i t h Mallory's trichrome. the first T h i s photograph documents f o r time evidence of the r u p t u r e f o l l i c u l a r w a l l (arrow) f o l l o w i n g ovulation. Stage I p o s t - o v u l a t o r y corpus luteum a l s o s t a i n e d w i t h Mallory's trichrome. Here t h e r e i s no evidence o f the r u p t u r e o f the f o l l i c l e a f t e r o v u l a t i o n . This i s mainly due to the s e c t i o n i n g o f the o v a r i a n t i s s u e . A d e t a i l e d photograph o f the stage I p o s t - o v u l a t o r y corpus luteum showing the h y p e r t r o p h y o f g r a n u l o s a c e l l s (c) as compared t o the f o l l i c u l a r c e l l s , b o t h t h e c a l and granul o s a o f the normal ovary ( f ) . The r a d i a t e n a t u r e o f t h e zona r a d i a t a (z) i s e v i d e n t . Stage I I p o s t - o v u l a t o r y corpus luteum ( a r r o w s ) . l o s a and t h e c a l c e l l s The granu- c o l l a p s e d i n t o an i r r e g u l a r mass o f cells. A d e t a i l e d photograph o f t h e s t a g e I I corpus luteum. The c e l l s e s p e c i a l l y t h e n u c l e i a r e l e s s h y p e r t h r o p h i e d than those o f the s t a g e I corpus luteum ( F i g . 2 5 ) . The stage I I I p o s t - o v u l a t o r y corpus luteum s t a i n e d w i t h haematoxylin and e o s i n . the T h i s stage i s c h a r a c t e r i z e d by d i f f e r e n t i a t i o n o f some c e l l s i n t o o o g o n i a (arrow). T a b l e 4: A summary o f the h i s t o c h e m i c a l r e a c t i o n o f h y d r o x y s t e r o i d dehydrogenases i n o v a r i a n follicles. STEROID SUBSTRATE STAGES IN OOGENESIS ENZYME 1 s t growth Vesicle Early yolk Vesicle Late yolk Vesicle Yolk granule DtUycU ue . i •T Control Diaphorase Dehydroepiandrosterone 33-HSD Androsterone 3a-HSD 17a-hydroxy pregnenolone 33-HSD o r 17a-HSD 17a-hydroxyprogesterone 17a-HSD Estradiol 173-HSD Testosterone 176-HSD + + 4- 4-4-44 4- ++7 4-4- I n t e n s i t y o f r e a c t i o n s were graded (-) t o ( M i l ) ; (-) denotes no demonstrable a c t i v i t y and a maximal a c t i v i t y . 1 HSD i s h y d r o x y s t e r o i d T dehydrogenase. (MM) 46 much reduced when t h e t e t r a z o l i u m s a l t , MTT, was used a s t h e f i n a l electron acceptor. No 3 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was e v i d e n t i n ooc y t e s a t the l a t e y o l k g r a n u l e stages activity ( F i g . 30) although some enzyme was d i s c e r n a b l e i n e a r l y y o l k g r a n u l e stage o o c y t e s . 3 3 - h y d r o x y s t e r o i d dehydrogenase. i n a medium used f o r the demonstration O v a r i e s r e a c t e d when i n c u b a t e d of 33-hydroxysteroid dehydro- genase w i t h e i t h e r NAD o r NADP as a c o - f a c t o r b u t the r e a c t i o n was weaker w i t h NADP as t h e c o - f a c t o r . d r o s t e r o n e o r pregnenolone. follicular c e l l s of f i r s t i m p o s s i b l e t o determine The s u b s t r a t e was dehydroepian- T h i s 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 growth phase oocytes ( F i g . 29) but i t was whether the r e a c t i o n was o f t h e c a l o r granu- l o s a l o r i g i n s i n c e these t i s s u e s a r e not w e l l d i f f e r e n t i a t e d . s m a l l e r second The growth phase o o c y t e s , i n the y o l k v e s i c l e s t a g e , gave p o s i t i v e r e s u l t s w i t h the r e a c t i o n l o c a t e d i n t h e g r a n u l o s a cells; o c c a s i o n a l l y 3 3 - h y d r o x y s t e r o i d dehydrogenase was a l s o d e t e c t e d i n t h e theca c e l l s during t h i s stage. No 3 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was e v i d e n t i n o o c y t e s a t the l a t e y o l k g r a n u l e s t a g e s ( F i g . 30) a l t h o u g h some enzyme a c t i v i t y was d i s c e r n i b l e i n e a r l y y o l k g r a n u l e stage o o c y t e s . 3ot-Hydroxysteroid dehydrogenase a c t i v i t y . The d i s t r i b u t i o n o f 3 a - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n g o l d f i s h o v a r i e s , was s i m i l a r t o t h a t o f 3 3 - h y d r o x y s t e r o i d dehydrogenase. The b e s t r e s u l t s were o b t a i n e d w i t h NAD as a c o - f a c t o r ; NADP gave a much weaker r e a c t i o n . 47 T h i s 3 a - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y was periphery of f i r s t growth phase oocyte as was s t e r o i d dehydrogenase ( F i g . 29). l o c a l i z e d i n the the case f o r 33-hydroxy- Formazan d e p o s i t s w i t h androsterone as s u b s t r a t e were d e t e c t e d i n the g r a n u l o s a o f y o l k v e s i c l e stage ooc y t e s b u t t h e a c t i v i t y o f 3 a - h y d r o x y s t e r o i d dehydrogenase was much weaker than 3 3 - h y d r o x y s t e r o i d dehydrogenase. As p r e v i o u s l y d e s c r i b e d f o r 33- h y d r o x y s t e r o i d dehydrogenase, no 3 a - h y d r o x y s t e r o i d dehydrogenase ty was d e t e c t e d i n the f o l l i c u l a r c e l l s o f y o l k g r a n u l e stage 17a^-Hydroxysteroid dehydrogenase. activi- oocytes. 17a-hydroxy p r o g e s t e r o n e was s u c c e s s f u l l y used as a s u b s t r a t e f o r the h y d r o x y s t e r o i d dehydrogenase reaction. T h i s r e a c t i o n i n d i c a t e s the presence of 1 7 a - h y d r o x y s t e r o i d dehydrogenase enzymes i n g o l d f i s h o v a r i e s . The d i s t r i b u t i o n of t h i s enzyme was 3 3 - h y d r o x y s t e r o i d dehydrogenases. s i m i l a r t o t h a t o f 13a- and In s h o r t , 17a-hydroxysteroid dehydro- genase o c c u r r e d i n the p e r i p h e r y o f f i r s t growth phase o o c y t e s and i n the g r a n u l o s a o f y o l k v e s i c l e stage oocytes but y o l k g r a n u l e stage showed no s i g n o f 1 7 a - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y . oocytes The 17a- h y d r o x y s t e r o i d dehydrogenase a c t i v i t y i n the o v a r i e s i s l e s s i n t e n s e than 3 3 - h y d r o x y s t e r o i d dehydrogenase and s i m i l a r i n i n t e n s i t y w i t h 3a-hydroxyteroid dehydrogenase. 173-Hydroxysteroid dehydrogenase. E s t r a d i o l and t e s t o s t e r o n e were s u c c e s s f u l l y used as s u b s t r a t e s f o r the demonstration dehydrogenase a c t i v i t y i n the ovary. ase r e a c t i o n s NAD of 173-hydroxysteroid As i n the h y d r o x y s t e r o i d dehydrogen- gave a b e t t e r r e a c t i o n than NADP. 48 F i g u r e 29: Histochemical demonstration of 33-hydroxysteroid genase i n o v a r i e s o f n o r m a l f i s h . dehydro- The formazan d e p o s i t s (arrow) a r e l o c a l i z e d i n t h e g r a n u l o s a c e l l s o f o o c y t e s . The n u c l e i (n) a r e l a b e l l e d t o p r o v i d e e a s i e r i n t e r p r e t a t i o n of t h e i l l u s t r a t i o n . F i g u r e 30: O v a r i e s o f g r a v i d f i s h s t a i n e d w i t h 3 ( 3 - h y d r o x y s t e r o i d dehydrogenase. There were no a c t i v i t i e s i n t h e f o l l i c u l a r c e l l s ( f r e e arrow) o f t h e y o l k granulestage F i g u r e 31: oocytes. O v a r i e s o f 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 1 7 3 - h y d r o x y s t e r o i d ase. dehydrogen- A c t i v i t y , as i n d i c a t e d by t h e d e p o s i t i o n o f formazan (arrow) was l o c a l i z e d i n y - s t a g e 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 p r e s e n t i n f o l l i c u l a r c e l l s of t h e f i r s t growth phase o o c y t e s . F i g u r e 32: Histochemical demonstration o f 3 [ 3 - h y d r o x y s t e r o i d dehydro- genase i n the p o s t - o v u l a t o r y f o l l i c l e s o f r e c e n t l y o v u l a t e d goldfish. luteum H i g h enzyme a c t i v i t y was d e t e c t e d i n t h e corpus (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 i n the f i r s t 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 dehydrogenase activity. 50 HYDROXYSTEROID DEHYDROGENASES IN HYPOPHYSECTOMIZED FISH T a b l e 5 summarizes the d i s t r i b u t i o n o f the v a r i o u s h y d r o x y s t e r o i d dehydrogenases i n the o v a r i e s of hypophysectomized One week a f t e r hypophysectomy, observed but no q u a n t i f i c a t i o n goldfish. o n l y a-stage a t r e t i c oocytes were o f the r a t e o f a t r e s i a was because of the g r e a t v a r i a b i l i t y as i n d i c a t e d e a r l i e r . attempted Hydroxysteroid dehydrogenases were not d e t e c t e d i n c e l l s o f e i t h e r a-stage oocytes in or i n f i r s t growth phase o o c y t e s . the f o l l i c l e s o f f i r s t The l a c k o f enzyme a c t i v i t y growth phase oocytes of hypophysectomized g o l d f i s h atretic c h a r a c t e r i z e d the o v a r i e s at a l l times. Most y o l k y oocytes have become a t r e t i c by the end o f the second o r t h i r d weeks of hypophysectomy. Y-stage d u r i n g t h i s p e r i o d . oocytes, they were mainly A t r e s i a progressed Although as f a r as the t h e r e were some a-stage atretic i n t h e 3 - or y-stages and no s i g n i f i c a n t dif- f e r e n c e s were noted i n t h e o v a r i e s o f f i s h hypophysectomized f o r two o r t h r e e weeks. Hydroxysteroid dehydrogenase a c t i v i t y was f i r s t growth phase oocytes In s t r i k i n g as w e l l as « - and was granulosa ( F i g . 31). oocytes. This was used as The o t h e r h y d r o x y s t e r o i d dehydrogenases, 3a-, 17a-hydroxysteroid oocytes. 173-hydroxysteroid o b t a i n e d whether t e s t o s t e r o n e or e s t r a d i o l the s u b s t r a t e . atretic i n f o l l i c l e s of 3-stage a t r e t i c c o n t r a s t , y-stage a t r e t i c oocytes had dehydrogenase, a c t i v i t y i n the h y p e r t r o p h i e d activity absent 33-, dehydrogenases, were n o t d e t e c t e d i n t h e y-stage and T a b l e 5. H i s t o c h e m i c a l R e a c t i o n o f H y d r o x y s t e r o i d Dehydrogenase i n O v a r i e s o f Hypophysectomized Goldfish. STEROID SUBSTRATE ENZYME Control Diaphorase Dehydroepiandrosterone 33-HSD Androsterone 3a-HSD 17a-hydroxyprogesterone 17a-HSD Estradiol 173-HSD Testosterone 173-HSD 1 s t growth phase o o c y t e a corpus l u t e u m s t a g e s 3 y 6 1 I n t e n s i t y o f h y d r o x y s t e r o i d dehydrogenase r e a c t i o n and ( M M ) maximal. a c t i v i t y graded (-) t o ^HSD denotes h y d r o x y s t e r o i d dehydrogenase enzymes. ( M i l ) ; (-) denotes no demonstrable- 2 No enzyme a c t i v i t y was d e t e c t e d i n second growth phase o o c y t e s when t h e y were p r e s e n t . Ln 52 No intact second growth phase oocytes were found i n ovaries of fish k i l l e d four weeks after hypophysectomy. A l l yolk oocytes had become a t r e t i c and some of them reached the 6-stage while others were at the a-stage. These a-stage a t r e t i c oocytes were much smaller than those observed e a r l i e r ; the majority of oocytes were at the 3- or ystages. As i n the ovaries of f i s h hypophysectomized f o r 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 a t r e t i c f o l l i c l e s . Although 173-hydroxysteroid dehydrogenase a c t i v i t y was detected i n y-stage atret i c oocytes, 3a-, 33-> and 17a-hydroxysteroid dehydrogenases were not evident. The ovaries of f i s h k i l l e d i n the f i f t h to eight week a f t e r hypophysectomy had only f i r s t growth phase oocytes and 6-stage a t r e t i c cles. folli- 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 i n the y-stage a t r e t i c follicles. 