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Gonadotropin involvement in the control of oocyte maturation and ovulation in coho salmon (Oncorynchus… Van Der Kraak, Glen John 1984

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GONADOTROPIN INVOLVEMENT IN THE CONTROL OF OOCYTE MATURATION AND OVULATION IN COHO SALMON (ONCORHYNCHUS KISUTCH): NEUROENDOCRINE CONTROL OF GONADOTROPIN SECRETION, EFFECTS ON STEROID PRODUCTION AND PROPERTIES OF OVARIAN GONADOTROPIN BINDING SITES. by GLEN JOHN VAN DER KRAAK B.Sc. (Hons), The University of Manitoba, 1976 M.Sc. The University of Manitoba, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June 1984 © Glen Van Der Kraak, 1984 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an a d v a n c e d degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f ^r&OL>OC'y  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main M a l l V a n c o u v e r , Canada V6T 1Y3 Date - i -ABSTRACT The involvement of gonadotropin in the regulation of reproductive development in coho salmon (Oncorhynchus kisutch) was studied by monitoring changes in plasma gonadotropin and steroid hormone levels and the induction of oocyte maturation and ovulation following the administration of gonadotropin releasing-hormones (Gn-RH). Further studies examined the properties of gonadotropin binding sites in ovaries from adult coho salmon and immature chum salmon (0. keta). Intraperitoneal injections of luteinizing hormone-releasing hormone (LH-RH), i ts active analog des-GlylO[D-Ala6]LH-RH-ethylamide (LH-RHA DAIa&) and chum salmon Gn-RH caused an increase in plasma gonadotropin levels. LH-RHA DAla^, which had a more prolonged effect on the maintenance of elevated plasma gonadotropin levels than LH-RH and chum salmon Gn-RH, induced oocyte maturation and ovulation. Intraperitoneal injections of pimozide, a dopamine receptor antagonist, also increased plasma gonadotropin levels suggesting that endogenous dopamine may inhibit gonadotropin secretion. Pimozide potentiated the effects of LH-RHA DAla& on gonadotropin release. Studies comparing the effects of part ia l ly purified salmon gonadotropin (SG-G100) and LH-RHA DAla^ on the induction of ovulation demonstrated that the induction of ovulation was dependant on the duration rather than the magnitude of the i n i t i a l increase in plasma gonadotropin levels. Plasma testosterone and 17a,206dihydroxy-4-pregnene-3-one (17a2O0P) levels were increased and 17B-estradiol levels decreased following an elevation of plasma gonadotropin levels . High levels of 17a20BP were associated with both spontaneous and gonadotropin induced oocyte maturation. Declining 178-estradiol production appears to determine the time of oocyte maturation and ovulation by having a permissive effect on 17a20BP production. - i i -The presence o f a gonadotropin receptor was demonstrated f o r the f i r s t time in the salmonid ovary. Gonadotropin binding to immature chum salmon o v a r i e s was a s a t u r a b l e process as the uptake of - ^ I - l a b e l e d salmon gonadotropin was reduced .in a dose dependant manner by unlabeled gonadotropin. Gonadotropin b i n d i n g was a t t r i b u t e d to a s i n g l e c l a s s of high a f f i n i t y binding s i t e s present i n l i m i t e d numbers. S i m i l a r s i t e s were not present i n l i v e r , kidney or muscle. The a b i l i t y of various t e l e o s t and mammalian gonadotropins to decrease t h e b i n d i n g of j - l a b e l e d salmon gonadotropin to ovarian t i s s u e was i n agreement with the a b i l i t y of these hormone preparations to s t i m u l a t e s t e r o i d p r o d u c t i o n i n v i t r o . Gonadotropin b i n d i n g s i t e s were demonstrated in thecal and granulosa c e l l l a y e r s from adult coho salmon, although the l e v e l s of binding were lower than those f o r immature chum salmon o v a r i e s . ACKNOWLEDGMENTS I wish to thank my research supervisor, Dr. E.M. Donaldson, for his constant support and encouragement. I would also l ike to express my gratitude to Dr. D.J. Randall who served as chairman of my supervisory committee at U.B.C. and the other members including Drs. R. L i l ey , D. Lyster and A.M. Perks for their constructive comments during my studies. A special thanks is extended to Helen Dye for her assistance when doing hormone assays, but more importantly for her company in the lab which made my stay at West Vancouver so enjoyable. Additionally, the patience and understanding displayed by Morva Young when typing this manuscript is gratefully acknowledged. F inal ly , I especially wish to thank my wife Debbie, whose enthusiastic support during my studies has made i t a l l worthwhile. - iv -TABLE OF CONTENTS PAGE CHAPTER 1 - INTRODUCTION 1 CHAPTER 2 - MATERIALS AND METHODS 11 A. Experimental Animals 11 B. Hormone Injections, Tissue and Blood Sampling 11 C. Stimulation of Steroid Production In Vitro 13 D. Gonadotropin Receptor Studies 14 E. Gonadotropin RIA 17 F. Steroid Hormone RIA 19 G. Radiation Counting 21 H. Stat ist ics 21 CHAPTER 3 - GONADOTROPIN CHANGES ASSOCIATED WITH SEXUAL MATURATION IN FEMALE COHO SALMON 23 A. Introduction 23 B. Experimental Protocol 23 I. Gonadotropin and Ovarian Changes During Sexual Maturation 23 II. Preovulatory Gonadotropin Changes 24 III. Effects of LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Oocyte Development 24 IV. Effects of Chum Salmon Gn-RH, LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation 25 V. Effects of LH-RHA DAla 6 and SG-G100 on Plasma Gonadotropin Levels and Ovulation 25 VI. Effects of Pimozide and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation 26 C. Results 26 I. Gonadotropin and Ovarian Changes During Sexual Maturation 26 II. Preovulatory Gonadotropin Changes 28 III. Effects of LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Oocyte Development 28 VI. Effects of Chum Salmon Gn-RH, LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation 31 V. Effects of LH-RHA DAla 6 and SG-G100 on Plasma Gonadotropin Levels and Ovulation 34 VI. Effects of Pimozide and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation 39 D. Discussion 42 CHAPTER 4 - 178-ESTRADIOL, TESTOSTERONE, AND 17a208P CHANGES ASSOCIATED WITH SEXUAL MATURATION IN FEMALE COHO SALMON 54 A. Introduction 54 B. Experimental Protocol 54 I. Preovulatory Steroid Changes 54 II. Steroid Changes in Response to LH-RH and LH-RHA DAla 6 55 - v -III. Steroid Changes in Reponse to LH-RHA DAI a 6 and SG-G100 55 IV. In vitro Steroid Production by Ovarian Fo l l i c l e s During Sexual Maturation 55 V. Effects of 17B-estradiol on 17a20BP Production in vitro 55 C. Results 56 I. Preovulatory Steroid Changes 56 II. Steroid Changes in Response to LH-RH and LH-RHA DAla 6 56 III. Steroid Changes in Response to LH-RHA DAla 6 and SG-G100 63 A. Temporal steroid changes following injections of LH-RHA DAla 6 and SG-G100 63 B. Steroid changes in relation to the time of ovulation 66 IV. In vitro Steroid Production by Ovarian Fo l l i c l e s During Sexual Maturation 73 A. 17B-estradiol 73 B. Testosterone 76 C. 17a20BP 79 V. Effects of 17B-estradiol on 17a20BP Production in vitro 81 D. Discussion 81 Preovulatory Steroid Changes 81 Steroid Changes in Response to Elevated Plasma Gonadotropin Levels 87 Steroid Changes Associated with Induced Oocyte Maturation and Ovulation 92 CHAPTER 5 - THE FUNCTIONAL PROPERTIES OF GONADOTROPIN RECEPTORS IN ADULT COHO SALMON AND IMMATURE CHUM SALMON 98 A. Introduction 98 B. Experimental Protocol 98 I. Effects of Teleost and Mammalian Gonadotropins on the Stimulation of Steroid Production In Vitro 98 II. Effects of Iodination on the Biological Act iv i ty of Gonadotropin 99 III. Properties of Gonadotropin Binding Sites in Immature Chum Salmon Ovaries 99 IV. Properties of Gonadotropin Binding Sites in Adult Coho Salmon Ovaries 101 C. Results 101 I. Effects of Teleost and Mammalian Gonadotropins on the Stimulation of Steroid Production In Vitro 103 II. Effects of Iodination on the Biological Act iv i ty of Gonadotropin 108 III. Properties of Gonadotropin Binding Sites in Immature Chum Salmon Ovaries 108 IV. Properties of Gonadotropin Binding Sites in Adult Coho Salmon Ovaries 119 D. Discussion 122 CHAPTER 6 - SUMMARY AND CONCLUSIONS 132 - vi -LITERATURE CITED 136 APPENDIX 1 - Gonadotropin RIA: Assay Validation 159 APPENDIX 2 - 173-estradiol, testosterone and 17a203P RIAs: Assay Validation 167 - v i i -LIST OF TABLES TABLE PAGE 1. Effects of LH-RH and LH-RHA DAla 6 on body weight, gonadosomatic index, oocyte diameter and oocyte maturity in coho salmon. Values represent the mean + standard error 33 2. Effects of LH-RH, LH-RHA DAla 6 and chum salmon Gn-RH on plasma gonadotropin levels and ovulation in coho samon. Gonadotropin levels (mean ± standard error) which are similar, at each sampling time, as determined by Duncan's Multiple Range Test (P > 0.05) are identified by a similar superscript. The number of f ish which had ovulated was determined on day 10 35 3. The effects of pimozide and LH-RHA DAla 6 on ovulation in coho salmon. The results represent the cummulative number of f ish in each group which ovulated 4, 6 and 8 days after injection (A) and after correction to eliminate f ish which were at an advanced stage of maturity at the time of injection (B) 43 4. Oocyte development determined 96 hr following the injection of LH-RH, LH-RHA DAla 6 or saline. Fish were assigned to specific categories which correspond to the position of the germinal vesicle. A maturation index was calculated to numerically describe the average oocyte class i f icat ion in each of the treatment groups 58 5. Effects of LH-RHA DAla 6 and SG-G100 on plasma 173-estradiol levels. At each sampling period, plasma 178-estradiol levels (mean ± standard error) which are similar as determined by Duncan's Multiple Range test (P > 0.05) are identified by a similar superscript 64 6. Effects of LH-RHA DAla 6 and SG-G100 on plasma 17a206P levels. At each sampling period, plasma 17a203P values (mean ± standard error) which are similar (P > 0.05) as determined by Duncan's Multiple Range test are identified by a similar superscript 65 7. The number of f ish which ovulated during different time periods in response to various combinations of LH-RHA DAla 6 and SG-G100 administered in a single injection or two separate injections 72 hr apart. Each treatment group contained 8 f ish 67 8. Gonadal characteristics and plasma sex steroid levels in coho salmon ut i l i zed for in vitro steroid production studies. . 77 - v i i i -LIST OF FIGURES FIGURE PAGE 1. Changes in the gonadosomatic index and average oocyte diameter during sexual maturation of coho salmon. Each value represents the mean ± standard error of measurements from 10 fish 27a 2. Changes in plasma gonadotropin levels during sexual maturation of coho salmon. Each value represents the mean ± standard error of the indicated number of samples. Gonadotropin levels in fish which contained ovulated oocytes ( • ) are reported separately from the levels in f ish which had not ovulated ( • ) 29a 3. Preovulatory changes in plasma gonadotropin levels in coho salmon. Each value represents the mean ± standard error of the indicated number of samples 30a 4. Plasma gonadotropin levels (mean ± standard error, N = 7-8) in coho salmon following single intraperitoneal injections of LH-RH (A) or LH-RHA DAla 6 (B). At each sampling period, plasma gonadotropin levels which are similar (P > 0.05) as determined by Duncan's Multiple Range Test are identified by the same superscript 32a 5. Effects of intraperitoneal injections of LH-RHA DAla 6 (A) and SG-G100 alone or in combination with LH-RHA DAla 6 (B) on ovulation in coho salmon. Values represent the cummulative percentage of the number of f ish which ovulated in each of the hormone treated groups. N = 17 or 18 36a 6. Effects of intraperitoneal injections of LH-RHA DAla 6 (A) and SG-G100 alone or in combination with LH-RHA DAla 6 (B) on plasma gonadotropin levels in coho salmon. Values represent the mean ± standard error of measurements from 8 f i sh . At each sampling period, plasma gonadotropin levels which are similar (P > 0.05) as determined by Duncan's Multiple Range Test are identified by the same superscript 38a 7. Plasma gonadotropin levels in f i sh , which ovulated by day 10 ( A ) and after day 10 ( ^ ) in response to single intraperitoneal injections of 0.02 (A) and 0.2 mg/kg LH-RHA DAla 6 (B). In each case, values represent the mean ± standard error of measurements from 4 f i sh . Gonadotropin levels in f ish ovulating by day 10 and at later times were compared using the t-test (P < 0.05*, P < 0.01**) 40a 8. Effects of pimozide and LH-RHA DAla 6 on plasma gonadotropin levels (mean ± standard error) in coho salmon. At each sampling period, plasma gonadotropin levels which are similar (P > 0.05) as determined by Duncan's Multiple Range Test are identified by the same superscript 41a - ix -9. Preovulatory changes in plasma 178-estradiol, testosterone and 17a208P levels in coho salmon. Values represent the mean ± standard error of measurements from 8 fish 57a 10. Changes in plasma 178-estradiol levels in response to single intraperitoneal injections of saline, LH-RH and LH-RHA DAla 6 . LH-RHA DAIa 6-injected f ish have been separated into two groups based on whether the f ish had completed GVBD at the 96 hr sampling. Values represent the mean ± standard error with the number of f ish per group indicated. At each sampling time, plasma 178-estradiol levels which are similar as determined by Duncan's Multiple Range Test (P > 0.05) are identified by the same superscript 60a 11. Changes in plasma 17a208P levels in response to single intraperitoneal injections of saline, LH-RH and LH-RHA DAla 6 . Additional information is provided in the legend to F ig . 10 61a 12. Changes in plasma testosterone levels in response to single intraperitoneal injections of saline, LH-RH and LH-RHA DAla 6 . Additional information is provided in the legend to F ig . 10 62a 13. Changes in plasma 178-estradiol (A) and 17a208P ( B ) levels in relation to the time of ovulation for saline- injected f i sh . Steroid levels in individual f i sh have been grouped according to the time of ovulation ( O 8-10, • 12-14, O > 14 days post injection). Values represent the mean ± standard error, where applicable, with the number of f i sh indicated in parenthesis 68a 14. Changes in plasma 178-estradiol (A) and 17a208P ( B ) levels in relation to the time of ovulation for coho salmon receiving a single injection of LH-RHA DAla 6 , SG-G100 or combined injections of LH-RHA DAla 6 and SG-G100 (see Table 4). Steroid levels in individual f ish have been grouped according to the time of ovulation ( • 6-7, O 8-10, • 12-14, O > 14 days post injection). At each sampling period, plasma steroid levels (mean ± standard error, N) which are similar as determined by Duncan's Multiple Range Test (P > 0.05) are identified by the same superscript 70a 15. Changes in plasma 178-estradiol (A) and 17a208P (B) levels in relation to the time of ovulation for coho salmon receiving two separate injections of LH-RHA DAla 6 or SG-G100 followed by LH-RHA DAla 6 (see Table 4). For additional information see the legend to F ig . 14 71a 16. Temporal changes in plasma 178-estradiol (A) and 17a20BP (B) levels in two fish which fa i led to ovulate by day 14 in response to two injections of LH-RHA DAla 6 over a 72 hr period. Values are based on measurements from one f ish receiving 0.02 mg LH-RHA DAla 6/kg ( • ) and a second f ish receiving 0.2 mg LH-RHA DAla 6/kg ( • ) 72a - X -17. In vitro 173-estradiol production by ovarian f o l l i c l e s of coho salmon at different stages of sexual maturity. Fo l l i c le s were incubated with Ringer's alone (0) or with various doses of SG-G100 for 24 hr at 10 °C . Values represent the levels of hormone in the media (mean ± standard error) based on three replicate incubations 75a 18. In vitro testosterone production by ovarian f o l l i c l e s of coho salmon at different stages of sexual maturity. Fo l l i c l e s were incubated with Ringer's alone (0) or with various doses of SG-G100 for 24 hr at 10 °C . Values represent the levels of hormone in the media (mean ± standard error) based on three replicate incubations 77a 19. In vitro 17a203P production by ovarian f o l l i c l e s of coho salmon at different stages of sexual maturity. Fo l l i c l e s were incubated with Ringer's alone (0) or with various doses of SG-G100 for 24 hr at 10 °C . Values represent the levels of hormone in the media (mean ± standard error) based on three replicate incubations. 17a20BP levels which were less than 50 pg/ml were considered to be non-detectable 80a 20. Effects 173-estradiol on the production of 17a20BP in vitro in response to graded amounts of SG-G100. Values represent the mean ± standard error based on three determinations using f o l l i c l e s characterized by a central germinal vesicle (A) and by a peripheral germinal vesicle (B) 82a 21. Time course of the effects of SG-G100 on 178-estradiol production by chinook salmon ovarian f o l l i c l e incubated in vitro at 10° (A) and 20°C (B). Values represent the mean ± standard error of the amounts of 173-estradiol released to the media based on three replicate incubations when incubated with Ringer's alone ( O ) or 1 yg/ml SG-G100 ( • ) 102a 22. Effects of various salmon gonadotropin preparations on the stimulation of 173-estradiol production by chinook salmon ovarian f o l l i c l e s . Values are expressed as the mean ± standard error of the amounts of 173-estradiol released to the media based on three replicates except for controls which were based on nine replicates 104a 23. Effects of SG-G100, ovine LH and ovine FSH on the stimulation of 17B-estradiol production by chinook salmon ovarian f o l l i c l e s incubated in v i t ro . Values represent the mean ± standard error of the amounts of 173-estradiol released to the media based on three replicate incubations 105a - xi -24. Effects of SG-G100, acetone dried pituitary powder, ovine LH, ovine FSH and hCG on 17a20gP production by coho salmon postovulatory f o l l i c l e s incubated in v i t ro . Values represent the mean ± standard error of the amounts of 17a208P released to the media based on three replicates 106a 25. Testosterone production by chum salmon testicular tissue in vitro in response to untreated and various forms of iodinated salmon gonadotropin. Values represent the mean levels of testosterone released to the media based on three replicate incubations using untreated ( • ) or 125i_]abeled ( • ) gonadotropin and gonadotropin subjected to the iodination conditions but with the iodide (O)or H 2 O 2 ( • ) omitted. The average coefficient of variation was 0.19 107a 26. Testosterone production by coho salmon ovarian f o l l i c l e s in  vitro in response to untreated and iodinated salmon gonadotropin. Values represent the levels of testosterone released to the media (mean ± standard error) based on three replicate incubations 109a 27. Total binding (open-bar) and nonspecific binding (hatched-bar) of 125i_i abeled salmon gonadotropin to immature chum salmon ovary fractions equivalent to 20 mg wet weight of tissue pel let . Tissue pellets were prepared by centrifugation of ovarian homogenates at 3000 g, 20,000 g and the supernatant f lu id from the 3000 g pellet recentrifuged at 20,000 g. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three replicate determinations 110a 28. Total binding (open bar) and nonspecific binding (hatched bar) of 125j-"labeled salmon gonadotropin to the 3000 g particulate fraction of immature chum salmon ovarian homogenates following chromatography of the label on Con A Sepharose and Sepharyl S-200. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three determinations 111a 29. Time course of specific binding of 125j_1abeled salmon gonadotropin to the 3000 g particulate fraction of immature chum salmon ovary homogenates at 4°C ( • ) , 10°C ( • ) and 20°C ( O ). Each point is the mean of duplicate determinations 113a 30. The specific binding of 12 5I-labeled salmon gonadotropin to increasing quantities of the 3000 g particulate fraction prepared from immature chum salmon ovary homogenates. Each point is the mean of duplicate determinations 114a - x i i -31. The binding of l^^ j- i a beled salmon gonadotropin to the 3000 g particulate fraction prepared from 50 mg original wet weight of ovary, l i ve r , kidney, muscle and testes from immature chum salmon. Values represent the percentage of added radioactivity bound to the tissue (mean t standard error) when incubated with 0, 0.1 and 10 yg SG-G100. Results were based on three replicates 115a 32. Effect of tissue concentration on the determination of the af f inity constant and number of gonadotropin binding sites in the immature chum salmon ovary. A. Specific binding of 125i_labeled salmon gonadotropin as a function of tissue concentration. B. Competition curves for the specific binding of 125j_-]abeled salmon gonadotropin as a function of increasing amounts of unlabled SGA-2359. C. Scatchard plot for the competition data shown in B 117a 33. The specific binding of 125I_-Jabeled salmon gonadotropin to the 3000 g particulate fraction prepared from immature chum salmon ovarian homogenates as function of increasing amounts of 125I_Tabeled salmon gonadotropin (A) and Scatchard analysis of the binding data (B). Values represent the mean of duplicate determinations 118a 34. Effects of teleost and mammalian gonadotropin preparations on the specific binding of 125j_-|abeled salmon gonadotropin to the 3000 g particulate fraction of immature chum salmon ovarian homogenates. Values were expressed as a percentage of the radioactivity speci f ica l ly bound to ovarian tissue in the absence of competitor. Result are the mean of duplicate determinations 120a 35. Total binding (open bars) and nonspecific binding (hatched bars) of 125i_iabeled salmon gonadotropin to the 3000 g particulate fraction of adult coho salmon ovaries. Binding studies were conducted at three levels of ovarian tissue (1.25, 5 and 20 mg/tube) obtained from fish at differing stages of maturity. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three determinations 121a 36. Total binding (open bars) and nonspecific binding (hatched bars) of 125i_iabeled salmon gonadotropin to intact ovarian f o l l i c l e s and isolated thecal ce l l layer (TCL) and granulosa cel l layers (GCL) from adult coho salmon at different stages of maturity. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three determinations 123a - x i i i -LIST OF APPENDIX TABLES TABLE PAGE APPENDIX I 1. Relative potency of various pituitary fractions as determined by RIA and bioassay 166 - xiv -LIST OF APPENDIX FIGURES FIGURE PAGE APPENDIX I 1. RIA (logit-1og) dose response curves for gonadotropin standard and serial dilutions of coho salmon plasma samples and pituitary extracts. Each point represents the mean of three determinations 161a 2. Recovery of known amounts of gonadotropin added to plasma samples as determined by RIA. Values represent the mean ± standard error of 4 determinations 162a 3. Testosterone production by minced testicular tissue from adult coho salmon in response to SG-G100 pretreated with normal rabbit serum (NRS) or gonadotropin antiserum (SG-RS). Values represent the mean ± standard error of 3 determinations 163a 4. Testosterone production by ovarian f o l l i c l e s from adult coho salmon in response to SGA-2360 pretreated with normal rabbit serum ( O ) or antiserum to salmon gonadotropin ( • ) . Values represent the mean ± standard error of 3 determinations.. 164a 5. RIA (logit/log) displacement curves for acetone dried pituitary powder, SG-G100, SGA-2360 and SGA-2359. Each point represents the mean of two determinations 165a APPENDIX II 1. RIA (logit-log) dose response curves for 17B-estradiol standard and dilutions of heated coho salmon plasma. Each point represents the mean of two determinations • 170a 2. RIA (logit-log) dose response curves for testosterone standard and dilutions of heated coho salmon plasma. Each point represents the mean of two determinations 171a 3. RIA (logit-log) dose response curves for 17a20BP standard and dilutions of heated coho salmon plasma. Each point represents the mean of two determinations 172a 4. Relationship between the levels of 17B-estradiol in plasma samples determined using the direct method and following diethyl ether extraction and chromatography on LH-20 Sephadex. The line represents the relationship Y = X 173a 5. Relationship between the levels of testosterone in plasma samples determined using the direct method and following diethyl ether extraction and chromatography on Celite columns. The line represents the relationship Y = X 174a XV -6. Relationship between the levels of 17a208P in plasma samples determined using the direct method and following diethyl ether extraction. The line represents the relationship Y = X 175a 7. Recovery of known amounts of 17B-estradiol added to plasma samples prior to heating as determined by RIA. Each point represents the mean ± standard error of three determinations 176a 8. Recovery of known amounts of testosterone added to plasma samples prior to heating as determined by RIA. Each point represents the mean ± standard error of three determinations 177a 9. Recovery of known amounts of 17a208P added to plasma samples prior to heating as determined by RIA. Each point represents the mean ± standard error of three determinations 178a - 1 -CHAPTER 1 - INTRODUCTION Reproduction in teleosts is controlled by the hypothalamic-pituitary-gonadal axis (see Peter, 1983a; Donaldson and Hunter, 1983; Idler and Ng, 1983; Fostier et  a l . , 1983). The predominant pituitary hormone regulating gonadal function in teleosts is gonadotropin. Gonadotropin is a glycoprotein with a molecular weight of about 30,000 and consists of two subunits (Donaldson, 1973; Burzawa-Gerard, 1982; Idler, 1982; Idler and Ng, 1983) as do the gonadotropins in other vertebrate classes (Licht et a l • , 1977a). In female teleosts, gonadotropin stimulates cAMP production in the ovary, steroidogenesis, oocyte maturation and ovulation (see Idler, 1982; Idler and Ng, 1983). Idler and associates have identified a second pituitary hormone direct ly involved in gonadal development which is distinguishable from the glycoprotein gonadotropin on the basis of a lower carbohydrate content and in terms of i ts biological act iv i ty (see Idler, 1982; Idler and Ng, 1983). This hormone termed the low carbohydrate or vitellogenic gonadotropin participates in vitellogenesis by promoting the uptake of the yolk precursor vitellogenin into oocytes (Ng and Idler, 1983). Recent data suggests that the glycoprotein gonadotropin may also mediate vitellogenin uptake (Sundararaj et a l . , 1982; Breton and Derrien-Guimard, 1983). In the present context the term gonadotropin is taken to refer to the glycoprotein gonadotropin. The diverse functions controlled by gonadotropin result from its stimulatory effects on the production of ovarian hormones which in turn mediate vitellogenin synthesis, oocyte maturation and ovulation. Estrogens, in particular 176-estradiol, act in the l iver to stimulate the production of vitellogenin (see Wallace and Selman, 1981; Wiegand, 1982; Ng and Idler, 1983). An increase in plasma 17B-estradiol levels during vitellogenesis has been correlated with the growth of vitellegenic oocytes (see Fostier et a l . , 1983). Furthermore, - 2 -gonadotropin stimulates the production of 173-estradiol in vivo (Crim and Idler, 1978; B i l lard et a l . , 1978) and in vitro (Yaron and Barton, 1980; Kagawa et a l . , 1982a,b, 1983; Zohar et a l . , 1982). Progestins, in particular 17a,203dihydroxy-4-pregnene-3-one (17a208P), have been identified as the most potent maturation inducing steroids in the majority of teleost species (see Jalabert, 1976; Goetz, 1983). High levels of 17a208P were found in the incubation media when rainbow trout (Fostier et a l . , 1981a) and amago salmon (Young et a l . , 1982a) f o l l i c l e s were induced to mature in response to gonadotropin in v i t ro . Additionally, high levels of 17a203P were found in the plasma of Atlantic salmon, coho salmon and rainbow trout induced to ovulate following injections of crude pituitary extracts (Wright and Hunt, 1982; Scott et a l . , 1983). These findings coupled with the high levels of 17a203P in the plasma of salmonids during the spawning period (Idler et a l . , 1960; Campbell et a l . , 1980; Fostier et a l . , 1981b; Fostier and Jalabert, 1982; Scott et a l . , 1982, 1983; Young et a l . , 1983a) are consistent with the hypothesis that 17a203P mediates oocyte maturation in salmonids. Less is known of the endocrine control of ovulation. It appears that ovulation is mediated by prostaglandins in that indomethacin (a prostaglandin synthesis inhibitor) blocks gonadotropin-induced ovulation and prostaglandins stimulate ovulation in vivo and in vitro (Stacey and Goetz, 1982; Goetz, 1983). However, the identity of the actual prostaglandin responsible for ovulation is not known (Goetz, 1983). Catecholamines may also mediate ovulation, however, i t is not known whether catecholamines influence ovulation direct ly or act indirectly by stimulating prostaglandin synthesis (Jalabert, 1976; Stacey and Goetz, 1982; Goetz, 1983). Extensive functional evidence indicates that gonadotropin secretion in teleosts is mediated by a gonadotropin releasing hormone (Gn-RH) (see Ba l l , 1981; Peter, 1982a,b, 1983a,b). Gn-RH act iv i ty has been demonstrated in crude - 3 -hypothalamic extracts of several teleost species (Breton and Weil, 1973; Crim et a l . , 1976; Crim and Evans, 1980). Additional studies in goldfish indicate that gonadotropin release is also regulated by a gonadotropin-release inhibitory factor (GRIF). The involvement of a GRIF in the control of gonadotropin secretion was suggested by the large increase in plasma gonadotropin levels in goldfish following lesions of the anterior ventral preoptic region or the pituitary stalk (Peter et  a l . , 1978; Peter and Paulencu, 1980; Nagahama and Peter, 1982). Strong evidence has been presented suggesting that dopamine acts as a GRIF in