Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effects on inhibiting estrogen production on sexual development in coho salmon (Oncorhynchus kisutch) Afonso, Luis Orlando Bertolla 1997

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1997-250075.pdf [ 9.56MB ]
Metadata
JSON: 831-1.0088288.json
JSON-LD: 831-1.0088288-ld.json
RDF/XML (Pretty): 831-1.0088288-rdf.xml
RDF/JSON: 831-1.0088288-rdf.json
Turtle: 831-1.0088288-turtle.txt
N-Triples: 831-1.0088288-rdf-ntriples.txt
Original Record: 831-1.0088288-source.json
Full Text
831-1.0088288-fulltext.txt
Citation
831-1.0088288.ris

Full Text

11 ABSTRACT The effects of inhibiting estrogen production on sexual development in coho salmon {Oncorhynchus kisutch) were investigated in this study through in vivo and in vitro experiments, using an aromatase inhibitor. In the in vivo studies female were injected with the aromatase inhibitor Fadrozole during vitellogenesis and close to final maturation, and males during spermatogenesis. In both females and males the effects of Fadrozole were investigated by determining the changes in plasma sex steroid levels (17p-estradiol, 17a-20p-P, testosterone and 11-ketotestosterone) before and several times after intraperitonial injection with different doses of Fadrozole, and by evaluating the reproductive development. In the in vitro studies the effects of Fadrozole, gonadotropins (GTH I and GTH II), and steroid hormone precursors (testosterone and 17a-hydroxyprogesterone) on steroid hormone secretion (17p-estradiol, 17a-20p-P, and testosterone) by coho salmon ovarian follicles were determined during vitellogenesis ( immature and central germinal vesicle) and close to final maturation (peripheral germinal vesicle). The in vivo experiments in females close to final maturation showed that injection of the aromatase inhibitor Fadrozole was effective in lowering plasma 17p-estradiol levels, and an associated increase in plasma 17a-20P-P levels occurred, indicating that the shift between plasma 17p-estradiol biosynthesis and 17a-20p-P biosynthesis, which is characteristic of maturating fish, was advanced by treatment with Fadrozole. It was also shown that Fadrozole can be used as a tool to induce ovulation in coho salmon. In vitellogenic females Fadrozole reduced plasma 17p-estradiol levels in a dose and time dependent manner, and increased plasma 17a-20p levels prematurely also in a dose and time dependent manner. The switch from 17P-estradiol to 17a-20p-P biosynthesis was transient in the iii groups that received only one injection. The study also showed that multiple injections with Fadrozole arrested ovarian development, indicating Fadrozole may have a potential as a tool to regulate sexual development in salmon. In males, injection with Fadrozole inhibited 17(3-estradiol secretion in the brain and caused a premature and transient increase in plasma 17cx-20p-P levels. Multiple injections caused further increases in plasma 17cx-20p-P, and 16 days after the beginning of the experiment the treatment groups which received the highest doses of Fadrozole (10 mg/kg and 5 x 10 mg/kg) started to spermiate, suggesting that the administration of aromatase inhibitors such as Fadrozole may provide a means of accelerating spermiation in salmonids and potentially other species of fish. The series of in vitro experiments demonstrated that Fadrozole is capable of inhibiting 17p-estradiol secretion throughout the periovulatory period, and that inhibition of 17p-estradiol per se does not elicit 17a-20P-P secretion, showing that gonadotropin is necessary for its secretion. It was also evident that 17p-estradiol secretion is dependent on availability of the substrate testosterone, which in turn is secreted in response to the gonadotropins. On the other hand 17a-20P-P secretion is directly dependent on stimulation of 20P-hydroxysteroid dehydrogenase by the gonadotropins, especially GTH II. These experiments demonstrated that the aromatase inhibitor Fadrozole can be used as tool to study mechanisms involved in the regulation of sexual development in fish. Also an aromatase inhibitor such as Fadrozole could be used close to final maturation to advance ovulation in females and spermiation and males and during vitellogenesis to inhibit reproductive development in females. iv T A B L E OF CONTENTS Page A B S T R A C T ii T A B L E O F C O N T E N T S iv LIST O F F I G U R E S vi LIST O F T A B L E S xi A C K N O W L E D G E M E N T S xii D E D I C A T I O N xiii C H A P T E R 1 - General Introduction 1 C H A P T E R 2 - General Materials and Methods 2.1. Experimental animals 10 2.2. Blood collection .10 2.3. Organs and oocyte collection 11 2.4. Ovulation and spermiation 11 2.5. Aromatase inhibitor inj ection 11 2.6. Hormone measurements 12 2.7. In vitro culture 16 2.8. Statistical Analysis 18 C H A P T E R 3 - Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and ovulation rate in female coho salmon close to final maturation. 3.1. Introduction 20 3.2. Materials and Methods 21 3.2.1. Experiment 1 21 3.2.2. Experiment 2 22 3.3. Results 22 3.3.1. Experiment 1 22 3.3.2. Experiment 2 26 3.4. Discussion 59 C H A P T E R 4 - Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon during vitellogenesis. 4.1. Introduction 70 4.2. Materials and Methods 71 4.3. Results 72 4.4. Discussion 106 CHAPTER 5 - Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion in male coho salmon during sexual maturation. 5.1. Introduction 115 5.2. Materials and Methods 117 5.3. Results 118 5.4. Discussion 137 CHAPTER 6 - Effects of Fadrozole, GTH I, GTH II, testosterone and 17a-hydroxyproges-terone on in vitro steroid secretion by coho salmon ovarian follicles. 6.1. Introduction 145 6.2. Materials and Methods 146 6.3. Results 6.3.1. Experiment 1. The effect of the aromatase inhibitor Fadrozole on 17p-estradiol secretion by ovarian follicles of coho salmon. 147 6.3.2. Experiment 2. The effects of the aromatase inhibitor Fadrozole, GTH I and GTH II on sex steroid secretion by ovarian follicles of coho salmon in vitro throughout the periovulatory period. 147 6.3.3. Experiment 3. The effects of GTH I, GTH II, testosterone, and 17oc-hydroxy progesterone on sex steroid secretion by ovarian follicles of coho salmon in vitro throughout the periovulatory period. 162 6.4. Discussion 171 CHAPTER 7 - General Summary and Conclusions 186 LITERATURE CITED 192 v i LIST OF FIGURES Page CHAPTER 1 Figure 1. Hormonal regulation of vitellogenesis and oocyte maturation in fish. 2 Figure 2. Pathways of steroid biosynthesis in the ovary of salmonid. 5 Figure 3. Mechanism of aromatization 6 CHAPTER 3 Figure 4. Mean plasma concentration of 17P-estradioI, 17a-20p-dihydroxy-4-pregnen-3-one (17cx-20P-P), and testosterone in female coho salmon from the control group in the Experiment 1. 24 Figure 5. Mean plasma concentration of 173-estradiol, 17a-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected withO.lmgof Fadrozole/ kg body weight in the Experiment 1. 27 Figure 6. Mean plasma concentration of 17P-estradiol, 17cc-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 1.0 mg of Fadrozole/ kg body weight in the Experiment 1. 29 Figure 7. Mean plasma concentration of 17P-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 10.0 mg of Fadrozole/ kg body weight in the Experiment 1. 31 Figure 8. Mean plasma concentration of 17P-estradiol in female coho salmon from Experiment 1, 3 hours, 24 hours, and 96 hours after injection. 33 Figure 9. Mean plasma concentration of 17cc-20p-dihydroxy-4-pregnen-3-one (17a-20P-P) in female coho salmon from Experiment 1, 6 hours, and 48 hours after injection. 36 Figure 10. Mean plasma concentration of 17P-estradiol, 17a-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon from the control group in the Experiment 2 (vehicle injected). 40 Figure 11. Mean plasma concentration of 17P-estradiol, 17cc-20p-dihydroxy-4-pregnen-3-one (17cx-20P-P), and testosterone in female coho salmon injected with 0.1 mg of Fadrozole/ kg body weight in the Experiment 2 43 vii Figure 12. Mean plasma concentration of 17P-estradiol, 17ct-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 1.0 mg of Fadrozole/ kg body weight in the Experiment 2. 45 Figure 13. Mean plasma concentration of 17,6-estradiol, 17cx-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 10.0 mg of Fadrozole/ kg body weight in the Experiment 2. 48 Figure 14. Mean plasma concentration of 17P-estradiol at 3 hours, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P) at 6 hours, and testosterone at 48 hours in female coho salmon from Experiment 2. 50 Figure 15. Effects of Fadrozole on the cumulative ovulation in female coho salmon from Experiment 2. 53 Figure 16. Effects of Fadrozole on the fertilization rate at hatching (%) in alevins from ovulated females in Experiment 2. 55 Figure 17. Effects of Fadrozole on the gonadosomatic index (GSI) in female coho salmon from Experiment 2. 57 Figure 18. Schematic representation of the hypothalamic-pituitary-gonadal axis in fish. 67 CHAPTER 4 Figure 19. Mean plasma concentration of 17p-estradiol, 17oc-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in vitellogenic female coho salmon from the control group (vehicle injected). 73 Figure 20. Mean plasma concentration of 17p-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in vitellogenic female coho salmon injected with 1.0 mg of Fadrozole/ kg body weight. 76 Figure 21. Mean plasma concentration of 17p-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in vitellogenic female coho salmon injected with 10.0 mg of Fadrozole/ kg body weight. 78 Figure 22. Mean plasma concentration of 17p-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in vitellogenic female coho salmon injected weekly with 10.0 mg of Fadrozole/ kg body weight (5x10 mg/kg). 81 Figure 23. Mean plasma concentration of 17P-estradioi in vitellogenic female coho salmon at 6 hours, and 192 hours after injection. viii 83 Figure 24. Mean plasma concentration of 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P) in vitellogenic female coho salmon at 6 hours, and 192 hours after injection. 86 Figure 25. Mean plasma concentration of testosterone in vitellogenic female coho salmon at 48 hours, 96 hours and 768 hours after injection. 88 Figure 26. Oocyte maturation index in vitellogenic female coho salmon injected or not with Fadrozole 91 Figure 27. Effects of Fadrozole on the oocyte diameter from vitellogenic female coho salmon. 93 Figure 28. Effects of Fadrozole on the gonadosomatic index (GSI) from vitellogenic female coho salmon. 96 Figure 29. In vitro 17P-estradiol production by ovarian follicles of vitellogenic female coho salmon at the end of the experiment (768 hours). 98 Figure 30. In vitro 17P-estradiol production by the brains of vitellogenic female coho salmon at the end of the experiment (768 hours) 100 Figure 31. Representative micrographs of ovarian section (5iu). 102 Figure 32. Representative micrographs of the periphery of the oocytes (5\x). 104 CHAPTER 5 Figure 33. In vitro 17P-estradiol production by the brains of male coho salmon at the end of the experiment (768 hours). 119 Figure 34. Mean plasma concentration of 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), 11 -ketotestosterone and testosterone in male coho salmon from the control group (vehicle injected). 121 Figure 35. Mean plasma concentration of 17cx-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), 11-ketotestosterone and testosterone in male coho salmon injected with 1.0 mg of Fadrozole/ '.g body weight. 123 Figure 36. Mean plasma concentration of 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), 11-ketotestosterone and testosterone in male coho salmon injected with 10.0 mg of Fadrozole/ kg body weight. 126 ix Figure 37. Mean plasma concentration of 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), 11-ketotestosterone and testosterone in male coho salmon injected weekly with 10.0 mg of Fadrozole/ kg body weight (5x10 mg/kg). 128 Figure 3 8. Mean plasma concentration of 17cc-20P-dihydroxy-4-pregnen-3 -one (17a-20P-P) in male coho salmon at 6 hours, and 192 hours, and 384 hours after injection. 130 Figure 39. Mean plasma concentration of 11-ketotestosterone and testosterone in male coho salmon at 96 hours after injection. 133 Figure 40. Effects of Fadrozole on the cumulative spermiation in male coho salmon. 135 CHAPTER 6 Figure 41. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 hours period in the presence of Fadrozole (10 or IOOUJM). 148 Figure 42. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 hours period in the presence of exogenous testosterone (0.10, 0.20, or 0.40 uM). 150 Figure 43. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 hours period in the presence of Fadrozole (10 or lOOuM) and with or without exogenous testosterone (0.10, 0.20, or 0.40 uM). 152 Figure 44. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 hours period in the presence of Fadrozole (10 p.M) and with or without GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. 155 Figure 45. 17o>20P-dihydroxy-4-pregnen-3-one levels (17a-20P-P) secreted by cultured ovarian follicles over an 18 hours period in the presence of Fadrozole (10 uM) with or without GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. 158 Figure 46. Testosterone levels secreted by cultured ovarian follicles over an 18 hours period in the presence of Fadrozole (10 uM) and with or without GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. 160 Figure 47. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 hours period in the presence or absence of exogenous testosterone (100 ng/mL), 17a-hydroxy-progesterone (100 ng/mL) and GTH I or GTH II (100 ng/mL) during different stages of the reproductive development 163 X Figure 48. 17a-20p-dihydroxy-4-pregnen-3-one levels (17a-20p-P) secreted by cultured ovarian follicles over an 18 hours period in the presence or absence of exogenous testosterone (100 ng/mL), 17a-hydroxyprogesterone (100 ng/mL) and GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. 166 Figure 49. Testosterone levels secreted by cultured ovarian follicles over an 18 hours period in the presence or absence of exogenous testosterone (100 ng/mL), 17a-hydroxy-progesterone (100 ng/mL) and GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. 169 xi LIST OF TABLES Page CHAPTER 3 Table 1. Effects of the aromatase inhibitor Fadrozole on gonadosomatic index and egg diameter in female coho salmon. Values represent the mean ± standard error. 38 Table 2 . Number of dead fish ovulated or not ovulated at different times during the experimental period. Each treatment had 10 fish at the beginning of the experiment. 39 CHAPTER 4 Table 3. Oocyte maturation index in vitellogenic female coho salmon injected or not with Fadrozole (mg/kg body weight). 90 CHAPTER 6 Table 4. Plasma hormone levels of 17P-estradiol, 17tx-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in individual female coho salmon at different times during sexual maturation. 154 Xll ACKNOWLEDGEMENTS I wish to express my gratitude to my supervisors Dr. Edward M. Donaldson and Dr. George Iwama for their constant support and guidance, and also for making possible this important step in my life. I wish to thank the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico -Brasil (CNPq) and the Brazilian people for the scholarship allowing me to have such a worthwhile experience. I am also grateful to Jessy Alves Pinheiro from CNPq, who made my connection with my sponsor wonderful. Muito Obrigado! I would like to thank the Department of Fisheries and Oceans - Canada for providing the excellent facilities at the West Vancouver Laboratory - Vancouver and for the opportunity to meet so many interesting people who worked there. I would like to thank specially Helen Dye for her support and friendship throughout this 3.5 years, which made all my lab work possible. I am also in debt to Jack Smith, for his assistance, advice and friendship during all my studies, to Igor Solar, and Miles Stratholt. I would like to thank all my friends and colleagues from George's Laboratory for their help and support. Thanks to Honora Cooper-Eckhardt from Novartis (NJ) for kindly providing the aromatase inhibitor Fadrozole. Finally, I want to express my gratitude and love to my wife Susana, for her courage, support and love, and to my daughters Betina and Fernanda. Eu amo voces. Xlll DEDICATION I wish to dedicate this work to my parents, to whom I owe what I am in my life. I am really luck to have you guys. I love you. Eu dedico este trabalho aos meu pais, a quern eu devo tudo que sou na vida. Eu tenho muita sorte de ter voces. Eu amo voces. 1 C H A P T E R 1 - General Introduction It is well recognized that in teleost fish gametogenesis is mediated by gonadal steroid hormones, which in turn are biosynthesized in response to pituitary gonadotropins (see Nagahama, 1987a). In the female, oogenesis can be divided in two phases: growth (endogenous and exogenous vitellogenesis) and final maturation (Figure 1). The gonadotropins (GTH), secreted from the proximal pars distalis in the pituitary gland, direct these two processes (see Nagahama, et al., 1993). Two chemically distinct GTHs: GTH I and GTH II, have been identified in the salmon pituitary which are chemically and biologically similar to tetrapod follicle-stimulating hormone (FSH) and luteinizing hormone (LH), respectively (Suzuki, et al., 1988ab; Swanson, et al., 1991). These glycoproteins consist of two subunits a and p. The a subunit structure is highly conserved in tetrapods and fish, when compared with the p subunit. The P subunit of salmon GTH II is about 30% homologous in its amino acid sequence with the GTH I P subunit (Nagahama, 1993). In salmonids, elevated plasma levels of GTH I are seen during the period of oocyte growth and probably function to stimulate ovarian growth and steroidogenesis at this stage. On the other hand, plasma GTH II increases during the period of final maturation (Swanson, 1991). The action of the GTHs on oogenesis in the teleost gonad is mediated by steroid hormones from the ovarian follicle cells (Nagahama, 1987a) (Figure 1). The teleost ovary is composed of oocytes and each one is surrounded by a follicle which consists of an outer thecal cell layer and an inner granulosa cell layer (Nagahama et al, 1993). Studies which have revealed the role of each cell layer and gonadotropin in steroidogenesis by the ovarian follicular layers will be discussed below. Hypothalamus 1 Pituitary I Gonadotropins O O C Y T E VITELOGENESIS O O C Y T E FINAL M A T U R A T I O N 17p-Estradiol — y Liver ) I O lOocyte Vitellogenin "0 I7a-20P-P | Germinal vesicle T H E C A L C E L L S Testosterone 17a-Hydroxyprogesterone GRANULOSA C E L L S Aromatase^**^'''^ 17p-Estradiol —) 20P-hydroxysteroid dehydrogenase >M7cx-20p-P( ) 17P-estradiol levels '"•«N S ^ „ * 17a-20P-P levels » * * * * * * «• ** Figure 1. Hormonal regulation of vitellogenesis and oocyte maturation in fish. 3 In non mammalian vertebrates oocytes grow while the oocyte nucleus is arrested in the first meiotic prophase (Nagahama et al, 1987a). In order to grow, the oocyte accumulates metabolic energy as "yolk" and in most vertebrates yolk is not synthesized in situ but is derived from a precursor protein called vitellogenin, synthesized in the liver (Ho, 1987). The sequence of events involved in the production of yolk and its accumulation into the oocyte, is referred to as vitellogenesis. It involves: l)the induction of vitellogenin synthesis in the liver and its subsequent release into the blood, 2) the transport of vitellogenin in the blood circulation, 3) the uptake of vitellogenin by the oocyte and 4) the conversion of vitellogenin into storage forms (Ho, 1987). Vitellogenesis is controlled by a two step process whereby GTH stimulates synthesis of 17p-estradiol, which in turns stimulates vitellogenin synthesis and secretion (Wallace, 1985; Tyler, 1991). The uptake of vitellogenin by the oocyte is stimulated by GTH I (Tyler, et al., 1991). During the process of oocyte growth the vitellogenic envelope develops between the granulosa cell layer and the oocyte, which consists of the egg membrane, vitelline membrane, a thin outer zona pellucida and a thick inner zona radiata (Nagahama, 1983). The synthesis of the major glycoprotein constituent of the zona radiata occurs in the liver under the influence of 17p-estradiol (Hamazaki, et al., 1989; Oppen-Berntsen, et al, 1992). In all teleost species studied to date, plasma 17P-estradiol is elevated during vitellogenesis (Fostier et al., 1983). The site of 17p-estradiol synthesis in the teleost ovary is in the follicle cells which surround vitellogenic oocytes (Nagahama et al., 1993). A two cell type model has been described for the production of 17p-estradiol in the ovarian follicle cells of salmonids (Nagahama, 1987a). In this model, during the vitellogenic stage, the thecal layer, under the influence of GTH, secretes mainly the aromatizable androgen testosterone, which diffuses into the granulosa cell layer and is converted to 17p-estradiol by the aromatase enzyme, which is located exclusively in this cell 4 layer (Figure 1). The activity of the aromatase enzyme varies with the developmental stage of the follicle (Young et al, 1983a). Once the oocyte has completed vitellogenesis, the new phase called final maturation (resumption of meiosis) starts, this occurs prior to ovulation and is essential for successful fertilization (Nagahama, 1987a). However, prior to oocyte maturation, there is a switch in the steroidogenic pathways in the ovarian follicles from 17P-estradiol biosynthesis to 17a,20p-dihydroxy-4-pregnen-3-one (17a-20P-P) biosynthesis (refer to Figure 1). This switch is essential for the growing oocyte to enter the final maturation stage (Nagahama, 1993). 