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 p o r c i n e l u t e i n i z i n g hormone (NIH-LH-S8). toneal injections, dehydrogenases. (Table 6 ) . w i t h 100 ug/g Two days a f t e r t h e i n t r a p e r i - t h e 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 T h e i r o v a r i e s gave s t r o n g r e a c t i o n s f o r a l l f o u r enzymes Apparently, the l u t e i n i z i n g hormone s t i m u l a t e s f o r m a t i o n o f t h e s e enzymes s i n c e h y d r o x y s t e r o i d dehydrogenases were not d e t e c t e d i n u n t r e a t e d hypophysectomized g o l d f i s h . f i s h the d i s t r i b u t i o n I n l u t e i n i z i n g hormone-treated o f the formazan d e p o s i t s was normal e x c e p t f o r the presence of a l l f o u r h y d r o x y s t e r o i d dehydrogenases i n y-stage atretic oocytes. As i n the o v a r i e s o f hypophysectomized g o l d f i s h , dehydrogenase enzymes were d e t e c t e d i n the a-, oocytes o f t h e hormoner:treated f i s h . no hydroxysteroid and 6-stage atretic By c o n t r a s t , t h e y-stage atretic o o c y t e s had a c t i v i t i e s o f 3 a - , 3(3-, 17a-, and 1 7 3 - h y d r o x y s t e r o i d dehydro- genases; o f t h e s e , 1 7 3 - h y d r o x y s t e r o i d active. This i s d i f f e r e n t steroid dehydrogenase was from hypophysectomized f i s h where o n l y the most 173-hydroxy- dehydrogenase was d e t e c t e d i n t h e y-stage a t r e t i c o o c y t e and a g a i n argues f o r s t i m u l a t i o n o f enzyme f o r m a t i o n by LH. T a b l e 6. E f f e c t o f LH Treatment on H i s t o c h e m i c a l l y Demonstrable H y d r o x y s t e r o i d Dehydrogenases i n O v a r i e s o f Hypophysectomized G o l d f i s h Steroid Substrate Enzyme Control diaphorase Dehydroepiandrosterone 33-HSD Androsterone 3a-HSD 17a-hydroxyprogesterone 17a-HSD Estradiol 176-HSD Testosterone 176-HSD 1 s t growth phase oocytes Stages o f a t r e s i a a g y * •6 + ++++ Ul 55 HYDROXYSTEROID DEHYDROGENASES IN POST-OVULATORY CORPUS LUTEUM The h i s t o c h e m i c a l d e t e c t i o n o f h y d r o x y s t e r o i d dehydrogenases was c a r r i e d out a t t h r e e times a f t e r o v u l a t i o n : on the day o f o v u l a t i o n and on the seventh and t h i r t i e t h days a f t e r o v u l a t i o n . a f t e r o v u l a t i o n showed v e r y a c t i v e h y d r o x y s t e r o i d i n their ovaries. c e l l s although The formazan d e p o s i t s were m a i n l y i n o c c a s i o n a l l y the t h e c a l c e l l s Except f o r t h i s v e r y a c t i v e h y d r o x y s t e r o i d post-ovulatory dehydrogenase r e a c t i o n s B l u e formazan d e p o s i t s developed i n t h e p o s t - o v u l a t o r y f o l l i c l e s w i t h a l l the s u b s t r a t e s . the g r a n u l o s a Fish k i l l e d shortly stained also. dehydrogenase r e a c t i o n i n t h e c o r p o r a l u t e a ( F i g . 32) t h e ovary l o o k s l i k e a normal v i t e l l o g e n i c ovary. First growth phase oocytes developed formazan i n the f o l l i c u l a r c e l l s w i t h a l l the s t e r o i d s u b s t r a t e s . Likewise, i n the y o l k v e s i c l e stages had dehydrogenases but a l l the hydroxysteroid these were c l e a r l y c o n f i n e d t o the g r a n u l o s a o v a r i e s , no h y d r o x y s t e r o i d dehydrogenase a c t i v i t y l a r c e l l s during the y o l k granules The cells. As i n normal occured i n the f o l l i c u - stages. d i s t r i b u t i o n o f the v a r i o u s h y d r o x y s t e r o i d seem t o change d u r i n g the f i r s t oocytes dehydrogenases d i d not seven days a f t e r o v u l a t i o n . p o s t - o v u l a t i o n , t h e r e were v e r y few stage I p o s t - o v u l a t o r y A t seven days corpora l u t e a but most o f t h e post o v u l a t o r y f o l l i c l e s had reached stage I I . The stage I p o s t o v u l a t o r y corpus luteum showed a l l f o u r hydroxy- s t e r o i d dehydrogenases (Table 7) w i t h formazan d e p o s i t s i n the g r a n u l o s a cells. strates. These d e p o s i t s were observed w i t h Only s l i g h t a l l s t e r o i d s t e s t e d as sub- a c t i v i t y was observed i n t h e stage I I post ovulatory T a b l e 7: 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 o f H y d r o x y s t e r o i d Dehydrogenases i n the O v a r i e s o f Ovulated G o l d f i s h Steroid Substrate Enzyme Stages l g t Y growth Control Diaphorase o l i n Oogenesis k vesicle Y o l k Post O v u l a t o r y g t a g e I g t Follicles ^ a g e Granule + + + + 4- +++ 4+++ + 4-H- + 4+ + Dehydroepiand androsterone 33-HSD Androsterone 3a-HSD +4- 444- + 17a-hydroxyprogesterone 17a-HSD 4-4- 4+ +• 4- +• Estradiol 17B-HSD 4+4- +4-f + +4+ + ON 57 c o r p u s luteum. The r e a c t i o n was n o t much g r e a t e r t h a n i n t h e c o n t r o l s e c t i o n s and suggested t h a t the r e a c t i o n was due to diaphorase 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 o f these enzymes i n the activity. other o v a r i a n t i s s u e s i s s i m i l a r t o 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 s m a l l oocytes i n the f i r s t growth phase had h y d r o x y s t e r o i d dehydrogenase a c t i v i t y . with a l l steroid substrates. T h i s h i g h a c t i v i t y was Y o l k v e s i c l e stage oocytes 36-, 17a- and 1 7 3 - h y d r o x y s t e r o i d high showed 3a-, 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 o f the normal o v a r y . o t h e r o v a r i e s examined, t h e r e was obtained As i n the no h y d r o x y s t e r o i d dehydrogenase a c t i v i - t y i n the f o l l i c l e s o f y o l k g r a n u l e s s t a g e o o c y t e s . Only stage I I p o s t - o v u l a t o r y c o r p o r a 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 t o the 6-stage p r e - o v u l a t o r y c o r p o r a 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 d e t e c t e d i n them. The distribu- t i o n o f h y d r o x y s t e r o i d dehydrogenase i n the o t h e r o v a r i a n t i s s u e s was s i m i l a r t o 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 c e l l s o f f i r s t growth phase oocytes c l e stage o o c y t e s . as w e l l as the g r a n u l o s a o f y o l k v e s i - A g a i n , no h y d r o x y s t e r o i d dehydrogenase a c t i v i t y c u r r e d i n y o l k g r a n u l e stage I t i s concluded follicular oocytes. t h a t the p o s t - o v u l a t o r y corpus l u t e u m i s c a p a b l e s t e r o i d hormone s y n t h e s i s . oc- of V e r y a c t i v e s t e r o i d s y n t h e s i s o c c u r s i n the c o r p u s l u t e u m as w e l l as i n the g r a n u l o s a c e l l s o f p o s t - o v u l a t o r y o v a r i e s . The Fate o f P o s t o v u l a t o r y Corpus Luteum as Determined by T r i t i a t e d thymidine Autoradiography l a b l e s were d e t e c t e d i n f o l l i c u l a r c e l l s o f 3 k i l l e d one day a f t e r t h y m i d i n e - H i n j e c t i o n ( F i g . 3 3 ) . More o f t h e s e fish cells 58 were l a b e l l e d n e a r t h e o v a r i a n p e r i p h e r y t h a n i n the c e n t e r o f o v a r i e s . Follicular ment. 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 o f t h e s t a g e o f ovum d e v e l o p - None o f t h e abundant s t a g e I p o s t - o v u l a t o r y c o r p o r a l u t e a were labelled. 3 The p a t t e r n o f t h y m i d i n e - H i n c o r p o r a t i o n was s i m i l a r i n o v a r i e s o f 3 f i s h k i l l e d two days a f t e r t h y m i d i n e - H i n j e c t i o n w i t h f o l l i c u l a r cells 3 l a b e l l e d randomly i n the o o c y t e s . On t h e f o u r t h day a f t e r t h y m i d i n e - H i n j e c t i o n , however, l a b e l s were observed i n t h e c e l l s ovulatory corpora l u t e a o f stage I I post- ( 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 . none of s t a g e I c o r p o r a l u t a c e l l s were l a b e l l e d . Again, The m a j o r i t y o f t h e s t a g e IT c o r p o r a l u t e a were l a b e l l e d i n o v a r i e s on f i s h k i l l e d e i g h t days 3 a f t e r t h y m i d i n e - H i n j e c t i o n . The i n c r e a s e i n number o f s t a g e I I c o r p o r a l u t e a . was accompanied by a r e d u c t i o n i n s t a g e I c o r p o r a l u t e a . 3 The d i s t r i b u t i o n o f t h y m i d i n e - H l a b e l s i n o v a r i e s o f f i s h 16 days a f t e r t r i t i a t e d t h y m i d i n e 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 t o t h a t o f f i s h k e p t f o r 8 days w i t h some l a b e l l e d c e l l s s t a g e IT c o r p o r a l u t e a . killed The f o l l i c u l a r of t h e s t a g e o f t h e o o c y t e . i n the majority of c e l l s were l a b e l l e d , irrespective The o n l y d i f f e r e n c e was t h e absence o f stage I corpora l u t e a i n the o v a r i e s of these f i s h . The f o l l i c u l a r c e l l s o f o o c y t e s as w e l l as c e l l s o f s t a g e IT c o r p o r a l u t e a were l a b e l l e d i n o v a r 3 i e s o f f i s h k i l l e d 32 days a f t e r t h y m i d i n e - H i n j e c t i o n . many o o g o n i a ( F i g . 35) and some o o c y t e s were l a b e l l e d In addition ( F i g . 36). The c o n c l u s i o n o f t h e experiments d e s c r i b e d i n t h i s s e c t i o n i s t h a t both f o l l i c u l a r and s t a g e I I c o r p o r a l u t e a c e l l s are a c t i v e l y multiplying. One o f t h e s e two c e l l types d i f f e r e n t i a t e s i n t o o o g o n i a and u l t i m a t e l y oocytes. 59 F i g u r e 33: A u t o r a d i o g r a p h y o f the o v a r y seven.days a f t e r the 3 i n j e c t i o n o f t h y m i d i n e - H. The f o l l i c u l a r cells (arrow) were l a b e l l e d . F i g u r e 34: A u t o r a d i o g r a p h y o f the o v a r y , seven days a f t e r the 3 i n j e c t i o n o f t h y m i d i n e - H. In addition to the l a b e l - l i n g o f f o l l i c u l a r c e l l s ( F i g . 33) c e l l s o f 5-stage corpus luteum were a l s o l a b e l l e d ( a r r o w s ) . F i g u r e 35: A u t o r a d i o g r a p h y o f g o l d f i s h o v a r i e s 30 days a f t e r t h y 3 midine- H i n j e c t i o n . Some o o g o n i a w i t h i n o o g o n i a l c y s t s 3 were l a b e l l e d by t h y m i d i n e - H ( a r r o w ) . 