goldfish (Chang and Peter, 1983a,b; Chang et a l . , 1983). Reproductive fa i lure , prespawning mortality and asynchrony of ovulation in captive broodstock have been major problems in f ish culture operations (see Donaldson and Hunter, 1983). As a solution to these problems, attention has focused on the use of hormones to accelerate reproductive development. Crude and purified gonadotropin preparations have been used for many years to accelerate oocyte maturation and ovulation (see Harvey and Hoar, 1979; Lam, 1982; Donaldson and Hunter, 1983) but are costly owing to the collection and processing of pituitary glands. The search for a replacement to the use of gonadotropin has resulted in the testing of synthetic molecules which operate at different levels in the hypothalamic-pituitary-ovarian axis (Lam, 1982; Donaldson and Hunter, 1983). These compounds either operate at a higher level in the axis by stimulating the production and/or release of gonadotropin (e.g. antiestrogens and Gn-RH) or operate at a lower level in the axis by replacing hormones which would normally be produced in response to endogenous gonadotropin (e.g. 17a206P and prostaglandins). The most promising results have been achieved using Gn-RH. It is well established that synthetic luteinizing hormone-releasing hormone (LH-RH) and i ts "superactive" analogs stimulate gonadotropin secretion in salmonid - 4 -and cyprinid fishes (see Peter and Crim, 1979; Peter, 1982a,b, 1983a,b). However, attempts to stimulate reproductive development using Gn-RH have met with varying degrees of success. LH-RH induces ovulation in the ayu (Hirose and Ishida, 1974), goldfish (Lam et a l . , 1975, 1976) and plaice (Aida et a l . , 1978), although the need for excessively high doses or multiple injections negates the practical application of this technique. In other species, including coho salmon (Donaldson et a l . , 1981), common carp (Weil et a l . , 1980; Sokolowska, 1982; Breton et a l . , 1983), goldfish (Peter, 1982a,b; Chang and Peter, 1983a; Sokolowska et a l . , 1984) and pike (Bil lard and Marcel, 1980) attempts to induce ovulation using LH-RH and in some cases i t s active analogs have been unsuccessful. Researcher's in the People's Republic of China reported that the superactive analog des-Gly!0[D-ALa6]LH-RH-ethylamide (LH-RHA DAla 6) induces ovulation in several Chinese carp species but the conditions for the successful induction of ovulation and the rate of ovulation in control f i sh are not c learly stated (Cooperative Team for Hormone Application in Pisciculture, 1977; Fukien-Kiangsu-Chekiang-Shanghai Cooperative Group, 1977; also see Peter, 1982a; Donaldson and Hunter, 1983). The inab i l i ty to routinely use Gn-RH to accelerate reproductive development presumably relates to i t s transitory influence on gonadotropin secretion. However, few studies have attempted to evaluate the magnitude and duration of gonadotropin changes required to accelerate reproductive development. It is necessary to consider several factors when attempting to use Gn-RH to accelerate ovulation in teleosts. These include not only the dosage and type of releasing hormone, but factors which may influence the responsiveness of the pituitary to Gn-RH and the ab i l i ty of the ovary to respond to gonadotropin. Several factors must be considered when evaluating the pituitary response to Gn-RH. A major consideration is the effect of different types of Gn-RH on - 5 -gonadotropin secretion. In goldfish (Peter, 1980) and common carp (Breton et a l . , 1983), LH-RH analogs promote a longer duration increase in plasma gonadotropin levels than LH-RH. However, Crim et a l . (1981a) reported no major difference in the potency of LH-RH and several of i t s active analogs on the stimulation of gonadotropin secretion in brown trout. Studies in the coho salmon suggest that LH-RHA DAla 6 has a greater potency than LH-RH. LH-RHA DAla 6 administered following an i n i t i a l injection of par t ia l ly purified salmon gonadotropin (SG-G100) was more effective in accelerating ovulation in coho salmon than SG-G100 alone or in combination with LH-RH (Donaldson et a l . , 1981). Recent evidence indicates that the structure of teleost Gn-RH differs from LH-RH (King and M i l l e r , 1980; Barnett et a l . , 1982; Sherwood et a l . , 1983), raising the poss ib i l i ty that these molecules may have different effects on gonadotropin secretion. Sherwood et a l . (1983) isolated a hypothalamic peptide from chum salmon which is believed to represent a Gn-RH. The chum salmon Gn-RH decapeptide differs from LH-RH with respect to two amino acids; the replacement of tryptophan for leucine in position 7 and leucine for arginine in position 8. Although l i t t l e difference was found between the activity of chum salmon Gn-RH and LH-RH in the goldfish (R.E. Peter, C S . Nahorniak and M. Sokolowska, unpublished data cited in Peter, 1983b), i t is not known whether this is the case in other species. Differences in the af f inity of teleost Gn-RH receptors for teleost Gn-RH and LH-RH may account for the re lat ively high doses of LH-RH required to stimulate gonadotropin secretion in teleosts. In mammals, multiple injections or long-term infusion of Gn-RH can result in self-suppression of gonadotropin release due to pituitary desensitization (Rivier et a l . , 1978; Clayton and Catt, 1981). In addition, high doses of Gn-RH disrupts reproductive function in mammals due to a direct inhibitory action on the ovary and testis (Catt et a l . , 1980; Hsueh and Jones, 1981). Suppression of gonadotropin release has been - 6 -reported in goldfish (Peter, 1980) and carp (Sokolowska, 1982) at high doses of mammalian Gn-RH. These observations may be of importance in studies with salmonids as they tend to be less sensitive to LH-RH than carp (Crim and Evans, 1980; Crim et  a l . , 1981a; Breton et a l . , 1983) and therefore require relat ively large amounts of releasing hormone. Pituitary responsiveness to Gn-RH varies on a seasonal basis, with the most responsive period found immediately prior to spawning (Crim and Cluett, 1974; Weil et a l . , 1975, 1978). Therefore, unlike the situation when using gonadotropin, the selection of f ish with the most advanced maturity, may be an important consideration when attempting to use Gn-RH to accelerate ovulation. An additional consideration is that pituitary responsiveness to Gn-RH is diminished owing to the influence of a GRIF. In this regard, blocking the act iv i ty of dopamine by using the dopamine receptor antagonist pimozide, potentiates the pituitary responsiveness to Gn-RH in the goldfish (Chang and Peter, 1983a; Sokolowska et a l . , 1984). To be effective in promoting oocyte maturation and ovulation, Gn-RH must evoke an increase in plasma gonadotropin levels which is of sufficient magnitude and duration to stimulate 17a20BP and prostaglandin synthesis. The necessary gonadotropin changes are not well defined and may vary depending on the species and the state of maturity. In f ish which have a marked ovulatory surge of gonadotropin release such as the carp (Fish Reproductive Physiology and Peptide Hormone Research Group, 1978) and goldfish (Stacey et a l . , 1979a), i t may be necessary to duplicate this surge to induce ovulation. Salmonids by comparison show only a slow and gradual increase in gonadotropin levels during the preovulatory period with l i t t l e evidence for a surge associated with ovulation (Fostier et a l . , 1978; Jalabert et  a l . , 1978a,b; Fostier and Jalabert, 1982; Scott et a l . , 1983). In this case, the duration rather than the magnitude of the increase in circulating gonadotropin - 7 -levels may be a more important consideration. The sensi t iv i ty of the f o l l i c l e to gonadotropin as determined by the amount of gonadotropin required to induce oocyte maturation in vitro varies inversely with the state of maturity (Fostier and Jalabert, 1982; Jalabert and Fostier, 1983). Larger amounts of gonadotropin were required to induce maturation of rainbow trout oocytes obtained 4-6 weeks prior to the expected time of ovulation than for oocytes obtained one week prior to the expected time of ovulation. The sensi t ivi ty of the f o l l i c l e to gonadotropin may depend on the number of gonadotropin receptors. It is well established that the actions of gonadotropins in mammals are mediated by their binding to specific high af f inity receptors located in the plasma membrane of target ce l l s (see Catt and Dufau, 1976, 1977; Dufau and Catt, 1978; Catt et a l . , 1980). In mammals, changes in the number of gonadotropin receptors modify the sensit ivity of gonadal tissue to gonadotropin (Richards, 1979; Catt et a l . , 1979, 1980). L i t t l e is known of the properties of gonadotropin receptors in teleosts or of changes in their number during ontogeny. In one study, Cook and Peter (1980a) reported that ovaries from goldfish having completed vitellogenesis or undergoing recrudesence bound more 125i_-|abe-|ecj carp gonadotropin than ovaries from goldfish undergoing regression. Autoradiographic analysis indicated that the greatest accumulation of label within the ovary occurred in the special thecal cel l s (Cook and Peter, 1980a). These cel l s are considered to represent the major site of steroidogenesis in the ovary (Hoar and Nagahama, 1978; Nagahama, 1983). Recent work suggests that gonadotropin has two sites of action during 17a203P biosynthesis. Both the thecal and granulosa cel l layers contribute to the synthesis of 17a20SP in rainbow trout and amago salmon (Nagahama, 1983; Young et  a l . , 1983b). Gonadotropin acting on the thecal ce l l layer stimulates the production of 17a-hydroxyprogesterone which is converted to 17a203P in the - 8 -granulosa ce l l layer by a 206-hydroxysteroid dehydrogenase (20B-HSD). Young et a l . (1983b) reported that gonadotropin increases the act ivi ty of 20B-HSD in isolated granulosa ce l l layers from the amago salmon. These results suggest that gonadotropin receptors may be present in the thecal and granulosa ce l l layers. The amount of gonadotropin required to induce oocyte maturation and ovulation may also depend on the biosynthetic capacity of the f o l l i c l e at the time of injection. Although l i t t l e is known of the prostaglandin changes in f i sh which ovulate spontaneously (see Goetz, 1983), 17a20BP levels in salmonids remain low until a few days prior to ovulation (Fostier et a l . , 1981b; Fostier and Jalabert, 1982; Scott et a l . , 1983; Young et a l . , 1983a). The poss ib i l i ty exists that attempts to accelerate ovulation may f a i l as the f o l l i c l e has only a limited capacity to produce 17a20BP and prostaglandins. Two injections of crude pituitary extracts over a 72 hr period have been shown to increase 17a206P production and induce ovulation in rainbow trout, Atlantic salmon and coho salmon (Scott et a l . , 1982; Wright and Hunt, 1982). In these cases, 17a20BP levels remain low for several days following injection, which presumably reflects the time lag to augment the act iv i ty of steroid converting enzymes in the f o l l i c l e . Previous studies have shown that 17a20BP synthesis depends on mRNA and protein synthesis as actinomycin D and puromycin block the gonadotropin induced maturation of rainbow trout oocytes in  vitro (Jalabert, 1976). This delay may be an important consideration when using Gn-RH in that injections of crude pituitary extracts or gonadotropin preparations which induce ovulation generally result in a much larger increase in plasma gonadotropin levels than injections of Gn-RH (see Bieniarz et a l . , 1980; Breton et  a l . , 1983). In addition to considering the effects of gonadotropin on 17a20BP production, attention must be given to other steroid hormones which may influence oocyte - 9 -maturation and ovulation. The preovulatory period in rainbow trout, amago salmon and coho salmon in characterised by declining plasma 173-estradiol levels and high testosterone levels (Fostier and Jalabert, 1982; Sower and Schreck, 1982; Scott et a l . , 1983; Kagawa et a l . , 1983). Although 176-estradiol is ineffective and testosterone has a limited effectiveness in promoting oocyte maturation (Jalabert, 1975; Young et a l . , 1982a), the maintenance of low 173-estradiol and high testosterone levels may contribute to the proper steroid environment for 17a203P synthesis and ovulation. It has been proposed that declining plasma 173-estradiol levels may determine the time of ovulation by f ac i l i t a t ing the release of gonadotropin necessary for 17a203P production and ovulation (Fostier et a l . , 1978; Scott et a l . , 1983). 173-estradiol may also direct ly influence the production of 17a203P. High levels of 173-estradiol reduce the effectiveness of gonadotropin on the promotion of oocyte maturation in vitro (Jalabert, 1975; Theofan, 1981 cited in Goetz, 1983; Jalabert and Fostier, 1983). The basis of this action is poorly understood, although in amphibians, 173-estradiol reduces the conversion of pregnenelone to progesterone (Speigel et a l . , 1978). L i t t l e is known of the involvement of 173-estradiol during induced ovulation. As gonadotropin stimulates 178-estradiol production during vitellogenesis (see above) the control of estrogen biosynthesis during attempts to accelerate ovulation may be c r i t i c a l . The major objective of this thesis has been to investigate the involvement of gonadotropin in the regulation of ovarian function in adult coho salmon. The possible use of mammalian and chum salmon Gn-RH as a means of accelerating reproductive development was investigated by studying their effects on plasma gonadotropin levels and ovulation (Chapter 3). Additionally, plasma gonadotropin levels in f i sh injected with LH-RHA DAla 6 and salmon gonadotropin were compared with the levels in f ish which ovulate spontaneously to evaluate the importance of - 10 -the magnitude and duration of the increase in gonadotropin levels on ovulation. The significance of steroid hormone changes during the preovulatory period was investigated by studying the profiles of 178-estradiol, testosterone and 17a20BP in the plasma of f ish during spontaneous and induced ovulation (Chapter 4). Further studies were conducted to determine the sequence of changes in steroid biosynthesis during development by comparing the ab i l i ty of ovarian f o l l i c l e s of various stages of maturity to produce these steroids in response to gonadotropin in v i t ro . F ina l ly , Chapter 5 deals with an investigation of the properties of gonadotropin receptors in ovarian f o l l i c l e s from adult coho salmon and immature chum salmon. - 11 -CHAPTER 2 - MATERIALS AND METHODS A. Experimental Animals Chinook and chum salmon were obtained at ages 2-3 years from stocks raised at the West Vancouver Laboratory, Dept. of Fisheries and Oceans. These f ish were held outdoors in 3 m-diameter fiberglas tanks supplied with aerated saltwater at 8 - l l ° C . Fish were fed Oregon Moist Pellets (Moore-Clarke Co. L t d . , La Conner, Wash.) twice daily to satiation. Female chinook salmon used in experiments weighed about 1.5 kg and had an average oocyte diameter of 1.5-2.5 mm. Chum salmon weighed 400-600 g and contained oocytes of 0.6-1.0 mm in diameter. Adult coho salmon were obtained from the Capilano Salmon Hatchery following their anadromous migration. Fish were transferred to the West Vancouver Laboratory and held outdoors in 3m-diameter fiberglas tanks supplied with aerated freshwater at 10 °C . Adult coho salmon were starved as they do not feed during this stage of their l i fecycle . B. Hormone Injections, Tissue and Blood Sampling Adult coho salmon were acclimated to the laboratory conditions for a minimum of four days prior to blood sampling or hormone injections. During a l l handling procedures, f i sh were anaesthetized by immersion in 0.02% 2-phenoxyethanol. Fish that were to receive hormone injections were screened on the day prior to injection to determine sex and maturity on the basis of the morphology of oocytes which were expelled following abdominal massage. Only those f ish which had not undergone germinal vesicle breakdown (GVBD) were used for hormone injection. At this time, individual f i sh were weighed and identified by means of a coded numbered tag inserted into the musculature below the dorsal f in (Floy Tag and Manufacturing Inc., Seattle, Washington). LH-RH and LH-RHA DAla 6 were purchased from Peninsula Laboratories Inc., San - 12 -Carlos, Cal i fornia or provided by Syndel Laboratories L t d . , Vancouver. Chum salmon Gn-RH was provided by Drs. J . Rivier and W. Vale, The Salk Institute, La Jol1 a, California and part ia l ly purified chinook salmon gonadotropin (SG-G100; Donaldson et a l . , 1972; Donaldson, 1973) was provided by Dr. E.M. Donaldson, West Vancouver, Brit ish Columbia. These hormones were dissolved in 0.65% saline immediately prior to injection. Pimozide (Janssen Pharmaceuticals L t d . , Beerse, Belgium) was obtained as a gift from Dr. R.E. Peter, University of Alberta, Edmonton, Alberta. Pimozide was suspended in saline containing 0.1% sodium metabisulphite. Hormones were administered intraperitoneally at the base of the pectoral fins using a 1 - ml tuberculin syringe (needle size; 22G). Injections volumes were adjusted to 0.4ml/kg bw. Details of the dosage and injection schedule are provided in the protocols to the experiments in Chap. 3. Blood was removed from the caudal vessels using a heparinized 1 or 3 ml tuberculin syringe (needle size; 21G). Blood samples (generally 0.75 ml) were expelled into 1-ml polypropylene centrifuge tubes and held on ice prior to centrifugation at 4000 rpm for 10 min at 4°C (Sorval, Model RS-5). The plasma was then divided among several tubes and stored frozen at - 4 0 ° C . Oocyte maturity was determined according to the c r i t e r i a established by Jalabert et a l . (1978b). In l ive f i sh , oocyte maturity was assessed on the basis of oocytes expelled by abdominal massage. At terminal samplings, oocyte maturity was assessed by visual examination of oocytes within the ovary after the f ish had been k i l l e d . Ovulation was indicated by the release of a stream of oocytes following the application of gentle pressure to the abdomen. In most experiments, the gonosomatic index (GSI) was calculated, based on the ovary weight expressed as a percentage of the total body weight. The average oocyte diameter was calculated on the basis of 10-20 oocytes. - 13 -C. Stimulation of Steroid Production In Vitro Chinook salmon at the mid-vitellogenic stage of development were k i l l ed by decapitation and the ovary quickly removed, weighed and placed in cold f i sh Ringer's (Donnen, 1976). Ten individual oocytes with intact f o l l i c l e layers ( fo l l ic les ) were transferred to borosilicate glass culture tubes together with 200 yl of f i sh Ringer's. Hormones to be tested were dissolved in f ish Ringer's and added to the incubation tubes in 200 yl aliquots. Incubations were routinely conducted for 24 hr in a water bath at 10°C in an atmosphere of 95% 02:5% C O 2 . Following incubation, 600 yl of Ringer's containing 0.1% gelatin was added, the media collected by aspiration and stored frozen at -40°C prior to 178-estradiol measurement by RIA. Results were expressed as the amount of 17B-estradiol produced per tube. A similar approach was used to examine the effects of gonadotropins on steroid production by f o l l i c l e s obtained from adult coho salmon. These studies were based on the incubation of 5 f o l l i c l e s per ml Ringer's, owing to their large size. Additionally, postovulatory f o l l i c l e s were dissected free from surrounding ovarian tissue and incubated in a similar fashion. In these studies, the amounts of 17B-estradiol, testosterone and 17a20BP released to the media were determined by RIA. Studies were also conducted to evaluate the effects of gonadotropins on steroid production by testis pieces from adult coho salmon incubated in v i t ro . Testis were placed in cold f ish Ringer's, minced with scissors into small pieces and divided into groups containing about 10 mg of tissue. The testis pieces were then rinsed with fresh Ringer's and incubated together with gonadotropin in 400 yl of Ringer's. After incubation for 20-24 hr at 10 °C , tubes were placed on ice and 600 yl of cold Ringer's containing 0.1% gelatin added. Tubes were then centrifuged - 14 -at 4000 rpm for 10 min at 4°C to compact the testicular tissue. The media were collected and stored frozen prior to testosterone determination by RIA. The pellet of testicular tissue was dried, digested with 0.1 N NaOH and analysed for protein content by the Lowry method (Lowry et a l • , 1951) using bovine serum albumin (BSA) as the standard. Results were expressed as the amount of testosterone produced per mg of protein. In most studies, gonadal tissues were incubated with Ringer's alone or with graded amounts of SG-G100 using 3-4 replicates at each hormone concentration. Hormone speci f ic i ty was examined by comparing the act iv i ty of f ish and mammalian gonadotropin preparations. Additional chinook salmon gonadotropin preparations were obtained from Syndel Laboratories. These included acetone dried pituitary powder (ADP) and two preparations (SGA-2359 and 2360) prepared in a similar fashion to that described for G-75 II gonadotropin (Idler et a l . , 1975a). These latter fractions were identified during purification on the basis of their ab i l i ty to augment cAMP production in immature rainbow trout ovaries and were estimated to have at least twice the potency of SG-G100 (Dr. T. Owen, pers. comm.). Ovine LH (NIH-LH-S-19) and ovine FSH (NIH-FSH-S-9) were obtained from the National Institutes of Health, Bethesda, Maryland) and hCG (3000 IU/MG) was obtained from Sigma, St. Louis, Missouri. D. Gonadotropin Receptor Studies Immature chum salmon were k i l l ed by decapitation and their ovaries removed and weighed. Ovaries from 2-4 chum salmon were chopped using scissors and homogenized with a loose f i t t ing glass-teflon homogenizer in 10 volumes of buffer which consisted of 0.01 M phosphate, pH 7.5 containing 0.1 M NaCl, 2.5 mM CaCl2> and 0.1% BSA. The homogenate was f i l t e red through 3 layers of cheesecloth and the f i l t r a t e centrifuged at 3000 g for 10 min at 4 ° C . The pellet was resuspended in - 15 -buffer and the centrifugation step repeated. The second pellet was resuspended at a concentration of 50-150 mg original wet weight of tissue per 0.2 ml and used for binding studies. The same procedure was used to prepare tissue pellets from kidney, l i ve r , muscle and testes for studies which examined the tissue distribution of gonadotropin binding sites. For a preliminary study to determine the distribution of gonadotropin binding sites in subcellular fractions of the ovary, tissue pellets were prepared by centrifugation of ovarian homogenates at 3000 g, 20,000 g and from the supernatant f lu id obtained at 3000 g centrifuged at 20,000 g. In this case, the tissue pellets were weighed and resuspended in buffer at a concentration of 20 mg wet weight of pellet per 0.2 ml. Adult coho salmon were k i l l e d by concussion and ovarian f o l l i c l e s separated from the connective tissue. Yolk was expressed by the application of pressure to rupture the oocyte and rinsed from the fo l l i cu l a r tissue prior to homogenization. Conditions for homogenization and centrifugation were similar to that described for immature chum salmon ovaries. The 3000 g particulate fraction was weighed and resuspended in buffer at 20 mg wet weight of pellet per 0.2 ml. In other studies, intact ovarian f o l l i c l e s were used to study the binding of 125I_-Jabeled salmon gonadotropin in a manner similar to that described for the determination of steroid production in v i t ro . Additionally, thecal and granulosa cel l layers were separated from preovulatory f o l l i c l e s as described by Kagawa et a l . (1982b) to study binding to specific ce l l types in the ovarian f o l l i c l e . 125I-"!abeled salmon gonadotropin was prepared by the lactoperoxidase method as described in Section E. For receptor binding studies, 10 yg of SGA-2359 and 0.5-1.0 mCi 125j w e r e u s e c j f o r iodination. In most experiments, the 125i_iabeled salmon gonadotropin was further purified by group specific af f inity chromatography on Con A Sepharose (Pharmacia) by modification of the procedure described by Dufau - 16 -et a l . (1972). Columns containing Con A Sepharose to a height of about 1.2 cm were prepared in disposable 5-ml syringe barrels (inside diameter 1.1 cm) f i t ted with sintered glass discs. Gonadotropin was added to the column in 400 yl of elution buffer (0.05 M phosphate buffer, pH 7.5 containing 0.85% NaCl and 0.1% bovine gamma globulin or BSA). After equilibration for 10 min, damaged gonadotropin was eluted with 6 ml of buffer. Gonadotropin was displaced from the column by the addition of 0.1 M 1-o-methyl-a-D-glucopyranoside (Sigma) in elution buffer. Fractions which eluted from the column with 2 - 3 ml of buffer containing competing sugar were used for binding studies. In an additional study, 125j_-|abeled salmon gonadotropin was subjected to f i l t r a t i o n on a much larger column (1.3 x 32 cm) containing Sephacryl S-200 (Pharmacia). Binding studies were conducted by combining 50 yl of unlabeled hormone solution or buffer alone, 50 yl of 125i_-|abeled salmon gonadotropin (40,000-60,000 cpm) and 200 yl of tissue preparation in borosilicate glass tubes. Samples were routinely incubated for 18-24 hr at 2 0 ° C . Following incubation, 1 ml of cold assay buffer was added to each tube and the tubes centrifuged at 4000 rpm for 10 min at 4 ° C . The pellets were washed by resuspension in 1 ml of cold assay buffer and the centrifugation step repeated. The f inal supernatant was decanted and the radioactivity in the pellet counted. Groups of 10 intact f o l l i c l e s or thecal and granulosa ce l l layers from preovulatory f o l l i c l e s were incubated in borsil icate glass tubes for 18 hr at 20 °C . In these studies, the incubation volume was adjusted to 1 ml by the addition of 900 yl of buffer together with 50 yl 125i_iabeled salmon gonadotropin and 50 yl of unlabeled competitor. Following incubation, the contents of the tubes were decanted on to f i l t e r paper and rinsed twice with 5 ml of incubation buffer. Ovarian tissue was removed from the f i l t e r paper and placed in new tubes for counting. - 17 -In a l l binding studies, determinations at each dose of unlabeled hormone were made in duplicate or t r ip l i ca te . Reaction tubes containing 125I_Tabeled salmon gonadotropin and ovarian tissue were incubated to determine total binding. Additional tubes containing 10 yg of SG-G100 in addition to the 1^5j_-jabe 1 ed salmon gonadotropin and ovarian tissue were incubated to determine non-specific binding. The difference between total binding and non-specific binding was designated as specific binding. E. Gonadotropin RIA Plasma gonadotropin levels were measured by RIA using an antiserum directed against chinook salmon DEAE-1 gonadotropin (Pierce et a l . , 1976) provided by Dr. J. Pierce, University of Cal i fornia , Los Angeles. SGA-2360 gonadotropin was used for iodination and chinook salmon gonadotropin (Drs. R. B i l lard and B. Breton, Institut National de la Recherche Agronomique, Jouy en Josas, France) was used as the RIA standard. Validation of this system for the measurement of gonadotropin in coho salmon plasma is provided in Appendix 1. 125i-labeled salmon gonadotropin was prepared for the RIA by either modification of the chloramine-T (Licht et a l . , 1977b) or 1actoperoxidase (Thorell and Johannson, 1972) methods. Iodination by the chloramine-T method involved combining 40 yl 0.5 M phosphate buffer pH 7.5 (PB) with 500 yCi 1 2 5 I (5 y l , Amersham) and 4 yg SGA-2360 (20 y l ) . The reaction was init iated by addition of 1 yg chloramine-T in 0.05 M PB (10 y l ) . After 12 min, the reaction mixture was diluted with 200 yl of 0.25 % BSA in 0.05 M PB and immediately chromatographed (see below). For 1actoperoxidase iodination, 40 yl of 0.5 M PB was mixed with 5 yl 125i } 20 yl gonadotropin and 10 yl 1actoperoxidase (100 IU/ml; Calbiochem, La Jo l la , Cal i fornia) . Iodination was achieved by the addition of 10 yl hydrogen peroxide (30% H 2 O 2 diluted 1:30,000). After 2 min, the addition of H 2 O 2 - 18 -was repeated. Following a second 2 min incubation, the reaction mixture was diluted with 500 yl 0.05 M PB containing 0.85% NaCl (PBS) and chromatographed. Separation of labeled gonadotropin from unreacted iodide involved chromatography through a 1 X 18 cm column containing G-50 (fine) Sephadex (Pharmacia). Prior to chromatography, the column was primed with 1 ml PBS containing 5% BSA to reduce adsorption of labeled hormone to the column and then rinsed with 20 ml PBS. The gonadotropin fractions were diluted in PBS containing 0.1% BSA and stored frozen for up to 3 weeks. The specific act ivi ty of labeled gonadotropin used in the RIA was 60-80 yCi/yg. The gonadotropin content of plasma samples measured by RIA did not differ using 125J_Tabeled gonadotropin prepared by the chloramine-T or 1actoperoxidase methods. However, the incorporation of 125j i n t o gonadotropin was more consistent when the 1actoperoxidase method was used and was the method of choice. A l l steps in the gonadotropin RIA were conducted at 4°C using PBS containing 0.1% ovalbumin as the diluent buffer. For hormone measurement, 50 yl of standard or plasma was combined with 50 yl of diluent and 200 yl of dilute antibody containing 2.5% Normal Rabbit Serum (Calbiochem). At 24 hr, 6000 cpm of 125i-labeled gonadotropin in 100 yl of diluent was added. At 48 hr, 100 yl of goat anti-rabbit gamma globulin (Calbiochem) was added. Following an additional 24 hr incubation, samples were centrifuged at 4000 rpm for 15 min. The resulting supernatants were then poured off and the pellets containing the antibody-bound hormone counted. Nonspecific binding was assessed by the same protocol except that the antibody was omitted. Non-specific binding generally represented about 5-8% of the added radioactivity. Using this format, approximately 85% of the added 125j_iabeled gonadotropin bound to excess antibody. The working dilution of antibody was adjusted so that 30-35% of the 125J_Tabeled gonadotropin was bound - 19 -in the absence of competitor. Sensit ivity of the assay was 0.05 to 0.1 ng/tube (1.0-2.0 ng/ml when using 50 yi plasma). Intraassay var iab i l i ty was 0.04-0.07 (N=6 determinations) and the interassay var iab i l i ty was 0.06 - 0.11 (N = 4 determinations). F. Steroid Hormone RIA 178-estradiol, testosterone and 17a208P were measured by RIA in plasma samples obtained from adult female coho salmon and in the media from ovary and test is incubations in v i t ro . The steroid content of plasma samples was determined direct ly and did not depend on the extraction and chromatographic isolation of individual steroids. Plasma samples (50 yl) were combined with 1 - 3 ml of assay buffer (0.05 M phosphate, pH 7.6 containing 0.1% gelatin; Scott et a l . , 1982) and heated at 70°C for 1 hr as described by Scott et a l . (1982, 1983). Aliquots of the media obtained from f o l l i c l e incubations were extracted with eight volumes of diethyl ether and dried under nitrogen. The dried extracts were dissolved in assay buffer and measured direct ly by RIA. No correction was applied for losses incurred during the extraction as the recovery of radiolabeled steroids were greater than 95%. Media from testis incubations were assayed direct ly for testosterone content. In preliminary studies, media from the incubation of ovarian f o l l i c l e s and testicular pieces were assayed using 0.05 M tris-HCl at pH 8.0 containing 0.1 M NaCl, 0.1% NaN3 and 0.1% gelatin as the RIA buffer. The validation of these protocols for steroid measurement is provided in Appendix 2. 