17a-20p-P was first isolated from female sockeye (Idler et al., 1960), and this steroid has since been shown to be the most potent oocyte maturation inducer in many fishes. In salmonids, 17oc,20p-P is the maturation inducing hormone (MIH), and there is also a two cell type model to explain MIH biosynthesis (Nagahama, 1987a). GTH stimulates production of 17a-hydroxyprogesterone that transverses the basal lamina and is converted to MIH by the 20p-hydroxysteroid dehydrogenase (20P-HSD) enzyme in the granulosa cell, where GTH stimulates the activity of this enzyme (Suzuki et al., 1988c; Nagahama, 1993). Suzuki et al. (1988c) demonstrated that GTH I and GTH II were able to stimulate conversion of 17a-hydroxyprogesterone to MLH by granulosa cells, however GTH II was more potent than GTH I. The pathways of steroid biosynthesis in the salmonid ovary can be seen in Figure 2. The details of the aromatization mechanism are shown in Figure 3. The steroid substrate binds to the active site of the aromatase enzyme and one of the three hydrogen atoms at the C-19 position becomes hydroxylated giving rise to 19-hydroxy-testosterone. This is then followed by a second hydroxylation yielding the 19,19-hydroxytestosterone which may reversibly dehydrate to 19-oxotestosterone. Either or both of these intermediates may undergo a concerted 5 Cholesterol 20,22 hydroxylase desmolase OH Pregnenolone 3B-HS D O ^ 1>- Progesterone 17a-hydroxylas e CH 3 HO-C-H OH O^yP^y 20p-HSD 17a-hydroxyprogesterone 17a,208-Dihydroxy-4-pregnen-3-one 17,20-lyase OH 17p-oxireductase Androstenedione Testosterone Aromatase OH Aromatase OH 17p-oxireductase Estrone HO 17P-estradiol Figure 2. Pathways of steroid biosynthesis in the ovary of salmonid. 3P-HSD, 3p-hydroxysteroid dehydrogenase; 20P-HSD, 20p-hydroxysteroid dehydrogenase. 6 19-oxotestosterone 19,19-hydroxy-testosterone OH 17p-estradiol Figure 3 . Mechanism of aromatization. 7 conformational change with a simultaneous breaking of the C10-C19, C1-H1(3 and C2-H2P bonds. This process by itself occasions the release of the newly-formed estrogen from the enzyme active site (Schulster et al., 1976). Knowledge of the control mechanisms for the steroidogenic pathways during oogenesis is important both from basic and practical points of views. It has been shown that GTHs stimulate the production of the steroid hormones in the ovary which are essential to the oogenesis process (see text above). It is not known, however, if a mechanism occurring at the ovarian level could by itself trigger changes in steroidogenic pathways. It is important therefore, to conduct experiments to test the hypothesis that inhibition of 17P-estradiol production in the ovarian follicles will trigger the activity of the enzyme 20P-HSD (responsible by the synthesis of the maturation inducing hormone 17a-20pP) without the influence of GTH. An alternative hypothesis is that the reduction in 17P-estradiol by aromatase inhibitor will reduce the feedback inhibition of 17p-estradiol on hypothalamic secretion of GnRH and pituitary secretion of GTH. Thus increasing plasma levels of GTH II and stimulating 17a-20pP secretion. Since 17p-estradiol mediates vitellogenesis, the inhibition of the synthesis of 17P-estradiol by inhibiting the activity of the aromatase enzyme during early stages of oocyte growth may cause a retardation of ovarian development and vitellogenesis. Consequently there may be a delay in oocyte final maturation, the development of secondary sexual characteristics, and detrimental changes in flesh quality associated with final maturation in salmonids. Since prior to oocyte maturation there is a switch in the steroidogenic pathways from 17p-estradiol biosynthesis to 17a-20PP biosynthesis it is also possible to hypothesise that the inhibition of 17P-estradiol synthesis during late vitellogenesis may trigger the final maturation process and cause ovulation. 8 It has recently become possible to reduce estrogen biosynthesis by inhibiting the activity of the aromatase enzyme, which catalyses the conversion of aromatizable androgens to estrogens using non-steroidal aromatase inhibitors such as Fadrozole (CGS 169494A)(Steele et al, 1987). Fadrozole has been described as a reversible competitive inhibitor (Steele et al., 1987). Whereby both substrate and inhibitor compete for the same site on the enzyme. The efficacy of this compound in reducing estrogen biosynthesis has been demonstrated in both in vivo and in vitro studies on mammals (Steele et al., 1987; Schieweck et al., 1988) and in vivo studies on chickens (Elbrecht et al., 1992). This compound has also been shown to influence sex differentiation in reptiles (Lance et al., 1992; Weenstrom, et al., 1995) and fish (Piferrer et al., 1994). Other compounds, such as natural and synthetic sex steroids and peptide hormones are presently being used by the aquaculture industry to manipulate sexual development in fish (Donaldson, 1996). The effectiveness of these drugs, however, varies in different species of fish (Piferrer et al., 1993; Goudie, 1994; Van Der Kraak, et al., 1983; Richter, et al., 1987). Therefore, it is valuable to look for alternative techniques. A potent inhibitory effect of Fadrozole on 17P-estradiol biosynthesis would indicate that this compound could potentially be used to manipulate sexual development in fish. The primary objective of this study was to conduct studies, using the aromatase inhibitor Fadrozole as a tool, to further understand the mechanisms involved in the biosynthesis of the sex steroid hormones. To that end, in the in vivo experiments the effects of inhibiting 17p-estradiol biosynthesis on plasma concentration of the reproductive steroid hormones (17p-estradiol, 17a-200-P, testosterone, and 11-ketotestosterone), and on gonadal development during vitellogenesis and close to final maturation in females and during spermatogenesis in males, were examined. The in vitro studies were performed to evaluate the effects of Fadrozole, gonadotropins, and steroid 9 hormone precursors (testosterone and 17a-hydroxyprogesterone) on the production of sex steroid hormones by ovarian follicles of coho salmon during vitellogenesis and close to final maturation at the following stages immature (germinal vesicle not visible), central germinal vesicle, and peripheral germinal vesicle. The secondary objective was to determine whether an aromatase inhibitor such as Fadrozole might offer a possible means of manipulating reproductive events in cultured fish. 10 C H A P T E R 2 - General Materials And Methods 2.1. Experimental animals Adult coho salmon, Oncorhynchus kisutch, that had completed their anadromous migration from the Pacific ocean were obtained in hatcheries from the Department of Fisheries and Oceans Canada. These fish were brought to the West Vancouver Laboratory (Department of Fisheries and Oceans -Canada), and held outdoors in 3 meter diameter fiberglass tanks , which were constantly supplied with water from Cypress Creek freshwater. As coho salmon at this stage of their life cycle do not feed, fish were starved. The anesthetic protocol described below was used during all handling procedures. After 7 days of acclimation to the laboratory conditions fish were anesthetized by immersion in water containing 100 mg tricaine methanesulfonate (MS-222) per liter (Syndel Laboratories, Vancouver, B.C.), buffered with sodium bicarbonate (100 mg/L). The fish were tagged with passive inductive transponder (PIT) tags inserted in the ventral muscle, weighed, measured, and screened morphologically to determine the sex and maturity on the basis of body coloration, shape and size of the abdominal region, and massage of the abdominal region to verify if the fish were expelling gametes. After these procedures fish were randomly divided in groups in accordance with the experimental protocol, and each group was kept in a 800 L tank constantly supplied with creek freshwater. 2.2. Blood Collection A sample of blood (3 mL) was collected from the caudal vasculature using a 10 mL heparinized vacutainer (Becton Dickinson) with a 21 gauge needle, and held on ice before centrifugation at 4,000 rpm for 10 min at 4 °C. The plasma was aliquoted into 1.5 mL plastic microfuge tubes and stored frozen at -20 °C until assayed. 11 2.3. Organs and oocyte collection To determine the gonadosomatic index (GSI), the anesthetized fish were weighed, measured and killed by decapitation. The ovaries were removed and weighed and expressed as a percent of the total body weight (GSI= weight of gonads x 100/weight of fish). Oocyte diameter was estimated by dissecting from the ovary and measuring the diameter of 20 eggs using a dissecting microscope fitted with an eyepiece micrometer. The oocyte developmental stages were defined on the basis of the position of the germinal vesicle in accordance with Van Der Kraak et al. (1984), and were determined by visual examination of the oocytes within the ovary and further confirmed by immersion of a portion of the ovary in an egg clearing solution (Stockard's solution - Syndel Laboratories) which clarifies the oocytes allowing better visualization. When histological examination was necessary a portion of the ovary was removed and preserved in 10% buffered formalin (Fischer Scientific), dehydrated in ethanol, embedded in paraffin wax, sectioned in 5 LI sections and stained with hematoxylin-eosin (Wax-It Histology Services, Vancouver, B.C.). 2.4. Ovulation and Spermiation Maturity was verified periodically before and after the beginning of the experiments through gentle abdominal massage. Ovulation and spermiation were revealed by the release of oocytes or sperm, respectively, after gentle abdominal massage. The motility of sperm was determined under microscopy by placing a small aliquot of sperm on a slide and mixing with water. 2.5. Aromatase Inhibitor injection These procedures were carried out at least 3 days after fish had been transferred to the 800 L tanks. The non-steroidal aromatase inhibitor Fadrozole (CGS 16949A) (4-(5,6,7,8-12 tetrahydroimidazol[l,5]-pyridin-5-yl)benzonitrile monohydrochloride), which was a gift from Novartis (Honora Cooper-Eckhardt, Summit, NJ) was dissolved in the vehicle propylene glycol (Steele, et al., 1987) (Fisher Scientific). Stock solutions which contained 0.1, 1.0, 10.0 mg of Fadrozole/mL were prepared. Fish were injected intraperitonially at the basis of the right ventral fin using individual 4 mL syringes fitted with a 18.5 gauge needle. Control fish were not injected, or injected with the vehicle propylene glycol only (1.0 mL/kg body weight). 2.6. Hormone measurements Plasma 17P-estradiol, testosterone, and 17a-20p-dihydroxy-4-pregnen-3-one (17a,20P-P) levels were measured by radioimmunoassays described and validated by Van Der Kraak et al. (1984), and plasma 11-ketotestosterone was measured by a radioimmunoassay described and validated by Dye et al. (1986). In preparation for assay, plasma samples (50uJ) were diluted twenty fold in steroid assay buffer (0.05 M phosphate buffer pH 7.6), and incubated at 70 °C in water bath for 1 h in covered 12 x 75 mm borosilicate tubes (Scott et al., 1982, 1983). The tubes were removed from the water bath, and after cooling to room temperature they were centrifuged at 2500 rpm for 15 min at 4°C. The supernatant then was decanted into another 12 x 75 mm borosilicate tube, capped and placed in the refrigerator for immediate analysis or the supernatant was transferred to 1.5 mL plastic tubes and stored at -20 °C when not immediately assayed. The steroid assay buffer (0.05 M phosphate buffer pH 7.6) was made up of: 5,75 g/L dibasic sodium phosphate, 1.315 g/L monobasic sodium phosphate and 1.0 g/L gelatin, in distilled water; Scott et al., 1982). Whenever necessary 1 L of buffer was prepared. Initially, the phosphates and the gelatin were dissolved in distilled water, and this solution was heated to 37 °C for approximately 1 h to completely dissolve the gelatin. After cooling to room temperature, the pH was adjusted to 7.6, then 65 mg sodium azide /L was added to prevent bacterial growth. 13 In the case of the in vitro experiments, the steroids were extracted from the ovarian follicle or brain culture incubation medium using a similar method to that described by Pickering and Pottinger (1983) and Campbell (1992). Aliquots of the incubation medium (500 u.1) were placed in a plastic tube and ethyl acetate (900 jai) was added, the tubes were capped and the two phases mixed by shaking. After 10 min extraction, the tubes were centrifuged for 2 min at 3000 g to separate the two phases. Then, 200 fil of the supernatant phase was placed in 12 x 75 mm borosilicate tubes, evaporated to dryness overnight in a fumehood, and redissolved in 100 \x\ buffer. From the redissolved solution, 50 ul was removed, and dissolved ten fold in steroid assay buffer (1:10). Antibody for 17P-estradiol and testosterone was provided by ICN Biomedical, Inc., Costa Mesa, California. The rabbit anti-17p-estradiol serum (Cat. N°: 61-305) cross reacts with: 17P-estradiol (100%), 17oc-estradiol (0.05%), estrone (10%), estriol (0.6%), and other steroids less than 0.01%. The rabbit anti-testosterone serum (Cat. N°: 61-315) cross reacts with: testosterone (100%), 5a-dihydrotestosterone (18.75%), 5a-androstane-3a, 17(3-diol (3.0%), 5p-androstene-3p-17P-diol (1.0%), androstenedione (0.56%), 5a-androstane-3,17-dione (0.18%), 5p-androstane-3,17-dione (0.13%), androsterone (0.09%), 17p-estradiol (0.08%), 11 P-hydroxyandrostenedione (0.07%), progesterone (0.06%), dehydroepiandrosterone (0.04), and other steroids less than 0.01%. The antibody for 11-ketotestosterone was provided by Dr. T.G. Owen (Helix Biotech Ltd., Richmond, B.C.) This anti serum cross reacts with: 11-ketotestosterone (100%), testosterone (6.25%),lip-hydroxytestosterone (6.25%), andrenosterone (1.4%) and other steroids less than 0.2%(Dye et al. 1986). The antiserum for 17a-20p-dihydroxy-4-pregnen-3-one (17a-20p-P) was a sheep anti 17a-20p-3-carboxymehtyl-oxime-BSA serum provided by Dr. A.P. Scott (Scott et al., 1982). It has been demonstrated that this antiserum cross reacts insignificantly with other 14 steroids (Scott et al., 1982). Stock solutions of testosterone and 17P-estradiol antibodies were prepared by adding 5 mL of steroid assay buffer to 1 vial of lyophilized antibody. Aliquots were removed and diluted to 1:20 and 1:50-1:90 for 17p-estradiol and testosterone, respectively. The working dilution for 11-ketotestosterone antibody was 1:3500 or 1:4000. The 17a-20p-P antibody, was first diluted at 1:100 and then stored, and the working dilution was 1:4000 and 1:16000. Standards of 17P-estradiol (Sigma), testosterone (Sigma), 11-ketotestosterone (Sigma), and 17a-20p-P (Steraloids), were prepared initially by weighing 1 mg of each steroid and diluting them in 1 mL of ethanol. From this stock they were serially diluted in buffer at ten fold intervals to 10 ng/mL. From 10 ng/mL the steroids were further serially diluted at two fold intervals to 39 pg/mL. Therefore the working standards ranged from 10000 to 39 pg/mL. Radiolabeled (1,2,6,7-3H) testosterone, (2,4,6,7-3H) 17p-estradiol, (1,2,6,7-3H) 17a-hydroxyprogesterone, and 11-keto 1,2 (n)-3H testosterone were purchased from Amersham Canada Ltd. (Oakville, Ontario). Radiolabeled 17a-20P-P was prepared from radiolabeled 17a-hydroxyprogesterone incubated with 3a-20p-dehydrogenase (Sigma) using the method described by Scott etal. (1982). Each assay included a standard curve, followed by the samples. The first 3 pairs of tubes of the standard curve were: the non specific binding tubes (NSB) containing tracer and charcoal, in which antibody and unknown were replaced by buffer; the total counts tubes (TC), where the activity of the tritiated hormone was monitored by replacing the standard (unknown), antibody and charcoal by buffer; and the maximum binding tubes (MB), which contained tracer, antibody, charcoal, but the unknown was replaced by buffer. All points of the standard curve following the three first pairs of tubes contained antibody and tracer. In the radioimmunoassay of 17p-estradiol 15 and testosterone duplicate 17 x 75 mm borosilicate tubes contained 200 u,l denatured plasma previously diluted in buffer (unknown), 200 JJ.1 of antibody solution, and 200 ul of tritiated steroid (5,000 cpm). For 11-ketotestosterone and 17a-20P-P the duplicated tubes contained 100 u.1 of standard or denatured plasma, 50 u.1 of antibody, and 50 u.1 of tritiated steroid (2,000 cpm). After adding antibody and tracer to the all tubes they were vortexed, covered with aluminum foil and incubated overnight at room temperature. The following day the reaction was stopped by immersing the tubes to 2/3 of .their height in ice-water for 15 min. After that ice-cold dextran-coated activated charcoal (0.5 g/1 dextran T-70 [Pharmacia (Canada) Ltd., Dorval, Quebec] and 5.0 g/L activated charcoal [C-5260, Sigma] in assay buffer was added to each tube (1000 p.1 for 17cx-20p-P and 11-ketotestosterone and 200 uL for 17p-estradiol and testosterone), except in the total counts tubes, to bind any unbound labeled steroid. During the adding of ice-cold dextran-coated activated charcoal the tubes remained in the ice-water. The outer surface of the tubes was then dried, and the tubes were vortexed, and placed in ice-water for 10 min, centrifuged at 2500 rpm for 10 min at 4 °C and then decanted into glass scintillation vials. After decanting, each vial received 10 mL of Ecolite (ICN Biomedicals), was mixed vigorously and, counted in a LKB Wallac 1214 RackBeta liquid scintillation counter (Wallac Oy, Turku, Finland) for 3 min, which then processed the radioimmunoassay data. In general terms the amount of radiolabeled steroid that bound to the antibody in the standard curve tubes was expressed as a percentage of the maximum binding tubes. The computer program constructed a standard curve, which by interpolation was used to calculate the percent binding for the unknowns using the following equation: % Binding = (unknown - NSB/MB -NSB) x 100 16 The intra-assay and inter-assay variability was calculated using a standard pool of coho salmon plasma samples. For all the assays, the intra-assay and inter-assay variation were less than 6 and 13%, respectively (Number of times that each assay was repeated = 5-10). 