3 F i g u r e 36: A u t o r a d i o g r a p h y o f o o c y t e l a b e l l e d w i t h t h y m i d i n e - H. t h i s o o c y t e was from t h e o v a r y o f g o l d f i s h i n j e c t e d w i t h 3 t h y m i d i n e - 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 , especiall y 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 c e l l s of normal oocytes, or as corpora lutea. In addition to steroid hormone synthesis, the f o l l i - cular c e l l s are responsible f o r the removal of a t r e t i c oocytes by phagocytosis . After a t r e s i a or ovulation the f o l l i c u l a r c e l l s developed into corpora lutea which l a t e r d i f f e r e n t i a t e into oogonia. Steroid Synthesis i n Fish Ovaries In the present study, hydroxysteroid dehydrogenases have only been found i n the ovarian granulosa. The a c t i v i t i e s of 3a-, 33-, 17a-, and 173-hydroxysteroid dehydrogenases increased with oocyte development or d i f f e r e n t i a t i o n , but at the yolk granule stage, a l l four hydroxysteroid dehydrogenases decreased so dramatically that during the l a t t e r part of t h i s stage, no hydroxysteroid dehydrogenases could be detected i n the granulosa. In post-ovulatory ovaries hydroxysteroid dehydrogenases were evident in f i r s t growth phase oocytes and i n corpora lutea, but were not detec- table i n 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 hormones synthesized are not known. The absence of hydroxysteroid dehydrogenase a c t i v i t y during the yolk granule stage was surprising. I t indicated an i n h i b i t i o n of steroid 61 synthesizing enzyme a c t i v i t y when oocytes reached the yolk granule stage. The steroid enzymes were evidently re-activated after ovulation. Hydroxysteroid dehydrogenase a c t i v i t y 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 v e s i c l e stage oocytes. Restoration of a c t i v i t y with bovine l u t e i n i z i n g hormone supports this argument. Although there are no comparable studies of hypophysectomy on hydroxysteroid dehydrogenases i n the ovaries of t e l e o s t s , Yamazaki and Donaldson (1968) demonstrated a decrease i n the 3g-hydroxysteroid dehydrogenase of i n t e r s t i t i a l c e l l s of goldfish t e s t i s a f t e r hypophysectomy. In their experiments, 33-hydroxysteroid dehydrogenase a c t i v i t y was restored with salmon gonadotropin, again confirming the p i t u i t a r y involvement. S i m i l a r l y , Yaron (1966) observed that 3B-hydroxy- steroid dehydrogenase decreased i n t e s t i s of T i l a p i a mossembica after hypophysectomy, while Wiebe (1969) found that methallibure administration decreased 33-hydroxysteroid dehydrogenase a c t i v i t y i n i n t e r s t i t i a l and Suertoli c e l l s 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 i s under the control of p i t u i t a r y gonadotropin. In the absence of gonadotropin a l l hydroxysteroid dehydrogenase a c t i v i t y ceases. A t r e s i a of Second Growth Phase Oocytes In this study, a t r e s i a was induced by hypophysectomy; i n nature, adverse environmental or physiological conditions probably induce a t r e s i a 62 i n y o l k y o o c y t e s ( Y a r o n , 1971). The h i s t o l o g y o f a t r e t i c follicles 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 W i t , 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 t h e h y p e r t r o p h y o f granu- l o s a c e l l s , o r t h e c a l c e l l s , o r b o t h as demonstrated (1967). by Chan e t a l . The e a r l y s t a g e s ( a - and 3-stages) i n v o l v e d t h e breakdown and r e s o r p t i o n o f moribund o o c y t e s . The p h a g o c y t i c a c t i o n o f f o l l i c u l a r c e l l s i n a t r e t i c o o c y t e s was g e n e r a l l y r e c o g n i z e d (see r e v i e w 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 o f t h e 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 o f g r a n u l o s a c e l l s i n g o l d f i s h c o n t i n u e s 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 o r two l a y e r s o f h y p e r t r o p h i e d c e l l s s u r r o u n d i n g an i n n e r c a v i t y , t h e a t r i u m ( F i g . 19). Though many i n v e s t i g a t o r s r e c o g n i z e d a y-rstage d u r i n g 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 o f 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 o u r s and I n v o l v e o n l y t h e i r r e g u l a r mass o f c e l l s n o t e d i n t h e 6-stage. These a u t h o r s may have f a i l e d t o observe an i n t e r m e d i a t e s t a g e b e f o r e the c o l l a p s e o f t h e a t r e t i c f o l l i c l e s . The p r e s e n t s t u d y i n d i c a t e s t h a t t h i s s t a g e , c h a r a c t e r i z e d b y g l a n d u l a r morphology, i s very important t o the p h y s i o l o g i c a l f u n c t i o n s o f a t r e t i c follicles. 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 t o be t r u e e p i t h e l i a l g l a n d s ( F i g . 2 0 ) . H i s t o c h e m i c a l d a t a ( T a b l e 5, F i g . 31) show c o n s i d e r a b l e 1 7 3 - h y d r o x y s t e r o i d dehydrogenase a c t i v i t y . T h i s enzyme 63. c a t a l y s e s e i t h e r the c o n v e r s i o n o f t e s t o s t e r o n e t o a n d r o s t e r o n e , e s t r a d i o l to e s t r o n e ( T a l a l a y , 1965) and i t s presence or i n y-stage c o r p o r a l u t e a a t r e t i c a s u p p o r t s Hoar's (1965) s u g g e s t i o n t h a t the tele- ost In 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 e s t r o g e n s . c o n t r a s t t o 1 7 3 - h y d r o x y s t e r o i d dehydrogenase, t h e r e was no evidence f o r 3 3 - h y d r o x y s t e r o i d dehydrogenase o r 3a- and 1 7 a - h y d r o x y s t e r o i d dehydro- genases i n y-stage a t r e t i c f o l l i c l e s . These o b s e r v a t i o n s a r e i n p a r t i a l agreement w i t h t h o s e o f Lambert (1970) and Y a r o n (1971) who absence of 3 g - h y d r o x y s t e r o i d r e p o r t e d an 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 for 3 17 ^-i,-. o r 1 7 3 - h y d r o x y s t e r o i d test dehydrogenases. Hypophysectomized g o l d f i s h i n j e c t e d w i t h LH developed d r o x y s t e r o i d dehydrogenases i n y-stage c o r p o r a l u t e a . a l l f o u r hy- This s t i m u l a t i o n o f h y d r o x y s t e r o i d dehydrogenases suggests t h a t 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 t h e a c t i v i t y o f t h e s e h y d r o x y s t e r o i d dehydrogenase enzymes i n t h e a t r e t i c f o l l i c l e s . Hypophysectomy i m p a i r e d p a r t o f t h e s t e r o i d synthesizing c a p a b i l i t y of a t r e t i c P o s t - o v u l a t o r y Corpora follicles. Lutea Stage I p o s t - o v u l a t o r y c o r p o r a l u t e a are s i m i l a r i n appearance t o 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 o f h y p e r t r o p h i e d g r a n u l o s a t h e c a l c e l l s s u r r o u n d i n g an empty c a v i t y . the presence of an opening f o l l i c l e ( F i g . 23 and 24). and The main d i f f e r e n c e noted was i n some, but not a l l o f the p o s t - o v u l a t o r y P o s t - o v u l a t o r y c o r p o r a l u t e a w i t h t h i s open- i n g w h i c h marks the p o i n t o f o v u l a t i o n have not been p r e v i o u s l y d e s c r i b e d . 64 In f a c t , most i n v e s t i g a t o r s have p a i d o n l y c u r s o r y a t t e n t i o n t o p o s t ovulatory f o l l i c l e s , r e p o r t i n g o n l y t h e i r r a p i d d i s i n t e g r a t i o n and d i s a p p e a r a n c e ( B a i l e y , 1943; McMillan, 1967). Barr (1967) m a i n t a i n e d Moser in teleost. B a r a , 1960; R a s t o g i , 1966; (1963), Lambert (1966), R a j a l a k s h m i (1966), and t h a t no p o s t - o v u l a t o r y corpus luteum e x i s t e d T h e i r c o n c l u s i o n s were based on random sampling which d i d not i n c l u d e o v a r i e s soon a f t e r o v u l a t i o n . fish Braekevelt & o v a r i e s from o v u l a t e d fish A detailed analysis of gold- showed t h a t the p o s t - o v u l a t o r y follicles always e x i s t e d and had a d e f i n i t e p a t t e r n o f h i s t o g e n e s i s . L o c a l i z e d hormone p r o d u c t i o n . The d i s t r i b u t i o n o f h y d r o x y s t e r o i d dehydrogenases i n stage I p o s t - o v u l a t o r y stage atretic follicles i nfish f o l l i c l e s was s i m i l a r t o y i n j e c t e d w i t h LH. I n s h o r t , the t e s t s were p o s i t i v e f o r 173-hydroxysteroid dehydrogenase as w e l l as 3a-, and 17a-hydroxysteroid dehydrogenases. - 33- However, these stage I p o s t - o v u l a t o r y c o r p o r a l u t e a w i t h complete s t e r o i d s y n t h e s i z i n g c a p a c i t i e s were r a t h e r t r a n s i e n t and l a s t e d no l o n g e r than a week. ovulation hydroxysteroid Seven days a f t e r dehydrogenase a c t i v i t y had d e f i n i t e l y d e c l i n e d . B a r a (1965) demonstrated weak 33-hydroxysteroid dehydrogenases i n t h e theca of post-ovulatory gested follicles t h a t stage I p o s t - o v u l a t o r y o f Scomber scomber and t h i s a l s o sugf o l l i c l e s were capable of steroid hormone s y n t h e s i s . The main d i f f e r e n c e between the p o s t - o v u l a t o r y b o d i e s described here and the mammalian corpus luteum i s the sparse v a s c u l a r i t y o f t h e t e l e o s t corpus luteum. As i n stage 1 p o s t - o v u l a t o r y f o l l i c l e s , stage a t r e t i c f o l l i c l e s were p o o r l y v a s c u l a r i z e d . Again, the t h i s poor y 65 v a s c u l a r i z a t i o n suggested t h a t t h e hormone s y n t h e s i z e d , most l i k e l y e s t r o g e n , a c t e d l o c a l l y and were p r o b a b l y n o t r e l e a s e d i n l a r g e amounts i n t o t h e c i r c u l a t o r y system. T h i s l o c a l i z a t i o n o f s t e r o i d s would r e s u l t i n h i g h c o n c e n t r a t i o n o f s t e r o i d hormones i n the corpus luteum. High l e v e l s o f e s t r o g e n s w i t h i n t h e o v a r y might be r e q u i r e d t o 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 a t the p o i n t o f a c t i o n would p r o v i d e a mechanism f o r d i r e c t a c t i o n o f e s t r o g e n on the t a r g e t tissues without a s i m i l a r r i s e i n b l o o d e s t r o g e n w h i c h might i n h i b i t t h e production of gonadotropin. L o c a l i z e d a c t i o n s o f s t e r o i d hormone p r o v i d e a p l a u s i b l e e x p l a n a t i o n o f t h e asynchronus development o f oocytes i n the ovary. I n h i s s t u d i e s on t h e t e s t i c u l a r c y c l e of a n o t h e r amniote, t h e r e p t i l e Naja naja, L o f t s (1972) found t h a t t h e S e r t o l i c e l l s , a homologue o f t h e g r a n u l o s a c e l l s , s y n t h e s i z e d androgens w h i c h were n o t r e l e a s e d i n t o t h e p e r i p h e r a l v a s c u l a r system b u t induced cells locally. the p r o p a g a t i o n o f germ I n a d d i t i o n the S e r t o l i c e l l s o f r e p t i l e s p l a y e d an im- p o r t a n t r o l e i n the s y n c h r o n i z a t i o n and maintenance o f t h e 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 o o g o h i a . Yamazaki (1965) observed numerous o o g o n i a i n o v a r i e s o f g o l d f i s h w i t h c o r p o r a l u t e a . Though o o g o n i a a r e d e t e c t e d i n the o v a r i e s o f many d i f f e r e n t t e l e o s t s throughout the y e a r , a sharp r i s e i n number o f f r e q u e n t l y observed s h o r t l y a f t e r spawning ( C r a i g - B e n n e t t , 1930; H i c k l i n g , 1935; Matthews, 1938; Mendoza, 1943; B u l l o u g h , 1942; B a r r , 1963; Yamazaki, 1965). o f o o g o n i a a f t e r o v u l a t i o n and t h e o c c u r r e n c e The sudden i n c r e a s e o f h y d r o x y s t e r o i d dehydro- genases i n p o s t - o v u l a t o r y f o l l i c l e s s u g g e s t s some r e l a t i o n s h i p between 66 the two processes and the p o s s i b i l i t y 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 i n the number of oogonia i n ovaries of Phoxirius l a e v i s and Oryzias l a t i p e s respectively. Yamamoto (1969) has reviewed the many d i f f e r e n t studies on sex d i f f e r e n t i a t i o n i n t e l e o s t that demonstrated 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 c e l l s to form oogonia. The Fate of Corpus Luteum C e l l s 3 The incorporation of thymidine- H into r e l a t i v e l y large numbers of stage I I post-ovulatory corpora lutea c e l l s indicated that these c e l l s were a c t i v e l y 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 c e l l s or corpora lutea c e l l s . The h i s t o l o g i c a l picture of intermediate stages between stage IT corpora lutea and oogonial cysts supports the suggestion that some c e l l s i n the stage IT corpora lutea d i f f e r e n t i a t e into oogonia. This postulation that oogonia were derived from corpora lutea c e l l s of recently ovulated or resorbed ova i s supported by the sudden increase i n oogonia after ovulation or resorption of ova. Yamazaki (1965) ob- served large numbers of oogonia i n ovaries with a t r e t i c oocytes. Other 67 workers have also noted a sharp r i s e i n 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 c y c l i c a l manner ( F i g . 