178-estradiol and testosterone were measured using rabbit anti-178-estradiol-6(0-carboxymethyl)-oxime-BSA and antitestosterone-7acarboxymethyl-thioether-BSA serum obtained from Miles Laboratories, Rexdale, Ontario. The anti-178-estradiol serum cross reacts with 178-estradiol, 17a-estradiol, estrone, e s t r i o l , testosterone and 17a208P, at 100, 1.6, 1.4, 0.8, < 0.01, and < 0.01% levels, - 20 -respectively. The antitestosterone serum cross reacts with testosterone, 5atestosterone, 11-oxotestosterone, 178-estradiol and 17a208P at 100, 17, 4.5, < 0.01 and < 0.01% levels, respectively. Radiolabeled (2,4,6,7-3H) 178-estradiol and (1,2,6,7-3H) testosterone were obtained from Amersham. For hormone measurement, 200 yl of appropriate standard (Sigma) or diluted sample was combined with 200 yl of 3H-steroid (4000 cpm) and 200 yl of diluted antiserum. Samples were incubated at room temperature for 16-20 hr. The samples were then cooled on ice for at least 15 min prior to the addition of 200 yl of a Dextran-coated charcoal suspension containing 0.5% Norit A charcoal and 0.05% Dextran T-70 in assay buffer. After a further 10 min incubation on ice, samples were centrifuged at 4000 cpm for 10 min at 4 ° C . The resulting supernatant f lu id was poured direct ly into sc int i l l a t ion vials and combined with 6 ml of s c in t i l l a t ion f lu id (667 ml toluene, 333 ml Triton X-100, 4 g PP0 and 0.2 g dimethyl P0P0P) for counting. 17a208P was measured by RIA using sheep anti 17a208-3-carboxymethyl-oxime-BSA serum provided by Dr. A.P. Scott (Scott et a l . , 1982). This antiserum shows insignificant cross reaction with 17a-hydroxyprogesterone, progesterone, 178-estradiol and testosterone (Scott et a l . , 1982). Radiolabeled 17a208P was prepared from (1,2,6,7-3H) 17a-hydroxyprogesterone (Amersham) by treatment with 3a,208-hydroxysteroid dehydrogenase (Sigma) as described by Scott et a l . (1982). The separation of radiolabeled 17a208P from the parent compound was accomplished by chromatography on a precoated prewashed s i l i c a gel plate (LK5DF, 250 ym thickness, Whatman) developed with a dichloromethane (50 ml): diethyl ether (20 ml) mixture. I n i t i a l l y , standard 17a-hydroxyprogesterone and 17a208 (Steraloids, Wilton, New Hampshire) were chromatographed and their positions on the chromatogram identified in an iodide chamber. Chromatography of radiolabeled 17a-hydroxyprogesterone following treatment with the 3a,20B-hydroxysteroid dehydrogenase resulted in over - 21 -90% of the radioactivity on the plate migrating to the expected position of 17a208P. This material was extracted from the chromatogram and stored in absolute ethanol for direct use in the RIA. For the RIA, 100 yl of standard of diluted sample was combined with 50 y l of tracer (1500 cpm) and 50 y l of antiserum at a f inal di lution of 1:25,000. The samples were incubated at room temperature for 16-20 hr. After incubation, the samples were cooled on ice for at least 15 min before the addition of 1 ml of cold Dextran-coated charcoal suspension. After standing for 10 min, the samples were centrifuged (4000 rpm for 10 min at 4 ° C ) , the supernatant poured into sc in t i l l a t ion vials and combined with 8 ml of s c in t i l l a t ion f l u i d . The antibody concentrations used in each of the foregoing RIA procedures were adjusted so that 45-55% of the radiolabelled steroids were bound in the absence of competitor. A l l samples were analyzed in duplicate. Intraassay and interassay variation were assessed using pooled plasma samples which corresponded to approximately 75 and 35% of the total binding observed in the absence of competitor. For a l l three assays, the intraassay and interassay variations were less than 7 and 14%, respectively. G. Radiation Counting Samples containing 125j_iabeled salmon gonadotropin were counted in a Picker Pace-1 automatic gamma counter and those containing ^H-steroids were counted in a Packard Tri-Carb 460 C Liquid Sc int i l l a t ion Spectrometer. A l l samples were counted for 10 min or 10,000 counts. H. Stat ist ics Al l data were expressed as the mean ± standard error. One way analysis of variance and Duncan's Multiple Range Test were used to determine differences between plasma gonadotropin and steroid values of the same experimental group at - 22 -different sampling times or differences between the values of experimental groups at the same sampling time. In these cases, log^O transformation was used to achieve homogeneity of variance. The same approach was used to calculate differences in the effectiveness of various gonadotropin doses on the stimulation steroid production in v i t ro . Comparisons based on two treatments were analyzed by Student's t-test . The effects of hormone treatments on oocyte maturation and ovulation were compared with the saline injected group using the Mann Whitney U test. Parallel l ine s tat i s t ics were used to compute the potencies of different goadotropins on the stimulation of steroid production in vitro and from competitive inhibition studies based on RIA or receptor binding. When non-parallelism was apparent, potencies were calculated graphically from the dose giving a 50% response or were discussed separately in the text. - 23 -CHAPTER 3 - GONADOTROPIN CHANGES ASSOCIATED WITH SEXUAL MATURATION IN FEMALE COHO SALMON A. Introduction Considerable attention has focused on the development of techniques to accelerate reproductive development in Pacific salmon owing to the marked asynchrony of ovulation and high prespawning mortality of captive broodstock (Donaldson et a l . , 1982; Donaldson and Hunter, 1983). The present studies were undertaken to evaluate the poss ib i l i ty of using mammalian and teleost Gn-RH to replace f ish pituitary extracts for the acceleration of ovulation in coho salmon. In Exp. I and II, plasma samples were taken at regular time intervals during the preovulatory period to determine the pattern of gonadotropin secretion in coho salmon at the time of oocyte maturation and ovulation. The effects of LH-RH, LH-RHA DAla 6 and chum salmon Gn-RH were investigated in Exp. I l l and IV by studying their short term effects on plasma gonadotropin levels and oocyte development. The effects of LH-RHA DAla 6 alone and in various combinations with SG-G100 on plasma gonadotropin levels and the acceleration of ovulation were examined in Exp. V. The possible involvement of dopamine in the regulation of gonadotropin secretion was investigated in Exp. VI by studying the effects of intraperitoneal injections of the dopamine receptor antagonist pimozide on plasma gonadotropin levels and ovulation. B. Experimental Protocol I. Gonadotropin and Ovarian Changes During Sexual Maturation. A group of 120 adult coho salmon were held at the West Vancouver Laboratory during the 1979 spawning season. Subgroups of 10 females were bled at weekly or biweekly intervals during a seven week period which commenced on September 18. Plasma samples were analyzed for gonadotropin content. After blood sampling, fish - 24 -were k i l l ed and their ovaries removed for the determination of GSI and oocyte diameter. II. Preovulatory Gonadotropin Changes A group of 12 adult coho salmon were bled at 2-day intervals throughout the final stages of the preovulatory period. Four f ish died prior to ovulation and were not considered for analysis. Gonadotropin was measured in plasma samples obtained from 8 fish which were sampled until ovulation. The time of ovulation was used as a reference for analysis, with data presented which covers the 12 day period preceding ovulation. III. Effects of LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Oocyte Development Adult coho salmon were divided into five groups after screening to select fish with oocytes containing a central germinal vesicle. Groups of 7-8 f ish received single intraperitoneal injections of LH-RH (0.2 or 1.0 mg/kg) or LH-RHA DAla 6 (0.02 or 0.2 mg/kg). Control f i sh were injected with saline. Blood samples were taken at the time of injection (1100-1130 hr, October 31, 1980) and at increasing intervals to 96 hr for gonadotropin measurement. At 96 hr, the f ish were k i l l ed and the GSI and oocyte diameter determined. The effects of the hormone treatments on oocyte development were assessed at 1.5 hr on the basis of oocytes expelled by abdominal massage and at 96 hr by examination of oocytes in the ovary. To f ac i l i t a te comparisons between treatments, oocytes were c lass i f ied numerically according to the stage of development: 1.0 for oocytes prior to maturation, in which the germinal vesicle occupied a central position; 2.0 for oocytes undergoing germinal vesicle migration, in which the germinal vesicle had migrated to a peripheral posit ion; and 3.0 for oocytes which had completed GVBD, in which the germinal vesicle was no longer v i s ib le and the yolk had become transparent. The - 25 -arithmetic mean of these ratings was used to calculate an oocyte maturity index. IV. Effects of Chum Salmon Gn-RH, LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation Six groups consisting of 5-8 adult coho salmon were given single intraperitoneal injections of chum salmon Gn-RH, LH-RH and LH-RHA DAla 6 at 0.02 or 0.2 mg/kg. Control f i sh were injected with saline. Fish were too immature to assess oocyte development using abdominal massage at the time of injection. In preinjection samples from 8 f i sh , the average oocyte diameter was 5.1 ± 0.2 mm while the GSI was 14.7 ± 1.1%. The germinal vesicle was not v i s ib le in oocytes obtained from fish sampled at the time of injection. Gonadotropin levels were measured in the preinjection samples (10:00 hr, September 28, 1982) and in samples obtained 1.5, 24 and 48 hr following injection. Fish were checked for ovulation at 10 days post injection (October 8). V. Effects of LH-RHA DAla 6 and SG-G100 on Plasma Gonadotropin Levels and Ovulation Coho salmon were divided into nine experimental groups after an i n i t i a l screening to eliminate f ish in the f inal stages of oocyte maturation. Five groups of f ish were given a single injection at time 0 (11:00-12:00 hr, October 15, 1981). Single treatment groups included a low and high dose of LH-RHA DAla 6 (0.02 and 0.2 mg/kg), SG-G100 (0.1 mg/kg) and combined injections of SG-G100 with the low and high dose of LH-RHA DAla 6 . Three additional groups were given a second injection at 72 hr. Two of these groups were injected at time 0 and 72 hr with either the low or high LH-RHA DAla 6 dose while the third group was injected with SG-G100 followed at 72 hr with the high LH-RHA DAla 6 dose. A control group was injected with saline. Experimental groups consisted of 17-18 f i sh , eight of which were bled at various times after injection for gonadotropin determination. Blood samples were removed from 12 randomly selected fish at the time of injection. Fish - 26 -in groups receiving a single injection were bled on days 1, 2 and 3. Blood samples were taken from f i sh in a l l treatment groups on days 4, 5, 6, 8 and 10. A l l f ish were checked for ovulation on a daily basis for 8 days and subsequently at 2-3 day intervals. VI. Effects of Pimozide and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation Four groups of 8 or 9 adult coho salmon were given single injections of saline, LH-RHA DAla 6 (0.02 mg/kg), pimozide (10 mg/kg) or a combination of pimozide and LH-RHA DAla 6 . Blood samples were taken at the time of injection (10:00, October 30, 1982) and 1.5, 6, 24, 48 and 96 hr after injection for gonadotropin measurement. Fish were checked for ovulation on days 4, 6, and 8. C. Results I. Gonadotropin and Ovarian Changes During Sexual Maturation Fig . 1 shows the average oocyte diameter and GSI in coho salmon sampled from mid-September to November. The average oocyte diameter increased s ignif icantly (P < 0.01) from 4.5 ± 0.1 mm in fish sampled in September to above 6.3 mm in f ish sampled at the end of October. Similar changes were observed for the GSI, which doubled during the sampling period. The GSI increased from 10.2 ± 0.8% in September to above 21% at the end of October. The germinal vesicle was not vis ible in oocytes obtained on September 18 or October 2. By October 16, the germinal vesicle was vis ible in a central position approximately 1 mm from the oocyte surface. Fish sampled at subsequent times were at various stages of maturity, although oocyte development in individual f i sh was synchronous. These included oocytes in which the germinal vesicle had migrated towards the periphery, mature oocytes in which the germinal vesicle was not v i s ib le and the yolk had become translucent, and ovulated oocytes. No significant differences were - 27 -FIG. 1 . Changes in the gonadosomatic index and average oocyte diameter during sexual maturation of coho salmon. Each value represents the mean ± standard error of measurements from 1 0 f i sh . - 27a -OOCYTE DIAMETER (mm ) m—B - 28 -distinguished with respect to the stage of maturity and the average oocyte diameter or GSI in samples taken on October 23, 30 and November 6. F ig . 2 shows the gonadotropin levels in the plasma of female coho salmon sampled from September to November. Gonadotropin levels while similar in fish sampled on September 18 to October 16, increased (P < 0.05) at subsequent sampling times. The plasma gonadotropin concentration in one f ish which had ovulated by the October 23 sampling was 45 ng/ml (data not shown in F ig . 2). Gonadotropin levels in f ish which had not ovulated at this time were lower (mean 13.9 ± 4.1, range 4.9 - 29 ng/ml, N = 9). On October 30, the average gonadotropin concentration in the plasma of ovulated f ish was higher than the levels in non-ovulated f ish but the difference was not significant (Fig. 2). By November 6, plasma gonadotropin levels in ovulated f i sh were s ignif icantly higher (P < 0.01) than the levels in non-ovulated f i s h . II. Preovulatory Gonadotropin Changes The preovulatory changes in plasma gonadotropin levels are presented in F ig . 3. Plasma gonadotropin levels increased about 2-fold over the 12 day period preceding ovulation and peaked 2 days prior to ovulation. The gonadotropin levels 12 and 10 days prior to ovulation (6.0 ng/ml) were s ignif icantly lower (P < 0.05) than the levels at ovulation (12.2 ng/ml). Oocyte maturation, as defined by GVBD, occurred 2-4 days prior to ovulation based on the visual examination of oocytes expelled while checking for ovulation. III. Effects of LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Oocyte Development Gonadotropin levels in plasma samples taken immediately before injection were 4.5 ± 0.3 ng/ml (N = 38). Plasma gonadotropin levels in saline-injected fish did not change from this level during the 96 hr sampling period (Fig. 4). Gonadotropin - 29 -FIG. 2. Changes in plasma gonadotropin levels during sexual maturation of coho salmon. Each value represents the mean ± standard error of the indicated number of samples. Gonadotropin levels in f ish which contained ovulated oocytes ( • ) are reported separately from the levels in f ish which had not ovulated ( • ). - 29a -(|tu/6u) NldOillOaVNOO - 30 -FIG. 3. Preovulatory changes in plasma gonadotropin levels in coho salmon. Each value represents the mean ± standard error of the indicated number of samples. - 30a -DAYS PRIOR TO OVULATION - 31 -levels in groups injected with LH-RH and LH-RHA DAla 6 were s ignif icantly higher (P < 0.01) than the levels in the saline-injected group at 1.5 and 3 hr. Gonadotropin levels had returned to control levels by 11 hr in f ish injected with 0.2 mg/kg LH-RH and by 24 hr in f ish injected with 1.0 mg/kg LH-RH. LH-RHA DAIa 6-injected fish maintained higher (P < 0.05) gonadotropin levels than saline and LH-RH-injected f ish from 11-96 hr. No significant differences between saline and Gn-RH-injected groups were apparent in terms of body weight, GSI, oocyte diameter or oocyte maturity index at 1.5 hr post injection (Table 1). By 96 hr, 5 out of 7 f ish in groups injected with 0.02 and 0.2 mg/kg/LH-RHA DAla 6 had undergone oocyte maturation. An accelerated rate of ovarian development was apparent in these groups by their having a higher oocyte maturity index (P < 0.02) than saline-injected f i sh . No fish ovulated during the 96 hr experimental period. A comparison of LH-RHA DAla 6 - injected fish which fa i led to complete GVBD and those which completed GVBD revealed no significant difference with respect to plasma gonadotropin levels. IV. Effects of Chum Salmon Gn-RH, LH-RH and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation Gonadotropin levels were 6.4 ± 1.9 ng/ml in plasma samples from 8 fish at the time of injection. Plasma gonadotropin levels in a l l hormone-treated groups were signif icantly higher (P < 0.05) than those in saline-injected f ish at 1.5 hr (Table 2). Gonadotropin levels in groups injected with 0.02 and 0.2 mg/kg LH-RH were similar at 1.5 hr and declined to a level similar to saline-injected f ish by 24 hr. Fish injected with 0.02 mg/kg chum salmon Gn-RH had lower gonadotropin levels at 1.5, 24 and 48 hr than f ish receiving the high dose of chum salmon Gn-RH. Gonadotropin levels in f ish injected with 0.02 mg/kg chum salmon Gn-RH were similar - 32 -FIG. 4. Plasma gonadotropin levels (mean ± standard error, N = 7-8) in coho salmon following single intraperitoneal injections of LH-RH (A) or LH-RHA DAla 6 (B). At each sampling period, plasma gonadotropin levels which are similar (P > 0.05) as determined by Duncan's Multiple Range Test are identified by the same superscript. - 32a -HOURS POST INJECTION TABLE 1 - Effects of LH-RH and LH-RHA DAla 6 on body weight, gonadosomatic index, oocyte diameter and oocyte maturity in coho salmon. Values represent the mean ± standard error. Body weight Gonadosomatic Oocyte diameter Oocyte maturity index Dosage N kg index (%)1 (mm) 1.5 hr 96 hr 1.0 mg/kg LH-RH 82 1.79 ± 0.13 18.49 ± 1.10 5.92 ± 0.09 1.25 ± 0.16 1.57 ± 0.202 0.2 mg/kg LH-RH 8 2.08 ± 0.17 18.26 ± 0.83 5.87 ± 0.17 1.13 ± 0.13 1.38 ± 0.18 0.2 mg/kg LH-RHA DAla 6 7 2.31 ± 0.29 18.97 ± 0.94 5.95 ± 0.09 1.29 ± 0.19 2.57 ± 0.30* 0.02 mg/kg LH-RHA DAla 6 7 2.52 ± 0.32 20.47 ± 1.04 6.48 ± 0.12 1.29 ± 0.19 2.71 ± 0.18* saline 8 2.32 + 0.19 19.81 ± 0.99 5.93 ± 0.18 1.25 ± 0.16 1.50 ± 0.27 1 ovary weight/body weight x 100 2 N = 7 following 48 hr sample owing to a single mortality * Significantly different from the saline-injected group as determined by the Mann-Whitney U test (P < 0.02). - 34 -to the levels in saline-injected f ish by 24 hr. Fish injected with the high dose of chum salmon Gn-RH maintained higher plasma gonadotropin levels than saline and LH-RH injected f ish at 24 hr but were similar to these groups at 48 hr. Groups injected with LH-RHA DAla 6 had higher plasma gonadotropin levels (P < 0.01) than al l other groups at 24 and 48 hr. A l l of the treatments were ineffective in accelerating the rate of ovulation (Table 2). V. Effects of LH-RHA DAla 6 and SG-G100 on Plasma Gonadotropin Levels and Ovulation The effects of LH-RHA DAla 6 alone and in various combinations with SG-G100 on ovulation are reported in F ig . 5. These results represent the combined data from fish which had blood samples removed for gonadotropin measurement and f ish sampled only for ovulation; since blood sampling had no significant effect on ovulation. A l l hormone treatments accelerated the rate of ovulation by day 14 when compared to saline-injected f i sh (P < 0.05). In saline-injected f i s h , 50% ovulation occurred by day 32 whereas in the least effective of the hormone-treated groups (0.02 mg/kg LH-RHA DAla 6) 50% ovulation occurred by day 14. In most hormone-treated groups, 50% ovulation occurred 8-10 days after injection. The effectiveness of treatments involving the injection of LH-RHA DAla 6 alone on the acceleration of ovulation can be summarized as follows: double low = double high > single high > single low (Fig. 5A). The ovulatory response to a single injection of SG-G100 was similar to that observed following single injections of LH-RHA DAla 6 . Combined injections of LH-RHA DAla 6 and SG-G100 were more effective in promoting ovulation than SG-G100 alone (Fig. 5B). The ovulatory response to two injections of LH-RHA DAla 6 at either low or high doses was as effective as treatments involving the injection of SG-G100 and LH-RHA DAla 6 . No prespawning mortality occurred by day 14 in hormone-treated f i sh which had not ovulated. A l l hormone treated f ish which subsequently died prior to ovulation had completed oocyte maturation. Furthermore, TABLE 2 - Effects of LH-RH, LH-RHA DAla 6 and chum salmon Gn-RH on plasma gonadotropin levels and ovulation in coho salmon. Gonadotropin levels (mean ± standard error) which are similar, at each sampling time, as determined by Duncan's Multiple Range Test (P > 0.05) are identified by a similar superscript. The number of f i sh which had ovulated was determined on day 10. Treatment N Plasma gonadotropin (ng/ml) 1.5 24 48 hr Number ovulated Saline 8 6.9 ± 1.2a 9.2 ± 0.5a 8.6 ± l . i ab 0 LH-RH 0.02* 8 16.5 ± 1.7bc 7.0 ± l .Oa 8.7 ± l.Oab 1 0.2 8 19.5 + 2.4bcd 7.2 ± 0.5a 7.8 ± 0.7ab 0 LH-RHA DAla 6 0.02 8 21.3 + 1.5cd 26.7 ± 2.3C 26.5 ± 3.4C 2 0.2 7 26.0 ± 1.5d 35.6 ± 6.4C 35.5 ± 5.2C 1 Chum salmon Gn-RH 0.02 5 14.2 ± 0.9b 8.0 ± 0.9a 6.4 ± 0.7a 0 0.2 7 24.8 ± 4 .0 c d 14.2 + 2.4b 11.3 ± 1.6b 1 * Injected dose (mg/kg) - 36 -FIG. 5. Effects of intraperitoneal injections of LH-RHA DAla 6 (A) and SG-G100 alone or in combination with LH-RHA DAla 6 (B) on ovulation in coho salmon. Values represent the cummulative percentage of the number of f ish which ovulated in each of the hormone treated groups. N = 17 or 18. - 36a -100 Z o 3 > o DAY DATE 80 60 4 0 20 0.2 + 0.2 A N A L O G 0.02 A N A L O G 0.02 + 0.02 A N A L O G SALINE 0 5 10 15 2 0 2 5 30 35 4 0 45 O C T . 15 20 2 5 30 N O V . 4 9 14 19 2 4 29 100 r 8 0 z o 3 > o DAY DATE 6 0 40 20 B p—/a •-/ / 3 '\ -X- -X-x S G - G 1 0 0 • S G - G 1 0 0 . 0 . 0 2 A N A L O G A S G - G 1 0 0 ,0.2 A N A L O G • S G - G 1 0 0 + 0.2 A N A L O G 0 5 10 15 2 0 25 3 0 35 OCT. 15 20 25 30 N O V . 4 8 14 19 - 37 -based on the morphology of oocytes released while checking for ovulation, a l l but one of the hormone-injected fish had completed oocyte maturation by day 10. Plasma gonadotropin levels were 5.4 ± 0.8 ng/ml in 12 f ish at the time of injection. Plasma gonadotropin levels in saline-injected f ish showed l i t t l e change with respect to this i n i t i a l level during the 8 day sampling (Fig. 6a). A l l hormone treatments resulted in increased plasma gonadotropin levels when compared to saline-injected f i s h . Fish injected with 0.02 mg/kg LH-RHA DAla 6 maintained elevated plasma gonadotropin levels for 3 days when compared to saline-injected f i sh . Gonadotropin levels in f ish injected with 0.2 mg/kg LH-RHA DAla 6 were similar to f ish injected with 0.02 mg/kg LH-RHA DAla 6 on days 1 and 2, but remained higher than saline-injected f ish for 6 days. Fish receiving two separate injections of 0.02 or 0.2 mg/kg LH-RHA DAla 6 at 0 and 72 hr had s ignif icantly higher plasma gonadotropin levels on days 4 to 6 than f ish receiving a single injection of LH-RHA DAla 6 at comparable doses. Plasma gonadotropin levels in these groups did not decrease to the levels in saline-injected f ish until day 8. Injections of SG-G100 alone or in combination with LH-RHA DAla 6 resulted in higher plasma gonadotropin levels at 1 and 2 days post injection than did injections of LH-RHA DAla 6 alone (Fig. 6a and b). Although there was a rapid decrease in plasma gonadotropin levels in SG-G100 injected f i sh , the levels remained higher than those in saline-injected f ish until day 8. Fish receiving combined injections of SG-G100 and 0.02 or 0.2 mg/kg LH-RHA DAla 6 had higher plasma gonadotropin levels on days 2 to 5 than f ish injected with SG-G100 alone. Fish receiving an i n i t i a l injection of SG-G100 followed by 0.2 mg/kg LH-RHA DAla 6 at 72 hr had higher plasma gonadotropin levels on days 4 and 5 than SG-GlOO-injected f i sh . Gonadotropin t i t res in f ish which ovulated by day 10 and f ish which ovulated at later times were examined to determine i f the acceleration of ovulation - 38 -FIG. 6. Effects of intraperitoneal injections of LH-RHA DAla 6 (A) and SG-G100 alone or in combination with LH-RHA DAla 6 (B) on plasma gonadotropin levels in coho salmon. Values represent the mean ± standard error of measurements from 8 f i s h . At each sampling period, plasma gonadotropin levels which are similar (P > 0.05) as determined by Duncan's Multiple Range Test are identified by the same superscript. .1 120-110- A * • Saline I Q Q - A 0.02 Analog • 0.2 Analog OQ . a 0.02 + 0.02 Analog • 0.2 -i- 0.2 Analog DAYS POST - 39 -was related to the magnitude or duration of the increase in plasma gonadotropin levels. Figure 7 i l lustrates these data for f ish receiving single injections of LH-RHA DAla 6 at 0.02 and 0.2 mg/kg. Fish which ovulated by day 10 in response to 0.02 mg/kg LH-RHA DAla 6 had higher plasma gonadotropin levels on days 2-5 than fish which ovulated at later times (Fig. 7A). Fish which ovulated by day 10 following the injection of 0.2 mg/kg LH-RHA DAla 6 had higher plasma gonadotropin levels on days 1-4 than f ish which ovulated after 10 days (Fig. 7B). For other treatments, i t was not possible to distinguish significant differences in plasma gonadotropin levels that related to the time of ovulation. VI. Effects of Pimozide and LH-RHA DAla 6 on Plasma Gonadotropin Levels and Ovulation The effects of pimozide and LH-RHA DAla 6 on plasma gonadotropin levels are shown in F ig . 8. Gonadotropin levels in saline-injected fish did not change during the sampling period. Gonadotropin levels in pimozide-injected f ish were similar to saline-injected f ish until 24 hr, but were higher at 48 and 96 hr. Groups injected with LH-RHA DAla 6 alone or in combination with pimozide had higher gonadotropin levels than saline-injected fish at 1.5 to 96 hr. Fish receiving the combination of pimozide and LH-RHA DAla 6 had higher gonadotropin levels than LH-RHA DAIa 6-injected f ish at 6-96 hr. In the pimozide-injected group, 3 out of 9 f ish had ovulated by day 4 compared to 1 out of 8 or 9 fish in the other groups (Table 3A). By day 8, only one additional f ish had ovulated in the pimozide and saline-injected groups. In contrast, 7 out of 8 fish receiving LH-RHA DAla 6 and 8 out of 9 f ish receiving pimozide and LH-RHA DAla 6 had ovulated by day 8. The high variation in the rate of ovulation for f i sh in the pimozide-injected group suggested that this may have resulted from the differing stages of maturity of f ish at the time of injection. - 40 -FIG. 7. Plasma gonadotropin levels in f i sh , which ovulated by day 10 ( A ) and after day 10 ( A ) in response to single intraperitoneal injections of 0.02 (A) and 0.2 mg/kg/LH-RHA DAla 6 (B). In each case, values represent the mean ± standard error of measurements from 4 f i sh . Gonadotropin levels in fish ovulating by day 10 and at later times were compared using the t-test (P < 0.05*, P < 0.01**). - 40a -A . 5 0 r 0 ' 1 1 1 1 1 — L_ 1 2 3 4 5 6 D A Y S - 4 1 -FIG. 8. E f f e c t s o f p i m o z i d e and LH-RHA D A l a 6 on plasma g o n a d o t r o p i n l e v e l s (mean ± s t a n d a r d e r r o r ) i n coho salmon. At each s a m p l i n g p e r i o d , plasma g o n a d o t r o p i n l e v e l s which are s i m i l a r (P > 0.05) as determined by Duncan's M u l t i p l e Range T e s t a re i d e n t i f i e d by t h e same s u p e r s c r i p t . c CL Q \— O a < O O 60 50 40 30 20 10 D i a PIMOZIDE & LH-RHA D A l a 6 • LH -RHA D A l a 6 © PIMOZIDE o SALINE e A (8) > (9) 0 i-y/.