2.7. In vitro culture In vitro incubation has been extensively used to measure steroid secretion by a variety of tissues in fish. The secretion of steroids by ovarian follicles in salmonids can be consistently studied in in vitro systems, since most of the follicles are in a similar developmental stage. Also brain tissue and ovarian follicles when in culture remain viable for a period of time, without altering their secretory capacity, and ability to respond to external stimuli. The in vitro culture of the coho salmon brain tissue and ovarian follicles was carried out using Leibovitz L-15 culture medium (Gibco BRL Products, ) in liquid form consisting of (mg/L): CaCl2, 140.00; KC1, 400.00; KH2P04> 60.00; MgCl2 (army), 93.70; MgS04 (anhyd.), 97.67; NaCl, 8000.00; Na2HP04, 190.00; D(+) Galactose, 900.00; Sodium Pyruvate, 550.00; DL-Alanine, 450.00; L-Arginine (freebase), 500.00; L-Asparagine, 250.00; L-Cysteine, 120.00; L-Glutamine, 300.00; Glycine, 200.00; L-Histidine (freebase) 250.00; L-Isoleucine, 250.00; L-Leucine, 125.00; L-Lysine (freebase), 75.00; DL- Methionine, 150.00; DL-Phenylalanine, 250.00; L-Serine, 200,00; DL-Threonine, 600,00; L-Tryptophan, 20.00; L-Tyrosine, 300.00; DL-Valine, 200.00; D-Ca Pantothenate, 1.00; Choline Chloride, 1.00; Folic Acid, 1.00; i-Inositol, 2.00; Niacinamide, 1.00; Pyridoxine HC1, 1.00; Riboflavin-5'-phosphate, 2H20, 0.10; Thiamine Monophosphate, 1.00. Hepes (Gibco BRL Products), 10 mM was also added and the pH of the medium adjusted to 7.8 using 0.5 M NaOH. Chum Salmon GTH I and GTH II which were a gift from Dr. H. Kawauchi (School of Fisheries Science, Kitasato University - Japan), were dissolved in 0.2 % BSA RIA grade-17 Leibovitz L-15 (Penny Swanson personal communication). The aromatase inhibitor Fadrozole was dissolved in Leibovitz L-15, and testosterone (Sigma) and 17a-hydroxyprogesterone (Sigma) were initially dissolved in 95 % ethanol (Fisher), and then diluted in the incubation medium. The final concentration of ethanol in the medium did not exceeded 0.1%. The procedure used for follicle culture was modified from Campbell (1992). After sacrificing the female by decapitation, the ovaries were removed, weighed, and an anterior portion of the right ovary was removed and placed in a container containing L-15 on ice. The ovarian portion was transferred to a petri dish containing fresh L-15 on ice, where individual follicles were dissected from the surrounding tissue. The follicles were washed three times in fresh medium and then incubated in 24 well plates (Falcon, Becton Dickinson) containing 1 mL of L-15, in the presence or absence of various concentrations of the hormones tested for 18 h at 12 °C. At the end of the incubation period, the plate was placed on ice and the medium was immediately removed from each well using a Pasteur pipette and transferred to a plastic tube and stored at -20°C. The procedure used for brain culture was modified from Timmers et al.(1987). After decapitation the brains were removed and immediately transferred to a container with L-15 on ice. From each brain a transverse slice of approximately 2 mm containing parts of the diencephalon, mesencephalon and metencephalon which have been shown to contain aromatase activity (Timmers et al., 1987) was cut and transferred to a petri dish, washed three times, placed in the well plates and incubated as described above. 18 2.8. Statistical analysis When data failed to achieve either normal distribution or equal variance, data were Log 10 transformed to achieve either normality or homogeneity of variance. Group-dependent variations in plasma concentrations for each hormone were detected by analysis of variance (ANOVA) followed by all pairwise multiple comparison by Student-Newman-Keuls test. The Kruskal-Wallis ANOVA on ranks was performed when data failed to achieve either normality or equal variance, followed by all pairwise multiple comparison by Student-Newman-Keuls test Time-dependent variations within each group were studied by repeated measures analysis of variance, since individual fish were sampled repeatedly, followed by a Student-Newman-Keuls test. Friedman Repeated Measures ANOVA on ranks was performed when data failed to achieve either normality or equal variance followed by a Student-Newman-Keuls test. ANOVA was also used to detect variations in gonadosomatic index, and in egg diameter among groups. The effects of treatments on ovulation, and spermiation were analyzed comparing two treatments using Fisher Exact Test. . The Kruskal-Wallis ANOVA on ranks was performed on atresia and sperm motility (%) data. The Mann-Whitney Rank Sum Test and the Chi-square Test were used to verify the effects of treatments between two groups on oocyte maturation index and on fertilization rate at hatching, respectively. The degree of association between variables was determined by the Pearson product moment correlation coefficient or the Spearman rank order correlation. Comparisons between pairs of groups were made by Student's T-test. In the in vitro experiments variations among the different treatments were detected by analysis of variance (ANOVA) followed by all pairwise multiple comparison by Student-Newman-Keuls test. Unless indicated, statistically significant differences were determined at p < 0.05. In the Chapters 3, 4, and 5 comparisons among the groups in terms of hormone levels were carried out. The main objective of such comparisons was not to determine the differences 19 between the treatment groups at each time throughout the experimental period. Rather, the analysis was carried out to highlight periods which show important physiological responses among the groups. Therefore, the comparisons were accomplished in different times for each hormone. The criteria to define those times was based on the periods where the treatment group injected with the highest dose of Fadrozole showed significant values, increases or decreases in plasma hormone levels, in relation to the previous sample mean. 2 0 C H A P T E R 3 - Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and ovulation rate in female coho salmon close to final maturation. 3.1. Introduction As stated above, oogenesis in teleost fish consists of two phases, vitellogenesis and final maturation, which are mediated by sex steroid hormones produced by the gonads in response to the gonadotropins. Data from several salmonids indicates that vitellogenesis is mediated by GTH I, which has been shown to stimulate the uptake of vitellogenin into vitellogenic oocytes in vivo and in vitro (Tyler et al., 1991). Oocyte maturation is controlled by GTH II, which has been shown to stimulate 17a-20p-P synthesis in vitro (see Swanson, 1991, 1994). These findings are further supported by the fact that the levels of the two gonadotropins vary according to the reproductive stage. During vitellogenesis GTH I is the gonadotropin dominant in the blood, whereas close to final maturation plasma GTH I levels decrease and plasma GTH II increase (Suzuki, et al., 1988d; Swanson, 1991; Prat et al., 1996 ). In salmonid species studied to date, plasma 17p-estradiol levels have been reported to be elevated during vitellogenesis and drastically decreased during the final maturation period, when plasma 17a-20p-P increases rapidly (Scott et al., 1982; Young et al., 1983b; Van Der Kraak et al., 1984; Yamauchi et al., 1984; Dye et al., 1986). The transition from the vitellogenic stage to the maturation stage relies on a switch of steroidogenic pathways from 17p-estradiol to 17a-20p-P. The objective of this study was to investigate the hypothesis that inhibiting estrogen production, by blocking aromatase enzyme 21 activity, close to final maturation may advance the switch from 17p%estradiol to 17a-20|3-P biosynthesis, and as a consequence cause oocyte maturation leading to ovulation. 3.2. Materials and Methods Two experiments were carried out during two consecutive spawning seasons to investigate the effects of Fadrozole in female coho salmon close to final maturation. Adult female coho salmon were obtained in mid-October 1994 (Experiment 1) and in mid-September 1995 (Experiment 2) from the Chilliwack River Hatchery, Chilliwack, B.C. (Department of Fisheries and Oceans) subsequent to their migration from the Pacific ocean. For Experiment 1, body coloration was used as the criterion to select fish which were not sexually mature. Only those females which were silver-colored were retained. For Experiment 2, besides body coloration, body conformation was also used as a criterion to select fish to further improve the probability of capturing females, since the collection date was 1 month earlier than in the previous year and it was difficult to distinguish the sexes visually. The hatchery supplied 19 fish in 1994 and 60 fish in 1995. At the time of capture the weights and the length of the 1994 fish (n = 19) were 2.6 ± 0.1 kg and 60.4 ± 0.9 cm (mean ± SE), respectively, whereas the 1995 fish (n = 50) were 2.0 ± 0.4 kg and 55 ± 4.0 cm (mean ± SE), respectively. 3.2.1. Experiment 1 Fish from Experiment 1 were divided in five groups: control non injected (n=3), control vehicle injected (n = 4), group treated with 0.1 mg Fadrozole/kg (n = 4), group treated with 1.0 mg Fadrozole/kg (n = 4), group treated with 10.0 mg Fadrozole/kg (n = 4). Blood sample was collected at 0 h (just before injection with vehicle or Fadrozole), 3 h, 6 h, 24 h, 48 h, 96 h, and 192 h after injection. Plasma 17p-estradiol, testosterone, and 17a-20P-dihydroxy-4-pregnen-3-22 one (17a-20P-P) levels were measured using radioimmunoassay. After the last blood collection fish were killed and GSI and oocyte diameter were determined for each group. 3.2.2. Experiment 2 In Experiment 2, fish were divided in 4 groups: control vehicle injected (n = 12), group treated with 0.1 mg Fadrozole/kg (n =12), group treated with 1.0 mg Fadrozole/kg (n = 12), group treated with 10.0 mg Fadrozole/kg (n = 12). Blood samples were collected at 0 h (just before injection vehicle or Fadrozole), 3 h, 6 h, 24 h, 48 h, 96 h, and 192 , 288 h, 384 h and 480 h after injection. Maturity was checked at 0 h, 192 h (8 days), 240 h (10 days), 288 h (12 days), 384 h (16 days), and 480 h (20 days). To determine the fertilization rate four ovulated females from each group were used, and whenever a female had ovulated, 200 eggs were stripped into a sterile plastic bag. Four groups of 50 eggs from each female were fertilized, and incubated . Eggs were fertilized using the sperm of a single male which was collected from the hatchery with the females. The sperm motility of this male was checked before use. Whenever a male was identified in an experimental group it was removed from the tank. Indeed males were found in all experimental groups, but never more than two per group. Plasma 17p-estradiol, testosterone, and 17a-20P-dihydroxy-4-pregnen-3-one (17a,20p-P) levels were measured by radioimmunoassay. After the last blood collection the salmon were killed and GSI was determined for each group. 3.3. Results 3.3.1. Experiment 1 One fish from the vehicle group jumped out from the tank 24 h after the beginning of the experiment, and its preceding data were removed from tbe experiment. Also, one fish from the control group and one from the vehicle group which presented estradiol levels below 10 ng/mL at time 0 and had ovulated were removed from the experiment, since this value is close to that 23 from fish which would likely ovulate in the next 14 days (Fitzpatrick et al., 1987). The plasma steroid profiles in the control and vehicle groups were similar during all sampling times for all three steroids measured. As no statistical difference was apparent the data from the two remaining salmon in each of these two groups were combined into a single control group containing four salmon. Plasma 17p-estradiol levels in the control group (Figure 4) remained constant up to 48 h, and from 96 h to 192 h declined significantly (p<0.05). On the other hand, plasma 17a-20p-P levels increased significantly (p<0.05) at 48 h relative to initial levels and continued to increase until the end of the study period. Plasma testosterone concentration increased significantly (p<0.05) at 192 h after injection. In the group injected with 0.1 mg of Fadrozole/kg (Figure 5), plasma 17p-estradiol levels, decreased significantly (p<0.05) at 24 h after injection, remained constant up to 96 h, and then declined further (p<0.05). In this group plasma 17a-20p-P levels were significantly (p<0.05) higher 24 h after injection, and increased significantly (p<0.05) again between 96 h and 192 h. On the other hand, plasma testosterone levels did not increase significantly (p>0.05) throughout the period. In the group injected with 1.0 mg of Fadrozole/kg plasma 17P-estradiol levels had declined significantly by 3 h. A further significant decline occurred by 6 h, 17p-estradiol then remained constant up to 192 h (Figure 6). Plasma 17a-20p-P and testosterone levels increased significantly (p<0.05) in this group at 24 h after injection. In the group injected with 10.0 mg of Fadrozole/kg, plasma 17p-estradiol significantly declined at 3 h, and further significant declines occurred at each sampling up to 48 h, remained 24 Figure 4. Mean plasma concentration of 17p-estradiol, 17a-20p-dihydroxy-4-pregnen-3-one (17a-203-P), and testosterone in female coho salmon from the control group in the Experiment 1. Each value represents the mean ± standard error of measurements from 4 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. Hours after injection 26 constant up to 96 h, and rebounded significantly (p<0.05) at 192 h (Figure 7). This group showed a significant increase (p<0.05) in plasma 17tx-20P-P levels at 3 h, and in all subsequent blood collections up to 48 h. At 192 h there was another significant increase (p<0.05) in plasma 17a-20p-P levels. Plasma testosterone levels increased significantly (p<0.05) at 48 h and up to 96 h. Comparison among the groups at 3 h demonstrated that the injection with 1.0 and 10.0 mg of Fadrozole/kg significantly (p<0.05) decreased plasma 17P-estradiol levels when compared to the control group (Figure 8). At 24 h, all treated groups exhibited significantly lower plasma 17p-estradiol levels, and a dose-dependent effect was apparent, since the group injected with 10 mg of Fadrozole/kg presented a significantly lower plasma 17P-estradiol concentration than the other two Fadrozole treated groups. At 96 h after injection only the group treated with the highest dose of Fadrozole had a lower plasma 17P-estradiol level when compared with the vehicle group. At 6 h after injection, the group injected with 10 mg of Fadrozole/kg showed a significantly higher plasma 17a-20P-P level than the other groups, and at 48 h a dose dependent effect was evident, since this group presented a significantly (p<0.05) higher plasma 17a-20p-P concentration than the other groups (Figure 9). Plasma testosterone levels were not different (p > 0.05) among the groups at any sampling time. The GSI and egg diameter were not significantly different (p > 0.05) among groups at the end of the experiment (Table 1). 3.3.2. Experiment 2 At the beginning of this study, each group had 10 females, however, there were some mortalities throughout the experimental period in all groups. Most of the mortalities that occurred up to 12 days were due to the fact that fish jumped out of the tank. Table 2 shows the mortality in each 27 Figure 5. Mean plasma concentration of 17f3-estradiol, 17a-20f$-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 0.1 mg of Fadrozole per kg body weight in the Experiment 1. Each value represents the mean ± standard error of measurements from 4 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 120 28 O) c "D CD i_ GO LU • CO. 100 80 H 60 40 H 20 H 0 1 I I I I I I 0 3 6 24 48 96 192 1600 1400 1200 — 1000 f 800 S 600 ca o CN S E CO c o CD 200 500 400 H 300 H 200 H o w 100 cu 0 0 3 6 24 48 96 192 1 1 1 1 1 I ~ 0 3 6 24 48 96 192 Hours after injection 29 Figure 6. Mean plasma concentration of 17f}-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20f3-P), and testosterone in female coho salmon injected with 1.0 mg of Fadrozole/ kg body weight in the Experiment 1. Each value represents the mean ± standard error of measurements from 4 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 30 90 0 3 6 24 48 96 192 600 - i 0 3 6 24 48 96 192 0 3 6 24 48 96 192 Hours after injection 31 Figure 7. Mean plasma concentration of 17f3-estradiol, 17a-20J3-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 10.0 mg of Fadrozole/ kg body weight in the Experiment 1. Each value represents the mean ± standard error of measurements from 4 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 32 100 CJ) c TD CO i_ 00 LU 1 ca 1600 1200 800 E "9) i ca o CM i e 400 -\ 100 0 24 48 96 192 400 350 H — 300 E o) 250 af 200 o i— <D —^* (0 o 00 <D 150 100 50 0 0 n 1 i i i r~ 3 6 24 48 96 192 Hours after injection 33 Figure 8. Mean plasma concentration of 173-estradiol in female coho salmon from Experiment 1, 3 h, 24 h, and 96 h after injection. Each bar represents the mean ± standard error from females in each treatment group. Plasma 17P-estradiol concentrations which are similar (p>0.05) among the groups within each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 34 Control [•• j Fadrozole 0.1 mg/kg [ j Fadrozole 1.0 mg/kg F i l l Fadrozole 10.0 mg/kg 24 T a IT 96 Hours after injection 35 treatment group during the 20 day period. If, for example, a fish died at 8 days, the preceding plasma samples for that fish were removed from that particular experimental group, and this explains why there are different numbers of fish in each group. Similarly, if a fish died between the days that ovulation was checked and it had not ovulated, the next checking day had fewer fish. Even though the following graph show the mean plasma hormone level at the last blood collection (480 h), the data were not considered for statistical analysis, due to a small number of fish in each group. The control group showed a significant decrease in plasma 17P-estradiol levels at 6 h after injection (Figure 10). From 6 h to 192 h plasma 17P-estradiol levels remained constant, and between 192 h and 384 h declined again. The decrease in 17{3-estradiol production in the control group at 6 h after injection was not followed by an increase in plasma 17a-20(5-P levels. The levels of 17a-20(5-P remained low up to 96 h (approximately 3 ng/mL) then increased although not significantly, from 192 h to 288 h, and at 384 h after injection there was a significant increase on plasma 17a-20P-P production. The significant decline on plasma 17p-estradiol production observed in this group at 6 h after injection was not negatively correlated with an increase in 17a-20P-P. The decrease in plasma 17P-estradiol levels observed between 96 h and 384 h, however, was negatively correlated with the significant increase of 17a-20P-P (r = - 0.79). Plasma testosterone levels in the control group declined significantly (p<0.05) at 6 h, but after 24 h there was a significant rebound and at 96 h plasma testosterone levels were close to those observed before injection (Oh). Groups injected with Fadrozole showed significantly (p<0.05) lower levels of 17P-estradiol at 3 or 6 h after injection. In the group injected with 0.1 mg of Fadrozole/kg there was a significant decrease (p<0.05) in plasma 17P-estradiol levels at 3 h after injection (Figure 11), and a continued decline up to 24 h. At 96 h after injection there was a significant rebound (p<0.05) in 17p-estradiol production, which dropped significantly (p<0.05) again at 288 h after injection. In this group a 36 Figure 9. Mean plasma concentration of 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P) in female coho salmon from Experiment 1, 6 h, and 48 h after injection. Each bar represents the mean + standard error from females in each treatment. Plasma 17cc-20P-P concentrations which are similar (p>0.05) among the groups within each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 37 CD C ca o CM a 500 400 300 200 100 Control I .1 Fadrozole 0.1 mg/kg I .11 Fadrozole 1.0 mg/kg H H Fadrozole 10.0 mg/kg 6 48 Hours after injection 38 Table 1. Effects of the aromatase inhibitor fradozole on gonadosomatic index and egg diameter in female coho salmon. Values represent the mean ± standard error. Dosage N a Gonadosomatic Indexb Egg Diameter0 (%) (mm) Control 4 18.40 ± 0.76 7.01 ± 0.04 0.1 mg fadrozole/kg 4 20.00 ± 0.91 7.21 ± 0.05 1.0 mg fadrozole/kg 4 17.10 ± 1.30 6.72 ± 0.08 10.0 mg fadrozole/kg 4 17.10 ± 0.34 7.03 ± 0.05 "Number of females in each group. b Ovary weight/bodyweight x 100 cEgg diameter was averaged from 20 eggs from each female. 39 Table 2. Number of dead fish ovulated or not ovulated at different times during the experimental period Each treatment had 10 fish at the beginning of the experiment. Number of dead fish ovulated or not ovulated at various days3 8-10 10-12 12-17 17-20 Treatment Nov . Ov. Nov. Ov. Nov . Ov. Nov . Ov. Control 1 1* 1 3 Fadrozole 0.1 mg/kg 1 2 1 1 Fadrozole 1.0 mg/kg 3 Fadrozole 10.0 mg/kg 1 2 a Most of the fish died because they jumped out of the tanks. * Fish died after the blood collection at 384 h. 40 Figure 10. Mean plasma concentration of 17(5-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon from the control group in the Experiment 2 (vehicle injected). Each value represents the mean ± standard error of measurements from 9 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. The last sampling time (480 h) was not considered in the statistical analysis as there were fewer fish. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 41 42 significant increase (p<0.05) in plasma 17a-20p-P levels only occurred after 288 h. The drop in plasma 17{5-estradiol levels observed at 3 h up to 24 h was not correlated with an increase in plasma 17a-20P-P levels The decrease in plasma 17(}-estradiol concentration between 96 h and 288 h after injection, however, was negatively correlated with the significant increase in plasma 17a-20P-P concentration (r = - 0.69) Plasma testosterone levels remained,constant throughout the 20 days. In the group injected with 1.0 mg of Fadrozole/kg (Figure 12), plasma 17P-estradiol levels declined significantly at 6 h following the injection up to 24 h, and from 24 h up to 96 h there was a significant rebound (p<0.05) that reached the levels observed 3 h after injection. From 96 h to 384 h plasma 17P-estradiol levels significantly (p<0.05) decreased. Plasma 17a-20p-P levels increased significantly (p<0.05) at 96 h after the injection and remained constant up to 384 h. The decrease in plasma 17P-estradiol levels observed from 6 h to 24 h was not correlated with an increase in plasma 17a-20P-P levels. Plasma testosterone levels showed some alterations throughout the 20 day period, and there was a significant decrease (p<0.05) between 3 and 6 h and an increase (p<0.05) between 6 and 96 h, and from 96 h to 384 h plasma testosterone levels significantly (p<0.05) declined. In the group injected with 10.0 mg of Fadrozole/kg (Figure 13), plasma 17P-estradiol levels declined significantly (p<0.05) at 3 h after injection up to 24 h and the levels then remained low throughout the experimental period. Plasma 17o>20P-P levels increased significantly (p<0.05) at 3 h after injection and the levels remained constant up to 48 h. At 96 h there occurred another significant increase in plasma 17oc-20P-P which remained steady throughout the 20 day period. The decrease in plasma 17P-estradiol levels between 0 and 24 h after injection was negatively correlated with the significant increase in plasma 17o>20P-P levels (r = - 0.63). Plasma testosterone levels were constant up to 24 h after injection, then increased significantly (p<0.05) to reach a maximum at 96 h, before declining at 192 h. 43 Figure 11. Mean plasma concentration of 17(5-estradiol, 17a-203-dihydroxy-4-pregnen-3-one (17a-20p-P), and testosterone in female coho salmon injected with 0.1 mg of Fadrozole/ kg body weight in the Experiment 2. Each value represents the mean ± standard error of measurements from 6 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. The last sampling time (480 h) was not considered in the statistical analysis as there were fewer fish. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 44 50 45 Figure 12. Mean plasma concentration of 170-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in female coho salmon injected with 1.0 mg of Fadrozole/ kg body weight in the Experiment 2. Each value represents the mean ± standard error of measurements from 10 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. The last sampling time (480 h) was not considered in the statistical analysis as there were fewer fish. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 46 47 Comparison among the treatments showed that the groups injected with 1.0 and 10.0 mg of Fadrozole showed significant declines (p<0.05) on plasma 17p*-estradiol 3 h after injection (Figure 14), and the decline was also dose-dependent, since the plasma 17P-estradiol levels were lower in the groups injected with the higher doses (1.0 and 10.0 mg/kg) than with the lower dose of Fadrozole (0.1 mg/kg). At 6 h after injection the groups injected with 1.0 and 10.0 mg of Fadrozole/kg showed significantly higher levels of 17a-20p-P (p<0.05) than the vehicle group and the group injected with 0.1 mg/kg of Fadrozole (Figure 14). At 48 h after injection the groups injected with 1.0 and 10.0 mg of Fadrozole/kg showed significantly higher levels of testosterone (p<0.05) than the vehicle group. At 96 h after injection plasma testosterone levels was further higher (p<0.05) in the group injected with 10.0 mg of Fadrozole/kg, demonstrating a dose-dependent increase. One group of five fish, killed at 0 day, was used as a control where gonadal development i.e. GSI and ovulation rate were checked. The average GSI was 13.0+0.7 (mean + SEM), and no fish had ovulated. Fish injected with the two highest doses of Fadrozole started to ovulate before (p<0.05) the group injected with the lowest dose (Figure 15), and 10 days after injection 50% and 67% of the fish which received the two highest dose of Fadrozole, 1.0 and 10.0 mg/kg, respectively, had ovulated, in contrast with 0% in the group treated with 0.1 mg/kg. The ovulation rate was significantly higher (p<0.05) in the groups treated with 10.0 and 1.0 mg of Fadrozole/kg respectively 10 and 12 days after injection when compared with the control examined on day 0. In contrast the ovulation rate in the experimental control group (vehicle injected group) was only significant (p<0.05) at the end of the experimental period, and the group injected with 0.1 mg/kg never ovulated significantly, when compared to the control examined at 0 day. The fertilization rate of the eggs (Figure 16) varied between 96% in the control group and 85% in the group which received the highest dose of Fadrozole, 48 Figure 13. Mean plasma concentration of 17f}-estradiol, 17o>20p-dihydroxy-4-pregnen-3-one (17a-20fj-P), and testosterone in female coho salmon injected with 10.0 mg of Fadrozole/ kg body weight in the Experiment 2. Each value represents the mean ± standard error of measurements from 9 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. The last sampling time (480 h) was not considered in the statistical analysis as there were fewer fish. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 49 i r T T i i r 0 3 6 24 48 96 192 288 384 480 0 3 i r 6 24 48 96 192 288 384 480 i i i i i i i i r 0 3 6 24 48 96 192 288 384 480 Hours after injection 5 0 Figure 14. Mean plasma concentration of 17f5-estradiol at 3 h, 17cx-20P-dihydroxy-4-pregnen-3-one (17a-20P-P) at 6 h, and testosterone at 48 h in female coho salmon from Experiment 2. Each bar represents the mean ± standard error from females in each treatment group. Plasma hormone concentrations which are similar (p>0.05) among the groups at each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 51 300 250 200 150 100 -50 -25 20 0 H Control | Fadrozole 0.1 mg/kg 3 Fadrozole 1.0 mg/kg i Fadrozole 10.0 mg/kg 17p-Estradiol 3h 17a-20p-P 6h abb Testosterone 48 h Hormone levels (ng/ml) and hours after injection 52 and it was significantly higher (p<0.037) in the vehicle group than in the treated groups. At 20 days, the GSI was significantly higher (p<0.05) in all experimental groups when compared with the group examined at 0 h (Figure 17) indicating that gonadal growth had continued during the study, and that Fadrozole had no effect on gonadal growth. 53 Figure 15. Effects of Fadrozole on the cumulative ovulation in female coho salmon from Experiment 2. Values represent the cumulative percentage of the number of fish which ovulated in each group. The proportion of observations at each checking day which are similar (p>0.05) among the treatment groups, as determined by Fisher Exact Test, are identified by the same superscript letter. The asterisk symbol (*) means that the proportion of observation at each sampling day was significantly different (p<0.05) as determined by Fisher Exact Test, from the control group examined and killed on day 0 (n=5). 54 100 0 0 8 10 12 Days after injection 16 20 55 Figure 16. Effects of Fadrozole on the fertilization rate at hatching (%). Values represent the percent of alevins alive at hatching from 4 replicated groups of 50 eggs each from 4 female in each treatment group. The proportion of observations which are similar (p>0.05), as determined by chi-square and Yates correction for continuity, are identified by the same superscript letter. 56 120 0.0 0.1 1.0 10.0 Groups injected or not with Fadrozole (mg/kg) 57 Figure 17. Effects of Fadrozole on the gonadosomatic index (GSI) in female coho salmon. Each bar represents the mean ± standard error from females in each treatment group GSI which are similar (p>0.05), as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Gonadosomatic index = ovary weight/body weight x 100. Letter C means control examined and killed at the beginning of the experiment. 58 25 20 H 15 H 10 H 5 H 0 C 0.0 0.1 1.0 10.0 Groups injected or not with Fadrozole (mg/kg) 59 3.4. Discussion In both Experiment 1 and Experiment 2 the high levels of plasma 17P-estradiol and the low levels of 17a-20p*-P observed in all groups at time 0 are characteristic of late vitellogenesis prior to the onset of the final maturation process. These profiles are in accordance with those for saline injected female coho salmon, 49 days to 10 days prior to ovulation (Fitzpatrick et al., 1987). Plasma testosterone levels at time 0 in the present experiments are closer to the plasma testosterone levels for saline injected female coho salmon 49 days to 10 days prior to ovulation (Fitzpatrick et al., 1987). Levels similar to those found in this study for 17a-20P-P and testosterone at time 0 were reported by Van Der Kraak et al. (1984) in female coho salmon 12 days prior to ovulation. Plasma 17P-estradiol levels at time 0, however, were lower in the study reported by Van Der Kraak et al. (1984) than those found in this study. The decline in plasma 17P-estradiol level in the control group between 96 h and 192 h after the initiation of the study in the experiment 1 and after 288 h in the experiment 2, was accompanied by an increase on plasma 17a-20P-P concentration. This response is characteristic of the completion of vitellogenesis and the initiation of the final maturation process which is mediated by 17a-20P-P, which has been well established as the maturation inducing hormone (MIH) in salmonids and in several non salmonid fish (Nagahama, 1987a; Nagahama, 1987b; Nagahama and Yamashita, 1989). A similar endocrine profile has been observed in several salmonids including rainbow trout, Oncorhynchus mykiss (Scott et al., 1982), coho salmon (Van Der Kraak et al., 1984; Fitzpatrick et al., 1986), masu salmon, Oncorhynchus masou (Yamauchi et al., 1984) spring chinook salmon, Oncorhynchus tshawytscha (Slater et al., 1986), and pink salmon, Oncorhynchus gorbuscha (Dye et al., 1986) This profile can change over a matter of 60 days. For example, in coho salmon during spontaneous reproductive activity plasma 17f3-estradiol levels declined from 16 ng/mL 12 days prior to ovulation to 1-2 ng/mL 4 days prior to ovulation and plasma 17a-20|3-P levels increased form less than 10 ng/mL to 270 ng/mL in the same period (Van Der Kraak et al., 1984) The present studies clearly demonstrated that Fadrozole is capable of inhibiting estrogen biosynthesis in a teleost. The results indicate that Fadrozole accelerated the decrease in plasma 17fJ-estradiol levels in post vitellogenic female coho salmon and furthermore that it accelerated the shift to 17a-20P-P production leading to dramatic increases on plasma 17a-20p-P levels. The timing and the magnitude of the decline in plasma 17P-estradiol concentration and the increase in plasma 17a-20P-P levels were dose-dependent There are no previous investigations of the effect of Fadrozole on periovulatory steroidogenesis in fish. However, some studies using the same aromatase inhibitor have been conducted in mammals. Steele et al. (1987) reported that Fadrozole caused maximum inhibition of rat ovarian estrogen synthesis (94% depression of estrogen production) at an oral dose of 0.26 mg/kg 4 h after injection. The same authors showed that female rats treated orally with 4 mg of Fadrozole/kg for 14 days developed uterine atrophy due to aromatase inhibition. Fadrozole is considered to be a competitive inhibitor of aromatase activity (Steele et al., 1987) Fadrozole has also been shown to significantly reduce estrogen formation in the avian brain, zebra finch (Wade et al., 1994), and fish brain, salmo salar (Antonopoulou et al., 1995). Although the results of the main effects of Fadrozole were similar between Experiments 1 and 2 up to 8 days, there were some variations on the long term responses. For example, in both experiments the groups injected with 0.1 and 10 mg of Fadrozole/kg presented lower plasma 17p-estradiol concentrations at 24 h after injection In Experiment 1, plasma 17P-estradiol 61 remained low after 24 h, but in the second experiment there was a rebound in plasma 17(3-estradiol levels at 96 h after injection In Experiment 1, plasma 17a-20P-P level increased significantly at 24 h after injection in the groups injected with 0.1 and 1.0 mg of Fadrozole/kg. In the Experiment 2, however, the increase in plasma 17a-20p-P occurred at 288 h and 96 h in the groups injected with 0.1 and 1.0 mg of Fadrozole/kg, respectively On the other hand, in the group injected with 10.0 mg of Fadrozole/kg the plasma 17P-estradiol and 17a-20P-P profiles were similar between the two experiments. The differences in the long term results between the experiments are probably due to the fact that fish from Experiment 1 were closer to final maturation than the fish from Experiment 2. The Experiment 1 started on October 25 1994 and Experiment 2 started on September 29 1995. The results of both experiments, suggest that, in fish close to final maturation, the highest dose of Fadrozole suppressed 17P-estradiol biosynthesis for the duration of the study. On the other hand, the results of Experiment 2, demonstrated that the injection of 0.1 and 1.0 mg of Fadrozole/kg did not suppress plasma 17P-estradiol biosynthesis permanently as suggested by Experiment 1. The permanent decline on plasma 17P-estradiol level in experiment 1 can be explained by the fact that the fish were already by 96 h at a maturation stage where plasma 17P-estradiol level would have declined naturally as indicated by plasma 17P-estradiol in the control group. It has been demonstrated that testosterone is the major aromatizable androgen to be produced by the thecal cells in amago salmon, Oncorhynchus rhodurus (Nagahama, 1987a) Furthermore, testosterone is capable of increasing 17P-estradiol secretion in vitro by coho salmon ovarian follicles in a dose dependent manner (Young et al., 1983a). As testosterone is 62 probably the major substrate for 17P-estradiol production, is expected that inhibition of aromatase activity by Fadrozole would cause an increase in plasma testosterone levels. Fish treated with the highest doses of Fadrozole showed this trend in both experiments. In Experiment 2, however, plasma testosterone reached maximum levels at 96 h and then declined. At this point, i.e. 96 h, the highest dose of Fadrozole presented a significantly higher plasma testosterone levels than the other groups. In vitro studies in this thesis (Chapter 6) demonstrated that incubation of ovarian follicles with Fadrozole alone slightly increased testosterone production. On the other hand, the combination of Fadrozole with GTH I and GTH II dramatically increased testosterone production in different stages of the reproductive development. These results indicates that the increase in plasma testosterone levels in vivo, is probably due to the stimulation of testosterone production by the GTH. It is difficult to explain why, in the groups injected with the highest dose of Fadrozole, plasma testosterone levels decreased significantly after they had peaked, although plasma 17(3-estradiol levels were at 288 h and 384 h at least 15 times lower than the levels at 0 h. Since at this point, plasma 17a-20p-P levels were high and constant, it could be speculated that the decrease in plasma testosterone levels is associated with an increase in 20p-hydroxysteroid dehydrogenase activity, which uses the same substrate, i.e. 17cx-hydroxyprogesterone, to synthesize 17tx-20p-P as does C17-20 lyase, which catalyzes the synthesis of androstenedione, the testosterone precursor. It could also be speculated that a reduction in 17P-hydroxysteroid dehydrogenase activity, the enzyme responsible for the conversion from androstenedione to testosterone, occurred. The sharp decline in plasma 17p-estradiol levels observed in the Fadrozole treated groups contrasts with more gradual declines in plasma 17p-estradiol levels induced by LH-RHA or GTH. Thus in earlier studies on periovulatory coho salmon in this laboratory it was shown that 6 3 injection of GTH (SG-G100, 0.1 mg/kg) (Van Der Kraak et al., 1985) or LH-RHA (0.02 mg/kg) (Van Der Kraak et al., 1984, 1985) resulted in a significant decline in plasma 17p-estradiol levels after two and two to four days, respectively. This comparison between Fadrozole, GTH, and LH-RHA suggests that Fadrozole is acting first at the level of ovarian inhibiting 17(3-estradiol synthesis rather than directly stimulating GnRH or gonadotropins at the hypothalamic or pituitary level. This study also demonstrated the effectiveness of the aromatase inhibitor Fadrozole in promoting 17a-20(3-P production. The decrease in plasma 17p-estradiol levels was followed by an increase in the plasma 17a-20P-P levels in all groups. In the Fadrozole treated groups however, the higher the dose of Fadrozole administered, the faster was the decline of the plasma 17P-estradiol levels and the switch to the 17ct-20p-P biosynthesis. The shift from 17p-estradiol to 17a-20p-P biosynthesis observed in the Fadrozole treated groups contrasts with the pattern observed when LH-RHA or GTH are applied. The injection of LH-RHA in adult female coho salmon caused a significant increase in plasma 17a20p-P levels 3 h after injection, even though plasma 17p-estradiol levels remained constant up to 24 h (Van Der Kraak et al., 1984). The injection of GTH resulted in a significant increase in plasma 17a-20p-P levels 24 h after injection, while the plasma 17pestradiol concentration was unchanged at 24 h (Van Der Kraak et al., 1985). In coho salmon injected 4-8 days earlier with GTH (SG-100) or LH-RHA, respectively, plasma 17a-20P-P levels (Van Der Kraak et al., 1985) were similar to levels found in the present study between 24 and 96 h after administration of Fadrozole. Since the pre-injection hormonal profile exhibited by the fish in this experiment suggests that they were between 14 to 21 days prior to ovulation (Fitzpatrick et al., 1987) the results also indicates that the mechanism responsible for 17a-20p-P biosynthesis is already in place some days before final maturation. In the normal 64 process of ovarian maturation in salmonids the decline in aromatase activity correlates with an increase 20fJ-hydroxysteroid dehydrogenase (20P-HD) in the cells of the follicle and an increase in plasma 17a-20p-P levels (Nagahama et al., 1993). Young et al. (1983a) reported that aromatase activity in the ovarian granulosa cells of amago salmon increased during the period of vitellogenesis to reach a peak in late vitellogenesis, and then declined rapidly in association with the ability of the oocyte to mature in response to gonadotropin. In the same species both in vivo and in vitro studies showed that plasma GTH levels in mature and ovulated fish correlate with increases in plasma 17a-20(5-P levels (Young et al., 1983b). The same authors suggested that activation of the enzyme 20P-HD and subsequent 17a-20P-P synthesis is GTH dependent. The results from in vitro studies in this thesis (Chapter 6) demonstrated that Fadrozole per se does not elicit secretion of 17a-20f}-P in the incubation medium at any of the different stages during the periovulatory period. However, during the central and peripheral germinal vesicle stage, both GTH I and GTH II interact positively increasing 17a-20f}-P secretion. The synergism of GTH II with Fadrozole was stronger at the peripheral germinal vesicle stage and it was far more potent than Fadrozole with GTH I. Swanson (1991), analyzing the plasma GTH I and GTH II levels in maturing female coho salmon demonstrated that GTH II levels increases close to ovulation, and they were correlated with the increase in plasma 17cc-20P-P levels. A similar profile of plasma GTH II secretion was reported by Suzuki et al. (1988d) for chum salmon, Oncorhynchus keta, Slater et al. (1994) for spring chinook salmon, and by Prat et al. (1996) for rainbow trout. It is possible, therefore that the early increase in plasma 17cc-20P-P observed in the present study was due to a increase in plasma GTH II levels. Neuroendocrine studies in teleost fish have shown that 17P-estradiol and testosterone can have a positive feedback effect on the pituitary stimulating it to secrete GTH (Peter et al., 1986, 65 1991, in goldfish, Carassius auratus, Kah et al., 1995, Linard et al., 1995b, 1996, in rainbow trout). Sex steroids can also have a stimulatory effect over the catecholamine dopamine, which in turn inhibits GTH release (Kah et al. 1995). 