37) a f t e r each spawning. The reproductive cycle within the ovary. During the i n i t i a l ma- turation, primodial germ c e l l s d i f f e r e n t i a t e into oogonia which form yolk-ladened oocytes. developmental paths. A f t e r v i t e l l o g e n e s i s , oocytes follow one of two Under adverse conditions the yolk laden oocytes become a t r e t i c and develop into ing a - stage corpora lutea a t r e t i c a . Dur- this stage, f o l l i c u l a r c e l l s hypertrophy and phagocytose the a t r e t i c oocyte and form the g-stage corpora lutea which terminates the resorbtion of the aberrant oocyte. of the y s t a g e . The absorption of the oocyte marks the beginning Steroid (probably estrogen) synthesis occurs i n y-stage corpora lutea. The steroid synthesizing c a p a b i l i t y i n y-stage f o l l i c l e s were part i a l l y impaired by hypophysectomy. Under normal circumstances (with the adverse conditions removed and the p i t u i t a r y intact) the f u l l steroid synthesizing apparatus would be functioning as suggested by the occurrence of a l l four hydroxysteroid dehydrogenases i n corpora lutea of hypophysec- tomized f i s h injected with LH. Estrogen synthesized bv y-stage corpora lutea i s probably l o c a l i z e d within the 6-stage corpora lutea concerned with the d i f f e r e n t i a t i o n of 68 oogonia i n t o e-stage c o r p o r a l u t e a . r i s e to new I t i s suggested t h a t t h i s gave o o g o n i a l c y s t s t o r e p l a c e the germ c e l l s l o s t d u r i n g a t r e s i a . The m a j o r i t y of f i s h a t t a i n e d t h e i r f u l l m a t u r i t y and o v u l a t e d regularly. The o v u l a t e d f o l l i c l e s formed the stage I p o s t - o v u l a t o r y c o r p o r a l u t e a , comparable to the y-stage c o r p o r a l u t e a a t r e t i c a . f o l l i c l e s had full steroid synthesizing a b i l i t y . s i z e d e s t r o g e n s which were s t o r e d l o c a l l y . t h e s i s was lutea. completed They p r o b a b l y These synthe- By the time the s t e r o i d the c o r p o r a l u t e a developed syn- i n t o stage IT c o r p o r a E s t r o g e n s t h a t were s y n t h e s i z e d e a r l i e r , induced some stage I I c e l l s t o d i f f e r e n t i a t e i n t o oogonia. The newly d i f f e r e n t i a t e d oogonia w i t h i n the stage I I I c o r p o r a l u t e a r e p l a c e d oocytes l o s t d u r i n g o v u l a t i o n . 69 F i g u r e 37: Summary of the f a t e of p r e - and p o s t - o v u l a t o r y follicles (1) Adverse i n goldfish ovaries. c o n d i t i o n s cause y o l k y oocytes t o undergo atresia. F o l l i c u l a r c e l l s hypertrophy and became p h a g o c y t i c ; the h y p e r t r o p h i e d g r a n u l o s a a t the and 3-stages r e s o r b the a t r e t i c o o c y t e . oocyte i s removed the a t r e t i c f o l l i c l e s i n t o the Y ~ s t a S e corpus luteum. a- Once the develops T h i s has the appear- ance of an e n d o c r i n e gland and most l i k e l y s y n t h e s i z e s estrogens. The e s t r o g e n s s y n t h e s i z e d p r o b a b l y some of the d-stage i a as observed c e l l s to d i f f e r e n t i a t e i n t o i n the € - s t a g e corpus induce oogon- luteum. (2) The y o l k y oocytes n o r m a l l y mature and a r e o v u l a t e d ; the o v u l a t e d f o l l i c l e s (Stage I p o s t - o v u l a t o r y c o r p o r a lutea) synthesize steroids. duct of s t e r o i d metabolism I suspect t h a t the end i s estrogens, l o c a l i z e d with- i n the p o s t - o v u l a t o r y c o r p o r a l u t e a . These steroids p r o b a b l y induce some of the stage I I corpus luteum to d i f f e r e n t i a t e i n t o oogonia. some c e l l s i n t o oogonia e-stage corpus luteum. pro- cells This d i f f e r e n t i a t i o n of g i v e r i s e to the s t a g e I I I or 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 a r e v e r y few s t u d i e s on t h e e f f e c t s o f s t e r o i d s on o o g e n e s i s and o v a r i a n development i n s p i t e o f t h e e x t e n s i v e s t u d i e s o f t h e e f f e c t s o f 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 ( s e e r e v i e w by Yamamoto, 1969), t h e secondary s e x u a l c h a r a c t e r s and s e x u a l b e h a v i o u r ( s e e r e v i e w by L i l e y , 1969). There i s up t o now no adequate study on t h e e f f e c t s o f s t e r o i d s on o o g o n i a l m i t o s i s , though B u l l o u g h (1942) and Egami (1954) observed an abundance o f oogonia i n f i s h t r e a t e d w i t h e s t r o g e n and s p e c u l a t e d that e s t r o g e n induces i n c r e a s e oogonia f o r m a t i o n . 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 o f Phoxinus phoxinus ( B u l l o u g h , 1942), Xiphophorus maculatus ( T a v o l g a , 1 9 4 9 ) , P o e c i l i a r e t i c u l a t a ( B e r k o w i t z , 1951), and O r y z i a s l a t i p e s (Egami, 1955). The i n h i b i - t i o n o f 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 o v a r i a n r e g r e s sion. R e c e n t l y , Yamazaki (1972) demonstrated t h a t t e s t o s t e r o n e a l s o s u p p r e s s e d o v a r i a n development l e a d i n g t o f o l l i c u l a r a t r e s i a . I n 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 A i d a ejt a l . (1973) demonstrated t h a t e s t r o g e n s stimulated the l i v e r to synthesize y o l k precursors. K i r s h e n b l a t (1952) f i r s t demonstrated t h a t p r o g e s t e r o n e 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 e s t r o g e n s and androgens gave a n e g a t i v e r e s p o n s e . o f 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 c o n f i r m e d The i n d u c t i o n by Ramaswami (1962) , S u n d a r a r a j and Goswami (1966), Goswami and S u n d a r a r a j (1971) and H i r o s e (1972). I n t h i s s e c t i o n , I have examined t h e e f f e c t s o f s t e r o i d hormones on o o g o n i a l m i t o s i s , v i t e l l o g e n e s i s , m a i n t a i n e n c e o f second phase o o c y t e s and o v u l a t i o n o f mature eggs. growth 73 METHODS AND MATERIALS THE EFFECTS OF STEROIDS ON OOGONIAL MULTIPLICATION The e f f e c t s o f e s t r a d i o l , p r o g e s t e r o n e and d e o x y c o r t i c o s t e r o n e 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 t h e y had spawned, w i t h 1 yc t h y m i d i n e - H p e r 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 f o u r groups o f f i v e f i s h each. F i s h i n each of t h e f i r s t . t h r e e groups were i n j e c t e d t h r e e times per week f o r f o u r weeks w i t h 10 yg/g o f a m i c r o c r y s t a l l i n e s u s p e n s i o n of e i t h e r e s t r a d i o l , p r o g e s t e r o n e o r d e o x y c o r t i c o s t e r o n e . The f o u r t h group, t h e c o n t r o l s , were i n j e c t e d a t 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 t h e 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 a u t o r a d i o g r a p h y . O v a r i e s were r o u t i n e l y embedded i n p a r a f f i n , s e c t i o n e d ( a t 7 y) , mounted on albumen c o a t e d s l i d e s , dewaxed and h y d r a t e d t h r o u g h a s e r i e s o f alcohols. Hydrated s e c t i o n s were d i p - c o a t e d w i t h n u c l e a r t r a c k e m u l s i o n (Kodak NTB 2) i n t h e dark (Kopriwa & L e B l a n d , 1962); s l i d e s were a i r d r i e d and k e p t i n a l i g h t p r o o f box i n t h e 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 p r o c e s s e d w i t h p h o t o g r a p h i c de- v e l o p e r (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 h a e m a t o x y l i n and e o s i n (Beserga & Malamud, 1969), d e h y d r a t e d i n a l c o h o l , c l e a r e d i n x y l e n e , 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 p e r c e n t a g e o f o o g o n i a and c o r p o r a l u t e a cells 3 l a b e l l e d by t h y m i d i n e - H was based on 50 random h i g h power (x 450) f i e l d s o f v i e w from each o v a r y . The m i t o t i c f r e q u e n c y o f o o c y t e s was based on the t o t a l number o f o o c y t e s w h i c h showed m i t o t i c f i g u r e s i n f i v e c r o s s s e c t i o n s of each o v a r y . The m i t o t i c f r e q u e n c y 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 o f f o l l i c u l a r c e l l s showing f i g u r e s i n the t e n l a r g e s t o o c y t e s o f each o v a r y . mitotic The average o f f o l l i c u l a r c e l l s counted p e r o v a r y was 505 + 207. number I n a d d i t i o n 790 + 248 c o r p o r a l u t e a c e l l s , 53 + 35 o o g o n i a , and 469 + 245 o o c y t e s were counted p e r o v a r y . variance. The d a t a were a n a l y s e d s t a t i s t i c a l l y by a n a l y s i s o f The mean m i t o t i c f r e q u e n c i e s o f t h e v a r i o u s e x p e r i m e n t a l 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 ( S o k a l & R o h l f , 1969). EFFECTS OF STEROIDS ON VITELLOGENESIS Female g o l d f i s h were hypophysectomized a l l y o l k y o o c y t e s were r e a b s o r b e d . and k e p t f o r 5-6 weeks u n t i l A t t h e end o f t h i s p e r i o d , t h e y were d i v i d e d i n t o groups o f 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 m i x t u r e o f 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 s u s p e n s i o n s i n s a l i n e (0.7% NaCl) c o n t a i n i n g 2% Tween 80. was t r i t u r a t e d t o a f i n e powder i n a g l a s s homogenizer; Each s t e r o i d Tween 80 was then added and f o l l o w e d by 0.7% N a C l t o form t h e m i c r o c r y s t a l l i n e sion. suspen- 75 As i n the p r e v i o u s e x p e r i m e n t s , 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 o f s t e r o i d i n 0.1 ml o f 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 , p r e g n e n o l o n e , 17a-hydroxypregnenolone, p r o g e s t e r o n e , 17a- hydroxyprogesterone, dehydroepiandrosterone, androstenedione, e s t r a d i o l , e s t r o n e , 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 the s o l v e n t o n l y . injected with The i n j e c t i o n s c h e d u l e was once e v e r y 3 days f o r a p e r i o d of one month. A t 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 . sectomy was Completeness of hypophy- ensured by h i s t o l o g i c a l e x a m i n a t i o n of t h e heads. were f i x e d i n B o u i n ' s f i x a t i v e , d e c a l c i f i e d in paraffin. (Wiebe, 1968) The heads and embedded S e r i a l s e c t i o n s o f 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 tissue. EFFECTS OF LONG TERM STEROID INJECTIONS ON GRAVID GOLDFISH I n t h i s group o f e x p e r i m e n t s , the e f f e c t s o f c h r o n i c i n j e c t i o n s o f the f o l l o w i n g s t e r o i d s were compared: pregnenolone, p r o g e s t e r o n e , dehydroepiandrosterone, androstenedione, e s t r a d i o l , estrone, e s t r i o l , testos- t e r o n e , d e o x y c o r t i c o s t e r o n e , 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 controls. C a l c u l a t i o n of t h e p e r c e n t a g e o f y o l k y o o c y t e s t h a t were a t r e t i c was based on the assumption t h a t a l l a t r e t i c o o c y t e s were d e r i v e d from yolky oocytes. variance. The d a t a were t r e 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 o f The mean p e r c e n t a g e of a t r e t i c o o c y t e s i n d u c e d by e s t r o n e , 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 u s i n g Tukey's W- 76 procedure ( S o k a l & R o h l f , 1969). T h i s 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 t o compare t h e p e r c e n t a g e o f a t r e s i a i n t h e s e f o u r groups of e x p e r i m e n t a l f i s h were made a f t e r t h e r e s u l t s were known. EFFECTS OF STEROIDS ON OVULATION IN GRAVID GOLDFISH I n t h i s s e r i e s o f e x p e r i m e n t s , each group of f i s h was i n j e c t e d w i t h one o f t h e 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 , p r e g n e n o l o n e , 17ahydroxypregnenolone, progesterone, 17a-hydroxyprogesterone, dehydroepiandro. sterone, e s t r a d i o l , estrone, e s t r i o l , testosterone, deoxycorticosterone, c o r t i s o n e , and C o r t i s o l . As i n t h e p r e v i o u s s t u d y , groups o f 6-8 gravid g o l d f i s h were t r a n s f e r r e d t o 50 l i t e r e x p e r i m e n t a l t a n k s 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 w a t e r (12+1°C) and a d a i l y p h o t o p e r i o d o f 16 h o u r s . 