—i— 0 1.5 i i—• CU 24 96 HOUR - 42 -To investigate this poss ib i l i ty , preinjection samples were analysed for 17a20BP. Ini t ia l 17a20BP concentrations were highly variable, with the levels in some fish approaching the levels seen in f ish immediately prior to the completion of oocyte maturation (see Chap. 4, Exp. 1). To further examine the effects of these hormone treatments on ovulation, those f ish having high 17a20BP levels (> 50 ng/ml)at the time of injection were eliminated from consideration (Table 3B). Having applied this correction, i t was found that LH-RHA-DA1a6 injected alone or in combination with pimozide were more effective in accelerating ovulation than saline or pimozide injections. For example, 4 out of 6 f ish receiving LH-RHA-DA1a6 and 6 out of 7 f ish receiving pimozide and LH-RHA-DA1a6 had ovulated by day 6 whereas none of the pimozide or saline-injected f ish had ovulated. D. Discussion Although coho salmon undergo dramatic changes with respect to the GSI and average oocyte diameter following entry to freshwater (Fig. 1), plasma gonadotropin levels were unchanged until 1-2 weeks before ovulation with the highest levels seen at the time of f inal maturation and ovulation (Fig. 2 and 3). These results confirm and extend the observations of other workers (Crim et a l . , 1973, 1975; Bi l lard et a l . , 1978; Fostier et a l . , 1978; Jalabert et a l . , 1978a,b; Jalabert and Breton, 1980; Stuart-Kregor et a l . , 1981; Bromage et a l . , 1982; Fostier and Jalabert, 1982; Breton et a l . , 1983a; Scott and Sumpter, 1983; Scott et a l . , 1983; Young et a l . , 1983a) that gonadotropin levels increase during the preovulatory period in salmonids. The increase in plasma gonadotropin levels to 10-20 ng/ml in ovulated coho salmon (Fig. 2 and 3) was consistent with measurements of gonadotropin levels in ovulated brown trout and brook trout (Crim et a l . , 1975), rainbow trout (Jalabert et a l . , 1978a; Fostier et a l . , 1978; Fostier and Jalabert, 1982; Scott et a l . , 1983) and coho salmon (Jalabert et a l . , 1978b). Since the - 43 -TABLE 3 - The effects of pimozide and LH-RHA DAla 6 on ovulation in coho salmon. The results represent the cummulative number of f ish in each group which ovulated 4, 6 and 8 days after injection (A) and after correction to eliminate f ish which were at an advanced stage of maturity at the time of injection (B). Cummulative number ovulated Treatment N 4 Day 6 8 A. Uncorrected for maturity Saline 8 1 1 2 Pimozide 9 3 3 4 LH-RHA DAla 6 8* 1 6 7 LH-RHA DAla 6 and pimozide 9 1 8 8 B. Corrected for maturity Saline 7 0 0 1 Pimozide 6 0 0 1 LH-RHA DAla 6 6* 0 4 5 LH-RHA DAla 6 and pimozide 7 0 6 6 * Includes one non-ovulated f ish dead on day 6. - 44 -present studies were based on a laboratory-held population of f i sh , the gonadotropin changes may not be representative of wild f i sh . For example, Crim et a l . (1975) reported that gonadotropin levels in sockeye salmon sampled on the spawning grounds were about 300 ng/ml. Although gonadotropin levels were generally higher in ovulated f ish than fish undergoing maturation, this was only significant on the November 6 sampling (Fig. 2). Since these values were based on measurements from fish that were not ser ia l ly sampled, the precise time of ovulation was not recorded. This may be of importance as gonadotropin levels in rainbow trout continue to increase following ovulation and do not reach maximal values until several weeks after ovulation (Jalabert and Breton, 1980; Scott et a l . , 1983). In coho salmon ser i a l ly sampled during the preovulatory period, there was no difference in the gonadotropin levels in fish undergoing oocyte maturation and at ovulation (Fig. 3). The demonstration that LH-RH, LH-RHA DAla 6 and chum salmon Gn-RH stimulate gonadotropin secretion in coho salmon (Table 2; F ig . 4, 6 and 8) confirm and extend previous studies which indicate that gonadotropin secretion in teleosts is mediated by a hypothalamic releasing hormone (Peter and Crim, 1979; Ba l l , 1981; Peter, 1982a,b, 1983a,b). Furthermore, the demonstration that pimozide stimulates gonadotropin secretion in coho salmon (Fig. 8) suggests that a dopaminergic inhibitory system shown to regulate gonadotropin secretion in cyprinids (Chang and Peter, 1983a,b) may also exist in salmonids. Chum salmon Gn-RH, LH-RH and LH-RHA DAla 6 had similar effects on gonadotropin secretion at short-time intervals following injection (Table 2). The increase in plasma gonadotropin levels in coho salmon following injections of Gn-RH was similar to that observed in brown trout, rainbow trout and goldfish (Crim and Cluett, 1974; Weil et a l . , 1978; Peter, 1980; Crim et a l . , 1981a; Chang and Peter, 1983a). The - 45 -magnitude of the response in coho salmon was also similar to that in the common carp (Weil et a l . , 1975; Sokolowska, 1982; Breton et a l . , 1983) but considerably less than that described in an earl ier report on this species (Breton and Weil, 1973) or in Chinese carps (Remlian et a l . , 1980). Peter (1980) also observed no difference in the magnitude of the i n i t i a l increase in plasma gonadotropin levels in male goldfish following single intraperitoneal injections of LH-RH and LH-RHA DAla 6 . Additionally, Crim et a l . (1981a) found no differences in the magnitude of the increase in plasma gonadotropin levels in male brown trout at short-time intervals following injections of LH-RH, des-Gly i 0LH-RH-ethylamide and des-GlylO[D-Leu]6-LH-RH- ethylamide. Despite differences in their structure, i t appears that chum salmon Gn-RH, LH-RH and LH-RHA DAla 6 have a similar a f f ini ty for Gn-RH receptors in coho salmon. As a f u l l range of doses was not tested, the poss ibi l i ty remains that differences in the act iv i ty of the releasing hormones may exist , particularly at lower doses. There is an indication that this may be the case in that injections of LH-RHA DAla 6 at 0.02 mg/kg resulted in higher plasma gonadotropin levels than injections of chum salmon Gn-RH at the same dose. As there was a dose dependance when the duration of the response was considered (Table 2; F ig . 4 and 6), the fai lure to detect differences in the potency of different Gn-RHs at short-time intervals following injection may result from only a small proportion of pituitary gonadotropin reserves being available for release. Major differences in the act iv i t ies of chum salmon Gn-RH, LH-RH and LH-RHA DAla 6 were apparent when the duration of the increase in plasma gonadotropin levels was considered (Table 2 and Fig . 4). The effects of chum salmon Gn-RH and LH-RH were transitory, although the effects of chum salmon Gn-RH appeared to last for a s l ight ly longer duration than LH-RH (Table 2). This finding was consistent with data from the goldfish in which the potency of LH-RH and chum salmon Gn-RH were - 46 -r e p o r t e d t o be s i m i l a r (R.E. P e t e r , C.S. Nahorniak and M. Sokolowska, u n p u b l i s h e d d a t a c i t e d i n P e t e r , 1983b). The r e s p o n s e t o LH-RHA D A l a 6 was o f much l o n g e r d u r a t i o n than t h a t t o chum salmon Gn-RH o r LH-RH ( T a b l e 2 and F i g . 4 ) . The i n c r e a s e d a c t i v i t y o f LH-RHA D A l a 6 was i n d i c a t e d by t h e maintenance o f e l e v a t e d plasma g o n a d o t r o p i n l e v e l s f o r a t l e a s t 96 hr compared t o o n l y 11 hr f o l l o w i n g i n j e c t i o n s o f LH-RH ( F i g . 4 ) . S t u d i e s i n mammals (see V a l e e t a l 1 9 7 7 ) and i n o t h e r t e l e o s t s have a l s o shown t h a t LH-RH an a l o g s have a more p r o l o n g e d e f f e c t on g o n a d o t r o p i n s e c r e t i o n than LH-RH. F o r example, P e t e r (1980) r e p o r t e d t h a t LH-RHA D A l a 6 a d m i n i s t e r e d i n two i n j e c t i o n s 12 hr a p a r t o r i n t h r e e d a i l y i n j e c t i o n s promoted a l o n g e r d u r a t i o n i n c r e a s e i n plasma g o n a d o t r o p i n l e v e l s i n male g o l d f i s h than i n j e c t i o n s o f LH-RH. B r e t o n e t a l . (1983) r e p o r t e d t h a t s i n g l e i n j e c t i o n s o f des G l y 1 0 [ D - S e r 6 ] - L H - R H - e t h y l a m i d e had a l o n g e r e f f e c t than LH-RH i n t h e c a r p . A d d i t i o n a l s t u d i e s i n coho salmon have shown t h a t analogs o f chum salmon Gn-RH c o n t a i n i n g [D-Arg] o r [ D - A l a ] i n p o s i t i o n 6 and removal o f p o s i t i o n 10 g l y c i n e promote a l o n g e r d u r a t i o n i n c r e a s e i n plasma g o n a d o t r o p i n l e v e l s than t h e n a t i v e m o l e c u l e (Donaldson e t a l . , 1983). In t h i s c a s e , LH-RHA D A l a 6 and an a l o g s o f chum salmon Gn-RH were shown t o be e q u i p o t e n t (Donaldson e t a l . , 1983). The b a s i s f o r t h e p r o l o n g e d e f f e c t i v e n e s s o f LH-RHA D A l a 6 compared t o LH-RH and chum salmon Gn-RH i n coho salmon i s not known. E x p l a n a t i o n s i n c l u d e a p o s s i b l e l o n g e r m e t a b o l i c c l e a r a n c e r a t e f o r LH-RHA D A l a 6 o r a slower d e g r a d a t i o n upon b i n d i n g t o Gn-RH r e c e p t o r s i n t h e p i t u i t a r y . A l t h o u g h s e v e r a l f a c t o r s p r e c l u d e d i r e c t comparisons between s p e c i e s ( s t a t e of m a t u r i t y , s i t e o f i n j e c t i o n , dosage and t y p e o f r e l e a s i n g hormone), t h e d u r a t i o n o f r e s p o n s e t o LH-RHA D A l a 6 i n coho salmon was g r e a t e r than t h a t d e s c r i b e d f o r o t h e r t e l e o s t s p e c i e s . In t h e common c a r p , plasma g o n a d o t r o p i n l e v e l s were e l e v a t e d f o r l e s s t h a t 24 hr f o l l o w i n g i n t r a c a r d i a c i n j e c t i o n o f d e s - G l y ! 0 [ D - S e r 6 ] - 47 -LH-RH-ethylamide at 0.3 ug/kg (Breton et a l . , 1983). In adult female goldfish, plasma gonadotropin levels were elevated for 48 hr following intraperitoneal injections of LH-RHA DAla 6 at 0.1 mg/kg (Chang and Peter, 1983a). In coho salmon, plasma gonadotropin levels were elevated for up to 6 days following injections of LH-RHA DAla 6 at 0.2 mg/kg (Fig. 6). Additional evidence for the long-acting effects of LH-RHA DAla 6 in coho salmon was provided by the higher plasma gonadotropin levels on days 2-5 in f ish receiving combined injections of LH-RHA DAla 6 and SG-G100 when compared to f ish receiving SG-G100 alone (Fig. 5). To account for these species differences i t may be necessary to consider the rate of uptake into the circulation following injection and the duration of binding to Gn-RH receptors. In goldfish, the effects of LH-RHA DAla 6 on gonadotropin release was potentiated by two injections of LH-RHA DAla 6 over a 12 hr period (Peter, 1980; Chang and Peter, 1983a). There was no indication for the potentiation of gonadotropin release in coho salmon when LH-RHA DAla 6 was administered in two injections over a 72 hr period. The effect of a second LH-RHA DAla 6 injection was esentially additive to the effects of the f i r s t injection (Fig. 6). For example, the gonadotropin level measured on day 4 following two injections of LH-RHA DAla 6 was equivalent to the sum of the level measured on day 3 and the 25 ng/ml increase in gonadotropin level measured one day following a single injection of LH-RHA DAla 6 . Previous studies have shown that pituitary responsiveness to Gn-RH varies on a seasonal basis (Weil et a l . , 1975, 1978; Lin et a l . , 1984). L i t t l e evidence was found to indicate a major change in pituitary responsiveness to Gn-RH in coho salmon during the preovulatory period. However, a difference was found in that f ish injected with 0.02 mg/kg LH-RHA DAla 6 about one month prior to the expected - 48 -time of ovulation maintained elevated plasma gonadotropin levels for only 3 days (Fig. 6) whereas f ish of more advanced maturity maintained elevated plasma gonadotropin levels for at least 4 days (Fig. 4 and 8). Additionally f i sh which were induced to ovulate following single injections of LH-RHA DAla 6 showed a larger increase in plasma gonadotropin levels which persisted for a longer duration than that in f ish which fa i led to ovulate (Fig. 7). Whether these differences relate to changes in pituitary responsiveness or the subsequent effects of changing steroid feedback on gonadotropin secretion remains to be evaluated. The dopamine receptor antagonist pimozide was shown to stimulate gonadotropin secretion in coho salmon (Fig. 8). Following a delay of 1.5 hr, combined injections of pimozide and LH-RHA DAla 6 were more effective in promoting gonadotropin secretion than LH-RHA DAla 6 alone (Fig. 8). Although the effects of pimozide alone were not marked, these results suggest that pimozide potentiates the response to LH-RHA DAla 6 in coho salmon. Chang and Peter (1983a) reported that pimozide injected with the f i r s t or second of two LH-RHA DAla 6 injections (12 hr injection interval) , or prior to a single injection of LH-RHA DAla 6 greatly potentiated the gonadotropin release-response to LH-RHA DAla 6 in goldfish held at 12 °C . However, the simulataneous injection of pimozide and LH-RHA DAla 6 was no more effective in stimulating gonadotropin secretion than injection of LH-RHA DAla 6 alone in goldfish at 12°C (Chang and Peter, 1983a). Subsequent studies based on goldfish held at 20°C have shown, l ike the data for coho salmon, that the simultaneous injection of pimozide and LH-RHA DAla 6 was more effective than LH-RHA DAla 6 alone (Sokolowska et a l . , 1984). Recent studies in rainbow trout (Bil lard et a l . , 1983a) provide confirmation that pimozide stimulates gonadotropin secretion in salmonids, although in this case, injections of pimozide were more effective than LH-RHA DAla 6 . Furthermore, there was no potentiation of the response in groups - 49 -receiving pimozide and LH-RHA DAla 6 . The basis of the difference between the results in rainbow trout (Bil lard et a l . , 1983a) and coho salmon (Fig. 8) is not known, but may be a consequence of the lower doses of LH-RHA DAla 6 (1 yg/kg) used in studies with rainbow trout. Although i t appears that dopamine inhibit ion may regulate gonadotropin secretion in salmonids and cyprinids, physiological evidence for a GRIF has only been described for cyprinids (see Peter, 1982a,b, 1983a,b). It has been suggested that the preovulatory gonadotropin surge in the goldfish is regulated by the removal of dopamine inhibition on gonadotropin release and the stimulation of gonadotropin secretion by Gn-RH. The significance of a GRIF such as dopamine in the regulation of gonadotropin secretion in salmonids remains to be investigated. The results presented here (Fig. 5) confirm and extend previous studies on the use of LH-RHA DAla 6 to induce ovulation in salmonids. Consistent with recent studies (Donaldson et a l . , 1981; Sower et a l . , 1982), i t was shown that LH-RHA DAla 6 injected 72 hr following an i n i t i a l injection of SG-G100 accelerates ovulation in coho salmon. It was also shown that a combined single injection of 0.02 or 0.2 mg/kg LH-RHA DAla 6 together with SG-G100 accelerates ovulation in a higher percentage of f ish than SG-G100 alone. In addition, the present results also demonstrate that single intraperitoneal injections of LH-RHA DAla 6 induce ovulation in coho salmon. Furthermore, by the administration of LH-RHA DAla 6 in two injections over a 72 hr period, an ovulatory response was obtained which was comparable to that observed following combined injections of SG-G100 and LH-RHA DAla 6 . The effectiveness of two separate injections of LH-RHA DAla 6 on the promotion of ovulation in this study was comparable to recent studies in Atlantic salmon and rainbow trout in which LH-RH analogs were administered in cholesterol pellet implants which were designed for a long-term gradual release of hormone - 50 -(Crim et a l . , 1983a,b). LH-RHA DAla 6 alone and in combination with pimozide had similar effects on the acceleration of ovulation in coho salmon (Table 8). In contrast, B i l l a rd et a l . (1983a) reported that the combination of LH-RHA DAla 6 and pimozide was more effective in accelerating ovulation than pimozide or LH-RHA DAla 6 alone in rainbow trout and brown trout. The inabi l i ty to demonstrate an increased effectiveness of pimozide and LH-RHA DAla 6 in coho salmon may have resulted from the advanced state of maturity of f i sh at the time of injection. LH-RH was ineffective in accelerating oocyte maturation in coho salmon (Table 1). In contrast, LH-RHA DAla 6 had at least 50 x the potency of LH-RH based on i ts stimulation of oocyte maturation. Since LH-RH and LH-RHA DAla 6 have a similar effect on plasma gonadotropin levels at 1.5 and 3 hr (Fig. 4), a potency estimate based on gonadotropin measurement at short-time intervals following injection would not agree with this observation. The fa i lure of LH-RH injections to induce oocyte maturation suggests that short-lived changes in plasma gonadotropin levels are of insufficient duration to stimulate f inal maturation and ovulation. Although single injections of LH-RHA DAla 6 and SG-G100 have very different effects on plasma gonadotropin levels (Fig. 6), these treatments have a similar effect on the acceleration of ovulation (Fig. 5). These treatments differ in that the injection of SG-G100 causes a much larger increase in plasma gonadotropin levels at 1 and 2 days post injection than LH-RHA DAla 6 . It was previously shown that gonadotropin levels at 24 hr were not s ignif icantly different from the levels 1.5 hr following the injection of LH-RHA DAla 6 (Table 2 and Fig . 4 ). However, i t is l ike ly that measurements 24 hr after the injection of SG-G100 do not provide a measure of peak gonadotropin concentrations (see Cook and Peter, 1980b; Breton et  a l . , 1983). These results suggest that i t is not solely the magnitude of the i n i t i a l increase in plasma gonadotropin level that determines the success of - 51 -hormone treatments on the stimulation of ovulation. The duration of the increase in plasma gonadotropin level appears to be more important.In the case of f ish which ovulated following a single injection of LH-RHA DAla 6 , i t was possible to associate the duration of the increase in plasma gonadotropin levels with the acceleration of ovulation (Fig. 7).Additional evidence supporting this hypothesis was available from the higher plasma gonadotropin levels in f ish receiving two separate injections of LH-RHA DAla 6 and the greater number of f i sh which ovulated in these groups compared to f ish receiving single injections of LH-RHA DAla 6 (Fig. 5 and 6). Additionally, combined single injections of SG-G100 and LH-RHA DAla 6 resulted in higher plasma gonadotropin levels on days 2-5 when compared to SG-GlOO-injected f ish and were more effective in accelerating ovulation. However, with the exception of f ish receiving single injections of LH-RHA DAla 6 , i t was not possible to distinguish f ish in other hormone treated groups which would ovulate based solely on gonadotropin measurements. This suggests, that although gonadotropin can be increased to a level seemingly appropriate for ovulation, factors in addition to gonadotropin are responsible for ovulation. Ultimately, the acceleration of ovulation may depend on the capacity of the ovary to respond to gonadotropin by the synthesis of appropriate steroids and prostaglandins (see Chapter 4). The demonstration that single intraperitoneal injections of LH-RHA DAla 6 accelerate ovulation in coho salmon is in contrast with the majority of studies in other teleosts (see Introduction). The high rate of success using LH-RHA DAla 6 to accelerate ovulation in coho salmon compared to the carp and goldfish may relate to differences in the pattern of gonadotropin secretion at ovulation. In salmonids, there is a slow and gradual increase in plasma gonadotropin levels during the preovulatory period with no evidence for a gonadotropin surge immediately prior to - 52 -ovulation (Fig. 3; Fostier et a l . , 1978; Jalabert et al.,1978a,b; Fostier and Jalabert, 1982; Scott et a l . , 1983). In contrast, a dramatic ovulatory gonadotropin surge occurs in the goldfish (Stacey et al.,1979a) and carp (Fish Reproductive Physiology Research Group and Peptide Hormone Group, 1978). The ovulatory gonadotropin surge in the goldfish lasts at least 12 hr, during which gonadotropin levels increase about 10-fold above basal levels (Stacey et a l . , 1979a). The significance of differences in the pattern of gonadotropin secretion at ovulation is not fu l ly understood, but presumably reflects changes in reproductive strategy. In the goldfish, ovulation and spawning are precisely coordinated by environmental factors while salmonids apparently lack this precise coordination (Peter, 1981). Goldfish generally complete gonadal development at cold temperatures and retain fu l ly developed oocytes which ovulate 1-2 days after exposure to environmental conditions appropriate for spawning (warm temperature, suitable substrate) (Stacey et a l . , 1979a,b; Peter 1981). In salmonids, ovulation occurs at re lat ively cold temperatures, apparently as a consequence of completed ovarian development. Additionally once ovulated, oocytes can be held for several days prior to spawning (Bry, 1981; J. Stoss, personal communication). The inab i l i ty of injections of LH-RH analogs to accelerate ovulation in carp and goldfish presumably relates to a fa i lure to duplicate the preovulatory gonadotropin surge. For example, the implantation of cholesterol pellets containing d e s - G l y l ° [ D - T r p ] 6 L H - R H - e t h y l a m i d e in goldfish, results in a chronic elevation of plasma gonadotropin levels lasting at least 7 days, but f a i l s to induce ovulation (Sokolowska et a l . , 1984). By comparison, a similar approach in landlocked Atlantic salmon accelerates ovulation at least two weeks in advance of control f i sh (Crim et a l . , 1983a). It was recently shown that ovulation could be induced in the goldfish (Chang and Peter, 1983a; Sokolowska et a l . , 1984) and the - 53 -common carp (Bil lard et a l . , 1983b) by using pimozide and LH-RHA DAla 6 . It appears that the removal of dopamine inhibit ion by pimozide potentiates the effects of LH-RHA DAla 6 on gonadotropin release making i t possible to mimic or exceed the normal ovulatory gonadotropin surge (Chang and Peter, 1983a). However, the rate at which plasma gonadotropin levels increase appears to be as important as the magnitude of the increase in the goldfish. This appears to be in direct contrast with the situation in coho salmon. The highest rate of ovulation in coho salmon occurred 6 - 1 0 days following hormone treatments and was associated with declining plasma gonadotropin levels (Fig. 6 and 7). Jalabert et a l . (1978a,b) noted a similar relationship between plasma gonadotropin levels and ovulation in rainbow trout and coho salmon following the injection of gonadotropin preparations. In contrast to the situation in the goldfish, the duration of the increase in plasma gonadotropin level appears to be c r i t i c a l for the successful induction of ovulation in the coho salmon. In conclusion, the present results show that LH-RHA DAla 6 provides a suitable alternative to the use of gonadotropin preparations for the acceleration of ovulation in coho salmon when administered about one month prior to the expected time of ovulation. Although combined injections of LH-RHA DAla 6 and SG-G100 may be of particular benefit when handling stress is to be minimized, two injections of a low dose of LH-RHA DAla 6 may be more economical with respect to hormone usage. Additional research is warranted to optimize the dosage and to investigate the effects of varying the interval between injections of LH-RHA DAla 6 and to further investigate the potential application of pimozide to accelerate ovulation. - 54 -CHAPTER 4 - 178-ESTRADIOL, TESTOSTERONE, AND 17a20BP CHANGES ASSOCIATED WITH SEXUAL MATURATION IN FEMALE COHO SALMON A. Introduction In Chapter 3, i t was shown that oocyte maturation and ovulation in coho salmon were associated with the elevation of plasma gonadotropin t i t r e s . It is generally accepted that gonadotropin does not directly promote oocyte maturation but acts on the f o l l i c u l a r layers surrounding the oocyte to stimulate the production of steroidal mediators of maturation. Current evidence suggests that 17a208P functions as the maturation inducing steroid in salmonids (see Introduction). The role of other steroids, such as 178-estradiol and testosterone, is not completely understood. The present studies were conducted to assess the steroid changes associated with spontaneous and induced reproductive development, in particular, the steroid changes associated with the completion of vitellogenesis and the in i t i a t ion of oocyte maturation. Experiments I, II, and III represent a continuation of the experiments described in Chapter 3 by examining the changes in 178-estradiol, testosterone, and 17a208P during spontaneous reproductive development (Exp. I) and following injections of LH-RH, LH-RHA DAla 6 and SG-G100 (Exp. II and III). Additionally, ovarian f o l l i c l e s obtained throughout the preovulatory period and following ovulation were examined to determine their capacity to produce these steroids in response to gonadotropin in vitro (Exp. IV). The possible involvement of 178-estradiol in the regulation of 17a208P synthesis was investigated in Exp.V by studying the effects of 178-estradiol on the abi l i ty of SG-G100 to augment 17a208P production by f o l l i c l e s incubated in v i t ro . B. Experimental Protocol I. Preovulatory Steroid Changes 178-estradiol, testosterone and 17a208P were measured in plasma samples - 55 -obtained by serial sampling of coho salmon up until the time of ovulation. A fu l l description of the protocol is provided in Exp. II, Chapter 3. II. Steroid Changes in Response to LH-RH and LH-RHA DAla 6 178-estradiol, testosterone and 17a208P were measured in plasma samples obtained from coho salmon following single intraperitoneal injections of LH-RH and LH-RHA DAla 6 . Full details are provided in the protocol for Exp. I l l , Chapter 3. III. Steroid Changes in Reponse to LH-RHA DAla 6 and SG-G100 178-estradiol and 17a208P were measured in plasma samples obtained at time 0, 1, 2, 4, 6, 8 and 10 days following injections of varied combinations of LH-RHA DAla 6 and SG-G100. Details are provided in the protocol for Exp. IV, Chapter 3. IV. In Vitro Steroid Production by Ovarian Fo l l i c l e s During Sexual Maturation Ovarian f o l l i c l e s obtained at various times during the preovulatory period and following ovulation were incubated in vitro with or without SG-G100 to evaluate their capacity to produce 178-estradiol, testosterone and 17a208P. V. Effects of 178-estradiol on 17a208P Production In Vitro Two experiments were conducted to examine the effects of 178-estradiol on 17a208P production in v i t ro . The f i r s t experiment ut i l ized f o l l i c l e s from a fish prior to maturation which were characterized by a central germinal vesicle and the second experiment ut i l ized f o l l i c l e s from a f ish undergoing maturation which were characterized by a peripheral germinal vesicle. In each experiment, groups of five f o l l i c l e s were incubated with or without SG-G100 (0, 10, 100 and 1000 ng/ml) for 24 hr at 1 0 ° C . Additional groups of f o l l i c l e s were incubated in medium containing 178-estradiol at 25 or 250 ng/ml in addition to gonadotropin. Three replicate incubations were made for each concentration of SG-G100 and 178-estradiol. The amounts of 17a208P released to the media were determined by RIA. - 56 -C. Results I. Preovulatory Steroid Changes. Preovulatory changes in plasma 178-estradiol, testosterone and 17a208P levels are shown in F ig . 9. 178-estradiol levels decreased s ignif icantly from 16 ng/ml 12 days prior to ovulation to basal levels of 1-2 ng/ml 4 days before ovulation. Testosterone levels remained high (>125 ng/ml) during the preovulatory period reaching maximal levels about 6 days prior to ovulation and then gradually decreased until ovulation. 17a208P increased from basal levels of less than 10 ng/ml 12 days prior to ovulation to 270 ng/ml 4 days prior to ovulation and then declined. Oocyte maturation occurred 2-4 days prior to ovulation based on the visual examination of oocytes expelled when checking for ovulation. II. Steroid Changes in Response to LH-RH and LH-RHA DAla 6 No dose dependant differences were found when 178-estradiol, testosterone and 17a208P levels were compared in groups injected with 0.2 and 1.0 mg/kg LH-RH or in groups injected with 0.02 and 0.2 mg/kg LH-RHA DAla 6 . Subsequent comparisons between the effects of LH-RH and LH-RHA DAla 6 on plasma steroid levels (Figs. 10, 11 and 12) were based on the pooled data for a l l f ish injected with LH-RH and the pooled data for a l l f ish injected with LH-RHA DAla 6 . However, the steroid profiles in fish which completed GVBD by 96 hr were examined separately from those f a i l ing to complete GVBD (see Table 4). The one saline-injected fish which completed GVBD spontaneously showed similar steroid changes to those reported in F ig . 9 and was not included in subsequent comparisons. In this f i sh , 178-estradiol was 4.9 ng/ml i n i t i a l l y and decreased to 1.9 ng/ml at 96 hr. 17a208P increased from 39 ng/ml to 325 ng/ml over the 96 hr sampling period. Testosterone levels were above 240 ng/ml at al l sampling times. Plasma 178-estradiol levels following the injection of LH-RH and LH-RHA - 57 -FIG. 9. Preovulatory changes in plasma 178-estradiol, testosterone and 17ct20BP levels in coho salmon. Values represent the mean ± standard error of measurements from 8 f i sh . - 57a -B • 1 • i B • 1 \ \ CD c c NE RO UJ C2 h-C/> O O CN to 8 TE 400 200 100 0 L 20 e c 112 o o 18 < DAYS PRIOR TO OVULATION TABLE 4 - Oocyte development determined 96 hr following the injection of LH-RH, LH-RHA DAla 6 or saline. Fish were assigned to specific categories which correspond to the position of the germinal vesicle. A maturation index was calculated to numerically describe the average oocyte classif ication in each of the treatment groups. Injected peptide Dose (mg/kg) Central Germinal vesicle 1 Oocyte classif ication Peripheral Germinal vesicle 2 Germinal vesicle breakdown 3 Maturation index LH-RH 1.0 3 4 0 1.57 LH-RH 0.2 5 3 0 1.38 LH-RHA DAla 6 0.2 1 1 5 2.57* LH-RHA DAla 6 0.02 0 2 5 2.71* Saline 0 5 2 1 1.50 * Significantly different from saline group as determined by the Mann Whitney U test (P < 0.02). - 59 -DAla° are shown in F ig . 