17p-Estradiol also has a negative effect over y-aminobutyric acid (GABA), which has a stimulatory effect over the GnRH neuronal system and consequently over GTH secretion (Peter et al., 1991) If plasma 17P-estradiol levels are reduced, as occurs during late vitellogenesis in salmonids or as occurred in this study after injection with aromatase inhibitor, it may be that the negative effect of 17P-estradiol over GABA and the positive effect of 17P-estradiol over dopamine were reduced, which in turn could cause an increase in GTH II secretion (Linard et al., 1995b; Kah et al., 1995), which would subsequently trigger an increase in 17a-20p-P biosynthesis (Young et al., 1983b; Fitzpatrick et al., 1986; Nagahama, 1987a; Kagawa, 1994). Linard et al. (1995b) obtained evidence that the catecholamine dopamine inhibited GTH II secretion in trout when plasma 17P-estradiol levels are elevated, suggesting an interaction between 17P-estradiol and the enzyme tyrosine hydroxylase, which is the limiting enzyme of catecholamine synthesis. Linard (1995a) showed that the plasma GTH II levels increased significantly in sexually mature female rainbow trout treated with a-methyl-p-tyrosine (MPT), which is an inhibitor of tyrosine hydroxylase. It has been shown in mammals that estradiol inhibits (feedback mechanism) the secretion of the luteinizing hormone (Carrol and Baum 1989, and Bhatnagar, et al., 1993), the gonadotropin associated with maturation and ovulation, which is structurally similar to the maturational gonadotropin in salmonids, GTH II (Swanson, 1991). Furthermore, Bhatnagar et al (1993), showed that in female rats the secretion of the follicle stimulating hormone (FSH), structurally similar to the GTH I in salmonids (Swanson, 1991) is not under control of 17P-estradiol. 66 This study shows that injection of Fadrozole is effective in accelerating ovulation when administered approximately 20 days prior to normal time of ovulation. The highest rate of ovulation occurred at 8 days after injection. Considering the fact that the steroid hormones involved in gametogenesis are produced mainly at the gonadal level, Fadrozole is probably acting at ovarian level inhibiting 17(3-estradiol synthesis. This study suggests that the route used by Fadrozole to stimulate ovulation (gonad- hypothalamus-hypophysis-gonad) is different from the routes used by the traditional methods used to induce ovulation (Peter et al., 1993; Donaldson, 1996). These methods act through the hypothalamic-hypophyseal-gonadal axis, stimulating GnRH secretion which in turn stimulates gonadotropin secretion from the pituitary which then will circulate in the blood and will bind to receptors at the gonadal level stimulating steroid synthesis which in turn will affect gonadal maturation. In some species e.g. carps, ovulation is induced by combining GnRHa and antagonists of dopamine (e.g. domperidone or pimozide), a catecholamine which acts at the gonadotroph levels inhibiting GnRH-stimulated GTH II release in teleost fish (Peter et al., 1991, Peng, et al., 1994). Studies of the hormone profile of different salmonids have shown that close to ovulation there is a natural decline in 17P-estradiol and an increase in 17a-20p-P, which is probably due to a surge in GTH II. The use of Fadrozole to induce ovulation by inhibiting 17($-estradiol (Figure 18) biosynthesis in the ovary and in the central nervous system may provide an alternative method to the current use of GnRH or partially purified gonadotropin. Both methods result in increased 17a-20p-P levels, but in the latter technique the decline in 17p-estradiol is more gradual than when Fadrozole is used (Van Der Kraak et al., 1984, 1985). It would be interesting to study the combined effect or not of Fadrozole and GnRH or GTH on the ovulation rate of teleost fish. The hormone profile obtained in fish injected with the aromatase inhibitor Fadrozole 67 SENSORY STRUCTURES External stimuli BRAIN < Neurohormone < ±Lz ' -±/= HYPOTHALAMUS GnRH Dopamine Other pituitary hormones +/-GTH I, GTH II +/-Non pituitary factors—• V TESTIS/O VARY J 17B-estradiol +/- "^""7 \ Testosterone \ spermiation ovulation ^ 0 0 F A D R O Z O L E F A D R O Z O L E F i g u r e 18. Schematic representation of the hypothalamic-pituitary-gonadal axis in fish. 68 (reduction in plasma 17p-estradiol and increase in plasma 17a-20f}-P concentrations) correlates well with the hormone profile occurring naturally in salmonids, and with the observations made by Young et al. (1983b) and Kanamory et al. (1988) that there is a natural decline in aromatase activity towards final maturation. Considering that there is no limitation in terms of substrate for 17p-estradiol biosynthesis, since testosterone is naturally present in high levels, it could be speculated that there is a natural factor which could be inhibiting aromatase activity. Indeed, in mammals Al-Gubory et al. (1994) obtained evidence that a non-steroidal factor in the corpus luteum of pregnant sheep inhibited aromatase activity of ovarian follicles in vitro. Whether such a factor is present in ovarian follicles of fish it remains to be investigated. This study demonstrated that there was a reduction between 6 and 9% on the fertilization rate in fish injected with Fadrozole when compared to the control group (95%). It has been demonstrated that female coho salmon treated with SG-G100 and 17cx-20P-P or a combination of SG-G100+17cx-20P-P presented decreased fertilization rate, between 5 and 10%, when compared with the control group (Jalabert et al., 1978). The same authors observed that in another similar experiment there was no difference in the fertilization rate, however the fish from this experiment were closer to maturation than the first ones. Therefore this indicates that the fertilization rate may vary with the time when ovulation is induced, and suggests that the closer to the final maturation stage the better are the results in terms of fertilization rate. Mean GSI showed no significant change during Experiments 1 and 2. In pink salmon, Dye et al. (1986) were not able to detect a change in GSI over an interval of 12 days during the final part of the anadromous migration. Those authors also reported that the GSI on the spawning ground was 17.44 and very near maximum values. For coho salmon, Sower and Schreck (1982) reported that a maximum GSI of 16.8 and egg diameter of 6.3 when eggs were in the peripheral 69 germinal vesicle(PGV) stage Van Der Kraak et al. (1983) also did not find differences in GSI and egg diameter of female coho salmon injected or not with LH-RH analogs. The GSI values were very close to those reported in this experiment. Even though GSI was not different among groups, they reported that fish treated with LH-RHA Dala6 presented eggs undergoing GVBD. In experiment 2, GSI was not different among the experimental groups after 20 days, but confirming that there was gonadal growth during the 20 day period and that Fadrozole did not affect it. In summary, the present study provides the first evidence that injection of the aromatase inhibitor Fadrozole is effective in lowering plasma 17|3-estradiol levels in a teleost. Furthermore the study has demonstrated an associated increase in plasma 17ct-20P-P levels, indicating that the shift between plasma 17p-estradiol biosynthesis and 17a-20P-P biosynthesis, which is characteristic of maturating fish, was advanced by treatment with Fadrozole. Finally it has been shown that Fadrozole can be used as a tool to induce ovulation in coho salmon. 70 C H A P T E R 4 - Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon during vitellogenesis. 4.1. Introduction Vitellogenesis in teleosts is promoted by a two step mechanism in which, gonadotropin increases ovarian secretion of 17P-estradiol, which in turn stimulates the hepatic synthesis and secretion of vitellogenin (Wallace, 1985; Tyler, 1991). The phase following vitellogenesis, the oocyte maturation stage is also controlled by gonadotropin, which stimulates the production of a maturation inducing substance by the follicles cells 17a-20p-dihydroxy-4-pregnen-3-one (17a-20P-P) (Nagahama, 1987a; Kagawa, 1994), which in turns mediates the activation of the maturation promoting factor (MPF) in the oocytes (Nagahama and Yamashita, 1989). In salmonid fish the transition from the vitellogenic stage to the final maturation stage is accompanied by morphological, and physiological changes, which cause changes in pigment distribution in the skin, resulting in a darkening of the skin, changes in jaw morphology, decreased efficiency of the immune system, and interruption of growth, and also changes in proximate composition and in the organoleptic characteristics of the muscle. In terms of aquaculture these changes associated with maturation are not beneficial, since they result in decreased value in the market. Therefore the prevention of sexual maturation in species of fish such as salmonids that undergo deleterious changes during maturation could have a beneficial economic impact in fish culture. Since the experiments described in the previous Chapter demonstrated that it is possible to block estrogen in vivo using the aromatase inhibitor Fadrozole, the objective of this study was to 71 investigate the hypothesis that inhibiting estrogen production, by blocking aromatase enzyme activity, during vitellogenesis, may delay oocyte growth and maturation. 4.2. Materials and Methods One hundred adult coho salmon were obtained in mid-August 1996 from the Capilano Salmon Hatchery, Vancouver, B.C. (Department of Fisheries and Oceans) and transported to the West Vancouver Laboratory (Department of Fisheries and Oceans). Fish were individually identified with a PIT tag inserted in the ventral muscle. Since it was impossible to visually sex the fish by examining external morphology, a sample of blood was collected from each individual and the plasma 17P-estradiol level was determined in each fish. Fish in which 17(3-estradiol was undetectable were considered male and were used in the experiment described on the next Chapter. In females, plasma 17P-estradiol was always above 10 ng/mL. The exam of the gonads at the end of the experiment demonstrated that this technique was completely reliable. At the time of capture the weights and the lengths of the female fish were 1.2 ± 0.4 kg and 47.2 ± 4.0 cm (mean ± SE), respectively. Fish were divided in four groups: control vehicle injected (n = 8), group treated with 1.0 mg Fadrozole/kg (n = 8), group treated with 10.0 mg Fadrozole/kg (n = 8), and group treated weekly with 10.0 mg Fadrozole/kg (5 x 10 mg/kg) (n = 8). The experimental protocol consisted of determining the plasma sex steroid levels before injection with Fadrozole and during thirty two days after injection. Blood samples were collected at 0 hour (just before injection with vehicle or Fadrozole), 6 h, 24 h, 48 h, 96 h, and 192 h, 384 h, and 768 h after injection. Plasma 17P-estradiol, testosterone, and 17a-20P-dihydroxy-4-pregnen-3-one (17a,20P-P) levels were measured by radioimmunoassay. After the last blood collection fish were killed by decapitation, GSI and oocyte diameter were determined, a sample of the right ovary was 72 collected for histological examination (from four fish from each group) and a sample of the right ovary was also collected to carry out in vitro experiment (from each fish) to verify the steroid secretion capacity of the ovarian follicles. Besides, the brain of each fish was also collected to determine the in vitro secretion of 17(J-estradiol. Six untreated fish were killed at 0 hour, from which GSI and egg diameter were determined and a sample of the right ovary submitted to histology (from 4 fish) to compare with the fish after 32 days. Percentage atresia in the ovarian follicles was determined by examining one cross section containing approximately 15 oocytes from 4 fish per treatment group. Atresia was identified by hypertrophy of the granulosa cells, by an irregular inner surface or a breakdown or decrease in thickness of the zona radiata around the oocyte (Lambert, 1967; Nagahama, 1983, Morrison, 1990). 4.3. Results Some fish died during the course of the experiment, mostly because they jumped out of the tanks. Therefore, the exact number of fish in each group can be seen on the legend preceding the figure of each treatment group. In the control group, which was injected with the propylene glycol vehicle, plasma 170-estradiol levels 768 h after injection increased slightly but significantly (p<0.05) in relation to the first blood collection (Figure 19). Plasma 17a-20P-P levels remained undetectable up to 384 h and then increased significantly at 768 h. In this group fish showed a significant (p<0.05) elevation in plasma testosterone levels at 48 h, increasing from approximately 9 ng/mL at 0 hour to 16 ng/mL at 48 h. At 384 h plasma testosterone levels were significantly (p<0.05) higher in relation to 48 h (from 16 ng/mL to 32 ng/mL). At 768 h there was another significant increase (p<0.05), when levels reached approximately 53 ng/mL). 73 Figure 19. Mean plasma concentration of 170-estradiol, 17a-20f3-dihydroxy-4-pregnen-3-one (17a-20[}-P), and testosterone in vitellogenic female coho salmon from the control group. Each value represents the mean ± standard error of measurements from 7 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 74 Hours after injection 75 A single injection of 1.0 mg of Fadrozole was effective in lowering plasma 17p-estradiol concentration for 48 h (Figure 20). In this group plasma 17P-estradiol levels declined significantly (p<0.05) at 6 h up to 24 h after injection, and the levels remained low up to 48 h before rebounding significantly (p<0.05). At 384 h plasma 17P-estradiol levels were not significantly different (p>0.05) from concentrations observed at 0 hour (before injection). Plasma 17a-20P-P levels increased slightly but significantly (p<0.05) at 6 h after injection, and the increase was negatively correlated (p<0.004 and r = - 0.67) with the significant decrease observed in plasma 17P-estradiol levels at this time. From 48 hour to 384 h plasma 17a-20P-P levels decreased significantly (p<0.05), and the decrease was negatively correlated (p<0.01 and r = -0.44) with the increase in plasma 17P-estradiol levels observed during same period. Plasma testosterone levels remained constant up to 24 h after injection. Between 24 h and 48 h, when plasma 17P-estradiol levels were at a rriinimum, there was a dramatic increase in plasma testosterone level, from approximately 15 ng/ml to almost 60 ng/ml. From 48 h to 384 h, when plasma 17P-estradiol levels were rebounding, plasma testosterone levels declined. A single injection of 10.0 mg Fadrozole was effective in lowering plasma 17P-estradiol concentration for 96 h (Figure 21). In this treatment group plasma 17P-estradiol levels decreased significantly (p<0.05) at 6 h up to 24 h after injection. From 24 h to 96 h plasma 17P-estradiol levels remained low and steady, but after that rebounded significantly (p<0.05) up to 768 h after injection. At 192 h after injection plasma 17P-estradiol was similar to the levels observed before injection. Plasma 17tx-20P-P levels increased significantly (p<0.05) at 6 h after injection, and the increase was negatively correlated (pO.OOl and r = - 0.77) with the significant decrease observed in plasma 17P-estradiol levels at the same time. From 6 h to 96 h plasma 17a-20P-P levels remained steadily elevated, coinciding with the period that plasma 17P-estradiol remained low. Plasma 17a-20P-P levels 76 Figure 20. Mean plasma concentration of 17(3-estradiol, 17a-20(3-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in vitellogenic female coho salmon injected with 1.0 mg of Fadrozole/ kg body weight. Each value represents the mean ± standard error of measurements from 8 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 77 78 Figure 21. Mean plasma concentration of 17{3-estradiol, 17a-20f)-dihydroxy-4-pregnen-3-one (17a-20P-P), and testosterone in vitellogenic female coho salmon injected with 10.0 mg of Fadrozole/ kg body weight. Each value represents the mean ± standard error of measurements from 8 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 79 Hours after injection 80 decreased significantly (p<0.05) at 192 h, and the decline was negatively correlated (p<0.001 and r = -0.73) with the increase in plasma 17(3-estradiol levels at the same time. From 192 h to 384 h plasma 17cx-203-P levels remained low and then increased (p<0.05) slightly at 768 h after injection. Plasma testosterone levels began to increase slightly but significantly (p<0.05) at 24 h after injection. Plasma testosterone levels continued to increase significantly (p<0.05) up to 96 h when the levels reached approximately 135 ng/ml in contrast with approximately 8 ng/ml at time 0. After 96 h plasma testosterone levels declined significantly (p<0.05), but remained significantly (p<0.05) higher than the plasma testosterone levels observed between 0 hour and 24 h after injection. In the group injected weekly with 10.0 mg of Fadrozole, plasma 17|3-estradiol levels declined significantly (p<0.05) between 0 and 6 h and between 6 and 24 h, and remained steadily low throughout the remaining experimental period (figure 22). This treatment group exhibited a significant increase in plasma 17a-20f3-P levels 6 h after injection, which was negatively correlated (p<0.004 and r = - 0.67) with the significant decline in plasma 17p-estradiol levels at the same time. From 6 h to 24 h plasma 17a-20f3-P levels declined significantly (p<0.05). After they remained constant up to 768 h at a level higher than that observed at 0 hour. Subsequent injections did not cause another peak in plasma 17a-20P-P levels, however the plasma 17o>20P-P levels did not decline. Plasma testosterone levels increased significantly at 48 h after injection up to 96 h. After that plasma testosterone levels continued unchanged up to 384 h and then declined significantly (p<0.05) at 768 h after injection to the plasma testosterone levels observed before injection. Comparison among the groups at 6 h showed that the groups injected with Fadrozole presented significantly (p<0.05) lower plasma 17P-estradiol levels than the vehicle injected group (Figure 23). The decrease was dose-dependent, since the plasma 17P-estradiol levels were significantly 81 Figure 22. Mean plasma concentration of 173-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-20f3-P), and testosterone in vitellogenic female coho salmon injected weekly with 10.0 mg of Fadrozole/ kg body weight (5x10 mg/kg). Each value represents the mean ± standard error of measurements from 8 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Arrows from the left to the right indicate the first, second, third, fourth, and fifth injections, respectively. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 82 83 Figure 23. Mean plasma concentration of 17P-estradiol in vitellogenic female coho salmon at 6 h, and 192 h after injection. Each bar represents the mean ± standard error from females in each treatment group. Plasma 17P-estradiol concentrations which are similar (p>0.05) among the groups at each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 84 1 0 8 H 4 H 0 Control [.., . J Fadrozole 1.0 mg/kg a I i j Fadrozole 10mg/kg IHH Fadrozole 5x10 mg/kg 6 192 Hours after injection 85 (p<0.05) lower in the groups injected with 10 mg of Fadrozole/kg than in the group injected with 1.0 mg of Fadrozole/kg. At 192 h plasma 17P-estradiol levels in the group injected weekly were significantly (p<0.05) lower than the other groups. At 6 h after injection groups injected with Fadrozole presented significantly (p<0.05) higher plasma 17a-20p-P levels than the vehicle injected group (Figure 24). Comparison among the groups at 192 h showed that the vehicle injected group and groups injected once with Fadrozole presented significantly lower plasma 17a-20p-P levels than the group injected weekly with Fadrozole, which at this point had received 2 injections of 10 mg of Fadrozole/kg. The treatment groups injected with Fadrozole showed a significant (p<0.05) elevation in plasma testosterone levels at 48 h after injection (Figure 25) when compared with the vehicle group. At 96 h the two treatment groups injected with 10 mg/kg presented significantly (p<0.05) higher plasma testosterone levels than the other groups, and at 768 h the group injected weekly showed significantly reduced plasma testosterone levels when compared with the other three groups. The oocyte maturation index (Table 3 and Figure 26) showed that during the 32 day period there was a change in the developmental stage of the oocytes when compared with the developmental stage of the oocytes of the fish examined and killed at the beginning of the experiment, in which oocytes had germinal vesicles which were most frequently in the central position. A l l groups, except the group injected weekly, had significantly higher (p<0.05) oocyte maturation indices than the group examined and killed at the beginning of the experiment, and in most of the oocytes the germinal vesicle was migrating or was peripheral. Oocyte diameter measurement (Figure 27) demonstrated that there was significant increase in oocyte size during the experimental period. A l l groups, except the group injected weekly, presented a significantly higher (p<0.05) oocyte diameter than the group examined and killed at the beginning of the experiment. 