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 o f t h e approp r i a t e s t e r o i d suspended i n 0.1 ml o f 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 o f 0.7% N a C l 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 a t 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 a t 9:00 a.m. t h e n e x t day. Each f i s h was h e l d i n t h e l e f t hand so t h a t t h e abdomen was not grasped. O v u l a t e d eggs were s t r i p p e d out o f t h e o v i d u c t by g e n t l y moving t h e f i r s t two f i n g e r s o f t h e r i g h t hand o v e r t h e abdomen i n t h e d i r e c t i o n of the cloaca. E x c e s s i v e f o r c e was a v o i d e d as i t w i l l r u p t u r e t h e o v a r i e s and e x p r e s s u n o v u l a t e d o o c y t e s . I f nothing or only f l u i d i s s u e d from t h e c l o a c a t h e f i s h were r e p l a c e d and t e s t e d a g a i n a f t e r 24 hours. T h i s procedure was c a r r i e d o u t d a i l y f o r 5 days a f t e r the i n i t i a l steroid injection. O v u l a t e d eggs t h a t had been s t r i p p e d i n t h i s manner were t h e n p l a c e d 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 o f the experiments the f i s h were k i l l e d by d e c a p i t a t i o n and the o v a r i e s removed f o r h i s t o l o g i c a l examination. of each f i s h was determined. The gonadosomatic index 78 RESULTS THE EFFECTS OF STEROIDS ON MITOSIS T a b l e 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 mitosis i n goldfish ovaries. c o r t i c o s t e r o n e was injected on When e s t r a d i o l , p r o g e s t e r o n e , o r deoxyinto recently ovulated f i s h together with 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 incorporated i n the DNA. I t s h o u l d be emphasized t h a t the this c e l l s l a b e l l e d by label 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 o c c u r u n t i l o o c y t e s are ready t o o v u l a t e . f u l l y mature and 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 l u t e u m c e l l s , o o g o n i a o o c y t e s ( F i g . 36, between t h e c a l and 37, 38, 39). I t was not g r a n u l o s a c e l l s o f the p o s s i b l e to The and differentiate f o l l i c u l a r layers i n autora- diography . About 10% o f 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 e x p e r i m e n t a l or c o n t r o l . p r o g e s t e r o n e , and An analysis of v a r i a n c e i n d i c a t e d d e o x y c o r t i c o s t e r o n e had no that estrogen, s i g n i f i c a n t e f f e c t on the 3 p e r c e n t a g e 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 The s i t u a t i o n , however, was where s t e r o i d s quite different twice weekly i n j e c t i o n s f r e q u e n c i e s by the m i t o t i c i n the H. corpora lutea, i n d u c e d s i g n i f i c a n t i n c r e a s e i n m i t o s e s (p < 0.01). h i g h e s t l e v e l of m i t o s e s i n c o r p o r a l u t e a the thymidine- the (3.9 f o r f o u r weeks. + 1.0%) was found The after Comparison o f mean m i t o t i c least significant difference method i n d i c a t e d that f r e q u e n c y o f e s t r o g e n 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 h i g h e r than t h o s e of p r o g e s t e r o n e o r d e o x y 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 (p < 0.01) T a b l e 8: The e f f e c t s of e s t r a d i o l , p r o g e s t e r o n e , and ovaries. Treatment •(10 yg/g i n 0.1 ml. s a l i n e ) i n goldfish % follicular cells labelled % corpora l u t e a cells labelled 5 9.0 +.1.3 1.3 + 0.3 15.5 + 6.8 0.6 + 0.2 Estrogen 5 10.5 + 1.7 3.9 + 1.0 26.1 + 6.1 0.4 + 0.3 Progesterone 5 9.6 + 2.0 2.2 + 0.4 11.0 + 4.4 0.7 + 0.1 Deoxycorticosterone 5 9.7 + 1.0 2.5 + 0.7 7.9 + 3.0 0.7 + 0.3 Control (Saline) No. g o l d fish d e o x y c o r t i c o s t e r o n e on m i t o s i s % oogonia labelled % oocyte labelled Each f i s h was i n j e c t e d w i t h 1 uc/g t h y m i d i n e - H two days a f t e r o v u l a t i o n . F i s h was t h e n 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 a p p r o p r i a t e s t e r o i d t w i c e a week, f o r f o u r weeks. A u t o r a d i o graphs of o v a r i a n s e c t i o n s were examined to c a l c u l a t e the p e r c e n t a g e o f v a r i o u s o v a r i a n c e l l t y p e s l a b e l l e d by t h y m i d i n e ^ H . * S t a t i s t i c a l s i g n i f i c a n c e a g a i n s t c o n t r o l f i s h a t 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 Comparisons o f m i t o t i c steroids. f r e q u e n c i e s i n oogonia o f 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 o f e s t r o g e n . Estrogen treated f i s h had 26.1 + 6.1% o f l a b e l l e d as compared t o 15.5 + 6.8% group o f f i s h . Progesterone i n the and d e o x y c o r t i c o s t e r o n e had no e f f e c t on o o g o n i a l m i t o s i s w h i l e d e o x y c o r t i c o s t e r o n e may inhibitory. significant be slightly I n c o n t r a s t t o the oogonia, o o c y t e s showed no r e s p o n s e t o any of the s t e r o i d s t e s t e d . Only s m a l l p e r c e n t a g e o f o o c y t e s were l a - 3 b e l l e d by t h y m i d i n e - H (0.6%) a t any THE control time. EFFECTS OF STEROID ON VITELLOGENESIS IN HYPOPHYSECTOMIZED FISH A l l the e s t r o g e n s ( e s t r a d i o l , e s t r o n e , and e s t r i o l ) induced f o r m a t i o n of y o l k v e s i c l e s i n the o v a r i e s o f hypophysectomized (Table 9 ) . I n some o o c y t e s There was randomly throughout the no i n d i c a t i o n of y o l k g r a n u l e i n the o v a r i e s o f e s t r o g e n t r e a t e d hypophysectomized E s t r a d i o l was E s t r a d i o l was t r o g e n s was goldfish the y o l k v e s i c l e s appeared i d e n t i c a l t o n o r - 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 ooplasm ( F i g . 3 8 ) . the goldfish. the most p o t e n t i n d u c e r o f y o l k v e s i c l e f o l l o w e d by e s t r o n e . formation formation. The d i f f e r e n c e between the two es- e v i d e n t i n a r e s p o n s e o f f i v e out o f s i x f i s h w i t h e s t r a d i o l compared to t h r e e out o f s i x f o r e s t r o n e and e s t r i o l . percentage of oocytes Moreover, a g r e a t e r (6.9 + 1 . 9 % ) i n any one o v a r y developed yolk v e s i - c l e s w i t h e s t r a d i o l ( F i g . 4 1 ) ; 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 o o c y t e s w i t h y o l k v e s i c l e s (5.6 + 1.6%). E s t r o g e n induced y o l k v e s i c l e (v) f o r m a t i o n i n oocytes o f hypophysectomized g o l d f i s h . This section was s t a i n e d w i t h M a l l o r y ' s t r i c h r o m e . The ovary o f hypophysectomized g o l d f i s h s t a i n e d with Mallory's trichrome. first The ovary c o n t a i n s o n l y growth phase o o c y t e s . Pregnenolone induced y o l k granule f o r m a t i o n i n the o o c y t e s o f hypophysectomized g o l d f i s h . granules ment. Only y o l k (g) were formed a f t e r pregnenolone t r e a t - The o v a r i a n s e c t i o n was s t a i n e d w i t h M a l l o r y ' s trichrome. T a b l e 9: The e f f e c t s o f v a r i o u s s t e r o i d s ( i . p . , 10 ug/g once e v e r y 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 . Steroids Body wt. (g) Saline 20.6 + 7.8 1.9 + 0.8 6 0 0 Cholesterol 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 Estradiol 23.6 + 4.3 1.5 + 0.6 6 5 0 Estrone 25.0 + 8.7 2.2 + 0.7 6 3 0 Estriol 23.9 + 2.9 1.8 + 0.8 6 3 0 M i x t u r e o f above s t e r o i d s 23.5 + 3.2 2.1 + 0.6 6 0 0 (Control G.S. ,1. No. fish No. o v a r i e s w i t h yolk vesicles No. o v a r i e s w i t h y o l k granules ^ oo 83 Pregnenolone induced the f o r m a t i o n o f y o l k g r a n u l e s without the p r i o r f o r m a t i o n o f y o l k v e s i c l e s and t h i s c u r i o u s f i n d i n g i s i n sharp c o n t r a s t to normal v i t e l l o g e n e s i s where the v a r i o u s y o l k i n c l u s i o n s are formed i n sequence w i t h t h e y o l k v e s i c l e s always a p p e a r i n g b e f o r e yolk granules. physectomized Y o l k v e s i c l e s were never observed i n o o c y t e s o f hypog o l d f i s h treated with pregnenolone. The y o l k g r a n u l e s induced by pregnenolone o u t e r membrane o f o o c y t e s ( F i g . 40). Although were found c l o s e to the they were p o o r l y d i f f e r e n - t i a t e d i n h e a m a t o x y l i n and e o s i n s t a i n e d s l i d e s , they were w e l l d e f i n e d as dark r e d g r a n u l e s w i t h M a l l o r y ' s t r i c h r o m e . showed these pregnenolone Histochemical studies induced y o l k g r a n u l e s to have the same c h e m i c a l p r o p e r t i e s as normal y o l k g r a n u l e s ( T a b l e 10); i n s h o r t they c o n t a i n e d p r o t e i n s , p h o s p h o l i p i d s and t y p i c a l n e u t r a l l i p i d s . i n t e r e s t concerns the v i t e l l i n e membrane. A further point of Under normal c o n d i t i o n s t h i s i s w e l l developed by the time t h e y o l k g r a n u l e s appear. However, i n con- t r a s t w i t h 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 e v i d e n t i n o o c y t e s w i t h y o l k g r a n u l e s induced by pregnenolone. None of the o t h e r s t e r o i d s t e s t e d appear to a f f e c t y o l k f o r m a t i o n . The o v a r i e s remain t y p i c a l of those i n hypophysectomized s i m i l a r l y the f i s h i n j e c t e d w i t h a complete showed no changes i n v i t e l l o g e n e s i s . fish mixture o f gonadal ( F i g . 39); steroids T a b l e 10. Comparison of the h i s t o c h e m i c a l s t a i n i n g p r o p e r t i e s o f pregnenolone y o l k granules w i t h those of normal v i t e l l o g e n i c o v a r i e s Techniques Periodic Normal Y o l k Granules Pregnenolone-induced Granules induced Yolk acid-Schiff's D i n i t r o f l u r o b e n z e n e method +++ +++ T e t r a z o t i z e d o - d i a n i s i d i n e method +++ +++ ++ ++ +++ +++ Sudan Black B A c i d haematin for phospholipids CO 85 Figure 41: Comparison o f t h e e f f e c t s o f e s t r a d i o l , e s t r o n e , and e s t r i o l on y o l k v e s i c l e f o r m a t i o n i n hypophysectomized g o l d f i s h . The mean gonadosomatic i n d e x of the groups o f f i s h from l e f t t o r i g h t a r e : 1.5 +0.6, 2 . 