10. Plasma 178-estradiol levels in saline-injected f ish were similar to preinjection levels (27 ng/ml) at 24 hr but then declined to 5.9 ng/ml by 96 hr. Plasma 178-estradiol levels in LH-RH-injected f ish followed the same trend as the saline-injected group. Plasma 178-estradiol levels in LH-RHA DAIa 6-injected f i sh which completed GVBD were reduced s ignif icantly compared to saline-injected f ish at 48 to 96 hr. LH-RHA DAla 6-injected f i sh which fa i led to complete GVBD had higher plasma 178-estradiol levels than saline-injected f i sh at 48 hr but then declined at 72 and 96 hr to levels similar to saline-injected f i sh . Plasma 17a208P levels in saline-injected f ish were 9.1 ng/ml at the time of injection and increased s ignif icantly at 72 and 96 hr reaching 38 ng/ml (Fig. 11). Injections of LH-RH and LH-RHA DAla 6 resulted in elevated plasma 17a208P levels by 3 hr when compared to saline-injected f i sh . The response to LH-RH was transitory, at 24 hr plasma 17a208P levels had decreased to the levels in saline-injected fish and at 96 hr were s ignif icantly lower than the levels in saline-injected f i s h . LH-RHA DAIa 6-injected f ish which completed GVBD maintained high 17a208P levels at 24 hr and then showed a further increase reaching 480 ng/ml at 72 and 96 hr. LH-RHA DAla 6-injected f i sh which fai led to complete GVBD maintained elevated 17a208P levels , but t i t res were only 55 ng/ml at the 96 hr sampling. Testosterone levels in saline-injected fish were highly variable and showed l i t t l e change during the sampling period (Fig. 12). No significant differences were found between the testosterone levels in LH-RH- and saline-injected groups, although testosterone levels in LH-RH-injected f ish were decreased s ignif icantly at 72 and 96 hr when compared to preinjection levels. Testosterone levels in LH-RHA DAIa 6-injected f i sh which completed GVBD were s ignif icantly higher than the levels in saline-injected f ish at 24 to 96 hr. Testosterone levels in this group were - 60 -FIG. 10. Changes in plasma 173-estradiol levels in response to single intraperitoneal injections of saline, LH-RH and LH-RHA DAla 6 . LH-RHA DAI a 6-injected f ish have been separated into two groups based on whether the f ish had completed GVBD at the 96 hr sampling. Values represent the mean ± standard error with the number of f ish per group indicated. At each sampling time, plasma 173-estradiol levels which are similar as determined by Duncan's Multiple Range Test (P > 0. 05) are identified by the same superscript, 1. e . , A, B, and C. • LH-RHA D A l a 6 (GVBD) o LH-RHA D A l a 6 ( N O G V B D ) • S A L I N E _ ' • • I I I I I 0 1,5 3 6 11 24 48 72 96 HOURS - 61 -FIG. 11. Changes i n plasma 17a20gP l e v e l s i n response t o s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n s of s a l i n e , LH-RH and LH-RHA DA l a 6 . A d d i t i o n a l information i s provided i n the legend to F i g . 10. - 61a -600 r 500 — 400h \ D) c w 3001 • L H - R H A D A l a 6 ( G V B D ) o L H - R H A D A l a 6 ( N O G V B D ) • S A L I N E • L H - R H a. CQ O C N 250i 801 60 40 20 do) 6 11 HOURS 24 48 72 96 - 62 -FIG. 12. Changes in plasma testosterone levels in response to single intraperitoneal injections of saline, LH-RH and LH-RHA DAla 6 . Additional information is provided in the legend to F ig . 10. - 62a -400r 300 E \ CD C tu 200 o CK L U t— to O H— CO UJ 100 I- i B » ( 1 0 ) e LH-RHA D A l a 6 (GVBD) LH-RHA D A l a 6 ( N O G V B D ) • SAL INE • LH-RH J L 0 1.5 6 11 HOURS 24 48 72 96 - 63 -maximal at 48 hr and decreased s ignif icantly by 96 hr. Testosterone levels in LH-RHA DAla 6-injected f ish which fa i led to complete GVBD were elevated at 48 to 96 hr when compared to saline-injected f i sh . III. Steroid Changes in Response to LH-RHA DAla 6 and SG-G100 A. Temporal steroid changes following injections of LH-RHA DAla 6 and SG-G100 Plasma 178-estradiol and 17a208P levels were 17.2 ± 1.2 ng/ml and 8.1 ± 0.9 ng/ml, respectively, in 12 fish sampled at the time of injection. The effects of various hormone treatments on plasma 178-estradiol and 17a208P levels are reported in Table 5 and 6. No significant differences in plasma 178-estradiol concentrations were observed between hormone-injected and saline-injected f ish at 1 day post injection (Table 5). SG-G100 injected alone and in combination with LH-RHA DAla 6 signif icantly reduced plasma 178-estradiol levels by 2 days post injection when compared to saline-injected f i sh . Plasma 178-estradiol levels in a l l hormone treated groups were lower than the levels in saline-injected f i sh by day 4 and remained lower until day 10. Owing to the high variation in 178-estradiol levels between individual f i sh in each of the hormone-treated groups (see below), i t was not possible to discriminate major differences in the effects of the various hormone treatments on days 4-10. However, groups receiving single injections of LH-RHA DAla 6 at 0.02 mg/kg and SG-G100 tended to have higher 178-estradiol levels than other groups. A l l hormone treatments increased plasma 17a208P levels compared to saline-injected f ish by 1 day post injection and maintained higher 17a208P levels than saline-injected f i sh until day 8 (Table 6). Plasma 17a208P levels in the group injected with 0.02 mg/kg LH-RHA DAla 6 had decreased by day 10 to a level similar to the saline-injected group. A l l other hormone-treated groups maintained higher TABLE 5 - Effects of LH-RHA DAla 6 and SG-G100 on plasma 178-estradiol levels. At each sampling period, plasma 178-estradiol levels (mean ± standard error) which are similar as determined by Duncan's Multiple Range test (P > 0.05) are identified by a similar superscript. Plasma 178-estradiol (ng/ml) at various days post injection Treatment 1 2 4 6 8 10 Saline 17.5 + l . i a 20.9 ± 3.8a 19.1 + 2.7a 17.9 + 3.8a 12.8 + 3.9a 11.8 + 3.5a Single Injection 0.02 LH-RHA* 15.0 + 1.3a 14.8 ± 2.4ab 6.2 + 1.6b 3.2 + 0.8 b 3.8 + 1.0b 3.7 + 1.1b 0.2 LH-RHA 18.4 ± l . i a 19.4 ± 2.7a 8.3 ± 2.4b 3.4 ± 1.0b 2.0 ± 0.6bc 1.4 ± 0.7Cd SG-G100** 14.5 ± 2.5a 9.22 ± 2.3b 6.0 ± 2.1b 3.5 ± 1.5bcd 3.7 ± 1.9b 2.1 ± l.Obc SG-G100, 0.02 LH-RHA 17.8 ± 0.6a 10.7 ± 1.1b 2.7 ± 0.6b 0.85 ± 0.17d 1.6 ± 0.2bc 0.53 ± 0.05d SG-G100, 0.2 LH-RHA 17.2 ± 2.4a 11.0 ± 2.1b 3.2 ± 1.0b 1.5 ± 0.6cd 1.9 ± 0.4bc 0.84 ± 0.25cd Double Injection*** 0.02 + 0.02 LH-RHA 7.4 ± 2.9b 3.5 + 1.6bc 2.0 ± 0.6bc 0.67 + 0.09Cd 0 . 2 + 0 . 2 LH-RHA 2.5 + 0.5b 1.4 + O.lbcd 1.2 + O.ic 0.69 + 0.13Cd SG-G100 +0.2 LH-RHA 6.8 + 2.3b 1.4 + 0.5Cd 1.5 + 0.3bc 0.56 + 0.07Cd * LH-RHA DAla 6 (mg/kg body wt) * * SG-G100 (0.1 mg/kg body wt) * * * F irst injection at time 0 + second injection at 72 hr. TABLE 6 - Effects of LH-RHA DAla 6 and SG-G100 on plasma 17a208P levels. At each sampling period, plasma 17a20BP values (mean ± standard error) which are similar (P > 0.05) as determined by Duncan's Multiple Range test are identified by a similar superscript. Plasma 17a208P (ng/ml) at various days post injection Treatment 1 2 4 6 8 10 Saline 6.0 ± 0.7a 7.3 ± 2.6a 20.7 ± 5.5a 48.3 ± 20.2a 79.0 ± 32.6a 74.3 ± 26.3a Single Injection 0.02 LH-RHA* 46.8 ± 5.4C 90.9 ± 34.7&C 228 ± 7 9 bc 222 + 7lbc 136 ± 26b 95.6 + 21.6ab 0.2 LH-RHA 30.7 ± 4.2bc 47.0 ± 6.2b 197 + 67bc 386 + 97bcd 418 + 83cd 146 + 28bc SG-G100** 28.1 ± 3.9b 76.3 + 15.lbc 112 ± 54b 187 + 76b 298 + 86bc 380 + 97d SG-G100, 0.02 LH-RHA 40.8 ± 6.9&C 84.6 ± 14.0bc 245 ± 79bc 379 + 72bcd 487 + 56d 379 ± 54d SG-G100, 0.2 LH-RHA 41.5 ± 6.9bc 95.9 ± 19.2C 358 ±105C 410 + 102bcd 445 ± 79cd 357 + 52d Double Injection*** 0.02 + 0.02 LH-RHA 253 ± 80bc 454 ± 85cd 311 ± 54C 223 ± 53cd 0.2 + 0.2 LH-RHA 380 ± 93C 649 ± 73d 557 ± 61d 386 ± 67d SG-G100 +0.2 LH-RHA 162 ± 48bc 455 ± 87cd 649 ± 83d 417 ± 45d * LH-RHA DAla 6 (mg/kg body wt) * * SG-G100 (0.1 mg/kg body wt) * * * First injection at time 0 + second injection at 72 hr. - 66 -17a20BP levels than saline-injected f ish on day 10. The extreme variation in 17a208P levels between fish in each of the hormone treated groups made generalizations regarding the effectiveness of the hormone treatments d i f f i c u l t . Differences in 17a20BP levels between treatment groups appear to be unrelated to the maximal levels of 17a20BP but rather to the number of f ish which had high levels of 17a20BP and subsequently ovulated (see below and Table 7). B. Steroid changes in relation to the time of ovulation To determine i f ovulation was associated with specific steroid changes, plasma 178-estradiol and 17a20BP concentrations have been examined in relation to the time of ovulation (Fig. 13, 14, 15 and 16). Furthermore, to evaluate the effects of administering hormones in a single injection or in two injections over a 72 hr period, the steroid changes in the single and dual injected groups have been examined separately. Table 7 shows the number of f ish which ovulated during the periods of 6-7, 8-10, 12-14 or greater than 14 days post injection in each of the treatment groups. Plasma 178-estradiol levels in saline-injected fish which ovulated on day 10 and day 14 declined to basal levels of about 2 ng/ml by day 4 and day 6 respectively (Fig. 13A). Saline-injected fish which did not ovulate by day 14 showed a slight increase in plasma 178-estradiol levels followed by a decline to a level (15 ng/ml) s l ight ly below the i n i t i a l concentration. Saline-injected fish which ovulated on day 10 and day 14 showed large increases in plasma 17a208P levels by day 6 and day 8 respectively (Fig 13B). In contrast, saline-injected fish which did not ovulate by day 14 showed a gradual increase in plasma 17a206P levels to 30 ng/ml by day 10. The steroid changes, when related to the time of ovulation, were similar in f ish receiving single injections of LH-RHA DAla 6 , SG-G100 and SG-G100 in - 67 -TABLE 7 - The number of f ish which ovulated during different time periods in response to various combinations of LH-RHA DAla 6 and SG-G100 administered in a single injection or two separate injections 72 hr apart . Each treatment group contained 8 f i sh . Treatment Number of f ish ovulated at various days 6-7 8-10 12-14 >14 Saline 0 1 1 6 Single injection 0.02 LH-RHA* 2 2 0 4 0.2 LH-RHA 0 4 1 3 SG-G100** 2 0 4 2 SG-G100, 0.002 LH-RHA 1 5 2 0 SG-G100, 0.02 LH-RHA 3 2 1 2 Double Injection*** 0.02 + 0.02 LH-RHA 3 3 1 1 0.2 + 0.2 LH-RHA 2 5 0 1 SG-G100 +0.2 LH-RHA 1 4 3 0 * LH-RHA DAla 6 (mg/kg body wt) * * SG-G100 (0.1 mg/kg body wt) * * * First injection at time 0 + second injection at 72 hr. - 68 -FIG. 13. Changes in plasma 178-estradiol (A) and 17a208P (B) levels in relation to the time of ovulation for saline- injected f i sh . Steroid levels in individual f ish have been grouped according to the time of ovulation ( O 8-10, • 12-14, 0 > 14 days post injection). Values represent the mean ± standard error, where applicable, with the number of f ish indicated in parenthesis. \7Q- ESTRADIOL (ng/ml) o - P89 -- 69 -combination with LH-RHA DAla 6 . Analysis of variance indicated no significant differences between the minimal 173-estradiol and maximal 17a20SP values in those fish induced to ovulate by day 14. The data from these treatment groups have been pooled according to the time of ovulation (Table 7) and are presented in Fig . 14. 173-estradiol levels decreased from 18.4 ng/ml to 4.6 ng/ml in f ish which did not ovulate by day 14, but these levels were higher on days 2-10 when compared to fish which ovulated (Fig. 14A). Plasma 173-estradiol levels in f ish which ovulated by day 7 were lower on day 2 when compared to f ish which ovulated 8-10 days after injection and were also lower on days 2 and 4 when compared to f ish which ovulated 12-14 days after injection. Fish which ovulated 8-10 days after injection had lower 178-estradiol levels on day 4 than fish which ovulated 12-14 days after injection. Fish which did not ovulate by day 14 had lower 17a203P levels on days 2-10 than f ish which ovulated (Fig. 14B). Fish which ovulated by day 7 had higher plasma 17a208P levels until day 6 when compared to f ish which ovulated 8-10 days post injection and to day 8 when compared to fish which ovulated 12-14 days post injection. Fish which ovulated 8-10 days after injection had higher 17a208P levels on days 4 and 6 than f ish which ovulated 12-14 days after injection. The combined data for plasma 178-estradiol and 17a208P levels in fish receiving double hormone injections (Table 7) are i l lustrated in relation to the time of ovulation in F ig . 15 and 16. No significant differences in plasma 178-estradiol levels were observed for f ish which ovulated 6-7 and 8-10 days after injection (Fig. 15A). Both of these groups had lower plasma 178-estradiol levels on days 4, 6, and 8 than f ish which ovulated 12-14 days after injection.PIasma 17ct208P levels in f i sh which ovulated by day 7 were higher on day 4 when compared to f ish which ovulated 8-10 days after injection and were also higher on days 4 and 6 when compared to f ish which ovulated 12-14 days after injection (Fig,. 15B). Fish - 70 -FIG. 14. Changes in plasma 173-estradiol (A) and 17a206P (B) levels in relation to the time of ovulation for coho salmon receiving a single injection of LH-RHA DAla 6 , SG-G100 or combined injections of LH-RHA DAla 6 and SG-G100 (see Table 4). Steroid levels in individual f ish have been grouped according to the time of ovulation ( • 6-7, O 8-10, • 12-14, 0 > 14 days post injection). At each sampling period, plasma steroid levels (mean ± standard error, N) which are similar as determined by Duncan's Multiple Range Test (P > 0.05) are identified by the same superscript. 17<? - ESTRADIOL ( ng/ml ) 17a 203 P ( ng/ml ) - eoz -- 71 -FIG. 15. Changes in plasma 178-estradiol (A) and 17a208P (B) levels in relation to the time of ovulation for coho salmon receiving two separate injections of LH-RHA DAla 6 or SG-G100 followed by LH-RHA DAla 6 (see Table 4). For additional information see the legend to Fig. 14. 1 7 4 - ESTRADIOL (ng/ml) - nL -- 72 -FIG. 16. Temporal changes in plasma 178-estradiol (A) and 17a208P (B) levels in two fish which fa i led to ovulate by day 14 in response to two injections of LH-RHA DAla 6 over a 72 hr period. Values are based on measurements from one f ish receiving 0.02 mg LH-RHA DAla 6/kg ( • ) and a second fish receiving 0.2 mg LH-RHA DAla 6/kg ( • ). 17tf - ESTRADIOL (ng/ml ) - B 2 Z -- 73 -which ovulated on days 8-10 had higher plasma 17a208P levels on days 4 and 6 than fish which ovulated on days 12-14. The plasma 178-estradiol level in one fish which fai led to ovulate was 22 ng/ml on day 4 and declined to 1.1 ng/ml by day 10 (Fig. 16). In this case, plasma 17a208P remained low (< 100 ng/ml) during the sampling. Plasma 178-estradiol levels in a second f ish which did not ovulate, decreased to below 2 ng/ml by day 6 and showed a large surge in 17a208P to over 500 ng/ml at this time (Fig. 16). This f i sh had completed oocyte maturation by day 7 based on the visual examination of oocytes released while checking for ovulation, although ovulation did not occur until day 20. Fish which ovulated 6-7 and 8-10 days following a single (Fig. 14) or double injection (Fig. 15) had similar plasma 178-estradiol and 17a208P concentrations on days 4 to 10. Fish which ovulated on days 12-14 following two injections had higher (P < 0.01) plasma 178-estradiol levels on days 4 and 6 than f ish ovulating at this time but given only a single injection. No temporal differences in plasma 17a208P levels were apparent for f ish ovulating 12-14 days following single or dual hormone injections. IV. In Vitro Steroid Production by Ovarian Fo l l i c l e s During Sexual Maturation Figures 17, 18 and 19 show the levels of 178-estradiol , testosterone and 17a208P in the media following the incubation of ovarian f o l l i c l e s at various stages of maturity with or without SG-G100. The gonadal characteristics and plasma steroid levels in donor f ish are presented in Table 8. A. 178-Estradiol SG-G100 stimulated a dose-related increase in 178-estradiol production by ovarian f o l l i c l e s obtained in September (Fig. 17). 178-estradiol production by f o l l i c l e s from one of these f ish was-3.5 ± 0.2 ng/ml in the absence of gonadotropin and increased s ignif icantly (P < 0.05) to 4.6 ± 0.5 ng/ml in response to 62.5 ng/ml - 74 -TABLE 8 - Gonadal characteristics and plasma sex steroid levels in coho salmon ut i l ized for in vitro steroid production studies. Maturity Oocyte Plasma steroid concentration (ng/ml) Date status diameter (MM) 178-estradiol testosterone 17a208P September 16 Immature 4.1 12.0 7.6 < 1.0 September 16 Immature 4.3 15.4 9.4 < 1.0 October 15 Central GV* 5.1 22 108 5.6 October 15 Central GV 5.4 18 140 3.8 October 25 Migratory GV 5.6 9.4 275 18 October 25 Mature 5.5 2.0 185 220 October 27 Postovulatory - 0.8 110 240 * GV = germinal vesicle - 75 -FIG. 17. In vitro 17B-estradiol production by ovarian f o l l i c l e s of coho salmon at different stages of sexual maturity. Fo l l i c le s were incubated with Ringer's alone (0) or with various doses of SG-G100 for 24 hr at 10 °C . Values represent the levels of hormone in the media (mean ± standard error) based on three replicate incubations. 8 cn c < !/5 UJ I CO 0 15 62 250 1000 0 15 62 250 1000 4 September' » IM M A T U RE +rh 0 10 100 1000 0 10 100 1000 l Early O c t o b e r — — — * C E N T R A L cn 0 10 100 1000 0 10 100 1000 — Late October-PERIPHERAL M A T U R E 0 15 62 250 1000 POST OVULATORY S G - G I O O (ng /ml ) - 76 -SG-G100. Further increases occurred in response to 250 and 1000 ng/ml SG-G100 reaching 7.7 ± 0.3 ng/ml at the higher concentration. The pattern of 178-estradiol production by f o l l i c l e s from a second f ish obtained in September was similar. Basal 178-estradiol production was 4.0 ± 0.2 ng/ml in the absence of gonadotropin. SG-G100 at 250 ng/ml increased 178-estradiol production s ignif icantly (P < 0.05) with a further increase (P < 0.05) seen in response to 1000 ng/ml SG-G100 reaching 8.9 ± 0.5 ng/ml. Ovarian f o l l i c l e s characterized by a central germinal vesicle showed a diminished response to SG-G100 in terms of 178-estradiol production. Basal 178-estradiol production was 2.9 ± 0.6 ng/ml by f o l l i c l e s from one of these f ish and did not change in response to SG-G100 at doses up to 1000 ng/ml. 178-estradiol production by f o l l i c l e s from a second fish was unchanged from basal levels of 2.5 ± 0.2 ng/ml when incubated with SG-G100 at doses up to 100 ng/ml. However, SG-G100 at 1000 ng/ml promoted a significant increase (P < 0.01) in 178-estradiol levels to 4.3 ± 0.2 ng/ml. Fo l l i c le s obtained from coho salmon in late October were insensitive to SG-G100 at doses up to 1000 ng/ml in terms of 178-estradiol production. There was a progressive reduction in basal 178-estradiol production which appeared to relate to oocyte maturity. Fo l l i c l e s characterized by a peripheral germinal vesicle produced 1.2 ± 0.2 ng/ml 178-estradiol compared to 0.5 ± 0.1 ng/ml 178-estradiol by f o l l i c l e s obtained after maturation in vivo. 178-estradiol production by postovulatory f o l l i c l e s was 0.1 ng/ml when incubated with Ringer's alone. B. Testosterone Ovarian f o l l i c l e s obtained from coho salmon in September produced relat ively small amounts of testosterone in the absence of gonadotropin (0.07 ± 0.01 and 0.08 ± 0.01 ng/ml; Fig 18). Testosterone production by f o l l i c l e s from one of these fish was elevated (P < 0.05) in response to 62.5 ng/ml SG-G100 and showed - 77 -FIG. 18. In vitro testosterone production by ovarian f o l l i c l e s of coho salmon at different stages of sexual maturity. Fo l l i c l e s were incubated with Ringer's alone (0) or with various doses of SG-G100 for 24 hr at 10 °C . Values represent the levels of hormone in the media (mean ± standard error) based on three replicate incubations. 50 r 40 30 20 10 16 63 250 1000 16 63 250 1000 10 - September-I M M A T U R E 100 1000 0 10 100 1000 -Early O c t o b e r — — — * C E N T R A L 0 10 100 1000 0 10 100 1000 o • Late October-PERIPHERAL MATURE 0 15 62 250 1000 POST OVULATORY S G - G I O O ( n g / m l ) - 78 -a further increase (P < 0.01) to 0.43 + 0.02 ng/ml in response to SG-G100 at 1000 ng/ml. Testosterone production by f o l l i c l e s from a second fish increased in response to 1000 ng/ml SG-G100 (P < 0.01) reaching 0.59 ± 0.02 ng/ml. Ovarian f o l l i c l e s obtained from coho salmon in mid-October showed an increased capacity to produce testosterone. Although basal testosterone production by f o l l i c l e s from one of these fish was low (0.36 ± 0.03 ng/ml), SG-G100 at 10, 100 and 1000 ng/ml stimulated a dose related increase (P < 0.01) in testosterone production. The 14.5 ± 2.7 ng/ml of testosterone produced in response to 1000 ng/ml of SG-G100 was about 40-fold higher than the levels produced in the absence of SG-G100. Testosterone production by ovarian f o l l i c l e s obtained from a second f ish at this stage, showed a similar pattern of response. Testosterone production increased from 1.2 ± 0.1 ng/ml in the absence of SG-G100 to 24.4 ± 0.6 ng/ml in response to SG-G100 at 1000 ng/ml. Fo l l i c l e s characterized by a peripheral germinal vesicle produced greater amounts of testosterone than f o l l i c l e s of less advanced maturity. Testosterone production was increased in a dose related manner (P < 0.01) by 10, 100 and 1000 ng/ml SG-G100. In this case, testosterone production was increased from basal levels of 6.4 ± 0.4 ng/ml in the absence of SG-G100 to 44.4 ± 4.2 ng/ml in response to 1000 ng/ml SG-G100. Ovarian f o l l i c l e s obtained following maturation in vivo produced 4.8 ± 0.2 ng/ml of testosterone when incubated with Ringer's alone. Testosterone production was increased s ignif icantly (P < 0.05) in response to 10 ng/ml of SG-G100 and further increased (P < 0.05) to maximal levels in response to 100 and 1000 ng/ml SG-G100 reaching 24.4 ± 1,8 ng/ml at the higher concentration. Testosterone production by postovulatory f o l l i c l e s was 3.6 ± 0.2 ng/ml when incubated with Ringer's alone and increased signif icantly (P < 0.01) when incubated with SG-G100 at 62 ng/ml. No further increases in testosterone production occurred when incubated with SG-G100 at 250 or 1000 ng/ml. - 79 -C. 17a20BP Ovarian f o l l i c l e s obtained from one fish in September produced nondetectable levels of 17a20BP (less than 50 pg/ml) when incubated with Ringer's alone or SG-G100 at doses up to 1000 ng/ml (Fig. 19). Similar results were obtained for f o l l i c l e s obtained from a second f i sh , although detectable levels of 17a20BP were measured in 2 out of the 3 replicates for f o l l i c l e s incubated with 1000 ng/ml of SG-G100. In these cases, 17a20BP levels were 125 and 220 pg/ml respectively. Ovarian f o l l i c l e s obtained in mid-October showed increased 17a20BP production. Fo l l i c l e s from one of these f ish produced 1.2 + 0.2 ng/ml of 17a20BP in the absence of gonadotropin and did not change following incubation with 10 or 100 ng/ml of SG-G100. However, 17a20BP production in response to 1000 ng/ml of SG-G100 was s ignif icantly higher (P < 0.01) than the other groups reaching 3.8 ± 0.5 ng/ml. Fo l l i c l e s from a second f ish at this stage produced low levels of 17a20BP (0.6 ± 0.02 ng/ml) when incubated with Ringer's alone but increased s ignif icantly (P < 0.01) when incubated with SG-G100 at 100 or 1000 ng/ml. In this case, 17a208P levels in response to 1000 ng/ml SG-G100 were higher (P < 0.05) than the levels produced in response to 100 ng/ml SG-G100 reaching 5.3 ± 0.7 ng/ml at the higher dose. Ovarian f o l l i c l e s obtained in late-October in which the germinal vesicle had migrated towards the periphery produced greater amounts of 17a20BP than did previous stages. 17a20BP production by f o l l i c l e s increased s ignif icantly (P < 0.05) in response to 10 ng/ml SG-G100 from basal levels of 3.0 + 0.2 ng/ml to 5.8 ± 0.1 ng/ml. A further increase (P < 0.05) was observed in response to 100 and 1000 ng/ml SG-G100 to 10.6 ± 0.8 ng/ml at the higher concentration. Fo l l i c l e s obtained following maturation in vivo produced 13.2 ± 1.1 ng/ml of 17a20BP in the absence of gonadotropin and showed a significant elevation (P < 0.05) in response to 10 ng/ml of SG-G100. 17a20BP production further increased in response to 100 or 1000 ng/ml - 80 -FIG. 19. In vitro 17a20BP production by ovarian f o l l i c l e s of coho salmon at different stages of sexual maturity. Fo l l i c l e s were incubated with Ringer's alone (0) or with various doses of SG-G100 for 24 hr at 10 °C . Values represent the levels of hormone in the media (mean ± standard error) based on three replicate incubations. 17a20BP levels which were less than 50 pg/ml were considered to be non-detectable. 40 t 30 20 10 Not Detectable Not Detectable 0 15 62 250 1000 0 15 62 2501000 » S eptember . » I M M A T U R E 0 10 100 1000 0 10 100 1000 > Early October * C E N T R A L S G - G I O O ( n g / m l ) 0 10 100 1000 PERIPHERAL 0 10 100 1000 •Late October-MATURE 0 15 62 250 1000 POST OVULATORY - 81 -of SG-G100 reaching maximal levels of 37.3 ± 4.3 ng/ml in response to 100 ng/ml of SG-GlOO.Postovulatory f o l l i c l e s in comparison with other stages produced very large amounts of 17a208P. In this case, media 17a208P levels were 14.6 ± 6.2 ng/ml in the absence of SG-G100 and increased to 199 ± 21 ng/ml in response to 1000 ng/ml of SG-G100. V. Effects of 178-estradiol on 17a208P Production In Vitro F ig . 20A shows the levels of 17a208P released to the media by f o l l i c l e s characterized by a central germinal vesicle following gonadotropin stimulation in the presence or absence of 178-estradiol SG-G100 stimulated a dose related increase in 17a208P production from 0.4 ± 0 . 1 ng/ml in the absence of SG-G100 to 2.2 ± 0.2 ng/ml in the presence of 1000 ng/ml SG-G100 The addition of 178-estradiol at 25 and 250 ng/ml did not influence basal or SG-G100 at 10 and 100 ng/ml stimulated 17a208P production. The amounts of 17a208p produced by f o l l i c l e s incubated with 1000 ng/ml SG-G100 and 178-estradiol was lower (P < 0.01) than the levels produced by f o l l i c l e s incubated with 1000 ng/ml SG-G100 alone. F ig . 20B show the results of a similar experiment conducted on f o l l i c l e s characterized by a peripheral germinal vesicle. In this case, 17a208P production increased from basal levels of 4.5 ± 0.2 ng/ml to 20.6 ± 0.6 ng/ml in response to SG-G100 at 1000 ng/ml. The addition of 178-estradiol at 25 or 250 ng/ml did not influence basal or gonadotropin stimulated 17a208P production. D. Discussion Preovulatory Steroid Changes The preovulatory period in coho salmon was characterized by declining 178-estradiol levels 10 days prior to ovulation and high testosterone levels with peak levels evident 6 days prior to ovulation (Fig. 9). The preovulatory period was also characterized by a large increase in plasma 17a20BP levels coincident to - 82 -FIG. 20. Effects 176-estradiol on the production of 17a20BP in vitro in response to graded amounts of SG-G100. Values represent the mean ± standard error based on three determinations using f o l l i c l e s characterized by a central germinal vesicle (A) and by a peripheral germinal vesicle (B). S G -G100 ( n g / m l ) - 83 -the completion of oocyte f inal maturation. A similar temporal pattern of change in these hormones has recently been described during the preovulatory period in rainbow trout (Fostier and Jalabert, 1982; Scott et a l . , 1983). This suggests that the pattern of steroid secretion associated with oocyte maturation and ovulation is similar in coho salmon and rainbow trout. The high levels of 17a20BP in the plasma of coho salmon at the time of oocyte maturation (Fig. 9) was consistent with the concept that 17a20BP functions as the maturation inducing steroid in salmonids (Jalabert, 1976; Young et a l . , 1982a; Goetz, 1983; Nagahama et a l . , 1983). However, the functional significance of changes in 173-estradiol and testosterone during the preovulatory period remains unclear. Although 173-estradiol is ineffective and testosterone has only a limited effectiveness in promoting maturation in salmonids (Jalabert, 1975; Young et a l . , 1982a), the maintenance of low 17B-estradiol and high testosterone levels may contribute to the proper steroid environment for 17a20BP synthesis and oocyte maturation. Testosterone has been shown to enhance the effectiveness of gonadotropin and 17a20BP on the induction of oocyte maturation in vitro (Jalabert, 1976; Young et a l . , 1982a) and therefore may be direct ly associated with oocyte maturation. 173-estradiol and testosterone may also have important regulatory effects on the control of gonadotropin synthesis and release. Antiestrogens have been shown to stimulate gonadotropin secretion in teleosts (see Peter, 1982a,b) suggesting that declining 173-estradiol levels may permit increased gonadotropin secretion. The decline in 173-estradiol levels (Fig. 9) and the concomitant rise in gonadotropin levels (Fig. 3; Chap. 3) are suggestive of a cause and effect relationship, although proof that this represents release from a negative feedback is lacking. Aromatizable androgens and estrogens have been shown to increase pituitary gonadotropin levels in sexually immature salmonids (Crim and - 84 -Evans, 1979; Crim et a l . , 1981b; Geilen et a l . , 1982). The high levels of testosterone during the preovulatory period in coho salmon may function in a similar fashion to increase pituitary gonadotropin levels in preparation for increased gonadotropin secretion associated with oocyte maturation and ovulation and may also have a role in spawning behavior. Based on short-term studies which examined the pattern of steroid secretion by coho salmon ovarian fo l l i ce s in vitro (Fig. 17, 18 and 19), i t appears that the preovulatory changes in 178-estradiol, testosterone and 17a208P (Fig. 9) result primarily from changes in the act ivi ty of steroid converting enzymes located in the f o l l i c l e . Although results of the in vitro studies were based on determinations from only one or two f ish at each stage of maturity, these findings have been confirmed by recent reports detailing the pattern of steroid secretion by amago salmon f o l l i c l e s (Kagawa et a l . , 1983; Young et a l . , 1983a). Fo l l i c l e s from adult coho salmon upon entry to freshwater produce high levels of 178-estradiol but then show a progressive loss of sensi t ivi ty to SG-G100 and by the postovulatory stage produce negligible amounts of hormone (Fig. 17). Previous studies have also shown that gonadotropin stimulates 178-estradiol production during vitellogenesis (Bi l lard et a l . , 1978; Yaron and Barton, 1980; Kagawa et a l . , 1982a,b; Zohar et a l . , 1982) although recent evidence indicates that this action is a consequence of providing a suitable substrate for aromatization (Kagawa et a l . , 1982b; Young et a l . , 1982b,c, 1983c). For example, the conversion of exogenous testosterone to 178-estradiol by isolated granulosa ce l l layers from the amago salmon was not enhanced by the addition of gonadotropin (Kagawa et a l . , 1982b; Young et a l . , 1982b, 1983c). It is doubtful that the preovulatory decline in 178-estradiol is related to a lack of substrate since coho salmon have high levels of testosterone throughout the preovulatory period (Fig. 9 and 12) and produce - 85 -large quantities of testosterone in response to SG-G100 in vitro (Fig. 18). It is more l ike ly that the preovulatory decline in 178-estradiol levels results from declining aromatase act iv i ty . A reduction of ovarian aromatase act iv i ty during the preovulatory period in rainbow trout has been suggested on the basis of the reduced incorporation of ^- labe led androstendione into estrogens (van Boehman and Lambert, 1981). In addition, Young et a l . (1983c) reported that isolated granulosa cel l layers from preovulatory f o l l i c l e s and postovulatory f o l l i c l e s from the amago salmon f a i l to convert exogenous testosterone to 173-estradiol. Fo l l i c l e s obtained from coho salmon in September produce only low amounts of testosterone (Fig. 18) which may reflect i t s high rate of conversion to 178-estradiol. The high levels of testosterone in the plasma of maturing coho salmon (Table 5, F ig . 