86 Figure 24. Mean plasma concentration of 17a-20P-dihydroxy-4-pregnen-3-one (17a-20|3-P) in vitellogenic female coho salmon at 6 h, and 192 h after injection. Each bar represents the mean ± standard error from females in each treatment group. Plasma 17cx-20P-dihydroxy-4-pregnen-3-one concentrations which are similar (p>0.05) among the groups at each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 8 7 1 1 Control d i - 1 Fadrozole 1.0 mg/kg I" -}j Fadrozole 10.0 mg/kg 192 Hours after injection 88 Figure 25. Mean plasma concentration of testosterone in vitellogenic female coho salmon at 48 h, 96 h and 768 h after injection. Each bar represents the mean + standard error from females in each treatment group. Plasma testosterone concentrations which are similar (p>0.05) among the groups at each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 89 140 H Control i J Fadrozole 1.0 mg/kg i -m \ : HI Fadrozole 10.0 mg/kg Bj Fadrozole 5x10 mg/kg 48 96 768 Hours after injection 90 Table 3 . Oocyte maturation index in vitellogenic female coho salmon injected or not with fadrozole (mg/kg body weight). For each female a sample of 20 oocytes were examined, and classified in relation to the position of the germinal vesicle. A numerical index was attributed for each classification and an average index was calculated for each treatment group. Control 0 hour means fish examined and killed at the beginning of the experiment. Oocyte classification in relation to the position of the germinal vesicle Treatment groups Central 1 Migrating 2 Peripheral 3 Germinal vesicle breakdown 4 Maturation* index Control 0 hour 4 2 1.33 Control 2 5 2.71 Fadrozole 1.0 mg/kg 2 6 2.75 Fadrozole 10.0 mg/kg 6 2 3.25 Fadrozole 5x10 mg/kg 2 2 4 2.25 •Maturation index control 0 h = (4 x 1) + (2 x 2)16 = 1.33 91 Figure 26. Oocyte maturation index in vitellogenic female coho salmon injected or not with Fadrozole For each female a sample of 20 oocytes were examined, and classified in relation to the position of the germinal vesicle. A numerical index was attributed for each classification and an average index was calculated for each treatment group. Oocyte maturation indices which are similar (p>0.05), as determined by Mann-Whitney Rank Sum test, are identified by the same superscript letter. Number between brackets above the superscript letter means number of fish in each group. Letter C means control examined and killed at the beginning of the experiment. 92 0 1 10 Fadrozole (mg/kg) 5 x 10 93 Figure 27. Effects of Fadrozole on the oocyte diameter from vitellogenic female coho salmon. Each bar represents the mean + standard error from females in each treatment group . Oocyte diameters which are similar (p>0.05), as determined by Student-Newman-Keuls test among the injected groups (0, 1,10, and 5x10 mg/kg), or by unpaired t-test (control examined and killed at the beginning against to the other groups) are identified by the same superscript letter. Number between brackets above the superscript letter means number of fish in each group. Letter C means control examined and killed at the beginning of the experiment. 94 5 x 10 Fadrozole (mg/kg) 95 In the group injected weekly with 10 mg Fadrozole/kg, the gonadosomatic index (Figure 28) was significantly lower (p<0.05) than the gonadosomatic index in both the vehicle injected group and the group injected with 10.0 mg of Fadrozole/kg, and it was comparable to the gonadosomatic index of the fish which were examined at the beginning of the study Measurement of the in vitro production of 17(5-estradiol by ovarian follicles at the end of the experiment showed that the group injected weekly with 10 mg Fadrozole/kg secreted significantly (p<0.05) less 17P-estradiol than other groups (Figure 29). On the other hand, 17tx-20P-P was not detected in vitro in any of the groups. In vitro incubation of the brain tissue (Figure 30) showed that all groups injected with Fadrozole presented significantly (p<0.05) lower levels of 17P-estradiol than the vehicle injected group. Histological examination showed that control fish and fish injected with 1.0 or 10.0 mg Fadrozole /kg presented a low incidence (1-2%) of oocytes with atretic characteristics (Figures 31-32). On the other hand fish injected weekly with 10 mg Fadrozole/kg demonstrated a significant increase in the incidence of atresia of vitellogenic oocytes, approximately 80% of the oocytes. 96 Figure 28. Effects of Fadrozole on the gonadosomatic index (GSI) from vitellogenic female coho salmon. Each bar represents the mean + standard error from females in each treatment group. GSI which are similar (p>0.05), as determined by Student-Newman-Keuls test among the injected groups (0, 1,10, and 5x10 mg/kg), or by unpaired t-test (control examined and killed at the beginning against to the other groups) are identified by the same superscript letter. Number between brackets above the superscript letter means number of fish in each group. Gonadosomatic index = ovary weight/body weight x 100. Letter C means control examined and killed at the beginning of the experiment. 97 (6) a J L (7) b J L J (8) ab _L (8) b T 0 1 10 Fadrozole (mg/kg) 5 x 10 98 Figure 29. In vitro 17p-estradiol production by ovarian follicles of vitellogenic female coho salmon at the end of the experiment (768 h). Each bar represents the mean ± standard error from females (four replicates per female with 4 oocytes each) in each treatment group. Follicles were incubated with incubation medium in the presence of testosterone (100 ng/ml) by 18 h at 12°C. 17P-Estradiol released to the media which are similar (p>0.05) as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Number between brackets above the superscript letter means number of fish in each group. 99 1 10 Fadrozole (mg/kg) 5 x 10 100 Figure 30. In vitro 17fi-estradiol production by the brains of vitellogenic female coho salmon at the end of the experiment (768 h). Each bar represents the mean ± standard error from females in each treatment group. Brains were incubated with incubation medium in the presence of testosterone (100 ng/ml) by 18 h at 12°C. 17fi-Estradiol released to the media which are similar (p>0.05) as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Number of fish in each group is specified on previous figure. 101 1 10 Fadrozole (mg/kg) 5 x 10 102 Figure 31. Representative micrographs sections (5u) of the anterior part of the right ovary stained with hematoxylin-eosin, from (a) control group (vehicle injected) and (b) treatment group which received multiple injections (5 x 10 mg Fadrozole/kg) at the end of experiment, a, atretic vitellogenic follicles; v, vitellogenic follicles. (8 x). 103 104 Figure 32. Representative micrographs of the periphery of the oocytes stained with hematoxylin-eosin, from (a) control group (vehicle injected) and (b) treatment group which received multiple injections (5 x 10 mg Fadrozole/kg) at the end of experiment. Granulosa cells (gr) have considerably increased in size and the zona radiata (zr) is thinner in the treatment group multiple injected, (th) theca cells (400 x). 105 106 4.4. Discussion Plasma 17p-estradiol, 17a-20P-P, and testosterone levels presented by the fish at the beginning of the experiment, as well as by the control group throughout the 32 day period, were similar with those observed in 1982 by Fitzpatrick et al. (1986) for vitellogenic female coho salmon. Similar plasma 17(5-estradiol levels have also been observed in vitellogenic rainbow trout (Zohar et al., 1986) and vitellogenic female amago salmon (Nagahama, 1987a). Comparable to this study, Young et al. (1983b) reported low plasma 17a-20P-P levels in female amago salmon during vitellogenesis. This study demonstrated that the aromatase inhibitor Fadrozole is capable of reducing 170-estradiol biosynthesis in female coho salmon during vitellogenesis and that it caused a premature increase in plasma 17a-20P-P levels. The data showed that the effects of Fadrozole were dose and time dependent, and the higher the dose used the longer and stronger was the reduction in plasma 17(3-estradiol levels and the greater the increase in plasma 17ct-20(J-P levels, respectively. The immediate results observed in this study after injection with Fadrozole during vitellogenesis are in accordance with those seen in females close to final maturation (previous Chapter). In contrast, the long term results were different between the two maturational stages. First of all this study showed clearly that the ability of Fadrozole to inhibit 17P-estradiol biosynthesis during vitellogenesis are both dose and time dependent. The data from the previous Chapter suggested this, but not as clearly as in this study. Comparing the treatment groups injected with 10 mg Fadrozole/kg, it can be seen that whereas during vitellogenesis plasma 17P-estradiol began to rebound after 96 h, in fish close to final maturation plasma 17P-estradiol remained low throughout the period The long term response, also showed that during vitellogenesis the premature increase in plasma 17a-20P-P levels observed at 6 h up to 96 h was transient, while in fish close to maturation plasma 17cc-20P-P levels increased further after 96 h, reaching approximately 80 ng/mL at 96 h, compared to 6.5 ng/mL at 96 h 107 in vitellogenic fish. The long term response in fish injected weekly with 10 mg Fadrozole during vitellogenesis demonstrated that 17P-estradiol biosynthesis was reduced throughout the experimental period, and plasma 17a-20p-P levels prematurely increased and remained constant from 24 h to 768 h after injection, but at a relatively low level. The recovery in 17P-estradiol concentration observed in vitellogenic fish injected with 1.0 or 10.0 mg Fadrozole/kg can be reasonably explained for two reasons. First, during vitellogenesis, process mediated by 17p-estradiol (Nagahama, 1987a) salmon ovarian follicles presented elevated aromatase activity. Young et al. (1983a) demonstrated in vitro the high ability of amago salmon ovarian follicles of secreting 17p-estradiol in response to exogenous testosterone during vitellogenesis, which decreased close to final maturation. Van Der Kraak et al. (1986) also observed a similar pattern for 17p-estradiol secretion in coho salmon ovarian follicles in vitro during vitellogenesis. Second, Fadrozole is a specific competitive reversible aromatase inhibitor (Steele et al., 1987) that can combine non-covalently with the enzyme at the same site as the substrate and can be readily removed by dialysis. Consequently, as soon as the catalytic site is free, the enzyme is ready to convert the substrate in product again. This experiment showed that the premature increase in plasma 17a-20p-P levels during vitellogenesis was transient (in the treatment groups that received one injection) and limited. This demonstrates that a permanent shift to 17cx-20p-P production did not occur as occurred in fish close to maturation. During vitellogenesis, plasma 17a-20P-P levels are low or undetectable, and increase enormously close to final maturation, coincidentally with an increase in plasma GTH II levels, which has been shown to stimulate the activity of the enzyme 20P-HSD, responsible for 17cx-20p-P biosynthesis (Kanamori et al., 1988 and Swanson, 1991). In vitro studies in this thesis (Chapter 6) have shown that during vitellogenesis in follicles with central germinal vesicle both GTH I and GTH II had 108 slight ability of stimulating 17a-20p-P secretion. Late in vitellogenesis, in follicles with peripheral germinal vesicle, however only GTH II increases its potency. Considering the fact that in salmonids during vitellogenesis GTH I is the primary gonadotropin synthesised by the pituitary (Suzuki et al, 1988d) and is the predominant gonadotropin in the plasma (Swanson, 1991; Prat et al., 1996), it is possible that the increase in plasma 17a-20p-P was due to stimulation by GTH I. The constant and slight biosynthesis of 17a-20P-P by the multiple injected group can also be explained by the ability of GTH I of stimulating 17a-20p-P secretion in vitro. Analysing the steroidogenic pathways involved in the production of 17P-estradiol and 17a-20P-P from 17a-hydroxyprogesterone (Figure 2), 17a-20p-P biosynthesis is a single step process, which is mediated by the enzyme 20p-HSD. On the other hand, for 17p-estradiol production from 17a-hydroxyprogesterone, 3 steps are needed: androstenedione formation (enzyme C17-20 lyase), testosterone formation (enzyme 17P-HSD), and 17p-estradiol formation (aromatase enzyme). Considering that inhibition of 17P-estradiol production increases 17a-20P-P biosynthesis and that increase in 17p-estradiol biosynthesis decreases 17a-20p-P biosynthesis, it is possible to speculate that one of the mechanisms which controls the hormonal shift is that 17p-estradiol through a paracrine action at the ovarian level may control the activity of the enzyme 20P-HSD. This speculation correlates well with the normal shift in the hormone profile and with the changes in enzyme activity observed during sexual maturation in salmonids. Young et al. (1983a) and Kanamori et al. (1988) observed a reduction in aromatase activity, and consequently a decrease in 17p-estradiol production, in amago salmon ovarian follicles prior to oocyte maturation. At the same stage there was an increase in the activity of the enzyme 20p-HSD (Kanamori et al., 1988), caused by GTH II (Suzuki et al., 1988c) which increased the follicular ability to secrete 17a-20p~P. Furthermore, 17a-20p-P secretion was inhibited in vitellogenic coho salmon ovarian follicles incubated in vitro in the presence of exogenous 109 17f3-estradiol (Van Der Kraak, 1984). The same effect was observed in intact rainbow trout ovarian follicles or in granulosa cells incubated in vitro in the presence of 17p-estradiol (Jalabert and Fostier, 1984 and Fostier and Baek, 1994). In this thesis, in vitro experiments carried out in vitellogenic coho salmon ovarian follicles, demonstrated that the presence of an aromatase inhibitor, which inhibits 17p-estradiol secretion, together with GTH II increased 17cc-20p-P secretion above levels when follicles were incubated only with GTH II. It is also important to consider as well, the possible effects of inhibiting ovarian 17p-estradiol biosynthesis on the brain, since close to final maturation GTH JJ is the predominant gonadotropin synthesised by the pituitary (Suzuki et al., 1988d) and is the predominant gonadotropin in the plasma (Swanson, 1991). The effects of inhibiting 17P-estradiol on the brain were discussed on the Chapter 3. Plasma testosterone levels in the treatment group injected with 10 mg Fadrozole/kg peaked at 96 h, the point of maximal 17p-estradiol inhibition, and after that declined, but the levels remained higher than at the beginning of the experiment. Since testosterone is the immediate precursor of 17P-estradiol, the decline may be explained by the recovery in 17p-estradiol biosynthesis. However, in the multiple injected group, plasma testosterone levels declined drastically between 384 h and 768 h to the level observed at the beginning of the experiment, even though plasma 17P-estradiol levels were almost undetectable. This suggests that an unknown mechanism could be down regulating testosterone biosynthesis at that time. A paracrine secretion controlling the activity of the enzymes involved in the pathways of testosterone synthesis at the ovarian levels as discussed above or the increase in 20p-hydroxysteroid dehydrogenase activity and decrease in 17P-hydroxysteroid dehydrogenase activity as discussed in Chapter 3 can be used to explain the reduction in plasma testosterone levels. The oocyte maturation index revealed that multiple injections of Fadrozole retarded the development of the oocytes. This was confirmed by comparing the egg diameter and gonadosomatic 110 index in the group examined at the beginning of the experiment with the multiple injected group after 32 days. These two groups presented similar egg diameters and gonadosomatic indices. Since vitellogenesis is mediated by 17P-estradiol, which stimulates the hepatic synthesis of vitellogenin, which in turn is going to be captured by the developing oocyte (Nagahama, 1987a and Tyler, 1991) through stimulation by GTH I (Tyler et al., 1991), it would be expected that a continuous inhibition of 17C3-estradiol, would cause a retardation in oocyte growth. The other treatment groups did not have the above parameters affected, probably due to the transitory effect of the aromatase inhibitor on 17(3-estradiol secretion as shown by the hormone profile. Thus at the highest dose (10 mg Fadrozole/kg) plasma 17p-estradiol was maximally reduced for 96 h and after that the levels rebounded. It is interesting to notice that even though the oocyte maturation index in the multiple injected treatment group was similar to the fish examined 32 days before, it was not different from the other groups at the end of the experiment, indicating that inhibition of 17P-estradiol production did not cause a complete retardation in the pattern of developmental stages. This suggests that other factors are involved in the regulation of oocyte development. Even though, the precise mechanism which controls oocyte growth is not completely understood, several hormones (such as gonadotropins, thyroid hormones, growth hormone, insulin, and insulin like growth factors) have been involved (Tyler and Sumpter, 1996). In coho salmon following the anadromous migration, superactive analogs of LHRH advanced oocyte development within 96 h (Van Der Kraak et al., 1983). Whereas most of the oocytes in the control group were in the central germinal vesicle stage, in the LHRH analog injected group most of the oocytes were in the germinal vesicle breakdown stage (Van Der Kraak, 1984). For all the in vitro experiments discussed in this thesis unlabeled substrates (testosterone and 17a-hydroxyprogesterone) and radioimmunoassay, instead of radiolabeled substrates were used as a method for steroid identification and quantification. Therefore, the activity of the enzymes involved in I l l the conversion of the above substrates to 17P-estradiol and 17a-20P-P, (aromatase and 20p-hydroxysteroid dehydrogenase) were estimated indirectly. This method was chosen in order to quantify the total output of the hormones to give a temporal perspective of the activity of the enzyme in relation to hormone production, and also to allow the comparison of the results in this thesis with other studies, since this methodology has been extensively used in in vitro experiments in fish to quantify steroid production (Young et al, 1983a; Young et al, 1986, Nagahama, et al., 1985; Sakai et al., 1988, and Suzuki et al., 1988c). One limitation of this method that could result in an underestimation of hormone production, is that it does not allow the identification of the major metabolites of 17p-estradiol and 17a-20p-P, which can be formed at different rates, in different stages of reproductive development. In the ovary of rainbow trout, however, 17P-estradiol and estrone were the only estrogens synthesised in significant amounts, and twice as much 17P-estradiol was produced relative to estrone (Bohemen and Lambert, 1981). Considering that has been shown that 17P-estradiol and 17a-20P-P are the main steroids secreted by the ovary of different salmonid fish, it is unlikely that the metabolites contribute significantly to the regulation of follicle development. The in vitro incubation of the follicles at the end of the experiment demonstrated that the multiple injected group presented a reduced ability to secrete 17P-estradiol in response to the stimulation by testosterone. This can be explained by the fact that those fish had received the last injection 4 days before, at a time when Fadrozole was still inhibiting aromatase activity, considering the reduced plasma 17P-estradiol