2 + 0 . 7 , and 1 . 8 + 0 . 8 A n a l y s i s of v a r i a n c e i n d i c a t e d significant differences ments . t h a t t h e r e were no between t h e d i f f e r e n t treat- 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 o f l o n g term a d m i n i s t r a t i o n o f s t e r o i d s i n t o g r a v i d fish. T a b l e 11 summarized t h e r e s u l t s o f t h e experiment t o d e t e r m i n e t h e l o n g term e f f e c t s o f s t e r o i d a d m i n i s t r a t i o n i n g r a v i d g o l d f i s h , e s p e c i a l l y on t h e maintenance o f second growth phase o o c y t e s . Though e s t r o g e n s i n d u c e t h e s y n t h e s i s and m o b i l i z a t i o n o f y o l k p r e c u r s o r s Ho & Vanstone, 1961; been r e p o r t e d U r i s t & S c h j e i d e , 1961; ( B a i l e y , 1957; P l a c k e t a l . , 1971), i t has t h a t e s t r o g e n s i n h i b i t v i t e l l o g e n e s i s and cause o v a r i a n regression. Estrone, ug/g 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 administered a t 10 t w i c e a week f o r f o u r weeks, i n d u c e d a t r e s i a i n o v a r i e s o f about 80% o f t h e s e f i s h . A n a l y s i s of variance i n d i c a t e d that a l l three e s t r o - g e n i c s t e r o i d s ( e s t r o n e , 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 p o t e n t b u t t h a t t e s t o s t e r o n e 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 t h e e s t r o g e n s (p < 0.01). About 54% o f y o l k y o o c y t e s were a t r e t i c i n t e s t o s t e r o n e f i s h as compared t o 80% w i t h t h e e s t r o g e n s ( F i g . 4 6 ) . treated The a t r e s i a ap- peared t o be s i m i l a r t o t h a t observed i n hypophysectomized f i s h b u t det a i l e d h i s t o g e n e s i s was n o t s t u d i e d . and appeared c o m p l e t e l y Some y o l k y o o c y t e s remained i n t a c t normal ( F i g . 4 2 ) . By c o n t r a s t , no e v i d e n c e o f a t r e s i a was found a f t e r treatment w i t h p r e g n e n o l o n e , p r o g e s t e r o n e , d e h y d r o e p i a n d r o s t e r o n e , a n d r o s t e n e d i o n e , deoxycorticosterone, 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 t h a n 5% o f p o s t - o v u l a t o r y corpora corpora l u t e a i n t h e i r ovaries (Table 1 1 ) , b u t t h e s e l u t e a were d i s t i n c t from t h e a t r e s i a i n d u c e d b y e s t r a d i o l , e s t r i o l or t e s t o s t e r o n e . The p o s t - o v u l a t o r y corpora estrone, l u t e a were made up 87 o f 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 follicles, e s p e c i a l l y those at the e a r l y s t a g e s , c o n t a i n e d p a r t i a l l y d i g e s t e d cytoplasmic i n c l u s i o n s ( F i g . 42). These f i n d i n g s i n d i c a t e t h a t estrogens and t e s t o s t e r o n e induce a t r e s i a and o v a r i a n r e g r e s s i o n p r o b a b l y by n e g a t i v e feedback on the p i t u i t a r y gonadotropin. discussion. T h i s h y p o t h e s i s w i l l be c o n s i d e r e d i n the 88 F i g u r e 42: S e c t i o n of an o v a r y o f a g o l d f i s h i n j e c t e d w i t h 10 ug/g o f e s t r a d i o l , t w i c e a week f o r f o u r weeks. Note t h e e x t e n s i v e a t r e s i a (arrows) i n t h e ovary. F i g u r e 43: S e c t i o n o f an ovary i n j e c t e d w i t h 10 ug/g o f deo x y c o r t i c o s t e r o n e , t w i c e a week f o r f o u r 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 t h e r e were some p o s t - o v u l a t o r y c o r p o r a l u t e a ( a r r o w ) . F i g u r e 44: Ovary o f a g r a v i d g o l d f i s h t h a t i s ready t o o v u l a t e . The ovary c o n s i s t s o f w e l l developed o o c y t e s l a t e y o l k granule F i g u r e 45: i n the stage. Ovary o f g o l d f i s h where t h e oocytes have r e a c h e d o n l y t h e l a t e y o l k v e s i c l e s t a g e and hence were n o t ready f o r o v u l a t i o n . T a b l e 11: E f f e c t s of s t e r o i d s a d m i n i s t e r e d t o g o l d f i s h f o r one month. R e s u l t s were r e c o r d e d as t h e p r e s e n c e o r absence o f a t r e s i a , more d e t a i l e d d a t a a r e p r e s e n t e d i n F i g . 46. Treatment Body Wt. (g) .G.S.I. 1 No. f i s h tested No. f i s h w i t h - a t r e t i c oocytes Saline (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 Estradiol 48.1 + 6.0 15.9 + 5.0 5 5 Estrone 41.1 + 7.4 13.5 + 3.3 5 5 Estriol 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 Corticosterone 40.1+7.5 13.3 + 7.1 5 2 2 "Gonadosomatic Index (G.S.I.) determined a t the end o f experiment 'These f i s h had s t a g e I I o r 6-stage c o r p o r a l u t e a . These were p r o b a b l y t h e r e s u l t o f o v u l a t i o n . They were d i s t i n c t from e s t r o g e n induced a t r e s i a which c o n s i s t e d m a i n l y o f « - and g-stage c o r p o r a l u t e a . ,90 F i g u r e 46: Comparison o f t h e e f f e c t s o f e s t r o n e , e s t r a d i o l , and estriol t e s t o s t e r o n e on a t r e s i a o f y o l k y o o c y t e s i n g r a v i d goldfish. The mean gonadosomatic i n d e x o f t h e groups o f f i s h from l e f t t o r i g h t a r e : 13.5 + 3.3, 15.9 + 5.0, 12.5 + 6.3, and 10.4 + 2.4. Analysis of variance i n d i c a t e d t h a t t h e r e 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 o f the t r e a t m e n t s ( p < 0 . 0 1 ) . A com- p a r i s o n o f the mean p e r c e n t a g e o f a t r e t i c o o c y t e s i n d u c e d by v a r i o u s s t e r o i d s b y Tukey's W-procedure i n d i c a t e d t h a t 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 e s t r o g e n s (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 t h e v a r i o u s e s t r o g e n treatments. Estrone Estradiol Estriol Testosterone 91 THE EFFECTS OF STEROIDS ON OVULATION IN GOLDFISH The e f f e c t i v e n e s s o f v a r i o u s s t e r o i d s u s p e n s i o n i n i n d u c i n g ovu- l a t i o n i n g r a v i d g o l d f i s h , maintained are p r e s e n t e d i n T a b l e 12. at 12 + 1°C and l o n g p h o t o p e r i o d , I t should be r e c a l l e d t h a t g o l d f i s h do not o v u l a t e under these e x p e r i m e n t a l conditions. P r o g e s t e r o n e , d e o x 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 were found t o induce o v u l a t i o n i n g r a v i d f i s h . C o r t i s o n e was the most p o t e n t of t h e s e s t e r o i d s w i t h f o u r out of the f i v e f i s h r e s p o n d i n g 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 c o r t i s o n e ; w i t h i n one on day, o v u l a t e d f i s h developed o v a r i e s w i t h p o s t - o v u l a t o r y c o r p o r a l u t e a ( F i g . 43). The most advanced oocytes i n these f i s h were i n the e a r l y y o l k granule stage. The o v u l a t e d eggs were f u l l y developed as evidence by the f a c t that when a r t i f i c i a l l y fry within five f e r t i l i z e d they developed the next day and another injection. p r o g e s t e r o n e , one o v u l a t e d on the t h i r d day a f t e r the i n i t i a l The o t h e r t h r e e d i d not o v u l a t e . l a t e d f i s h had Two o v a r i e s w i t h incomplete v i t e l l o g e n e s i s . The most advanced 45). Three of the d e o x y 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 o v u l a t e d . o v u l a t e d each o f the f i r s t steroid out of the t h r e e unovu- o o c y t e s had j u s t reached the l a t e y o l k v e s i c l e stage ( F i g l o v u l a t i o n was into days. Of the f i v e f i s h i n j e c t e d w i t h 100 ug/g on and hatched t h r e e days o f the experiment. The comparable to that i n c o r t i s o n e t r e a t e d f i s h . eggs were a l s o 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 One fish extent o f These o v u l a t e d of the two unovu- l a t e d f i s h i n t h i s group had oocytes o n l y up to the l a t e y o l k v e s i c l e T a b l e 12. The response of g r a 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 o f s t e r o i d s u s p e n s i o n d a i l y . F i s h were examined f o r o v u l a t i o n b e f o r e each i n j e c t i o n . The experiment was t e r m i n a t e d a f t e r f o u r days. Treatment No. fish Body Wt. (g) G.S.I. 1 No. f i s h ovulated No. f i s h w i t h immature o v a r ies Saline (Control) 5 39.6 +5.3 24.3 + 6.6 0 1 Cholesterol 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 Estradiol 5 36.7 + 9.4 16.5 + 4.2 0 1 Estrone 5 57.1 + 9 . 1 . 19.9 + 6.9 0 1 Estriol 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 + 47. 0 Corticosterone 5 32.0 + 6.9 12.9 + 5.9 2 1 "''G.S.I.: Gonadosomatic i n d e x , as 4.2 determined at t h e end o f the experiment. VO hp 93 s t a g e s ; the o t h e r had oocytes i n the e a r l y y o l k granule s t a g e . The t h r e e f i s h t h a t o v u l a t e d had p o s t - o v u l a t o r y c o r p o r a l u t e a i n t h e i r ovaries ( F i g . 47). Only two o f the f i s h i n j e c t e d w i t h c o r t i c o s t e r o n e o v u l a t e d . One o v u l a t e d 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 f o l l o w i n g day. None o f the o t h e r t h r e e showed any s i g n o f o v u l a t i o n d u r i n g the experiment. One of the t h r e e u n o v u l a t e d f i s h had oocytes a t the l a t e y o l k v e s i c l e stage o n l y ; the o t h e r two had oocytes i n the e a r l y y o l k granule stage w i t h a few i n the l a t e y o l k granule s t a g e . of Ovaries t h e o v u l a t e d f i s h were t y p i c a l of o v u l a t e d f i s h and c o n t a i n e d many p o s t - o v u l a t o r y c o r p o r a l u t e a and o o c y t e s i n a l l stages o f o o c y t e ment except the l a t e y o l k g r a n u l e develop- stage. The m a t u r i t y o f oocytes i n u n o v u l a t e d f i s h o v a r i e s as p r e s e n t e d i n T a b l e 12, i n d i c a t e s t h a t although one of the c h o l e s t e r o l t r e a t e d f i s h had o o c y t e s i n the l a t e y o l k v e s i c l e s t a g e , the o t h e r f i s h t r e a t e d w i t h c h o l e s t e r o l had many f u l l y developed of b e i n g o v u l a t e d . one oocytes ( F i g . 