1 and 4) were consistent with the enhanced capacity of preovulatory f o l l i c l e s to produce testosterone in response to SG-G100 in vitro (Fig. 18). The increase in testosterone production by f o l l i c l e s in October presumbly relates to i t s decreasing rate of conversion to 178-estradiol but may also reflect an increase in the act iv i ty of enzymes associated with testosterone production. It is apparent from measurements of 17a208P production by f o l l i c l e s incubated in vitro that the capacity to produce this steroid develops 1-2 months prior to ovulation (Fig. 19). The low levels of 17a208P in the plasma of coho salmon prior to maturation (Table 8; F ig . 9, 11 and 14) were consistent with the limited capacity to produce this steroid in v i t ro . Additonally, basal 17a208P production by f o l l i c l e s from fish which matured in vivo was higher than the levels of 17a208P produced by f o l l i c l e s prior to maturation even when incubated with SG-G100. This observation suggests that oocyte maturation is associated with an increased capacity of the f o l l i c l e to produce 17a208P which presumably reflects increasing - 86 -amounts of 20B-HSD. Postovulatory f o l l i c l e s produced far greater amounts of 17a208P in response to SG-G100 than preovulatory f o l l i c l e s which had undergone maturation. This was somewhat suprising in view of the comparable plasma 17a208P levels in these f ish (Table 8) and the tendency for 17a208P levels to decline at ovulation (Fig. 9 and F ig . 13). Young et a l . (1983a) also found that postovulatory f o l l i c l e s from the amago salmon produced far greater amounts of 17a208P in response to SG-G100 than preovulatory f o l l i c l e s . The basis of the increased capacity of postovulatory f o l l i c l e s to produce 17a20BP may relate to hypertrophy of the granulosa ce l l layer which is the l ike ly source of the hormone (Young et a l . , 1983a,b). Testosterone production by f o l l i c l e s following maturation in vivo and by postovulatory f o l l i c l e s was decreasing while 17a20BP production was highest (Fig. 18 and 19). This finding was consistent with the tendency for plasma testosterone levels to decline at maturation (Fig. 9) and the decreasing plasma testosterone levels following LH-RHA DAla 6 injection in f ish which completed GVBD (Fig. 12). The basis for the diminished capacity of the f o l l i c l e to produce testosterone is not known, however, both testosterone and 17a208P are produced from 17ahydroxyprogesterone. Whether decreasing amounts of testosterone result from the predominance of 20B-HSD which mediates the conversion of 17ahydroxyprogesterone to 17a20BP or a concomitant reduction in the act ivi t ies of C19-21 desmolase and 17B-HSD which mediate the conversion of 17ahydroxyprogesterone to testosterone remains to be investigated. In mammals, 17ct20BP binds i r revers ib i ly to desmolase blocking testosterone production (Inano et a l . , 1967). A similar mechanism in coho salmon could contribute the decreasing production of testosterone when 17a20BP production is high. - 87 -Steroid Changes in Response to Elevated Plasma Gonadotropin Levels The changes in plasma 17a208P and testosterone levels following injections of LH-RH and LH-RHA DAla 6 (Fig. 11 and 12) were consistent with the differential effects of these treatments on plasma gonadotropin levels (Fig. 4, Chapt. 3). Injections of LH-RH promote a transient increase in plasma 17a20BP levels which persists for less than 24 hr (Fig. 11). Although 17a208P levels in LH-RHA DAIa 6-injected f i sh were highly variable, these levels were higher than those in LH-RH-injected f ish at 11-96 hr (Fig. 11). Injections of LH-RH and LH-RHA DAla 6 also had very different effects on plasma testosterone levels (Fig. 12). LH-RH-injected f ish showed a significant decrease in plasma testosterone levels at 48-96 hr when compared to preinjection values. In contrast, testosterone levels in LH-RHA DAla 6-injected f ish were increased at 24 hr and 48 hr in f ish which completed and fa i led to complete GVBD, respectively (Fig. 12). Furthermore, the testosterone levels in LH-RHA DAla 6-injected f ish were higher than the levels in saline and LH-RH-injected f ish at 48-96 hr. These differences were consistent with the short duration increase in plasma gonadotropin levels in LH-RH-injected fish and the longer duration increase in LH-RHA DAIa 6-injected f ish (Fig. 4, Chapt. 3). Why testosterone levels decrease in LH-RH-injected f ish is not understood. LH-RH-injected f ish also had lower 17a20BP levels than saline-injected f ish at 96 hr. Additional research is warranted to investigate the poss ib i l i ty that a transitory elevation of plasma gonadotropin levels depletes the supply of steroid precursors and a long-term increase is necessary to provide additional substrate for steroid synthesis. The effects of gonadotropin on plasma 178-estradiol levels were more d i f f i cu l t to interpret. Injections of saline and LH-RH had similar effects on plasma 178-estradiol levels (Fig. 10). In this case, plasma 178-estradiol levels - 88 -were decreased s ignif icantly relative to preinjection values by 48 hr. Furthermore, this decrease occurred in the apparent absence of changes in plasma gonadotropin levels (Fig. 4, Chapt. 3). The effects of LH-RHA DAla 6 on plasma 178-estradiol levels were highly variable. 178-estradiol levels in LH-RHA DAIa 6-injected f i sh which were induced to mature decreased at 48-96 hr to levels 's ignificantly lower than those in saline or LH-RH-injected f ish (Fig. 10). In contrast, LH-RHA DAIa 6-injected f ish which fa i led to mature had higher 178-estradiol levels than saline-injected f ish at 24 and 48 hr but then showed a sharp decrease to levels similar to those in saline-injected f ish by 72 and 96 hr. It is possible that these disparate results are a consequence of differences in the maturity of f ish at the time of injection. A comparison of the steroid profiles in saline-injected f ish (Fig. 10, 11 and 12) and those of spontaneously ovulating fish (Fig. 9) suggest that hormone injections were administered about 8-10 days prior to the expected time of ovulation. A similar conclusion was reached on the basis of the time of ovulation in laboratory held coho salmon. Owing to this advanced state of maturity, the decline in 178-estradiol levels in saline-injected f ish would be expected in the absence of gonadotropin stimulation. Although the difference was not s ignificant, LH-RHA DAla 6-injected f ish which fa i led to mature tended to have higher 173-estradiol levels than other groups at the time of injection which could account for the delay in reducing 178-estradiol levels. Results of a second experiment using f i sh of less advanced maturity provide further evidence for a gonadotropin induced decrease in plasma 178-estradiol levels (Table 5). The effects of gonadotropin on plasma 178-estradiol levels were dose-and time-dependent. The injection of SG-G100 alone or in combination with LH-RHA DAla 6 results in higher plasma gonadotropin levels on days 1 and 2 than injections of LH-RHA DAla 6 alone (Fig. 6, Chapt. 3) and promotes a significant reduction in - 89 -plasma 178-estradiol levels by day 2 compared to day 4 following LH-RHA DAla 6 injection (Table 5). Owing to the high variation in 178-estradiol levels between individual f i sh in each of the hormone treated groups (Fig. 14, 15 and 16) i t was not possible to discriminate major differences in the effects of the various hormone treatments on days 4 to 10. In general, f i sh which ovulated in response to the various hormone treatments showed a more pronounced reduction in plasma 178-estradiol levels than f ish which fa i led to ovulate. Studies based on the production of 178-estradiol in vitro by ovarian f o l l i c l e s from coho salmon (Fig. 17) and from other salmonids (van Boehman and Lambert, 1981; Nagahama and Kagawa, 1982; Kagawa et a l . , 1983; Young et a l . , 1983c) suggest that aromatase act ivity declines from maximal levels during vitellogenesis to very low levels at ovulation. In part, the high variation in plasma 178-estradiol levels in response to elevated plasma gonadotropin levels may reflect differences in the maturity of f ish at the time of injection. Sower et a l . (1983) recently provided confirmation of these results in that injections of salmon gonadotropin and LH-RHA DAla 6 were shown to decrease plasma 178-estradiol levels in adult female coho salmon and steelhead trout. Furthermore, Zohar et a l . (1982) demonstrated a gonadotropin induced inhibition of 178-estradiol secretion by rainbow trout oocytes incubated in an open-perifusion system. It is l ike ly that the gonadotropin-induced decrease in plasma 178-estradiol levels results from an inhibition of aromatase act ivity although the precise mechanism of this action is poorly understood. There are several aspects of the regulation of aromatase act ivity that remain unclear. F i r s t , i t appears that there is a gradual loss of aromatase act ivity throughout the preovulatory period (Fig. 9). However, elevated gonadotropin levels were only evident during the 1-2 week period preceding ovulation (Fig. 2 and 3, Chap. 3). Zohar et a l . (1982) suggest during the preovulatory period in rainbow - 90 -trout that gonadotropin levels show a gradual increase but more importantly show a marked daily fluctuation to very high levels which direct ly contribute to the decline in ovarian aromatase act iv i ty . However in coho salmon,there was no evidence to indicate a significant daily cycle in plasma gonadotropin levels (see Fig . 4, Chapt. 3). It is possible that the increase in plasma gonadotropin levels at the end of the preovulatory period (Fig. 2 and 3; Chapt 3) is secondary to an earl ier increase from the very low and often undetectable levels present in immature f i sh . In addition to gonadotropin, the possible involvement of other hormones in regulating aromatase act iv i ty can not be excluded. For example, FSH is required to maintain aromatase act ivi ty in mammals (Leung and Armstrong, 1980). The possible involvement of the "vitellogenic gonadotropin" (Idler, 1982) in controlling aromatase act ivity merits further investigation. A third aspect of the regulation of aromatase act ivity which is poorly understood is the mechanism whereby gonadotropin switches from having a stimulatory to an inhibitory effect on 17B-estradiol secretion. During vitellogenesis in the amago salmon, gonadotropin acting on the thecal ce l l layer stimulates the production of aromatizable androgens which are converted to 17B-estradiol in the granulosa ce l l layer which contains aromatase (Kagawa et a l . , 1982b; Young et a l . , 1982b, 1983c). Young et a l . (1983b) have shown that gonadotropin acting direct ly on the granulosa ce l l s induces 20J3HSO act iv i ty . The loss of aromatase act ivity (Fig. 17) appears to coincide with the ab i l i ty to produce 17a20BP in vitro (Fig. 19). It is possible that gonadotropin acting via a receptor which appears in the granulosa ce l l s in the latter stages of the preovulatory period provides a second site of action which in addition to having a stimulatory effect of 208HSD act ivity is inhibitory to 17B-estradiol production. The gonadotropin-induced decrease in plasma 178-estradiol levels in coho - 91 -salmon contrasts with data for carp (Weil et a l . , 1980) and goldfish (Stacey et a l . , 1984). In the adult carp, plasma 17B-estradiol levels increase following injections of LH-RH or crude pituitary extracts (Weil et a l . , 1980). In goldfish, plasma 178-estradiol levels increase during spontaneous and brain-lesion induced ovulation (Stacey et a l . , 1983). It is possible that this may relate to the synchronous pattern of oocyte development in coho salmon and the asynchronous pattern of oocyte development in the carp and goldfish. For example, when oocytes from maturing goldfish were subdivided according to size, small f o l l i c l e s respond to gonadotropin by increased 178-estradiol production whereas large f o l l i c l e s showed a reduced response (W. Garcia and R.E. Peter, personal communication). Plasma 17a208P levels were low (< 10 ng/ml) at the time of injection in Exp. II and III, but increase rapidly following hormone treatment. In Exp. II, plasma 17a208P levels were elevated 3 hr following injections of LH-RH and LH-RHA DAla 6 (Fig. 11). In Exp. I l l , plasma 17a208P levels were increased at the f i r s t sampling (24 hr) i n 5 a l l hormone-treated groups (Table 6, F ig . 14, and 15). These results suggest that the enzymes required for the synthesis of 17a208P were present at least one month prior to the expected time of ovulation. A similar conclusionwas reached on the basis of 17a208P production by ovarian f o l l i c l e s in vitro (Fig. 19). 17a208P levels in LH-RHA DAla 6 injected f ish were increased to an apparent plateau at 6-11 hr, with the large increase observed in f ish which completed GVBD evident by 48 hr (Fig. 11). In Exp. IV, the increase in plasma 17a208P levels to greater than 150 ng/ml was delayed up to 6 days in f ish which ovulated by day 14 (Fig. 14 and 15). Recent studies in rainbow trout, coho salmon and Atlantic salmon have also shown a delay prior to the large increase in 17a208P levels following injections of crude pituitary extracts (Scott et a l . , 1982; Wright and Hunt, 1982). It is l ike ly that the amounts or act iv i t ies of enzymes associated with - 92 -17a20BP synthesis are l imiting and direct ly contribute to this delay. For example, Jalabert (1976) reported that gonadotropin-induced maturation of rainbow trout oocytes in vitro was sensitive to actinomycin D and puromycin. 17a20BP-induced maturation was not sensitive to these inhibitors , suggesting that the actions of gonadotropin are mediated by mRNA and protein synthesis required for the production of enzymes necessary for 17a20BP synthesis (Jalabert, 1976). Suzuki et a l . (1981) reported that gonadotropin stimulated the act iv i ty or induced the formation of 20B hydroxysteroid dehydrogenase (20BHSD) in the ayu. It is l ike ly that the the delay associated with the surge in 17a20BP levels in coho salmon is a consequence of this action. Steroid Changes Associated with Induced Oocyte Maturation and Ovulation The induction of oocyte maturation was related to the duration and magnitude of the increase in plasma 17a20BP levels. A transient elevation of 17a20BP levels as seen following injections of LH-RH or a short-term increase at low t i t res (55 ng/ml) as seen in some LH-RHA DAIa 6-injected fish fa i led to promote GVBD (Fig. 11). LH-RHA DAla 6-injected fish which completed GVBD showed a long-term increase in 17a20BP reaching 480 ng/ml by 72 hr (Fig. 11). GVBD was induced in rainbow trout oocytes incubated in vitro within 60 hr of treatment with 1 pg/ml of 17a20BP for 30 sec to 15 min (Jalabert, 1976). Although this dose was higher than the levels reported here, continuous exposure of rainbow trout oocytes to 30 ng/ml of 17a20BP (Jalabert, 1976) or amago salmon oocytes to 5-30 ng/ml of 17a20BP (Young et  a l . , 1982a; Nagahama et a l . , 1983) was sufficient to promote maturation in 50% of the oocytes during a 3 day incubation. The apparent difference between the higher levels of 17a20BP necessary to promote GVBD in vivo and that required in vitro may relate to the binding of 17a20BP to plasma proteins in vivo. The binding of 17ot20BP to plasma proteins would presumably reduce the concentration of free - 93 -hormone a v a i l a b l e f o r b i n d i n g t o 17a20|3P r e c e p t o r s on t h e o u t e r s u r f a c e o f t h e o o c y t e . F o s t i e r and Br e t o n (1975) r e p o r t e d t h a t t h e a d d i t i o n o f carbon t r e a t e d plasma t o rainbow t r o u t o o c y t e s i n c u b a t e d i n v i t r o i n c r e a s e d t h e median e f f e c t i v e dose of 17a20BP r e q u i r e d t o s t i m u l a t e GVBD by about 1 0 - f o l d n e c e s s i t a t i n g t h e use of 100-200 ng/ml. Recent d a t a on 17a20BP changes d u r i n g i n d u c e d o v u l a t i o n i n g o l d f i s h ( S t a c e y et a l . , 1983) were v e r y d i f f e r e n t from t h e d a t a r e p o r t e d here f o r coho salmon. Oocytes from t h e g o l d f i s h matured w i t h i n 5 hr o f hy p o t h a l a m i c l e s i o n s which s t i m u l a t e a mass i v e i n c r e a s e i n plasma g o n a d o t r o p i n l e v e l s but show o n l y a t r a n s i e n t i n c r e a s e i n 17a208P l e v e l s t o 20 ng/ml at 5 hr p o s t l e s i o n i n g ( S t a c e y e t a l . , 1983). U n l i k e coho salmon which r e q u i r e a lo n g - t e r m e l e v a t i o n o f 17a208P at hi g h l e v e l s t o ind u c e o o c y t e m a t u r a t i o n , g o l d f i s h r e q u i r e o n l y a s h o r t - t e r m i n c r e a s e i n 17a208P at r e l a t i v e l y low l e v e l s t o promote o o c y t e m a t u r a t i o n . However, Nagahama e t a l . (1983) f a i l e d t o d e t e c t a major d i f f e r e n c e i n t h e a c t i v i t y of 17a206P on t h e s t i m u l a t i o n o f o o c y t e m a t u r a t i o n when o o c y t e s from rainbow t r o u t , amago salmon, ayu and g o l d f i s h were i n c u b a t e d i n v i t r o . As 17a208P was not d e t e c t e d i n t h e plasma o f g o l d f i s h which o v u l a t e s p o n t a n e o u s l y , i t i s p o s s i b l e t h a t 17a208P c o u l d e x e r t i t s e f f e c t l o c a l l y b e f o r e t h e r a t e o f s y n t h e s i s was s u f f i c i e n t t o i n c r e a s e plasma l e v e l s . Oocyte m a t u r a t i o n i s a p r e l i m i n a r y s t e p t o o v u l a t i o n i n t e l e o s t s . These events a re n o r m a l l y c l o s e l y l i n k e d , w i t h o o c y t e m a t u r a t i o n o c c u r r i n g 2-4 days p r i o r t o o v u l a t i o n i n coho salmon. These e v e n t s can be d i s s o c i a t e d when a t t e m p t i n g t o induce o v u l a t i o n i n t h a t f i s h can be ind u c e d t o mature but f a i l t o o v u l a t e at t h e exp e c t e d t i m e . An ex a m i n a t i o n o f o o c y t e s e x p e l l e d when c h e c k i n g f o r o v u l a t i o n i n Exp. I l l i n d i c a t e d t h a t a l l but one o f t h e hormone-treated f i s h had completed o o c y t e m a t u r a t i o n by day 10. However, many o f t h e s e f i s h d i d not o v u l a t e by day 14 - 94 -and in certain cases did not ovulate until after day 20. The basis of the dissociation between oocyte maturation and ovulation is poorly understood. It was possible to distinguish f ish which were induced to mature from fish which ovulate on the basis of 17a20BP levels. With one exception (see below), there was a marked difference in the maximal 17a208P levels in f ish which ovulated by day 14 (450 ng/ml) and f i sh which ovulated at later times (100 ng/ml) (Fig. 14, 15, and 16). These results suggest that a long-term elevation of plasma 17a208P at re lat ively low concentrations was sufficient to promote oocyte maturation. Since 17a208P levels in the former group were lower than the levels in spontaneously ovulating fish (Fig. 9 and 13) and f ish induced to ovulate (Fig. 14 and 15) i t appears that high levels of 17a208P may have a role in ovulation. Ovulation occurs following oocyte maturation and after the detachment of granulosa ce l l s from the oocyte (Jalabert and Szol loski , 1975; Jalabert, 1978; Fostier and Jalabert, 1982). In rainbow trout, low levels of 17a208P were required to promote oocyte maturation whereas higher levels of 17a208P were required to promote detachment of the granulosa cel l s from the oocyte in vitro (Jalabert, 1978). A similar requirement for high levels of 17a208P in vivo may in part contribute to the fa i lure of some fish to ovulate. As ovulation appears to be mediated by prostaglandins (Jalabert, 1976; Stacey and Goetz, 1982; Goetz, 1983), i t is l ike ly that low levels of 17a208P are not the sole reason for the fai lure of f ish to ovulate. Furthermore, high levels of 17a208P do not necessarily ensure ovulation. For example, the 17a208P profi le in one f ish which did not ovulate until day 20 (Fig. 16) was comparable to that observed in f ish which ovulated by days 8-10 (Fig. 14 and 15). Scott et a l . (1982) also found that injections of crude pituitary extracts which result in a large increase in plasma 17a208P levels did not always result in ovulation in rainbow trout. Previous studies have shown that the injection of - 95 -17a20SP promotes oocyte maturation in rainbow trout and coho salmon but has a variable effect on ovulation (Jalabert et a l . , 1976, 1978a,b). In f ish which were close to maturity and have high levels of endogenous gonadotropin, injection of 17a20BP is sufficient to induce oocyte maturation and ovulation (Jalabert et a l . , 1976). In less mature f i sh , characterized by lower endogenous gonadotropin levels, i t was necessary to supplement 17a206P with gonadotropin to ensure that both f inal maturation and ovulation occur (Jalabert et a l . , 1978a,b). As the dissociation between maturation and ovulation was most pronounced in groups receiving a single hormone injection (see Table 7) i t is l ike ly that a transitory elevation of gonadotropin contributes to both the low levels of 17a208P and the fa i lure to ovulate. Furthermore, 17a206P has been shown to decrease gonadotropin secretion in salmonids (Jalabert et a l . , 1978b; Jalabert and Breton, 1980) which could compound the effects of a gonadotropin deficiency required for ovulation. The preovulatory period in salmonids involves a switch in steroidogenesis from 178-estradiol to 17a20BP (Fig. 9; Fostier and Jalabert, 1982; Scott et a l . , 1983). Declining 178-estradiol levels appear to be the primary signal which determines the time of oocyte maturation and ovulation during both spontaneous and induced development. For f ish which ovulate spontaneously, the decline in 17B-estradiol levels in the plasma precedes the large increase in 17a20BP required for oocyte maturation (Fig. 9 and 13). Similarly, changes in 178-estradiol production appear to determine the time of oocyte maturation and ovulation following hormone treatments which elevate plasma gonadotropin levels. In short-term studies, f i sh which were induced to mature following injections of LH-RHA DAla 6 could be distinguished from fish which f a i l to mature on the basis of 178-estradiol levels (Fig. 10). In this case, f ish which fa i led to mature maintained higher 178-estradiol levels and did not show a large surge in 17a208P - 96 -levels (Fig. 10 and 12). For f ish which were induced to ovulate, the time of ovulation was related to the rate of decline in plasma 178-estradiol levels (Fig. 14 and 15). In this case, the drop in 176-estradiol to less than 2 ng/ml preceded or was concomitant to an increase in 17a208P levels to 450-500 ng/ml. Furthermore, an increase in 17a20BP to high levels (> 200 ng/ml) did not occur in the absence of a decline in 173-estradiol levels to less than 2 ng/ml (Fig. 13 and 16). These results raise the poss ib i l i ty that 173-estradiol may have a regulatory influence on 17a203P synthesis. High levels of 173-estradiol in vitro have been shown to reduce the effectiveness of gonadotropin on the stimulation of oocyte maturation in rainbow trout (Jalabert, 1975) and brook trout (Theofan, 1981 cited in Goetz, 1983). As 173-estradiol had no effect on 17a203P induced maturation in these species (Jalabert, 1975; Goetz, 1983) i t appears that 178-estradiol blocks the actions of gonadotropin on 17a206P synthesis. However, Young et a l . (1982a) reported that high levels of 178-estradiol had no influence on gonadotropin induced maturation of amago salmon oocytes in v i t ro . Interpretation of these results are complicated by the poss ib i l i ty that the sensi t ivi ty of the f o l l i c l e to 178-estradiol may change during development. The possible involvement of 178-estradiol on 17a20BP production in coho salmon was investigated by studying the effects of 178-estradiol on the gonadotropin induced stimulation of 17a208P production in vitro (Fig. 20). For f o l l i c l e s characterized by a central germinal vesicle, 178-estradiol reduced the effectiveness of gonadotropin on the stimulation of 17a208P synthesis. However, 178-estradiol was without effect in f o l l i c l e s with a peripheral germinal vesicle. These results provide further evidence that 178-estradiol can modify 17a208P production, but further studies wi l l be necessary to determine the mechanism of this action. Traditional methods used for the induction of ovulation in teleosts generally - 97 -rely on the administration of pituitary extracts in two successive injections (see Harvey and Hoar, 1979; Lam, 1982). Priming by the injection of a low dose of gonadotropin has been shown to increase the effectiveness of subsequent hormone injections on the induction of ovulation in coho salmon (Jalabert et a l . , 1978b; Donaldson et a l . , 1981; Hunter et a l . , 1981; Sower et a l . , 1982). However, the basis of this priming action in largely unknown. In the present study, 72.5 and 91.7% of the f ish ovulated by day 14 in groups receiving one and two injections respectively (Table 7). The high rate of response to a single injection and the variation in the time of ovulation make i t d i f f i cu l t to establish whether f ish ovulate as a consequence of the f i r s t or second injection. Fish which fai led to ovulate in groups receiving a single injection could be distinguished from fish which ovulated on the basis of higher 173-estradiol levels on days 2-10 (Fig. 14A). Fish which ovulated on days 12-14 in groups receiving a second hormone injection at 72 hr had similar plamsa 170-estradiol levels on day 4 to those fish which fa i led to ovulate in response to a single hormone injection (Fig. 14A and 15A). On subsequent sampling days, f i sh in the former group showed a marked decrease in plasma 173-estradiol level and a surge in plasma 17a208P level (Fig. 15). These results suggest that f ish which ovulated on days 12-14 in groups receiving two injections did so as a consequence of the second injection. Since steroid changes associated with induced ovulation appear to follow a progression, the f i r s t injection may in i t i a te this sequence by an inhibition of aromatase act iv i ty . The second injection would then be able to reduce aromatase act ivity to basal levels and stimulate the preovulatory 17a208P surge. However, a detailed examination of this hypothesis would necessitate the sampling of f i sh earl ier in the season when there may be a greater dissociation between f ish ovulating in response to one or two injections. - 98 -CHAPTER 5 - THE FUNCTIONAL PROPERTIES OF GONADOTROPIN RECEPTORS IN ADULT COHO SALMON AND IMMATURE CHUM SALMON A. Introduction The actions of gonadotropins in mammals are mediated by their binding to specific high af f inity receptors located in the plasma membrane of target ce l l s (see Dufau and Catt, 1978). Information on the nature of gonadotropin receptors has been derived from physiological studies which compare the effects of various types of gonadotropins on the stimulation of biological responses and from the direct binding of radiolabeled gonadotropins to gonadal tissue in an analogous fashion to RIA (see Licht , 1980). These approaches were used to determine the properties of gonadotropin receptors in Pacific salmon. Experiments in section 1 examine the speci f ic i ty of gonadotropin receptors by determining the effects of mammalian and teleost gonadotropins on steroid production in vi tro . The val id i ty of using 125i_iabeled salmon gonadotropin as a probe to study gonadotropin receptors was evaluated by determining the effects of iodination on the biological act iv i ty of salmon gonadotropin (section II). The properties of gonadotropin receptors were determined by the direct binding of 125j_] abeled salmon gonadotropin to ovarian tissue from immature chum salmon (section III) and adult coho salmon (section IV). B. Experimental Protocol. I. Effects of Teleost and Mammalian Gonadotropins on the Stimulation of Steroid Production In Vitro . The effects of chinook salmon and mammalian gonadotropins on the stimulation of steroid production by mid-vitellogenic ovarian f o l l i c l e s from chinook salmon and postovulatory f o l l i