of the weekly injected treatment group. The in vitro 17P-estradiol levels observed in the other treatment groups , when most of the oocytes where in the peripheral germinal vesicle stage were much higher than those observed by the in vitro experiments reported in Chapter 6 for follicles in the same developmental stage. This experiment was conducted in late September, with fish from a stock which returns earlier to the hatchery, but generally is the latest to spawn. The 112 experiments reported in the Chapter 6 which were conducted in November were carried out with fish which arrived in late October. These differences suggest that in the fish from this experiment the decrease in aromatase activity, which is characteristic in the peripheral germinal vesicle stage, had not been established yet. The plasma hormone profile at the end of the this experiment (768 h) demonstrated that 17p-estradiol was still at a high level and 17a-20P-P was at a low level. On the other hand, the plasma hormone levels of the fish used in the in vitro experiment in Chapter 4 demonstrated that the characteristic switch that occurs close to final maturation was taking place (plasma 17p-estradiol = 4.25 ng/mL and plasma 17a-20P-P = 29.90 ng/mL). This also demonstrates that fish from the same stock, but with a range of returning times can present distinct patterns of gonadal development. It is difficult to explain, however, why fish that arrive early at the hatchery spawn later than those which arrive after them. In vitro incubation of the brain showed that the only treatment group that secreted significant amounts of 17p-estradiol was the control group. This contrasts with the results from the in vitro incubation of the ovarian follicles, where, except for the multiple injected group, all treatment groups secreted significant amounts of 17p-estradiol. This suggests that the inhibition of the aromatase activity in the brain was stronger than in the ovary. The significance of this inhibition at this stage of the reproductive development is uncertain. Aromatase activity has been detected in the central nervous system of all major vertebrate groups from fish to mammals (Callard et al., 1978a). In the marine teleost Myoxocephalus octodecimspinosus (longhorn sculpin) activity is present in all major parts of the brain (Callard et al, 1978b), and exceeds that in gonads when compared on a unit basis. Formation of estrogens in the brain is dependent on exogenous aromatizable androgens (Lambert and van Bohemen, 1980), and the conversion of androgens to estrogens in the brain under in vitro conditions has also been determined in rainbow trout, Oncorhynchus mykiss (Lambert and Van Oordt, 1982; 113 Lambert et al., 1984) , in the goldfish, Carassius aiiratus (Pasmanik and Callard, 1985), African catfish, Clarias gariepitms (Timmers et al., 1987), and tilapia, Oreochromis mosambicus (Callard et al., 1988). Formation of estrogen from circulating aromatizable androgens in the brain is an important step in the regulation of several neuroendocrine and behavioural responses (Naftolin et al., 1975, McEwen et al., 1979,1982; and Arnold and Schlinger,1993). In teleosts it is well recognised that gonadal steroids mediates gametogenesis (Nagahama, 1987a), however it not known if, for example estrogen formed in the brain reaches peripheral tissue. Timmers (1988) provided evidence that estrogens formed in the African catfish brain entered the general circulation. On the other hand Callard et al (1981) demonstrated that high levels of estrogen synthesis and retention in the brain correlated neuroanatomically with the distribution of estrogen-binding cells in reproductive control centers in the teleost brain (preoptic area and telencephalon). High incidence of follicular atresia was observed mainly in the ovary of the treatment group injected weekly with 10 mg Fadrozole/kg. It is likely that the atretic process is related to the maintenance of low plasma 17fi-estradiol levels in this group. In female red sea bream, Pagrus major (Matsuyama et al., 1988), and redgurnard, Chelidonichthys kumu (Clearwater and Pahkhurst, 1997) ovarian atresia was related to the low plasma 17P-estradiol levels. It is known that during vitellogenesis 17j3-estradiol induces the hepatic synthesis of vitellogenin which is the main precursor of yolk proteins incorporated into the oocytes (Ng and Idler, 1983, Tyler, 1991), and the synthesis of vitelline envelope proteins (Hyllner and Haux, 1995; Oppen-Berntsen, et al., 1992). Therefore, a decrease in 17(3-estradiol biosynthesis may affect the above cited process. In mammals it has been suggested that change in follicular steroid levels are involved in the initiation of atresia (Hsueh et al., 1994). An increase in androgen to estrogen ratio has been observed in the follicular fluid of atretic follicles 114 (Carson et al., 1981). Also, non aromatizable androgens, for example 5a-dihydrotestosterone, induced follicular atresia in rats (Louvet et al., 1975). From a practical point of view, the preliminary results that Fadrozole retarded gonadal development are important. Technologies to control reproduction in fish have constantly challenged the aquaculture industry (Donaldson, 1996). In salmonids particularly, morphological and physiological changes occur during sexual maturation, which include: darkening of the skin, sexual dimorphism, increased susceptibility to diseases, change in the proximate composition organoleptic characteristics of the muscle and growth cessation, which are detrimental to product quality. Therefore, the development of techniques which could prevent or retard those changes, under culture conditions, are important. This present study, however, was conducted over a period of one month and were not evaluated morphological changes. It would be necessary to conduct a longer term study probably starting in a previtellogenic stage and extending to late vitellogenesis to further evaluate the effects of Fadrozole on ovarian development and secondary sexual characteristics. In summary this study demonstrated that Fadrozole reduced plasma 17P-estradiol levels during vitellogenesis in a dose and time dependent mariner, and increased plasma 17a-20p levels prematurely also in a dose and time dependent manner. The switch from 17(3-estradiol to 17a-20P-P biosynthesis was transient in the groups that received only one injection. The study also showed that multiple injections with Fadrozole arrested ovarian development. The 32 day study period demonstrated that aromatase inhibitors such as Fadrozole may have potential as a tool to regulate sexual development in salmon. However, a longer term study would be necessary to further extend and support the present findings. 115 C H A P T E R 5 - Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion in male coho salmon during sexual maturation. 5.1. Introduction In male salmonids the major two androgens and progestogen produced by the testis are testosterone, 11-ketotestosterone and 17a-20P-P, respectively (Fostier et al., 1983, Nagahama, 1987a). These androgens are believed to be involved in spermatogenesis, since plasma testosterone and 11-ketotestosterone increase during this period (Scott and Sumpter, 1989). In teleost testes the interstitial cells (Leydig cells) are the main source of androgens (Loir, 1988;1990a,b). On the other hand, 17a20P-P has been shown to be responsible for final sperm maturation (Miura et al., 1992). Plasma 17a-20p-P increases concomitantly with the increase in plasma G T H II (Swanson, 1991). Several researchers have demonstrated that sperm cells are responsible for 17a-20p-P synthesis (Ueda et al., 1984; Sakai et al., 1990, and Barry et al., 1990). More recently, however, it has been demonstrated that non flagellated germ cells from rainbow trout immature testes were able to synthesize 17a-20P-P, and 20P-hydroxysteroid dehydrogenase activity (enzyme which converts 17a-hydroxyprogesterone to 17a-20P-P) has been detected in these cells (Vizziano et al., 1995, 1996). Most of the in vitro studies which have investigated the steroidogenic ability of the testis, however, have demonstrated that it is incapable of secreting estrogens, and therefore lacks aromatase activity (rainbow trout: Depeche and Sire, 1982; sea bass: Colombo et al., 1978; longhorn sculpin, Myoxocephalus octadecimspinosus, and the elasmobranch fish winter skates, Raja ocellata: Callard et al., 1978b). On the other hand, estrogens can be formed in the brain, and aromatase activity has been detected in the fish brain independent of the sex (female rainbow trout: Lambert and van Bohemen, 116 1980 and Lambert and van Oordt, 1982; male Atlantic salmon, Salmo salar, Andersson et al., 1988, Mayer et al., 1991; male and female of three-spined stickleback, Gasterosteus aculeatus: Borg et al., 1987a,b; male African catfish, Clarias gariepinus: Timmers and Lambert, 1987, and Timmers et al., 1987; male and female goldfish: Pasmanik and Callard, 1985). Indeed, aromatization of androgens in the central nervous system has been demonstrated in representatives of all classes of vertebrates (Callard et al., 1978a, and Callard, 1983). Since it has been shown that the fish testis has minimal ability to synthesise estrogens (see text above), the presence of aromatase activity in the brain suggests that the formation of estrogens in the brain is responsible for the low levels of plasma estrogen (Fostier et al., 1983). Additional evidence that in males the brain is the major site of estrogens and that they are released into the general circulation has been provided in African catfish (Timmers, 1988), and zebra finch (Schlinger and Arnold, 1991; Arnold and Schlinger, 1993; and Schlinger and Arnold, 1993). Since there is good evidence that the formation of estrogens from circulating androgens in the brain is important for certain neuroendocrine and behavioural responses (Naftolin, et al., 1975; McEwen et al., 1979; Arnold and Schlinger, 1993, Zumpe et al., 1993, and Bonsall et al., 1992), it is possible that inhibition of estrogen formation in the brain may affect the normal neuroendocrine secretion during reproductive development in fish. As described in Chapter 3 there were some males among the females in all experimental groups, that could not be recognized, and therefore were injected with Fadrozole. Observations suggested that the males treated with the highest dose of Fadrozole started to spermiate earlier (data not shown due to a small number offish). Associating the importance of estrogen in males, as discussed above, with the observations that the males treated with the highest dose of Fadrozole started to spermiate earlier, the purpose of the present study was to determine the effects of inhibiting estrogen biosynthesis on plasma sex steroid secretion and on reproductive development. 117 5.2. Materials and Methods One hundred adult coho salmon were obtained in mid-August 1996 from the Capilano Salmon Hatchery, Vancouver, B.C. (Department of Fisheries and Oceans) transported to the West Vancouver Laboratory (Department of Fisheries and Oceans). Each fish was identified individually with a PIT tag inserted in the ventral muscle. Since it was impossible to visually sex the fish by examining external morphology, a sample of blood was collected from each fish and plasma 17fi-estradiol level was determined. Fish in which plasma 17fJ-estradiol level was undetectable were considered male. This technique proved to be highly reliable. At the time of capture the weights and the lengths of the male fish were 0.8 ± 0.2 kg and 42.3 ± 4.0 cm (mean ± SE), respectively. Fish were divided in four groups each containing 8 fish: control vehicle injected, group treated with 1.0 mg Fadrozole/kg, group treated with 10.0 mg Fadrozole/kg, group treated weekly with 10.0 mg Fadrozole/kg (5 x 10 mg/kg). The experimental protocol consisted of determining the plasma sex steroid levels before injection with Fadrozole and during thirty two days after injection. Blood samples were collected at 0 h (just before injection), 6 h, 24 h, 48 h, 96 h, and 192 h, 384 h, and 768 h after injection. Plasma 17p-estradiol, testosterone, 11-ketotestosterone, and 17a-20P-dihydroxy-4-pregnen-3-one (17a,20p-P) levels were measured by radioimmunoassay. After the last blood collection fish were killed by decapitation, and GSI was determined. Maturity was checked periodically through gentle abdominal massage and when sperm was released, it was collected into a sterile plastic bag and the motility verified. Ten untreated fish were killed at 0 h, from which GSI was determined to compare with the fish after 32 days. After completion of the in vivo study, an in vitro experiment to determine cerebral aromatase activity 118 (indirectly through conversion of testosterone to 17(3-estradiol) was carried out using a portion of the brain, as described in the Chapter 2 (General Materials and Methods). 5.3. Results 17p-Estradiol in the male plasma was below the detection limit (niinimum detectable amount by the assay = 0.1 ng/mL) at any time. However, it was possible to detect 17P-estradiol in the brain at the end of the experiment. The results showed that only brain tissue from the vehicle injected group was able to secrete significant (p<0.05) amounts of 17p-estradiol into the incubation medium (Figure 33). Plasma 17a-20P-P levels in the vehicle injected group remained low and constant from 0 h to 192 h (Figure 34). At 384 h and 768 h there was a significant (p<0.05) increase in plasma 17a-20P-P levels. Plasma 11-ketotestosterone levels also remained constant from 0 to 192 h, and after that the levels increased significantly (p<0.05) up to the end of the experiment in relation to levels observed at 0 h. Plasma testosterone levels remained almost unchanged during the 32 day period. It declined significantly (p<0.05) at 96 h after injection in relation to levels at 0 h, but at 192 h there was a recover, and the concentration then remained constant throughout the rest of the experimental period. In the group treated with 1.0 mg of Fadrozole/kg plasma 17a-20p-P levels were variable during the experimental period showing both significant increases and decreases (Figure 35). Plasma 17a-20p-P increased slightly but significantly (p<0.05) at 6 h after injection, but after that and up to 192 h the levels were variable. A more consistent trend was observed from 192 h to 768 h when plasma 17a20P-P increased significantly (p<0.05). Plasma 11-ketotestosterone and testosterone levels were also varied during the period and the levels of both hormones showed a significant (p<0.05) decline at 24 h after injection. At 48 h both hormones recovered in relation to 24 h after injection. After 48 h, whereas plasma 11-ketotestosterone tended to increase, plasma testosterone levels decreased significantly (p<0.05), and at 384 h have reached the levels observed at 24 h after injection. 119 Figure 33. //; vitro 17p-estradiol production by the brains of male coho salmon at the end of the experiment (768 h). Each bar represents the mean ± standard error from males in each treatment group. Brains were incubated with incubation medium in the presence of testosterone (100 ng/mL) by 18 h at 12 °C. 17P-Estradiol released to the media which are similar (p>0.05) as determined by Dunn's Method, are identified by the same superscript letter. Number between brackets above the superscript letter means number of fish in each group. 120 "9) Q . g T3 OJ 1_ -•—* 00 UJ I c a f--0 1 10 Fadrozole (mg/kg) 5 x 10 121 Figure 34. Mean plasma concentration of 17a-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), 11-ketotestosterone and testosterone in male coho salmon from the control group. Each value represents the mean ± standard error of measurements from 6 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scales for each hormone, as well as different scales for the same hormone among the treatment groups. 122 18 0 6 24 48 96 192 384 768 0 6 24 48 96 192 384 768 Hours after injection 123 Figure 35. Mean plasma concentration of 17oc-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), 11-ketotestosterone and testosterone in male coho salmon injected with 1.0 mg of Fadrozole/ kg body weight. Each value represents the mean ± standard error of measurements from 8 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scales for each hormone, as well as different scales for the same hormone among the treatment groups. 124 O) c c a o C M i a i 1 T 1 1 1 1 r 0 6 24 48 96 192 384 768 i r 24 48 96 192 384 768 i 1 1 1 1 r 0 6 24 48 96 192 384 768 Hours after injection 125 In the group treated with 10.0 mg of Fadrozole/kg plasma 17cc-20P-P increased significantly (p<0.05) 6 h after injection, and decreased at 24 h (Figure 36). After that the plasma 17a-20(5-P levels remained almost constant throughout the period. Plasma 11-ketotestosterone and testosterone levels showed a similar trend throughout the period to that observed in the treatment group injected with 1.0 mg Fadrozole/kg. The profiles for both hormones presented significant (p<0.05) declines at 24 h, and rebounded after 48 h. After 96 h when both hormones peaked, 11-ketotestosterone tended to remain constant up to the end, whereas plasma testosterone decreased significantly (p<0.05). The treatment group injected weekly with 10.0 mg of Fadrozole/kg showed similar hormone profiles to the group which received a single injection of 10.0 mg of Fadrozole/kg from 0 h up to 96 h (Figure 37). Plasma 17a-20f3-P levels increased significantly (p<0.05) at 6 h after injection and declined significantly (p<0.05) at 24 h remaining steady up to 96 h. At 192 h, i.e. one day after the second injection, however, plasma 17a-20P-P levels slightly but significantly (p<0.05) increased in relation to the levels at 0 h, increased significantly (p<0.05) one day after the third injection (384 h) and remained high up to 768 h. Plasma 11-ketotestosterone and testosterone levels increased significantly (p<0.05) 96 h after injection. After that the data indicated a high variation among the individuals in terms of plasma 11-ketotestosterone levels. Plasma testosterone levels also were highly variable among the individuals, but a more steady profile was demonstrated throughout the experimental period. Comparison among the groups demonstrated that at 6 h after injection the groups injected with 10.0 mg Fadrozole/kg presented significantly (p<0.05) higher plasma 17a-20|3-P levels than the vehicle injected group and the group injected with 1.0 mg Fadrozole/kg (Figure 38). At 192 h and 384 h after injection only the group injected weekly with 10.0 mg Fadrozole/kg showed significantly (p<0.05) higher plasma 17a-203-P levels when compared to the other groups. In all the treatment 126 Figure 36. Mean plasma concentration of 17a-20fJ-dihydroxy-4-pregnen-3-one (17a-20|3-P), 11-ketotestosterone and testosterone in male coho salmon injected with 10.0 mg of Fadrozole/ kg body weight. Each value represents the mean ± standard error of measurements from 8 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 127 128 Figure 37. Mean plasma concentration of 17a-20p-dihydroxy-4-pregnen-3-one (17a-20P-P), 11-ketotestosterone and testosterone in male coho salmon injected weekly with 10.0 mg of Fadrozole/ kg body weight (5x10 mg/kg). Each value represents the mean ± standard error of measurements from 5 fish. At each sampling period, plasma hormone concentrations which are similar (p>0.05) as determined by Student-Newman-Keuls test are identified by the same superscript letter. Arrows from the left to the right indicate the first, second, third, fourth, and fifth injection, respectively. Note that there are different scale for each hormone, as well as different scales for the same hormone among the treatment groups. 129 40 0 6 24 48 96 192 384 768 100 0 6 24 48 96 192 384 768 Hours after injection 130 Figure 38. Mean plasma concentration of 17ct-20P-dihydroxy-4-pregnen-3-one (17a-20P-P) in male coho salmon at 6 h, and 192 h, and 384 h after injection. Each bar represents the mean ± standard error from males in each treatment group. Plasma 17a-20P-dihydroxy-4-pregnen-3-one concentrations which are similar (p>0.05) among the groups at each sampling period, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 131 24 22 -20 -18 16 14 -12 -10 -8 6 H 4 2 H 0 Control Fadrozole 1.0 mg/kg Fadrozole 10.0 mg/kg Fadrozole 5 x 10.0 mg/kg :rjil 192 384 Hours after injection 132 groups injected with Fadrozole, plasma 11-ketotestoterone and plasma testosterone levels increased significantly (p<0.05) at 96 h after injection (Figure 39). Fish which received 10.0 mg Fadrozole weekly started to spermiate before the vehicle injected group (p<0.08) and before the group which received 1.0 mg of Fadrozole/kg (p<0.032) (Figure 40). Comparison between the treated groups and the fish which were examined and killed at the beginning of the experiment showed in the vehicle injected group and the group injected with 1.0 mg of Fadrozole/kg significant spermiation (p<0.015 and p<0.0035, respectively) at 32 days. On the other hand in the other two groups injected with 10 mg/kg Fadrozole and 10 mg Fadrozole/kg weekly a significant increase in spermiation (p=0.077 and p=0.007) occurred at 16 days. Examination of sperm motility revealed that there was no difference among the groups (p>0.05) and the mean sperm motility was 89%. The GSI was not different among the groups (p>0.05). 