44) which were S i m i l a r l y one o f the androstenedion e s t r a d i o l t r e a t e d f i s h , one e s t r i o l capable treated f i s h , t r e a t e d f i s h and one f i s h t r e a t e d w i t h t e s t o s t e r o n e had i n c o m p l e t e l y mature o v a r i e s but the remainder o f t h e s e e x p e r i m e n t a l f i s h had o v a r i e s w i t h f u l l y developed l a t e y o l k granule In oocytes a t the stage. summary, p r o g e s t e r o n e , d e o x 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 induced i n v i v o o v u l a t i o n 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, d e h y d r o e p i a n d r o s t e r o n e , dione, e s t r a d i o l , estrone, e s t r i o l , ovulation. androstene- and t e s t o s t e r o n e had no e f f e c t on 94 III DISCUSSION T h i s study demonstrates t h a t , i n a d d i t i o n t o 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 r e v i e w by Yamamoto, 1969), s e x u a l b e h a v i o u r , and secondary s e x u a l c h a r a c t e r s (see r e v i e w by L i l e y , 1969), ovarian steroids also stimulate oogonial m i t o s i s , v i t e l l o g e n e s i s , ovulation. U n l i k e male f i s h where t e s t o s t e r o n e s t i m u l a t e s complete s p e r m a t o g e n e s i s i n hypophysectomized f i s h ( L o f t s e t a l . , 1966; and N a y y a r , 1967; their effects. Sundararaj 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 E s t r o g e n s w h i c h induced o o g o n i a l m i t o s i s had no 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 o f y o l k y o o c y t e s . d i n g l y , progesterone effect Correspon- and c o r t i c o s t e r o i d s w h i c h induced o v u l a t i o n had e f f e c t on o o g o n i a l m i t o s i s . and t h e f o r m a t i o n o f 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 g r a n u l e f o r m a t i o n . Instead s t e r o i d hormone, p r e g n e n o l o n e , induced the f o r m a t i o n o f y o l k These new no Estrogens, which s t i m u l a t e d the synthesis o f y o l k p r e c u r s o r s by the l i v e r ( P l a c k et al_ 1971) i n oocytes. and f i n d i n g s on the e n d o c r i n o l o g y another granules of oogenesis will be d i s c u s s e d i n r e l a t i o n to the r e p r o d u c t i v e b i o l o g y of the female f i s h . E f f e c t o f s t e r o i d s on o o g o n i a l m i t o s i s I t has been shown f o r the f i r s t time t h a t e s t r o g e n s oogonial mitosis i n post-ovulatory goldfish. q u i t e s p e c i f i c s i n c e progesterone The stimulated T h i s e f f e c t appears t o be and d e o x y c o r t i c o s t e r o n e had no e x t e n t o f t h i s s p e c i f i c i t y i s not known as the p r e s e n t study l i m i t e d t o these t h r e e s t e r o i d s . effect. was I n a d d i t i o n t o the s t i m u l a t i o n o f oogon- i a l m i t o s i s , e s t r o g e n s induced m i t o s i s i n 6-stage c o r p o r a 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 o o g o n i a . Our findings 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 c o r r e s p o n d i n g i n c r e a s e i n oogonia. I n s u p p o r t o f t h i s c o n c l u s i o n i t may be noted t h a t B u l l o u g h (1942) and Egami (1954) observed an abundance o f o o g o n i a i n f i s h t r e a t e d w i t h e s t r o g e n s 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 e s t r o g e n a t the time o f spawning; i n plasma e s t r o g e n may this increase be r e l a t e d to the marked i n c r e a s e i n the number o f oogonia w h i c h many workers have noted i n post-spawning Bennet, 1930; Matthews, 1938; B u l l o u g h , 1942; B a r r , 1963 fish and (CraigYamazaki, 1965). As a w o r k i n g h y p o t h e s i s , i t i s proposed that corpora l u t e a synthe- s i z e e s t r o g e n s which i n d u c e the g e n e s i s o f oogonia from c o r p o r a l u t e a t o r e p l a c e o o c y t e s l o s t d u r i n g spawning. T h i s 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 c o r p o r a l u t e a i n t e l e o s t where o o g o n i a l m i t o s i s goes on throughout the l i f e cycle. E f f e c t o f s t e r o i d s on v i t e l l o g e n e s i s . The s t u d y on t h e e f f e c t s o f 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 goldfish indicated that o n l y c e r t a i n a s p e c t s o f v i t e l l o g e n e s i s were under t h e i n f l u e n c e o f steroids. The two types o f y o l k i n c l u s i o n s were i n d u c e d by q u i t e d i f - f e r e n t s t e r o i d s ; e s t r o g e n s i n d u c e d the f o r m a t i o n of y o l k v e s i c l e s w h i l e pregnenolone i n d u c e d the f o r m a t i o n of t h e second type o f y o l k i n c l u s i o n s — the y o l k g r a n u l e s . The s e q u e n t i a l f o r m a t i o n o f t h e two t y p e s of y o l k 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 e x p e r i m e n t a l l y achieved. I t seems s t r a n g e t h a t the presence of a l l t h e o v a r i a n s t e r o i d s , as i n the experiment u s i n g a m i x t u r e o f o v a r i a n s t e r o i d s , had no effect 96 on y o l k f o r m a t i o n . However, t h e s e q u e n t i a l f o r m a t i o n o f y o l k i n c l u s i o n s may depend on t h e s e q u e n t i a l s y n t h e s i s o f s t e r o i d s o r some p r e c i s e comb 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 t h a t e s t r o g e n administration stimulated the s y n t h e s i s o f a complex p r o t e i n i n t h e l i v e r (Egami, 1955; I s h i i & Yamamoto, 1970; P l a c k e t a l . , 1971; and A i d a e t a l . , 1973). i s normally blood This p r o t e i n absent i n males and immature females b u t appears i n t h e i r f o l l o w i n g estrogen administration. T h i s complex p r o t e i n i n the b l o o d i s t h e p r e s u m p t i v e y o l k p r o t e i n ( P l a c k e_t a l . , 1971) . The uptake o f y o l k p r e c u r s o r s been s t u d i e d i n t e l e o s t s . from t h e plasma by o o c y t e s have n o t I n comparable s t u d i e s o f v i t e l l o g e n e s i s i n a m p h i b i a n s , F o l l e t t e t a l . (1968), W a l l a c e and J a r e d (1968), and Redshaw (1972) r e p o r t e d t h a t no a c t i v e u p t a k e o f y o l k p r e c u r s o r s by o o c y t e s o c curred i n the presence of high l e v e l s of estrogen. T h i s r e s p o n s e was v e r y 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 o f y o l k s o r s was o b s e r v e d . Gonadotropin treatment, u n l i k e estrogen precur- administration, s t i m u l a t e d b o t h the s y n t h e s i s o f yolk p r e c u r s o r s by t h e l i v e r and the subsequent i n c o r p o r a t i o n o f t h e s e y o l k p r e c u r s o r s i n t o oocytes. In contrast to the synthesis o f y o l k precursors immature f i s h , t h e a d m i n i s t r a t i o n o f e s t r o g e n a t r e s i a i n second growth phase o o c y t e s . i n the l i v e r of to g r a v i d f i s h induces Many w o r k e r s 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 leading t o f o l l i c u l a r a t r e s i a i n v a r i o u s f i s h : P h o x i n u s phoxinus ( B u l l o u g h , 1942), Xiphophorus m a c u l a t u s ( T r a v o l g a , 1949), P e o c i l i a r e t i c u l a t a ( B e r k o w i t z , 1951), and O r y z i a s l a t i p e s (Egami, 1954, 1955). I n a d d i t i o n , Yamazaki (1972) found t h a t 97 t e s t o s t e r o n e i n d u c e d a t r e s i a i n second growth phase o o c y t e s o f Oncorhynchus gorbuscha. Our f i n d i n g s i n d i c a t e d t h a t t h e responses t o e s t r o g e n s and t e s t o s t e r o n e were s p e c i f i c and n o t d u p l i c a t e d by pregnenol o n e , progesterone, dehydroepiandrosterone, androstenedione, d e o x 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 . D u r i n g v i t e l l o g e n e s i s , e s t r o g e n s appeared t o have d i v e r s e e f f e c t s . E s t r o g e n s s t i m u l a t e d the s y n t h e s i s o f y o l k p r e c u r s o r s by t h e l i v e r and i t s a c c u m u l a t i o n i n t h e plasma ( B a i l e y , 1957; Ho & Vanstone, U r i s t & S c h j e i d e , 1961). We observed t h a t e s t r o g e n s t i m u l a t e d t h e f o r m a t i o n o f yolk v e s i c l e s i n oocytes. The f o r m a t i o n o f y o l k v e s i c l e s i s n o t r e l a t e d to t h e s y n t h e s i s . o f y o l k p r e c u r s o r i n t h e l i v e r . of m u c o p o l y s a c c h a r i d e s of Yolk v e s i c l e s comprised and n o t l i p o p r o t e i n which i s t h e major c o n s t i t u e n t t h e y o l k p r e c u r s o r s y n t h e s i z e d by t h e l i v e r . I t i s t h e second type of y o l k i n c l u s i o n , t h e y o l k g r a n u l e s , t h a t s h a r e s t h e same c h e m i c a l comp o s i t i o n as y o l k p r e c u r s o r s d e s c r i b e d by P l a c k et_ a l . (1971). We found t h a t the d e p o s i t i o n o f y o l k g r a n u l e s i n o o c y t e s i s n o t mediated by e s t r o gens b u t by another o v a r i a n hormone, pregnenolone. Yamazaki and Donaldson (1968) have i n d u c e d complete v i t e l l o g e n e s i s i n hypophysectomized with salmon g o n a d o t r o p i n . T h i s f i n d i n g was c o n f i r m e d 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, ( S u n d a r a r a j e t a l . , 1972b). goldfish 1969) and H e t e r o p n e u s t e s fossilis The d e m o n s t r a t i o n t h a t b o t h g o n a d o t r o p i n and o v 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 g o n a d o t r o p i n i n d u c t i o n o f 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 o f O v u l a t i o n by S t e r o i d s . The p r e s e n t indicated that progesterone, deoxycorticosterone, findings c o r t i s o n e , and corti- c o s t e r o n e were e f f e c t i v e i n i n d u c i n g 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 fish. These o b s e r v a t i o n s were s i m i l a r t o those o b t a i n e d 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 t o i n v i v o o v u l a t i o n , Goswami and S u n d a r a r a j (1971) demonstrated i n v i t r o o v u l a t i o n o f o v a r i a n fragments by c o r t i c o s t e r o i d s . served that deoxycorticosterone was They ob- the most p o t e n t i n d u c e r . o n in vitro ovulation. R e c e n t l y 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 t h a t the e n d o c r i n e c o n t r o l of o v u l a t i o n a c t s by p i t u i t a r y g o n a d o t r o p i n s t i m u l a t i o n o f 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 post u l a t e d by S u n d a r a r a j and Goswami (1966). There i s no d e f i n i t i v e proof i n f a v o u r o f any o f t h e s e two p o s t u l a t i o n s . The Mechanism o f E n d o c r i n e C o n t r o l o f Oogenesis i n T e l e o s t . The i n t e r a c t i o n between p i t u i t a r y g o n a d o t r o p i n and s t e r o i d o g e n e s i s , as suggested by our f i n d i n g s , p r e s e n t a p l a u s i b l e mechanism whereby one t r o p i n , as i s w i d e l y a c c e p t e d i n t e l e o s t (Burzawa-Gerard and 1972; Donaldson e t a l . , 1972) or a t most two The Fontaine, g o n a d o t r o p i n s , c o n t r o l the d i v e r s e t e m p o r a l changes i n o v a r i a n f u n c t i o n s d u r i n g the cycle. gonado- reproductive 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 s y n t h e s i s through the complex s t e r o i d metabolic pathway ( F i g . 