c l e s from coho salmon were investigated. A preliminary study investigated the time course of the effects of SG-G100 on 178-estradiol production - 99 -by chinook salmon ovarian f o l l i c l e s incubated at in vitro 10°C and 20 °C . Routinely gonadotropins were tested over a wide range of doses in t r ip l i ca te . Incubations were conducted for 20-24 hr at 10°C. The steroidogenic response was quantified in terms of the amounts of 17B-estradiol produced by chinook salmon f o l l i c l e s and the amounts of 17a20|3P produced by coho salmon postovulatory f o l l i c l e s . II. Effects of Iodination on the Biological Act iv i ty of Gonadotropin. The effects of iodination on the biological act ivi ty of salmon gonadotropin were determined by comparing the effects of untreated and 125l-labeled salmon gonadotropin on testosterone production by minced testicular tissue and ovarian f o l l i c l e s from coho salmon incubated in v i t ro . Iodination was performed by the lactoperoxidase method as described in Chapter 2 except that 0.6 nmoles of KI supplemented with 100,000 cpm 125j. w a s u s e f j -jn place of 1 mCi 125j_. After iodination, labeled gonadotropin and untreated gonadotropin were ser ia l ly diluted with Ringer's and incubated with gonadal tissue for 24 hr at 1 0 ° C . In one of these experiments, the act iv i t ies of gonadotropin subjected to the iodination conditions but with the iodide or H2O2 omitted were also evaluated. In order to establish whether the iodination conditions used in these studies resulted in the labeling of gonadotropin, trichloroacetic acid (TCA) precipitation was used to determine the incorporation of 125j t 0 gonadotropin. Aliquots of the iodination mixture (25 yl) and 200 yl of saline containing 1.0% BSA were combined with 1 ml of 10% TCA. After 10 min at 4 ° C , the mixture was centrifuged at 3000 g for 10 min.1 The incorporation of 125j. to gonadotropin was estimated from the percentage of radioactivity in the TCA pel let . III. Properties of Gonadotropin Binding Sites in Immature Chum Salmon Ovaries A series of competitive binding studies based on the ab i l i ty of ^-^5j_-|aibeled salmon gonadotropin to bind to ovarian tissue and for unlabeled gonadotropin to - 100 -compete for these binding sites were conducted to determine the properties of gonadotropin receptors in immature chum salmon ovaries. In i t ia l studies were conducted to localize the site of gonadotropin binding by studying the uptake of 125i_-|abeled salmon gonadotropin to various subcellular fractions obtained by centrifugation of ovarian homogenates. Further studies examined the effects of time, temperature and tissue concentration on gonadotropin binding. The binding of 125j _•] abel ed salmon gonadotropin to the ovary and testes and the l i v e r , kidney and muscle from female chum salmon was examined to determine the tissue distribution of gonadotropin binding s ites . The aff inity and number of gonadotropin binding sites in the chum salmon ovary were determined by two methods. For the f i r s t method, a constant amount of 125i_iabeled salmon gonadotropin (40,000 cpm) and increasing amounts of SGA-2359 were incubated with the 3000 g particulate ovarian fraction for 20 hr at 20 °C . For the second method, increasing amounts of 125i_i abeled salmon gonadotropin (0.26 to 9.82 x 106 cpm) were incubated alone or in combination with 10 yg SG-G100 and the 3000 g particulate fraction from 50 mg of ovarian tissue. The binding inhibition curves obtained from these experiments were converted to Scatchard plots to determine the aff inity and number of gonadotropin binding sites (Scatchard, 1949). The specific act ivi ty of 125j_-| abel ed salmon gonadotropin was determined by the self-displacement of increasing amounts of l 2 5 I - l abe led salmon gonadotropin in the gonadotropin RIA. The proportion of 125j _-j abel ed salmon gonadotropin that reacted specif ical ly with ovarian tissue was identified by incubation with an excess of ovarian tissue. As only radiolabeled gonadotropin that is capable of specific binding is believed to represent biologically active hormone (Dufau and Catt, 1978), specific act ivi ty was corrected for the maximal bindabil i ty of the tracer preparation. - 101 -The speci f ic i ty of gonadotropin binding sites was evaluated by determining the capacity of various teleost and mammalian gonadotropin preparations to compete with the binding of 125j_iabeled salmon gonadotropin to ovarian tissue. IV. Properties of Gonadotropin Binding Sites in Adult Coho Salmon Ovaries The properties of gonadotropin binding sites in adult coho salmon ovaries were determined by studying the binding of 125i_iabeled salmon gonadotropin to the 3000 g particulate fraction prepared from coho salmon ovaries at different stages of development. Additional studies examined the binding of ^^5j_-jabeled salmon gonadotropin to intact ovarian f o l l i c l e s and isolated thecal and granulosa cel l layers from preovulatory f o l l i c l e s at different stages of development. C. Results I. Effects of Teleost and Mammalian Gonadotropins on the Stimulation of Steroid Production In Vitro . Fig . 21 shows the time course of the effects of SG-G100 on 178-estradiol production by chinook salmon f o l l i c l e s incubated in vitro at 10 and 20"C. SG-G100 increased (P < 0.05) media 178-estradiol levels by 4 hr for f o l l i c l e s incubated at 10°C (Fig. 21a). The levels of 178-estradiol produced in response to SG-G100 continued to increase as a function of time and by 24 hr were about 6 fold-higher than the levels produced by f o l l i c l e s incubated with Ringer's alone. SG-G100 increased (P < 0.05) media 178-estradiol levels by 2 hr for f o l l i c l e s incubated at 20°C (Fig. 21b). Media 178-estradiol levels were increased to a maximum by 8 hr in response to SG-G100 and remained unchanged at 12 and 24 hr. The amounts of 178-estradiol produced by f o l l i c l e s in response to SG-G100 at 20°C were lower than the levels produced by f o l l i c l e s incubated at 10°C and represented only about a 3-fold increase in 178-estradiol production compared to f o l l i c l e s incubated with Ringer's alone. - 102 -FIG. 21. Time course of the effects of SG-G100 on 178-estradiol production by chinook salmon ovarian f o l l i c l e s incubated in vitro at 10° (A) and 20°C (B). Values represent the mean ± standard error of the amounts of 178-estradiol released to the media based on three replicates when incubated with Ringer's alone ( O ) or 1 yg/ml SG-G100 ( • ) . 5r i i i i • • I _J i i 1 1 1— 1 2 4 8 12 24 1 2 4 8 12 24 HOURS - 103 -Fig. 22 shows the effects of various salmon gonadotropin preparations on 176-estradiol production by chinook salmon ovarian f o l l i c l e s . Each of the four gonadotropin preparations tested stimulated a dose related increase in 176-estradiol production. No difference was found with respect to the maximal response e l l i c i t e d by the different gonadotropin preparations. Upon comparing the ascending portion of the dose response curves, i t was found that S6A-2359 was about 20 times and SGA-2360 about 15.1 times as active as the acetone dried pituitary powder. SG-G100 was 7.7 times as active as the acetone dried pituitary powder. Fig. 23 shows the effects of SG-G100, ovine LH and ovine FSH on 176-estradiol production by chinook salmon ovarian f o l l i c l e s . SG-G100 at 50 ng/ml stimulated a significant increase (P<0.05) in 176-estradiol production with the maximal response evident at 500 ng/ml. Ovine LH and FSH were inactive at 5 and 20 yg/ml as the amounts of 176-estradiol produced by these hormones did not differ (P>0.05) from the levels produced by f o l l i c l e s incubated with Ringer's alone. Fig . 24 shows the effects of SG-G100, acetone dried pituitary powder, ovine FSH, ovine LH and hCG on 17a20BP production by adult coho salmon postovulatory f o l l i c l e s . SG-G100 and acetone dried pituitary powder stimulated a dose related increase in 17a20BP production. SG-G100 was about 9.5 times as active as the acetone dried pituitary powder. Mammalian gonadotropins were inactive at doses up to 25 yg/ml. II. Effects of Iodination on the Biological Act iv i ty of Gonadotropin. Untreated and 125i_i abeled salmon gonadotropin had similar effects on testosterone production by coho salmon testicular tissue incubated in vitro (Fig. 25). Additionally, the act ivity of gonadotropin subjected to the iodination conditions but with the iodide or H2O2 omitted did not differ from untreated gonadotropin. In a separate experiment, iodinated gonadotropin was shown to be - 104 -22. Effects of various salmon gonadotropin preparations on the stimulation of 17B-estradiol production by chinook salmon ovarian f o l l i c l e s . Values are expressed as the mean ± standard error of the amounts of 17B-estradiol released to the media based on three replicates except for controls which were based on nine replicates. 6h n g / m l - 105 -FIG. 23. Effects of SG-G100, ovine LH and ovine FSH on the stimulation of 178-estradiol production by chinook salmon ovarian f o l l i c l e s incubated in v i t ro . Values represent the mean ± standard error of the amounts of 178-estradiol released to the media based on three replicate incubations. r - ^ SG-G100 Ovine LH Ovine FSH I l _ _ l i I l 10 50 100 250 500 1000 SG-G100 (ng/ml) < 1 5000 20,000 Ovine LH and FSH (ng/ml ) - 106 -FIG. 24. Effects of SG-G100, acetone dried pituitary powder, ovine LH, ovine FSH and hCG on 17a20BP production by coho salmon postovulatory f o l l i c l e s incubated in v i t ro . Values represent the mean ± standard error of the amounts of 17a208P released to the media based on three replicates. 21 Or 15 62 250 1000 5000 25000 - 107 -FIG. 25. Testosterone production by coho salmon testicular tissue j_n vitro in response to untreated and various forms of iodinated salmon gonadotropin. Values represent the mean levels of testosterone released to the media based on three replicate incubations using untreated ( • ) or ^25i_Tabeled ( • ) gonadotropin and gonadotropin subjected to the iodination conditions but with the iodide ( O ) or H2O2 ( • ) omitted. The average coefficient of variation was 0.19. - 108 -equipotent to untreated hormone in terms of i t s effect on testosterone production by coho salmon ovarian f o l l i c l e s incubated in vitro (Fig. 26). In these experiments, the iodinated gonadotropin was estimated to contain 0.89 (Fig. 25) and 1.09 (Fig. 26) molecules of iodide per molecule of gonadotropin. III. Properties of Gonadotropin Binding Sites in Immature Chum Salmon Ovaries The binding of 125i_^abeled salmon gonadotropin to various subcellular fractions prepared by centrifugation of immature chum salmon ovarian homogenates is shown in F ig . 27. Saturable binding was demonstrated in the 3000 g, 20,000 g and 3000-20,000 g fractions by a decrease in the percentage of radioactivity bound to ovarian tissue when coincubated with 10 yg of SG-G100. Saturable binding was highest in the 3000 g pellet accounting for 2.2% of the added radioactivity. In this case, SG-G100 displaced approximately 40% of the total radioactivity bound to the 3000 g pel let . As the level of saturable binding was low in the i n i t i a l experiments, additional chromatographic purification of 125j_iabeled salmon gonadotropin was attempted in an effort to selectively isolate gonadotropin fractions with higher binding act iv i ty . Chromatography of ^25j _]abel ed salmon gonadotropin on either ConA Sepharose or Sephacryl S-200 increased the proportion of added radioactivity that bound speci f ica l ly to the 3000 g particulate fraction from immature chum salmon ovarian homogenates (Fig. 28). This increase was attributed primarily to a reduction in the amount of hormone that bound non-specifically to ovarian tissue. Whereas 2.6% of the original 125i_i abeled salmon gonadotropin preparation showed specific binding, the level of specific binding increased to 4.4% following chromatography on ConA Sepharose and 3.5% following chromatography on Sepharcyl S-200. Further, chromatographic purification of the label on Con A Sepharose and Sephacryl S-200 increased the proportion of radioactivity displaced by SG-G100 from - 109 -FIG. 26. Testosterone production by coho salmon ovarian f o l l i c l e s in vitro in response to intact and iodinated salmon gonadotropin. Values represent the levels of testosterone released to the media (mean ± standard error) based on three replicate incubations. - 109a -i Intact lodi noted A' o _J ! 1 1 15 62.5 2 5 0 1000 GONADOTROPIN (ng/ml) - 110 -FIG. 27. Total binding (open-bar) and nonspecific binding (hatched-bar) of 125I_Tabeled salmon gonadotropin to immature chum salmon ovary fractions equivalent to 20 mg wet weight of tissue pel let . Tissue pellets were prepared by centrifugation of ovarian homogenates at 3000 g, 20,000 g and the supernatant f lu id from the 3000 g pellet recentrifuged at 20,000 g. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three replicate determinations. - 110a -3 0 0 0 g 20,000 g 3000 20, TISSUE PELLET - I l l -FIG. 28. Total binding (open bar) and nonspecific binding (hatched bar) of 125i_iabeled salmon gonadotropin to the 3000 g particulate fraction of immature chum salmon ovarian homogenates following chromatography of the label on Con A Sepharose and Sephacryl S-200. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three determinations. - 111a -Not Con A Sephacryl Purified Sepharose S-200 - 112 -45 to 70% of the total radioactivity bound in the absence of competitor. The effects of time and temperature on the specific binding of l 2 5 I-labe1ed salmon gonadotropin to the 3000 g particulate fraction prepared from 100 mg of ovarian tissue are shown in F ig . 29. Temperature influenced both the rate and proportion of 125i_i at, e-| e c | salmon gonadotropin that bound speci f ical ly to ovarian tissue. Specific binding increased more rapidly at 20°C than 10 °C . Specific binding was maximal by 20 hr for ovarian tissue incubated at 20°C whereas specific binding showed a slight increase at 36 hr relative to the level at 20 hr for ovarian tissue incubated at 10°C. The amount of 125j_-|abeled salmon gonadotropin that bound speci f ica l ly to ovarian tissue at 20°C (4.0%) was about 25% higher than the amount speci f ica l ly bound to ovarian tissue at 10 °C . Specific binding at 4°C was low (0.5%) when compared to ovarian tissue incubated at 10° or 2 0 ° C . Figure 30 shows the specific binding of 1 2 5 I - l abe led salmon gonadotropin as a function of increasing amounts of ovarian tissue. Specific binding increased as a linear function of tissue concentration when incubated with the 3000 g particulate fraction prepared from 25-300 mg of ovarian tissue. Specific binding accounted for 6.1 ± 0.1% of the added radioactivity incubated with the particulate fraction derived from 300 mg of ovary but declined at higher tissue concentrations. SG-G100 at 0.1 and 10 yg reduced the binding of 1 2 5 I - l abe led salmon gonadotropin to the 3000 g particulate fraction prepared from immature chum salmon ovarian homogenates but had no effect on the binding of 125j_-|abeled salmon gonadotropin to similar fractions prepared from l iver , kidney or muscle (Fig. 31). The binding of 1^ 5 j _ -| abel ed salmon gonadotropin to the 3000 g particulate fraction prepared from immature chum salmon testes homogenates was also reduced in a dose dependent fashion by SG-G100 (Fig. 31). The af f inity and number of gonadotropin binding sites in the 3000 g - 113 -FIG. 29. Time course of specific binding of 125j_iabeled salmon gonadotropin to the 3000 g particulate fraction of immature chum salmon ovary homogenates at 4°C ( • ), 10°C ( • ) and 20°C ( O ). Each point is the mean of duplicate determinations. % SPECIFIC BINDING - e e n -- 114 -FIG. 30. The specific binding of 125J_-Jabeled salmon gonadotropin to increasing quantities of the 3000 g particulate fraction prepared from immature chum salmon ovary homogenates. Each point is the mean of duplicate determinations. 7 Tissue Concentration (mg) - 115 -FIG. 31. The binding of 125i_-| ab ei e ( j salmon gonadotropin to the 3000 g particulate fraction prepared from 50 mg original wet weight of ovary, l i ver , kidney, muscle and testes from ' immature chum salmon. Values represent the percentage of added radioactivity bound to the tissue (mean ± standard error) when incubated with 0, 0.1 and 10 yg SG-G100. Results were based on three replicates. 6 LIVER MUSCLE KIDNEY OVARY TESTES In • • Ici'ci ti a ci | • D ti a ti I n II II I a u u • a a tl II II II i) II n n ti ti u ti 0 0.1 10 0 0.1 10 0 0.1 10 G100 ( jug) - 116 -particulate fraction of the ovary determined from competition studies using a constant amount of ^-25j_-|abeled salmon gonadotropin and increasing amounts of unlabeled SGA-2359 are shown in F ig . 32. To evaluate whether the amount of ovarian tissue influenced the estimation of the binding parameters, competition studies were performed at three levels of ovarian tissue. Specific binding increased from 1.3 to 4.2% of 125j._-|abeled salmon gonadotropin incubated with the 3000 g particulate fraction prepared from 38-188 mg of ovarian tissue (Fig. 32a). SGA-2359 effected a dose related reduction of the specific binding of 125j._iabeled salmon gonadotropin (Fig. 32b). The ab i l i ty of SGA-2359 to displace labeled gonadotropin was influenced by the amount of ovarian tissue, as the sensit ivity to SGA-2359 increased at low tissue levels. The af f inity and number of gonadotropin binding sites were estimated by Scatchard analysis (Fig. 32c) from the competition data shown in F ig . 32b. Calculation of the binding parameters was done following correction to account for the proportion of 125j _ -j abel ed salmon gonadotropin capable of specific binding. This level (6.0%) was determined from incubations containing 300 and 450 mg of ovarian tissue. The estimation of the binding capacity was not influenced by the amount of ovarian tissue. The binding capacity of the 3000 g particulate fraction was 37-44 pg gonadotropin per mg of tissue. The estimation of the af f inity of the gonadotropin binding sites was influenced by the amount of tissue as the aff inity constant ranged from 1.4 - 3.5 x 109M-1 when incubated with the 3000 g particulate fraction prepared from 38-188 mg of ovarian tissue. Figure 33a shows the results of a saturation experiment in which increasing amounts of 125i_-|abeled salmon gonadotropin were incubated with the 3000 g particulate fraction prepared from 50 mg of ovarian tissue. Specific binding increased a linear function of 125i_iabeled salmon gonadotropin concentration over - 117 -FIG. 32. Effect of tissue concentration on the determination of the af f inity constant and number of gonadotropin binding sites in the immature chum salmon ovary. A. Specific binding of 125i_iabeled salmon gonadotropin as a function of tissue concentration. B. Competition curves for the specific binding of -'-2^ 1 — 1 abe Ted salmon gonadotropin as a function of increasing amounts of unlabled SGA-2359. C. Scatchard plot for the competition data shown in B. - 117a GtH BOUND (ng ) - 118 -FIG. 33. The specific binding of 125j_-]abeled salmon gonadotropin to the 3000 g particulate fraction prepared from immature chum salmon ovarian homogenates as function of increasing amounts of ^-^^I-l abel ed salmon gonadotropin (A) and Scatchard analysis of the binding data (B). Values represent the mean of duplicate determinations. S P E C I F I C A L L Y B O U N D (cpm x 1 0 3 ) - B8 L L " - 119 -the range of 0.26 - 1.83 x 10*5 Cpm. Binding was saturated at 5.44 and 9.82 x 105 cpm of l 2 5 I-labe1ed salmon gonadotropin. Scatchard analysis of the binding data indicated a single class of high af f inity binding sites (Ka: 3.1 x 109M"1) with a binding capacity of 43 pg/mg tissue (Fig. 33b). Figure 34 shows the effects of various teleost and mammalian gonadotropin preparations on the binding of l 2 5 I - l abe led salmon gonadotropin binding to the 3000 g particulate fraction prepared from 200 mg of ovarian tissue. SGA-2359, SG-G100 and acetone dried pituitary powder caused a dose related decrease in the binding of l 2 5i_T a beled salmon gonadotropin. SGA-2359 was approximately 29 times and SG-G100 8.2 times more active than the acetone dried pituitary powder. The Con A]_ fraction was less effective than acetone dried pituitary powder. Based on the amounts required to cause a 50% reduction in the binding of l 2 5 I - l abe led salmon gonadotropin, the Con A, gonadotropin fraction had 0.5 times the act ivi ty of acetone dried pituitary powder. Mammalian gonadotropins (ovine LH, ovine FSH and hCG) reduced the binding of 125j_i abeled salmon gonadotropin but only at high concentrations. No major differences in the potencies of the mammalian gonadotropins were evident. IV. Properties of Gonadotropin Binding Sites in Adult Coho Salmon Ovaries Figure 35 shows the binding of 125j_-|abeled salmon gonadotropin to the 3000 g particulate fraction prepared from adult coho salmon ovaries. Specific binding was not detectable in preovulatory f o l l i c l e s characterized by central or peripheral germinal vesicle. Preovulatory f o l l i c l e s which had undergone GVBD showed specific binding of l 2 5 I - l abe led salmon gonadotropin when using 1.25 and 20 mg of tissue but not when using 5 mg of tissue. Postovulatory f o l l i c l e s showed specific binding of ^2^1-1abeled salmon gonadotropin. However, the level of specific binding by ovarian tissue from adult coho salmon was low accounting for at most 3.0% of the - 120 -FIG. 34. Effects of teleost and mammalian gonadotropin preparations on the specific binding of 125j_labeled salmon gonadotropin to the 3000 g particulate fraction of immature chum salmon ovarian homogenates. Values were expressed as a percentage of the radioactivity speci f ica l ly bound to ovarian tissue in the absence of competitor. Result are the mean of duplicate determinations. 5 10 100 1000 10000 20000 GONADOTROPIN (ng) - 121 -FIG. 35. Total binding (open bars) and nonspecific binding (hatched bars) of 125i_Tabeled salmon gonadotropin to the 3000 g particulate fraction of adult coho salmon ovaries. Binding studies were conducted at three levels of ovarian tissue (1.25, 5 and 20 mg/tube) obtained from fish at differing stages of maturity. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three determinations. - 122 -added radioactivity with the majority of the radioactivity associated with the ovarian tissue bound nonspecifically. A series of experiments which examined gonadotropin binding to various subcellular fractions prepared from preovulatory f o l l i c l e s and the influence of time, temperature and incubation buffer composition failed to demonstrate significant specific binding of gonadotropin to preovulatory f o l l i c l e s (data not reported). Additional tests done in conjunction with some of these studies showed that 5 - 8% of the 1 2 5 I - l abe led salmon gonadotropin bound speci f ica l ly to ovarian tissue from immature chum salmon. Figure 36 shows the binding of 125i_iabeled salmon gonadotropin to intact ovarian f o l l i c l e s and isolated thecal and granulosa ce l l layers from preovulatory f o l l i c l e s . Intact f o l l i c l e s showed negligible specific binding of 125J_Tabeled salmon gonadotropin. The uptake of ^ I - l a b e l e d salmon gonadotropin by isolated thecal ce l l layers and particularly isolated granulosa ce l l layers from preovulatory f o l l i c l e s was greater than that for intact f o l l i c l e s . Specific binding to thecal ce l l layers obtained from oocytes characterized by a central or peripheral germinal vesicle represented 0.6% of the added radioactivity. Specific-binding was not detected in isolated thecal ce l l layers from preovulatory f o l l i c l e s which had matured in vivo. Granulosa ce l l layers generally bound greater amounts of 125j _ -j abel ed salmon gonadotropin speci f ical ly than thecal ce l l layers. Granulosa ce l l layers obtained from f o l l i c l e s characterized by a peripheral germinal vesicle showed 3.2% specific binding of 125j_-| abel ed salmon gonadotropin. Granulosa ce l l layers from oocytes characterized by a central germinal vesicle or following maturation bound lesser amounts of gonadotropin speci f ica l ly . Thecal and granulosa ce l l layers could not be separated from postovulatory f o l l i c l e s . D. Discussion These studies have demonstrated gonadotropin binding sites in chum and coho - 123 -FIG. 36. Total binding (open bars) and nonspecific binding (hatched bars) of 1251_Tabeled salmon gonadotropin to intact ovarian f o l l i c l e s and isolated thecal ce l l layer (TCL) and granulosa ce l l layers (GCL) from adult coho salmon at different stages of maturity. Values represent the percentage of added radioactivity (mean ± standard error) bound to the ovarian tissue based on three determinations. - 123a -10 8 O Z I 6 0 L I INTACT TCL GCL C E N T R A L G E R M I N A L V E S I C L E I INTACT TCL GCL PERIPHERAL G E R M I N A L V E S I C L E O Z 4 Q Z CQ 2 INTACT TCL M A T U R E GCL INTACT P O S T O V U L A T O R Y - 124 -salmon ovaries which share several of the properties that characterize physiologically relevant gonadotropin receptors in other vertebrates. Gonadotropin binding to ovarian tissue was a saturable process as the uptake of 125j_-jabe1 ed salmon gonadotropin to ovarian tissue was reduced in a dose dependant manner by unlabeled gonadotropin. The ab i l i ty of different gonadotropin preparations to compete with the binding of 125j_labeled s a i m o n gonadotropin to ovarian tissue was in agreement with the effects of these gonadotropins on the stimulation of steroid production in v i t ro . These results suggest that gonadotropin binding sites identified by direct binding studies show a similar hormone speci f ic i ty as seen for physiological gonadotropin receptors which mediate steroid producton. Saturable gonadotropin binding was demonstrated in the ovary and testis but not in the l iver , kidney or muscle. These results suggest that saturable gonadotropin binding sites were restricted to the expected gonadotropin target tissues. Studies using immature chum salmon ovarian tissue have shown that saturable gonadotropin binding sites were localized in the 3000 g particulate fraction (Fig. 27). Mammalian gonadotropin receptors are found in the plasma membrane (see Saxena, 1976; Dufau and Catt, 1978) and as with the studies in chum salmon are localized in low speed pellets following homogenization (Catt et a l . , 1974, 1976; Dufau and Catt, 1978). The binding of 125i_iabeled salmon gonadotropin to chum salmon ovarian tissue was dependent on time, temperature and tissue concentration (Fig. 29 and 30). From 5 to 9% of the added ^25i_iabeled salmon gonadotropin bound speci f ical ly to ovarian tissue. This level of specific binding while low in comparison with the proportion of radiolabeled mammalian gonadotropins which bind speci f ical ly to gonadal tissue of mammalian origin compares favorably with the binding act ivi ty of non-mammalian tetrapod gonadotropins to gonadal tissue from a variety of tetrapod species. For - 125 -example, up to 60% of 1 2 5 I-1abeled hCG and 20-40% of 1 2 5 I - l abe led mammalian LH preparations wi l l bind speci f ical ly to mammalian gonadal tissue (see Catt et a l . , 1974, 1976). Labeled mammalian FSH preparations commonly exhibit less than 12% specific binding to mammalian gonadal tissue (Catt et a l . , 1976; Nimrod et a l . , 1976; Sairam, 1979) although in certain cases up to 40% specific binding has been reported (Darga and Reichert, 1978; Chiauzzi et a l . , 1982). Mammalian FSH preparations exhibit high binding act iv i ty (up to 40% specific binding) to gonadal tissue from birds, reptiles and amphibians (Licht and Midgley, 1976; Ishii and Farner, 1976; Adachi et a l . , 1979; Bona Gallo and Licht, 1979). Previous attempts to demonstrate specific binding of mammalian FSH preparations to gonadal tissue from teleosts have been unsuccessful (Adachi et a l . , 1979; Adachi and I sh i i , 1980). Studies using radiolabeled ostrich FSH, turkey FSH, sea turtle FSH and cobra gonadotropin have shown 5-16% specific binding when incubated with gonadal tissue from a variety of tetrapod species (Licht et a l . , 1977a,b, 1979; Bona Gallo and Licht, 1979; Bona Gallo et a l . , 1983). Unlike studies using FSH, attempts to demonstrate specific binding of LH type gonadotropins in non-mammalian vertebrates have met with limited success. Despite high biological act iv i ty , hCG and mammalian LH preparations f a i l to bind speci f ica l ly to gonadal tissue from birds, reptiles and amphibians (Licht et a l . , 1977a; Licht, 1980; Etches and Cheng, 1981). Similarity, studies usingl 2 5l-labeled sea turtle LH and ostrich LH did not show appreciable specific binding to gonadal tissues from a variety of tetrapod species (Licht, 1980; Bona Gallo et a l . , 1983). In other studies, 1 2 5 I - l abe led turkey LH was shown to bind speci f ical ly to gonadal tissue from birds and reptiles but this was represented by less than 10% of the added radioactivity (Bona Gallo and Licht, 1979; Bona Gallo et a l . , 1983). Recently, Schlaghecke (1983) reported that l 2 5 I - l abe led hCG binds speci f ical ly to testicular tissue from rainbow trout. In - 126 -these studies specific binding accounted for about 1% of the added radioactivity when incubated with testicular tissue at 37 °C . Low levels of specific binding have been attributed, in certain cases, to a decrease in the biological act ivi ty of gonadotropins following radiolabelling (see Catt et a l . , 1976; Birnbaumer, 1978). In the present studies, 1 2 5 I - l abe led salmon gonadotropin prepared by the lactoperoxidase method and untreated gonadotropin had similar effects on the stimulation of steroid production (Fig. 25 and 26) suggesting that iodination does not reduce the act ivi ty of salmon gonadotropin. In the present study, further chromatographic purification of 125i_-|abeled salmon gonadotropin on ConA Sepharose or Sephacryl S-200 increased the proportion of label that bound speci f ical ly to ovarian tissue (Fig. 28). These approaches have been applied to gonadotropin receptor studies in other vertebrate classes with similar results (Dufau et a l . , 1972; Catt et a l . , 1976; Licht and Bona Gallo, 1978; Dufau and Catt, 1978). Perhaps the most effective technique used to selectively increase the proportion of radiolabeled gonadotropin capable of receptor binding is receptor purif icat ion. In this procedure, radiolabeled gonadotropin bound to gonadal tissue is f i r s t dissociated at low pH or high temperature and then ut i l ized in a receptor binding assay. When applied to studies with 1 2 5 I - l abe led human FSH, receptor purification results in a several fold increase in the proportion of radiolabeled hormone capable of receptor binding and increased biological act ivity relative to the starting material (see Catt et a l . , 1976). Attempts to u t i l i z e receptor purification to increase the proportion of l 2 5l- labeled salmon gonadotropin capable of specific binding have been unsuccessful, however the procedure merits further investigation. Gonadotropin binding to ovarian tissue from immature chum salmon was temperature dependant. Specific binding increased more rapidly and was of higher - 127 -magnitude at 20°C than 10°C (Fig. 29). Ovarian tissue incubated at 4°C showed only low levels of specific uptake. Kubokawa and Ishii (1980) reported that testicular tissue from repti les and amphibians but not birds or mammals incubated at 0°C showed high specific binding of 125j_-|abeled rat FSH. The present results suggest that the ab i l i ty of gonadal tissues to bind gonadotropins speci f ica l ly at low temperatures may not be a feature common to a l l poikilotherms. The effects of temperature on binding differed from that reported for steroid production (Fig. 21). 178-estradiol production by chinook salmon ovarian f o l l i c l e s was higher at 10°C than at 2 0 ° C . The differing effects of temperature on binding and steroid production need not imply that binding sites identified using 125 j _ -j abel ed salmon gonadotropin are dist inct from physiological receptors which mediate steroid production. As temperature direct ly influences the act ivity of steroidogenic enzymes (see Kime, 1982; Fostier et a l . , 1983), differences between the temperature optima for steroid production and receptor binding may occur. Saturable gonadotropin binding sites appear to be restricted to the expected gonadotropin target tissues. Saturable gonadotropin uptake was found in particulate fractions prepared from ovaries and testes of immature chum salmon but not in l iver , kidney or muscle (Fig. 31). Sundararaj and Goswami (1977) suggest that gonadotropin mediates steroidogenesis in interrenal tissue from the Indian catf ish. Whether gonadotropin has a similar effect in salmonids is not known, although the kidney which would also contain interrenal tissue fa i led to show saturable binding of 125j_iabeled salmon gonadotropin. Scatchard analysis indicated that gonadotropin binding to immature chum salmon ovaries was due to a single class of high aff inity binding sites. The binding aff inity for these sites (1.7-3.5 X lO^M - !) was similar to that described - 128 -for LH and FSH receptor interactions in other vertebrate classes (Saxena, 1976; Catt et a l . , 1976; Bona Gallo and Licht , 1979). The binding capacity was also comparable to that reported for LH and FSH binding sites in other vertebrates. In the present studies, attempts were made to evaluate the speci f ic i ty of gonadotropin receptors by comparing the effects of different gonadotropins on the binding of 125i_]abeled salmon gonadotropin to ovarian tissue and their effect on the stimulation of steroid production (Fig. 22, 23, 24 and 34). However, i t was not possible to evaluate the speci f ic i ty of gonadotropin receptors in the immature chum salmon ovary by both of these approaches owing to the low steroidogenic capacity of the ovarian tissue. For example, in preliminary studies basal 178-estradiol production was variable, and also SG-G100 at doses up to 5 yg/ml caused less than a 2-fold increase in 178-estradiol production. However, separate studies which examined the pattern of steroid production by chinook salmon and coho salmon ovarian f o l l i c l e s in response to different gonadotropins were in agreement with the ab i l i ty of these hormones to compete with the binding of 125i_Tabeled salmon gonadotropin to immature chum salmon ovarian tissue. The limited ab i l i ty of mammalian gonadotropins to stimulate steroid production in vitro and to compete for 125i_i abeled salmon gonadotropin binding sites was in general agreement with other studies which examined the actions of these hormones in salmonids. Idler et a l . (1975b) reported that hCG, ovine LH and ovine FSH were ineffective in promoting cAMP production in immature rainbow trout gonads. Also, ovine LH and hCG were ineffective in accelerating the rate of oocyte maturation in rainbow trout and amago salmon (Nagahama et a l . , 1980). Kagawa et a l . , (1982) reported that very high doses of ovine LH stimulates 178-estradiol production in amago salmon f o l l i c l e s while FSH and hCG had limited effectiveness. Ng et a l . (1980) suggested that separate receptors for glycoprotein and - 129 -vitellogenic gonadotropins were present in the winter flounder ovary on the basis of immunofluorescence localization studies. It was not possible to directly address the question of whether gonadotropin binding sites identified in the present studies were specific for glycoprotein gonadotropin as vitellogenic gonadotropin was not available for comparison. However, a crude fraction not retained on ConA Sepharose (ConAi) which would be expected to contain vitellogenic gonadotropin had only a limited effectiveness in competing for l 2 5 I - l abe led salmon gonadotropin binding sites in chum salmon ovaries (Fig. 34). In preliminary studies, the ab i l i ty of the ConAi fraction to compete for gonadotropin binding sites was consistent with i t s relative act ivi ty as determined by RIA, which suggests that the act ivi ty present in this fraction may represent contamination by the glycoprotein gonadotropin. Further studies using purified vitellogenic gonadotropin would be necessary to provide a definitive statement on hormone speci f ic i ty . Unlike the studies with immature chum salmon, ovarian tissue from adult coho salmon either fa i led to show specific binding of 125i_i abeled salmon gonadotropin or showed only very low levels of specific binding (Fig. 35 and 36). These data contrast with data from mammals in that the number of LH receptors in the ovary increase as a function of f o l l i c u l a r size (Channing and Kammerman, 1973; Lee, 1976; Ryan and Lee, 1976) and development (Nimrod et a l . , 1977; Diekman et a l . , 1978; Richards, 1979; McNeilly et a l . , 1980). Also, Cook and Peter (1980a) reported that ovaries from goldfish having completed vitellogenesis or undergoing recrudesence bound more 125J_-Jabeled carp gonadotropin than ovaries from goldfish undergoing regression. There are several possible explanations which could account for the low levels of specific gonadotropin binding to ovarian tissue from adult coho salmon (and for the low specific binding of radiolabeled LH preparations in - 130 -nonmammalian vertebrates). It is possible that the proportion of ovarian tissue which contains gonadotropin receptors is low. A vast excess of gonadal tissue containing only non-specific gonadotropin binding sites could mask the presence of a limited number of specific gonadotropin binding sites. However, attempts to selectively increase the proportion of specific ce l l types by studying gonadotropin binding to isolated thecal and granulosa ce l l layers did not result in increased levels of specific binding (Fig. 36). Low levels of specific binding could result from degradation of either labeled gonadotropin or receptor during incubation. To test these poss ib i l i t i e s , short-term binding studies with a proteolytic enzyme inhibitor (phenyl methyl sulfonylflouride) included in the incubation buffer were conducted. These conditions did not influence the specific binding of 125i_i a|-, e-| e c| salmon gonadotropin (unpublished results) . An additional consideration is that separate gonadotropin receptors exist in immature and adult salmonids. A change in the speci f ic i ty of these receptors could account for differences in the amount of specific l^i.iabQiQd salmon gonadotropin binding. In this regard, Young et a l . (1983d) reported a change in the act iv i ty of mammalian gonadotropins on the stimulation of 173-estradiol production in vitellogenic amago salmon and 17a203P production.in preovulatory animals. In these studies ovine LH was less effective in stimulating 17a208P production than 173-estradiol. In the present studies, the relative act ivit ies of SG-G100 and acetone dried pituitary powder were similar in chinook and coho salmon ovarian f o l l i c l e s at different stages of development (Fig. 22 and 24). These results suggest that the speci f ic i ty of gonadotropin receptor interactions in Pacific salmon did not change during development. In the present studies, saturable gonadotropin binding sites were demonstrated in both thecal and granulosa ce l l layers from preovulatory coho salmon - 131 -ovarian f o l l i c l e s (Fig. 36). Recently, H. Kagawa and Y. Nagahama (pers. comm.) found a similar distribution of saturable gonadotropin binding sites in amago salmon ovarian f o l l i c l e s . The presence of gonadotropin binding sites in thecal and granulosa ce l l s is consistent with the recently proposed two-cell model for 17a20BP synthesis in salmonids (Nagahama, 1983; Young et a l . , 1983b). In this model, gonadotropin binding to a receptor located in the thecal ce l l layer would result in the activation of steroidogenesis leading to the production of 17a-hydroxyprogesterone. Gonadotropin binding to a separate receptor in the granulosa ce l l layer would increase the production of 20BHSD responsible for the conversion of 17ahydroxyprogesterone to 17a20BP. It would be predicted based on the pattern of 17a20BP production in vivo (Fig. 9 and 13; Chapt. 4) and in vitro (Fig. 19; Chapt. 4) that the number of gonadotropin receptors in granulosa cel ls would increase during development and reach their highest levels at the time of oocyte maturation and ovulation. The present results provide an indication that this may indeed be the case as f o l l i c l e s characterized by a peripheral germinal vesicle and f o l l i c l e s having undergone maturation showed higher levels of saturable gonadotropin binding than f o l l i c l e s characterised by a central germinal vesicle. However, the low levels of saturable binding and the high variation in uptake preclude a direct evaluation of gonadotropin receptor number by Scatchard analysis. A second question which remains unanswered is whether the speci f ic i ty of gonadotropin binding sites in thecal and granulosa ce l l layers di f fer . However, an accurate assessment of these questions wil l await further developments to selectively increase the binding act ivi ty of 125i_i a b e - | e c | salmon gonadotropin or to increase the proportion of ovarian tissue showing saturable gonadotropin binding. - 132 -CHAPTER 6 - SUMMARY AND CONCLUSIONS The preovulatory period in coho salmon was characterized by a gradual increase in plasma gonadotropin levels with no evidence for a defined surge associated with ovulation. 17B-estradiol levels declined ten days prior to ovulation, reaching basal levels four days prior to ovulation. Testosterone levels were high during the preovulatory period but decreased at ovulation. 17a20BP increased from very low levels ten days prior to ovulation to maximal levels four days prior to ovulation. The preovulatory increase in 17a20BP coincides with germinal vesicle breakdown which is consistent with the view that 17a20BP functions as the maturation inducing steroid in salmonids. A dual system controlling gonadotropin secretion was demonstrated in adult female coho salmon. Intraperitoneal injections of mammalian and piscine Gn-RH were shown to increase plasma gonadotropin levels. LH-RH, LH-RHA DAla 6 and chum salmon Gn-RH had similar effects on plasma gonadotropin levels i n i t i a l l y , although the response to LH-RHA DAla 6 was of longer duration. LH-RH and chum salmon Gn-RH maintained elevated plasma gonadotropin levels for up to 24 hr whereas the effects of LH-RHA DAla 6 persisted for up to six days. Intraperitoneal injections of pimozide, a dopamine receptor antagonist, also increased plasma gonadotropin levels suggesting that dopamine may function to inhibit gonadotropin release. Although pimozide was less effective than LH-RHA DAla 6 on the stimulation of gonadotropin secretion, pimozide potentiated the actions of LH-RHA DAla 6 . The acceleration of oocyte maturation and ovulation was related to the magnitude and duration of the increase in plasma gonadotropin levels. LH-RH having a transitory influence on plasma gonadotropin levels was ineffective whereas LH-RHA DAla 6 having a prolonged action accelerated oocyte maturation and ovulation. Two injections of LH-RHA DAla 6 over a 72 hr period were additive to the effects of a - 133 -single injection with respect to plasma gonadotropin levels and were a more effective means of accelerating ovulation. A single injection of SG-G100 which resulted in higher plasma gonadotropin levels than LH-RHA DAla 6 for 48 hr had similar effects on the acceleration of ovulation. These results suggest that the induction of ovulation was dependent on the duration rather than the magnitude of the i n i t i a l increase in plasma gonadotropin levels. Combined injections of SG-G100 and LH-RHA DAla 6 resulted in higher plasma gonadotropin levels than injections of SG-G100 alone and were more effective in accelerating ovulation. Two injections of LH-RHA DAla 6 alone or injections of LH-RHA DAla 6 in combination with SG-G100 represent effective means of inducing ovulation in coho salmon. The su i tab i l i ty of LH-RHA DAla 6 for the induction of ovulation relates to i t s prolonged effect on the maintenance of elevated plasma gonadotropin levels. Treatments which elevate plasma gonadotropin levels increased the production of testosterone and 17a206P. LH-RH caused a transient increase in 17a208P levels whereas LH-RHA DAla 6 and SG-G100 resulted in a long-term elevation of 17a206P levels. The induction of oocyte maturation and ovulation was related to the duration and magnitude of the increase in plasma 17a208P levels. A short-term increase in 17a208P at low levels (55 ng/ml) fa i led to induce oocyte maturation whereas a long-term increase in 17a206P at high t i t res (100 ng/ml) resulted in oocyte maturation but not ovulation. 17a20BP levels in coho salmon which were induced to ovulate were increased to 450-500 ng/ml. Gonadotropin decreased plasma 176-estradiol levels in a dose and time dependent manner. Declining 178-estradiol levels determined the time of oocyte maturation and ovulation as low levels of 178-estradiol were a requirement for maximal 17a206P production. An increase in 17a208P levels to above 200 ng/ml did not occur in the absence of a decline in 178-estradiol to less than 2 ng/ml. The - 134 -production of large amounts of 17a208P in response to S6-G100 in vitro occurred when 176-estradiol production was reduced. Furthermore, exogenous 178-estradiol reduced the production of 17a208P in response to SG-G100 in vitro when 20p HSD act ivity was low. Gonadotropin binding sites were identified in chum and coho salmon ovaries which share several of the properties wich characterize physiologically relevant gonadotropin receptors in other vertebrates. The binding of l ^ i - - ] ^ ] ^ s a lmon gonadotropin to immature chum salmon ovarian tissue incubated in vitro was a saturable process. The addition of unlabeled SG-G100 reduced the uptake of 125i-labeled salmon gonadotropin by ovarian tissue. Saturable gonadotropin binding sites were localized in the 3000 g particulate fraction. Gonadotropin binding sites with similar properties were identified in particulate fractions prepared from chum salmon testes but not in l i ve r , kidney or muscle. The saturable binding component was due to a single class of high aff inity binding sites present in limited numbers. These binding sites were characterised by a high aff inity (Ka: 1.3 - 3.5 x l O 9 M-1) and a binding capacity of about 40 pg/mg of tissue. Gonadotropin binding sites exhibited a high degree of hormone speci f ic i ty . Salmon gonadotropin preparations inhibit the binding of 125j_iabeled salmon gonadotropin to immature chum salmon ovarian tissue whereas mammalian gonadotropins including ovine LH, ovine FSH and hCG were ineffective. The ab i l i ty of different salmon * gonadotropin preparations to compete for 1 2 5I-1abeled salmon gonadotropin binding sites were consistent with the relative effects of these preparations on steroid production. These results suggest that gonadotropin binding sites identified by the binding of 125i_i abeled salmon gonadotropin share properties similar to physiologically relevant receptors which mediate steroidogenesis. A crude pituitary fraction expected to contain vitellogenic gonadotropin had limited - 135 -abi l i ty to compete for gonadotropin binding sites suggesting that these sites were directed to the glycoprotein gonadotropin. Relative to immature chum salmon, ovarian tissue from adult coho salmon showed only low levels of saturable gonadotropin binding. The gonadotropin binding sites in adult coho salmon ovary were present in both thecal and granulosa ce l l layers surrounding the f o l l i c l e . However, the low binding act ivity precludes a direct evaluation of the properties of these sites. - 136 -REFERENCES Adachi, T . , and I sh i i , S. (1980). Species specif ic i ty of the binding affinity of rat f o l l i c l e stimulating hormone to the vertebrate tes t i s . In: "Hormones, Adaptation and Evolution" (S. I sh i i , T. Hirano and M. Wada, eds.), p. 314, Japan Sc i . Soc. Press, Tokyo. Adachi, T . , Pandey, A . K . , and I sh i i , S. (1979). Follicle-stimulating-hormone (FSH) receptors in the testis of the frog, Xenopus laevis. Gen. Comp. Endocrinol. 37, 177-185. Aida, K., Izumo, R.S. , Satoh, H . , and Hibiya, Y. (1978). 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Pudoc, Wageningen. - 159 -APPENDIX 1 Gonadotropin RIA: Assay Validation The su i tab i l i ty of the gonadotropin RIA for the measurement of coho salmon plasma and pituitary gonadotropin was investigated by comparing the slopes of RIA displacement curves using gonadotropin standard and serial dilutions of plasma and pituitary extracts (Fig. 1). The slopes of the displacement curves for plasma and pituitary extracts were similar to the standard suggesting a high degree of immunorelatedness between standard and endogenous gonadotropin. The accuracy of the gonadotropin RIA was evaluated by comparing known amounts of gonadotropin added to aliquots of a female coho salmon plasma pool and the measured excess of hormone determined by RIA (Fig. 2). The slope of the resulting regression line between the added and measured amounts of gonadotropin (b = 1.05) does not differ from 1 and the intercept (-0.46) does not differ from 0 (P > 0.05). Thus the amounts of gonadotropin measured by RIA were correlated with the amount of added gonadotropin. To evaluate whether the gonadotropin antisera reacts with biological ly active hormone, studies were conducted to investigate the effects of pretreatment with gonadotropin antisera on the ab i l i ty of gonadotropin to stimulate steroid production. In one of these studies, 5 yg of SG-G100 was combined with either gonadotropin antiserum or normal rabbit serum in 100 yl of PBS. Following incubation for 24 hr at 10 °C , aliquots of the pretreated gonadotropin preparations were incubated with minced testicular tissue from adult coho salmon. The amounts of testosterone released into the media were determined by RIA. Gonadotropin preincubated with 2 or 20 yl of normal rabbit serum induced a dose related increase in testosterone production whereas pretreatment with 20 yl of gonadotropin antiserum resulted in lower (P < 0.01) testosterone production (Fig. 3). In a - 160 -separate experiment, 5 yg of SGA-2360 in 100 yl of PBS was combined with 25 yl of either normal rabbit serum or the gonadotropin antiserum. After incubation for 24 hours at 10 °C , 200 yl of goat anti-rabbit gamma globulin was added and the incubation continued for an additional 8 hours. Following centrifugation for 10 min at 3000 g, aliquots of the supernatant f lu id were incubated with ovarian f o l l i c l e s obtained from adult coho salmon. SGA-2360 pretreated with normal rabbit serum stimulated a dose related increase in testosterone production during a 24 hr incubation (Fig. 4). SGA-2360 pretreated with gonadotropin antisera produced signficantly less testosterone (P < 0.01) than did hormone pretreated with normal rabbit serum. These data suggest that the gonadotropin antisera is capable of neutralizing the act ivi ty of gonadotropin in male and female coho salmon. Since salmon pituitary hormones other than gonadotropin were not available for comparison, i t was not possible to direct ly evaluate the speci f ic i ty of the gonadotropin RIA. However, tests were conducted to investigate the relationship between the amounts of gonadotropin present in various salmon pituitary preparations as determined by RIA and bioassay. Fig . 5 shows the RIA displacement curves for four gonadotropin preparations using 125j labeled SGA-2360 as the radioligand. The relative potencies of these pituitary extracts as determined by RIA and bioassay are shown in Table 1. These data indicate a close agreement between the gonadotropin content determined by either RIA or bioassay. This suggests that pituitary factors other than gonadotropin which are present in the acetone dried pituitary powder but are removed during purification do not influence the measurement of gonadotropin by RIA. - 161 -RIA (logit-log) dose response curves for gonadotropin standard and serial dilutions of coho salmon plasma samples and pituitary extracts. Each point represents the mean of three determinations. - 162 -FIG. 2. Recovery of known amounts of gonadotropin added to plasma samples as determined by RIA. Values represent the mean ± standard error of 4 determinations. - 162a -2.5 5 10 20 30 ADDED GONADOTROPIN (ng/ml) - 163 -FIG. 3. Testosterone production by minced test icular tissue from adult coho salmon in response to SG-G100 pretreated with normal rabbit serum (NRS) or gonadotropin antiserum (SG-RS). Values represent the mean ± standard error of 3 determinations. 200o!-- 163a -j / m l - 164 -FIG. 4. Testosterone production by ovarian f o l l i c l e s from adult coho salmon in response to SGA-2360 pretreated with normal rabbit serum ( O ) or antiserum to salmon gonadotropin ( • ) . Values represent the mean ± standard error of 3 determinations. - 164a -1 6 r SGA-2360 (ng/ml ) - 165 -Fig. 5 - RIA (logit/log) displacement curves for acetone dried pituitary powder, SG-G100, SGA-2360 and SGA-2359. Each point represents the mean of two determinations. 025 05 1 25~ ~5 8 10 ~25~ ~50 ~10  ng/ tube | - 166 -TABLE 1 - Relative potency of various pituitary fractions as determined by RIA and bioassayl. Pituitary preparation RIA Bioassay l 2 Bioassay 2^ Acetone dried pituitary powder 1.0 1.0 1.0 SG-G100 9.1 7.7 7.1 SGA-2360 16.8 15.1 17.8 SGA-2359 23.4 20.0 25.0 1 Relative potencies calculated on the basis of acetone dried pituitary powder which is assigned the value of 1.0 2 Stimulation of 170-estradiol production in chinook salmon ovaries in v i t ro . 3 Stimulation of testosterone production in coho salmon testis pieces in v i t ro . - 167 -APPENDIX 2 178-Estradiol, Testosterone and 17a208P RIAs: Assay Validation The appl icabi l i ty of the 178-estradiol, testosterone and 17a208P RIAs to coho salmon plasma samples were evaluated by comparing the dose response curves for the steroid standards and serial dilutions of heated plasma (Fig. 1, 2 and 3). The slopes of the displacement curves for the steroid standards and coho salmon plasma were similar suggesting that plasma constituents did not effect the immunological properties of the endogenous steroids. As measurements of plasma steroid levels by RIA were based on direct measurements from heated plasma samples, i t was considered essential to evaluate the accuracy of these values by comparing the levels obtained following diethyl ether extraction and chromatographic purification of steroids. Plasma samples used for assay validation were obtained from maturing and ovulated coho salmon. For the 178-estradiol RIA, 10 plasma samples were measured following heating and after diethyl ether extraction and chromatography on LH-20 Sephadex as described by Haning et a l . (1979). 178-estradiol levels in plasma samples measured direct ly and after chromatography were highly correlated (correlation coefficient (r)= 0.98, slope (b)=1.01, intercept (a) 0.56; Fig 4). For the testosterone assay, comparisons were made between 8 plasma samples measured direct ly and following diethyl ether extraction and chromatography on Celite columns as described by Abraham et a l . (1972). The correlation coefficient between testosterone levels measured by the direct assay and after chromatography was 0.97 (b = 0.98, a = 1.48; Fig. 5). No difference was found with respect to 17a208P in plasma samples measured direct ly and following diethyl ether extraction (r = 0.99, b = 0.96, a = 3.8, n = 6; F ig . 6). The accuracy of the direct methods for the measurement of plasma steroid - 168 -levels by RIA was evaluated by comparing the recovery of known amounts of steroid added to plasma before heating. Three separate plasma pools were obtained by combining plasma samples of known t i t r e so that a 200 ul aliquot measured in the RIA would contain approximately 50 pg of either 176-estradiol, testosterone or 17a206P. Known amounts of steroid were added to aliquots of the plasma pools so that a 200 y l sample would contain an additional 12.5, 25, 50, 100 or 150 pg of steroid. The relationship between the amounts of steroid added and the recovery of added steroid as determined by RIA are shown in Fig . 7, 8 and 9. In each of the steroid assays, the regression line slope between expected and measured quantities does not differ from 1 and the intercept does not differ from 0 (P>0.05). Thus the amounts of steroid measured by RIA were closely related with the amount of added steroid. The su i tab i l i ty of the 176-estradiol, testosterone and 17a206P RIA procedures for the measurement of steroids produced by ovarian f o l l i c l e s incubated in vitro was evaluated in a similar fashion to that described for coho salmon plasma samples. It was found necessary to extract the incubation media from ovarian f o l l i c l e incubations with diethyl ether in order to obtain parallelism between dilutions of the incubation medium and the RIA steroid standards. Known amounts of steroid added to the incubation medium were highly correlated with the amounts of hormone determined by RIA as were the levels of 176-estradiol and testosterone measured with or without chromatography (data not shown). Since the recovery of labeled steroids added to the incubation medium prior to extraction was greater than 95%, no correction was used to account for losses incurred during extraction. - 169 -REFERENCES Abraham, G. E . , Buster, J . E . , Lucas, L. A . , Corrales, P. C , and Tel ler , R. C. (1972). Chromatographic separation of steroid hormones for use in radioimmunoassay. Anal. Letters 5, 509-517. Haning, R., Orczyk, G.P. , Caldwell, B .V. , and Behrman, H.R. (1979). Plasma estradiol , estrone, estr iol and urinary estr iol glucronide. In "Methods of Hormone Radioimmunoassay" (B.M. Jaffe and H.R. Behrman, eds.). pp. 675-700. Academic Press, New York. - 170 -FIG. 1. RIA (logit-log) dose response curves for 178-estradiol standard and dilutions of heated coho salmon plasma. Each point represents the mean of two determinations. 17B—ESTRADIOL (pg) T T - 1 7 1 -FIG. 2. RIA (l o g i t - l o g ) dose response curves for testosterone standard and dilutions of heated coho salmon plasma. Each point represents the mean of two determinations. TESTOSTERONE (pg) T • - 172 -FIG. 3. RIA (logit-log) dose response curves for 17a208P standard and dilutions of heated coho salmon plasma. Each point represents the mean of two determinations. - 173 -FIG. 4. Relationship between the levels of 178-estradiol in plasma samples determined using the direct method and following diethyl ether extraction and chromatography on LH-20 Sephadex. The line represents the relationship Y = X. - 173a -- 174 -FIG. 5. Relationship between the levels of testosterone in plasma samples determined using the direct method and following diethyl ether extraction and chromatography on Celite columns. The line represents the relationship Y = X. - 174a -TESTOSTERONE (ng/ml) DIRECT - 175 -FIG. 6. R e l a t i o n s h i p between the l e v e l s of 17a20BP i n plasma samples determined using the d i r e c t method and f o l l o w i n g d i e t h y l ether e x t r a c t i o n . The l i n e represents the r e l a t i o n s h i p Y = X. - 175a -17oc20BP (ng/ml) DIRECT - 176 -FIG. 7. Recovery of known amounts of 178-estradiol added to plasma samples prior to heating as determined by RIA. Each point represents the mean ± standard error of three determinations. - 1 7 6 a -12.5 25 50 100 150 A D D E D (pg) - 177 -FIG. 8. Recovery of known amounts of testosterone added to plasma samples prior to heating as determined by RIA. Each point represents the mean ± standard error of three determinations. - 177a -ADDED (pg) - 178 -FIG. 9. Recovery of known amounts of 17a20BP added to plasma samples prior to heating as determined by RIA. Each point represents the mean ± standard error of three determinations. - 178a -12 .5 2 5 5 0 1 0 0 1 5 0 ADDED (pg) 

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