48) may be brought about by the t i o n o r 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. inhibi- 99 F i g u r e 48: The major s t e r o i d m e t a b o l i c pathways i n t e l e o s t ovaries. Figure 48i The m a j o r steroid metabolic pathways i n teleost ovaries. ft Cholasftrol Pwgwnolone Progasterona fro-Hy4-oxypre nenolon<> fforHydroxyprogasti 8 Oahydrcepiondrosterone Androttenadiona Oaoxycorticosferona ll-Deoxycortijol Tastostercna Cortiewterone Cortisol ll^-Hydroxytestosterona H 0 Estrona Esfrodiol-17^ Ealriot ll-KetotesteMerone Dehydrocorticostarone Cortisona 100 At the i n i t i a l stages o f t e l e o s t r e p r o d u c t i v e c y c l e induces the s y n t h e s i s o f e s t r o g e n s which f i r s t gonadotropin induce the differentia- t i o n o f germ c e l l s i n t o oocytes and l a t e r the f o r m a t i o n o f y o l k v e s i c l e s w i t h i n oocytes. Some of the o v a r i a n e s t r o g e n s a r e r e l e a s e d i n t o the c i r c u l a t o r y system where they induce the l i v e r to s y n t h e s i z e y o l k precursors. E s t r o g e n s w i l l be s y n t h e s i z e d u n t i l they r e a c h a t h r e s h o l d l e v e l which i s m a i n t a i n e d by n e g a t i v e feedback gonadotropin. and Marcus, i n t e r a c t i o n with p i t u i t a r y I f as i n mammalian and b a c t e r i a l enzyme systems 1955; Goldman, 1965), (Talalay 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 o v a r i e s , then an a c c u m u l a t i o n o f pregnenol o n e , 17 <* -hydroxypregnenolone, The and d e h y d r o e p i a n d r o s t e r o n e w i l l i n c r e a s e d s y n t h e s i s of pregnenolone result. at t h i s stage w i l l induce d e p o s i t i o n of the y o l k p r e c u r s o r s i n o o c y t e s as y o l k g r a n u l e s . the The l o - c a l i z e d i n h i b i t i o n o f h y d r o x y s t e r o i d dehydrogenase by e s t r o g e n s i s supp o r t e d by our o b s e r v a t i o n s o f the l a c k o f h y d r o x y s t e r o i d dehydrogenase a c t i v i t i e s i n the g r a n u l o s a of y o l k g r a n u l e stage o o c y t e s . As the i n h i b i t i o n o f 3 g - h y d r o x y s t e r o i d dehydrogenase by a l s o p r e v e n t s the s y n t h e s i s of a d d i t i o n a l e s t r o g e n s . The estrogens i n h i b i t i o n of 3 6 - h y d r o x y s t e r o i d dehydrogenase, as w e l l as the n e g a t i v e feedback of e s t r o g e n s on p i t u i t a r y g o n a d o t r o p i n w i l l be removed when the e s t r o g e n s are c a t a b o l i z e d . The removal of n e g a t i v e feedback w i l l cause a surge o f g o n a d o t r o p i n s y n t h e s i s and s e c r e t i o n , i n c r e a s e d s t e r o i d o g e n e s i s throughout h i g h l e v e l s of pregnenolone, of 3 3 - h y d r o x y s t e r o i d dehydrogenase, w i l l J . effects stimulating The during estrogen i n h i b i t i o n result i n increased synthesis "no snfi tcrtico&iicioids _ s 3 u l t i ^ , '» existing the whole m e t a b o l i c pathway. which accumulated effects ^ 101 pcLfle doe* Kerf &fitsr< 102 o f p r o g e s t e r o n e 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 o v u l a t i o n of mature oocytes. A l l the s t e r o i d o g e n i c enzymes a r e a p p a r e n t l y tion. The post-ovulatory f o l l i c l e s s y n t h e s i z e estrogens to c o r t i c o s t e r o i d s . The estrogens l u t e a induce of a new the genesis b a t c h of oogonia and dehydrogenase r e p e a t s This estrogen ovula- i n addition s y n t h e s i z e d by p o s t - o v u l a t o r y i n a c t i v a t e the s t e r o i d o g e n i c enzymes. hydroxysteroid a c t i v e during corpora at the same time i n h i b i t i o n of the r e p r o d u c t i v e c y c l e . 33- 103 GENERAL DISCUSSION The ' p i t u i t a r y i s t h e p r i m a r y e n d o c r i n e g l a n d r e g u l a t i n g r e p r o d u c t i o n s i n c e hypophysectomy e l i m i n a t e s b o t h 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 s t u d y p r o v i d e s f u r t h e r e v i d e n c e 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 strongly regulated 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 t h e s e q u e n t i a l f o r m a t i o n and d e p o s i t i o n o f two t y p e s o f 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 e p o s i t e d , f o l l o w e d s u b s e q u e n t l y by y o l k g r a n u l e s . The h i s t o c h e m i c a l s t u d y demonstrates t h a t y o l k v e s i c l e s a r e comprised o f m u c o p o l y s a c c h a r i d e s whereas y o l k g r a n u l e s a r e more complex and c o n t a i n p r o t e i n s , p h o s p h o l i p i d s and n e u t r a l l i p i d s . This y o l k types i n g o l d f i s h . i s t h e f i r s t c h e m i c a l c h a r a c t e r i z a t i o n o f two I t i s suggested t h a t t h e y o l k v e s i c l e s may be i n v o l v e d i n t h e c o r t i c a l r e a c t i o n a t f e r t i l i z a t i o n as d e s c r i b e d by Yamamoto (1961) w h i l e the y o l k g r a n u l e s s e r v e as n u t r i e n t s f o r t h e d e v e l o p ing embryo. A f t e r hypophysectomy a l l o o c y t e s c o n t a i n i n g y o l k become a t r e t i c and a r e r e s o r b e d . of 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 t h e r e s o r p t i o n dead o o c y t e s as has been suggested so o f t e n but t h i s s t u d y p r o v i d e d no c o n v i n c i n g e v i d e n c e . A f t e r the cytoplasmic i n c l u s i o n s o f a t r e t i c o o c y t e s a r e c o m p l e t e l y removed, t h e f o l l i c u l a r c e l l s develop i n t o t h e p r e - o v u l a t o r y corpus luteum. The p r e - o v u l a t o r y corpus luteum s y n t h e s i z e s s t e r o i d hormones, p r o b a b l y e s t r o g e n s s i n c e o n l y 1 7 3 - h y d r o x y s t e r o i d dehydrogenase was d e t e c t e d i n t h e y - s t a g e . 104 T h i s study p r o v i d e s the f i r s t d e f i n i t e evidence of the r u p t u r e of t h e t e l e o s t o v a r i a n f o l l i c u l a r membrane d u r i n g o v u l a t i o n p r i o r to t h e i r f o r m a t i o n i n t o corpus luteum. Post-ovulatory corpora lutea have v e r y a c t i v e 3 a - , 3 3 - , 1 7 a - , and 1 7 3 - h y d r o x y s t e r o i d s u g g e s t i n g a c t i v e s t e r o i d hormone s y n t h e s i s . dehydrogenases A f t e r a stage o f a c t i v e hormone s y n t h e s i s , t h e p o s t - o v u l a t o r y 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 t h e 6-stage p r e - o v u l a t o r y corpus luteum. The s t e r o i d hormones s y n t h e s i z e d by e i t h e r the p r e - o r t h e p o s t o v u l a t o r y corpus luteum p r o b a b l y induce some of t h e c o r p o r a l u t e a l to form the next b a t c h o f oogonia. cells T h i s f o r m a t i o n o f oogonia from corpus luteum c e l l s i s s t r o n g l y supported by a u t o r a d i o g r a p h i c evidence and has not p r e v i o u s l y been d e s c r i b e d . T h i s study a l s o demonstrated h y d r o x y s t e r o i d dehydrogenases t h e presence o f 3 a , 3 3 - , 1 7 a - , and 173- i n the g r a n u l o s a c e l l s o f a l l o o c y t e s except those a t the y o l k g r a n u l e s t a g e s . The presence o f these s t e r o i d o g e n i c enzymes suggests t h a t the g r a n u l o s a i s i n v o l v e d i n the s y n t h e s i s o f s t e r o i d hormones. Hypophysectomy i n a c t i v a t e s a l l these enzymes, they a r e r e a c t i v a t e d by l u t e i n i z i n g hormone therapy demonstrating t h a t g e n e s i s i n t h e s e t i s s u e s i s r e g u l a t e d by p i t u i t a r y The s t e r o i d s y n t h e s i z e d by t h e f o l l i c u l a r steroido- gonadotropin. c e l l s o f normal oocytes or the c o r p o r a l u t e a , b o t h p r e - and p o s t - o v u l a t o r y , a f f e c t many a s p e c t s o f oogenesis i n f i s h . A d m i n i s t r a t i o n o f exogenous s t e r o i d s indicates t h a t e s t r o g e n s i n c r e a s e oogonia f o r m a t i o n i n p o s t - o v u l a t o r y g o l d f i s h . E s t r o g e n s a l s o induce the f o r m a t i o n o f y o l k v e s i c l e s i n hypophysectomized fish. Further, y o l k granule d e p o s i t i o n i n o v a r i e s of f i s h i s s t i m u l a t e d by pregnenolone. hypophysectomized A d i f f e r e n t series of steroids (pro- 105 g e s t e r o n e and t h e c o r t i c o s t e r o i d s ) i n d u c e d o v u l a t i o n i n g r a v i d fish. The v a r i o u s p r o c e s s e s i n o o g e n e s i s a r e s t 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 s u g g e s t i n g t h a t a c e r t a i n degree o f c h e m i c a l s p e c i f i c i t y i s required f o r b i o l o g i c a l action. 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 n a t i o n f o r the endocrine c o n t r o l o f reproductive cycles. I t i s proposed t h a t t h e s t e r o i d hormones s y n t h e s i z e d by ovarian tissues act l o c a l l y . the asynchronous T h i s p r o p o s a l p r o v i d e s an e x p l a n a t i o n o f development o f o o c y t e s i n f i s h o v a r i e s . T h i s demon- s t r a t i o n o f gonadal s t e r o i d s a c t i n g d i r e c t l y on t h e o v a r i e s i s n o v e l but n o t u n i q u e ; i n comparable s t u d i e s , L o f t s e t a l . ( 1 9 6 6 ) , Sundararaj and Nayyar (1967) , Donaldson and Yamazaki (1969) and Pandey (1969) demons 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 restored fish spermatogenesis. Possible Practical Applications. T h i s s t u d y o f t h e e n d o c r i n e con- t r o l o f o o g e n e s i s and v i t e l l o g e n e s i s s u g g e s t s 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 r o d u c t i o n i n a q u a c u l t u r e u s i n g s t e r o i d hormones. The i n d u c t i o n o f o v u l a t i o n by p r o - g e s t e r o n e 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 n o m i c a l method t o supplement the current p r a c t i c e o f i n j e c t i n g eco- individual f i s h w i t h g o n a d o t r o p i n o r p i t u i t a r y e x t r a c t s ( s e e r e v i e w s by P i c k f o r d and A t z , 1957; Clements, 1968; Shehadeh, 1970; Donaldson, 1973). Pro- g e s t e r o n e o r 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 t h e w a t e r o r t h e food e n a b l i n g a g r e a t e r number o f f i s h t o be t r e a t e d s i m u l t a n e o u s l y . The economics o f t h i s type o f a p p l i c a t i o n s h o u l d be examined. Further, ster- o i d s a r e c h e m i c a l l y more s t a b l e than g o n a d o t r o p i n s and t h i s may be an i m p o r t a n t f a c t o r when c o n s i d e r i n g t h e i n t r o d u c t i o n o f t h e s e t e c h n i q u e s 106 into r u r a l aquaculture. F i s h kept i n c a p t i v i t y , e i t h e r i n ponds o r a q u a r i a , f r e q u e n t l y undergo o v a r i a n d e g e n e r a t i o n . t h e i r brood Prowse (1966) r e p o r t e d t h a t 30-60% o f s t o c k of grass c a r p , Ctenopharyngodon i d e l l a , had o o c y t e s and were of no use as spawners. suggests used on t h a t pregnenolone, and perhaps The p r e s e n t atretic investigations the c o r t i c o s t e r o i d s , can be to induce y o l k d e p o s i t i o n and o v a r i a n m a t u r a t i o n . Our s t u d i e s the long-term e f f e c t s o f s t e r o i d s on g r a v i d f i s h demonstrated pregnenolone, p r o g e s t e r o n e , and the c o r t i c o s t e r o i d s d i d not that induce a t r e s i a whereas e s t r o g e n s and t e s t o s t e r o n e cause e x t e n s i v e a t r e s i a of yolky oocytes. trogens may T h i s adverse e f f e c t o f l o n g term a d m i n i s t r a t i o n o f e s - e x p l a i n the o v a r i a n d e g e n e r a t i o n 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 w i t h salmon gonadotropin and e s t r o g e n f o r an extended p e r i o d . T h i s study opens up a l o g y which may new area i n t e l e o s t reproductive endocrino- have s e v e r a l p r a c t i c a l a p p l i c a t i o n s . on the economics i s r e q u i r e d and However, r e s e a r c h the p o s s i b l e s p e c i e s d i f f e r e n c e should be t h o r o u g h l y i n v e s t i g a t e d b e f o r e t h i s e n d o c r i n e m a n i p u l a t i o n o f r e p r o d u c t i o n can be a p p l i e d commercially. hormones on. metabolism The p o s s i b l e e f f e c t s o f these and growth s h o u l d a l s o be investigated. 107 REFERENCES Ahsan, S.N., and Hoar, W. S. 1963. hormones on t h e t h r e e - s p i n e Some e f f e c t s o f g o n a d o t r o p i c s t i c k l e b a c k , Gasterosteus aculeatus. Can. J . Z o o l . 41: 1045-1053. A i d a , K., H i r o s e , K., Y o k o t e , M., and H i b i y a , T. 1973. Physiological s t u d i e s on gonadal m a t u r a t i o n o f f i s h e s - I I . 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