133 Figure 39. Mean plasma concentration of 11-ketotestosterone and testosterone in male coho salmon at 96h after injection. Each bar represents the mean ± standard error from males in each treatment group. Plasma 11-ketotestosterone or testosterone concentrations which are similar (p>0.05) among the groups at 96 h, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. 134 I I Control S I Fadrozole 10.0 mg/kg 11 -Ketotestosterone Testosterone 96 hours after injection 135 Figure 40. Effects of Fadrozole on the cumulative spermiation in male coho salmon Values represent the cumulative percentage of the number of fish which spermiated in each group. The proportion of observations at each checking day which are significantly different (p<0.05) among the treatment groups, as determined by Fisher Exact Test, are identified by different superscript letter. The asterisk symbol (*) means that the proportion of observation at each checking day is significantly different (p<0 05), as determined by Fisher Exact Test, from the control group examined and killed on day 0. 136 100 0 8 16 Days after injection 32 137 5.4. D iscuss ion It was not possible to detect 17p-estradiol in any of the male coho salmon plasma samples. Usually, in male salmonid and other species of fish the quantification of plasma 17p-estradiol levels by radioimmunoassay reveals low levels of 17P-estradiol (rainbow trout: Schreck et al., 1973 and Billard et al., 1978; Atlantic salmon: Idler et al., 1981, Mayer et al., 1990; coho salmon: Sower and Schreck, 1982, Patino and Schreck, 1986, Fitzpatrick et al., 1986; sockeye salmon: Truscott et al., 1986; amago salmon: Nagahama et al., 1982; goldfish, Carassius auratus: Schreck and Hopwood, 1974). Sower and Schreck (1982) and Fitzpatrick et al. (1986) who worked with the same species in a similar developmental stage quantified plasma 17p-estradiol levels, using similar radioimmunoassay, which ranged from 0.1 to 0.6 ng/mL. This demonstrates that if 17p-estradiol was present in the plasma of the fish used in this experiment it would be in amounts lower than the detection limit. Although several tissues in the body of the fish are able to produce steroid hormones and may contribute to the overall production of sex steroid hormones, the gonads are the main producer of sex steroid hormones (Fostier et al., 1983). Most of the in vitro studies which investigated the steroidogenic ability of the testis, however, have demonstrated it is incapable of secreting estrogens, and lacks aromatase activity (rainbow trout: Depeche and Sire, 1982; sea bass: Colombo et al., 1978; longhorn sculpin, Myoxocephalus octadecimspinosus, and the elasmobranch winter skate, Raja ocellata: Callard et al., 1978b). Estrogens were produced, however, in small amounts by the testis of another elasmobranch fish, spiny dogfish, Squalus acanthias (Callard et al., 1978b). On the other hand, estrogens can be formed in the brain, and aromatase activity has been detected in the fish brain independent of the sex (female rainbow trout: Lambert and van Bohemen, 1980 and Lambert and van Oordt, 1982; male Atlantic salmon: Andersson et al., 1988, Mayer et al., 1991; male and female three-spined stickleback, Gasterosteus aculeatus: Borg et al., 1987a,b; male 138 African catfish, Clarias gariepinus Timmers and Lambert, 1987, and Timmers et al., 1987, male and female goldfish: Pasmanik and Callard, 1985). Indeed, aromatization of androgens in the central nervous system has been demonstrated in representatives of all classes of vertebrates (Callard et al., 1978a, and Callard, 1983) Since it has been shown that the fish testes have rninimal ability to synthesise estrogens (see text above), the presence of aromatse activity in the brain suggests that the formation of estrogens in the brain is responsible for the presence of low levels of estrogens in the plasma of some species (Fostier et al., 1983). Additional evidence that in males the brain is the major site of estrogen biosynthesis and that estrogen is released into the general circulation has been provided in the African catfish (Timmers, 1988), and the zebra finch (Schlinger and Arnold, 1991; Arnold and Schlinger, 1993; and Schlinger and Arnold, 1993). This study demonstrated, by measuring the in vitro secretion of 17P-estradiol from slices of brain incubated with the aromatizable androgen testosterone, the presence of aromatase activity in the brain of male coho salmon. At the end of the experimental period, secretion of 17P-estradiol was only detected in the brains of the control fish, indicating that Fadrozole was capable of inhibiting aromatase activity at the brain level. Fadrozole also inhibited aromatase activity in the brain of male Atlantic salmon male parr (Antonopoulou et al., 1995), in zebra finch (Wade et al., 1994) in monkeys species (Zumpe et al., 1993), and in the rat (Bonsall et al., 1992). In these experiments aromatase activity was measured directly through radiometric assays, which quantify the aromatase activity using a labelled substrate for aromatase (androstenedione or testosterone), and the tritiated hydrogen is released during the conversion of the androgen into estrogens and quantified in the water or as part of the estrogen molecule. Other aromatase inhibitors, for example ATD (l,4,6-androstatriene-3,17-dione) have also been shown to inhibit brain aromatase activity in male fish (African catfish: Vermeulen, 1994; Atlantic salmon parr: Antonopoulou et al., 1995). On the other hand, there is evidence that ATD 139 inhibits other steroid enzymes in quail species (Alexandre and Balthazart, 1987) and inhibits androgen receptor binding (Kaplan and McGinnis, 1989). Since there is good evidence that the formation of estrogens from circulating androgens in the brain is important for certain neuroendocrine and behavioural responses (Naftolin, et al., 1975; McEwen et al., 1979; Arnold and Schlinger, 1993, Zumpe et al., 1993, and Bonsall et al., 1992), it is possible that inhibition of estrogen formation in the brain would affect the normal neuroendocrine secretion during reproductive development in fish. The present results indicated that inhibition of 17(3-estradiol production by the aromatase inhibitor Fadrozole caused a premature increase in plasma 17a-20(J-P levels in adult male coho salmon. Several studies have shown that in male fish, plasma 17a-20(3-P levels remain low during the period of testicular development (spermatogenesis) and then increase dramatically coincidentally with spermiation (rainbow trout: Scott and Baynes, 1982, Ueda et al., 1983, and Scott and Sumpter, 1989; amago salmon: Ueda et al., 1983, 1984; coho salmon: Fitzpatrick et al., 1986, and Planas and Swanson, 1995 ; sockeye salmon: Truscott et al, 1986; Atlantic salmon: Mayer et al., 1990). Miura et al. (1992) demonstrated that 17a-20f}-P is responsible for promoting sperm motility in masu salmon by increasing the pH of the sperm duct, which in turn elevates cAMP levels in the sperm, allowing the acquisition of sperm motility. This contrast with the situation in mammals, where the hormonal control of spermatogenesis involves mainly testosterone production (Schulster et al., 1976). The present study also showed that the treatment group injected weekly with 10 mg Fadrozole/kg started to spermiate earlier than the control group and at 384 h 80% (4 of 5 fish) of the fish were spermiating in contrast with 17 % (1 of 6 fish) in the control group and 12.5 % (1 of 8 fish) in the group injected with 1.0 mg Fadrozole/kg. It is interesting to notice that at this point the fish had received the third injection of Fadrozole and the plasma 17a-20P-P levels peaked again, increasing from approximately 4 ng/mL at 192 h to 18 ng/mL at 384 h and 768 h. The other groups, however 140 had plasma 17a-20(3-P levels, ranging from 1.5 ng/mL to 3.0 ng/mL. The plasma 17a-20P-P levels observed at the period of higher spermiation rate correlates well with those for spermiating coho salmon observed by Fitzpatrick et al. (1986) and Planas and Swanson (1995). Even though it was not possible to detect 17p-estradiol in the male plasma, which would allow comparison with the situation observed in females, where plasma 17P-estradiol significantly declined after injection with Fadrozole, the results in terms of the ability of the brain to secrete 17P-estradiol after stimulation with the aromatizable androgen testosterone was the same, where only the control group was able to secrete a measurable quantity of estrogen. Furthermore the premature increase in plasma 17a-20P-P levels observed in females during vitellogenesis or close to final maturation was also observed in males at a similar magnitude, but was different in terms of duration, whereas in males there was a significant drop at 24 h, in female plasma 17cc-20P-P levels remained elevated during 96 h. It is also interesting to notice that as the females injected with Fadrozole (10 mg/kg) close to final maturation, which presented decreased plasma 17p-estradiol concentration and increased plasma 17a-20P-P levels, started to ovulate earlier, the males injected weekly (10 mg/kg) started to spermiate before. Therefore this study suggests that in males the steps involved in sexual maturation which leads to spermiation include decrease in estrogen production, probably at the brain level. The necessity of reducing estrogen synthesis to lead to spermiation can be explained by the fact estrogens that have been formed are hydroxylated to catecholestrogens, which together with dopamine are both methylated by the enzyme catechol-O-transferase (COMT) forming inactive products (Timmers, et al., 1988 and de Leeuw, et al., 1985). Since cathecolestrogens have a higher affinity with COMT, less methylation of dopamine occurs, therefore increasing its concentration, which may inhibit the GnRH-induced GTH release. Therefore a reduction in estrogen biosynthesis would reduce the competition between catecholestrogens and dopamine for COMT, leaving dopamine be metabolized, therefore abolishing its 141 inhibitory effect over GTH II secretion, which in turn would stimulate 17a-20f}-P biosynthesis at the testes level, which in turn has been shown to cause sperm maturation (Miura, 1992). Ueda et al. (1985) demonstrated that a partially purified salmon gonadotropin preparation (SGA) stimulated spermiation in vivo in amago salmon, probably through the production of 17a-20f5-P. In vitro studies in salmonids have also demonstrated that the ability of the testes to produce 17a-20P-P in response to SGA or pituitary extract, changes during developmental stages, increasing during spermiation (amago salmon: Sakay et al, 1989, and rainbow trout: Schulz and Bliim (1990). More recently, Planas and Swanson (1995) using the purified coho salmon gonadotropins I and II have demonstrated in male coho salmon that the sensitivity of the testicular tissue to the gonadotropins changes during the course of sexual development, and as maturity progressed to spermiation GTH II increased its potency in stimulating 17a-20P-P, whereas the potency of GTH I decreased. This correlates well with the in vivo observations of Swanson (1991) that plasma GTH II increases close to spermiation, whereas GTH I decreased, and also with the appearance of specific GTH II receptors in the Leydig cells (Miwa et al., 1994). The same profiles in vitro and in vivo for the gonadotropins I and II have also been observed in female salmonids (see discussion in Chapter 3 and 4 in this thesis). From these studies, it can be postulated that the changes observed in this experiment in terms of 17a-20f}-P could be related to changes in gonadotropic hormones, mostly an increase in plasma GTH II levels. Linard et al.(1995a,b) obtained evidence that the catecholamine dopamine inhibited GTH II secretion in trout when plasma 17P-estradiol levels are elevated, suggesting an interaction between 17P-estradiol and the enzyme tyrosine hydroxylase, which is the limiting enzyme of catecholamine synthesis. The same authors demonstrated that plasma GTH II levels increased significantly in sexually mature female rainbow trout treated with a-methyl-p-tyrosine (MPT), which is an inhibitor of tyrosine hydroxylase, and they suggested that the increase in plasma GTH 142 II levels observed close to final maturation, could be due to a decrease in plasma 17P-estradiol, which in turn reduced the inhibitory effect of dopamine on GTH II secretion . The inhibitory effect of dopamine on gonadotropin release has also been described in goldfish ( Peter et al., 1991) and African catfish (de Leeuw et al., 1986a). Other studies that investigated the negative feedback effect of gonadal sex steroids on gonadotropin secretion, demonstrated that the administration of aromatizable androgens after castration of male African catfish, suppressed the elevated surge of gonadotropin (de Leeuw et al., 1986b). On the other hand non aromatizable androgen had no effect. A similar observation was made by Billard (1978) in castrated rainbow trout. Further evidence that conversion of aromatizable androgens to estrogens are important to the regulation of the negative feedback, is that antiestrogens, such as tamoxifen and clomiphene citrate increase the gonadotropin surge in several species of fish (Peter, 1983). Treatment with tamoxifen accelerated the time of ovulation in female coho salmon primed with a previous injection of partially purified salmon gonadotropin (SG-G100) (Donaldson et al., 1981/1982). In African catfish it has been suggested that catecholestrogens are the mediator of the estrogen in the brain. This can be explained by the fact that after estrogens have been formed they are hydroxylated to catecholestrogens, which together with dopamine are both methylated by the enzyme catechol-O-transferase (COMT) forming inactive products (Timmers, et al., 1988 and de Leeuw, et al., 1985). Since cathecolestrogens have a higher affinity for COMT, less methylation of dopamine occurs, therefore increasing its concentration, which may inhibit the GnRH-induced GTH release. It may be possible that inhibition of estradiol, and consequently decrease in the inhibitory effect of dopamine on gonadotropin secretion, which has been suggested to explain the hormonal changes close to maturation in females after treatment with Fadrozole (see Chapter 3), is occurring as well in males, which could explain the premature surge of 17oc-20P-P and spermiation in males injected weekly with 10 mg Fadrozole/kg. 143 All groups treated with Fadrozole presented significantly higher levels of testosterone and 11-ketotestosterone than the control group at 96 h after injection. In other salmonid fish a decrease in plasma testosterone and 11-ketotestosterone concomitantly with an increase in plasma 17a-20P-P occurs during the onset of spermiation (Scott and Baynes, 1982; Fitzpatrick et al., 1986; and Scott and Sumpter, 1989, Planas and Swanson, 1995). It has been suggested that a shift occurs from androgen to progestogen production in the testis prior to spermiation (Scott and Baynes, 1982, Sakai et al., 1989, and Barry et al., 1990). The shift in steroidogenic pathways was not evident in this experiment. Even though Planas and Swanson (1995) observed in vivo the changes in hormone profiles, their in vitro studies did not support the proposed idea of a clear shift in steroids biosynthesis close to spermiation, mostly because there was no decrease in androgens after basal or GTH II-stimulated production of 17a-20P-P. Similar to the observations in this study was the findings of Planas and Swanson (1995) that plasma testosterone and 11-ketotestosterone levels remained elevated for approximately four weeks after the plasma levels of 17a-20P-P started to increase. Although in this study plasma gonadotropin levels were not measured, it is possible that the increase in plasma androgens levels is related to an increase in plasma GTH II levels, which has been shown to be more potent in stimulating androgen production in vitro at the time of spermiation (Planas and Swanson, 1995). In summary this study demonstrated that Fadrozole inhibited 17P-estradiol secretion in the brain of male salmon. Injection with Fadrozole in males caused a premature and transient increase in plasma 17a-20P-P levels. Multiple injections caused further increases in plasma 17a-20P-P, and 16 days after the beginning of the experiment the treatment groups which received the highest doses of Fadrozole (10 mg/kg and 5 x 10 mg/kg) started to spermiate, suggesting that the administration of 144 aromatase inhibitors such as Fadrozole may provide a means of accelerating spermiation in salmonids and potentially other species of fish. 145 C H A P T E R 6 - Effects of Fadrozole, G T H I, G T H II, testosterone and 17a-hydroxyprogesterone on in vitro steroid secretion by coho salmon ovarian follicles. 6.1. Introduction It has been shown that gonadototropins the GTH I (FSH) and GTH II (LH) stimulate the biosynthesis of the steroid hormones by the salmonid ovary which are essential to the oogenesis process (Suzuki et al., 1988c; Swanson et al., 1989, and Sumpter et al., 1991; Planas and Swanson, 1995). A two-cell type model has been described for the production of 17P-estradiol and 17a-20P-P in the ovarian follicles of salmonids (Nagahama, 1987a; Kagawa, 1994). In this model, during the vitellogenic stage, the thecal layer under influence of GTH (GTH I), secretes the aromatizable androgen testosterone, which is converted to 17P-estradiol by the aromatase enzyme in the granulosa cells. During the oocyte maturation stage the thecal layer produces 17a-hydroxyprogesterone which is converted to 17a-20P-P by the enzyme 20P-hydroxysteroid dehydrogenase (20P-HSD) in the granulosa cells, under the stimulation of GTH II. It is not known, however, whether a mechanism occurring at the ovarian level could by itself trigger changes in steroidogenic pathways. It was therefore important to obtain a basic understanding of the mechanisms by conducting in vitro experiments to test the hypothesis that inhibition of 17P-estradiol production occurs at the ovarian follicle level. It is also important to know whether inhibition of 17P-estradiol production may trigger the activity of the enzyme 20P-HSD, which is responsible for the synthesis of the maturation inducing hormone 17a,20pP without the influence of GTH II. The alternative hypothesis is that the reduction in 17P-estradiol biosynthesis by the aromatase inhibitor may reduce the feedback inhibition of 17P-estradiol on hypothalamic secretion of GnRH and consequently 146 pituitary secretion of GTH II. Thus increasing plasma levels of possibly GTH II and stimulating 17ot,20pP secretion. To address these hypotheses a series of in vitro experiments were carried out using coho salmon as a model. 6.2. M a t e r i a l s a n d M e t h o d s The methodology was similar for the three in vitro experiments. The procedures are described in detail in the Chapter 2 General Materials and Methods. In experiment 1, one maturing female coho salmon, Oncorhynchus kisutch, was obtained from Inch Creek Hatchery, Fraser Valley, B.C. (Department of Fisheries and Oceans). Groups of 4 follicles per well were incubated in 1 mL medium with or without the addition of 10 or 100 uM of the aromatase inhibitor Fadrozole and or 0.10, 0.20, 0.40 uM of testosterone for 18 h at 12 °C. For Experiments 2 and 3, fish were obtained from Capilano Salmon Hatchery, Vancouver, B.C. (Department of Fisheries and Oceans) at different times as described in Table 4. One female was used at each of the three developmental stage and from each female sufficient ovarian follicles were dissected to carry out Experiments 2 and 3 at the same time. In Experiment 2, groups of 4 follicles per well were incubated in 1 mL medium with or without addition of 10 uM of Fadrozole and 100 ng of GTH I or GTH II per mL, and a combination of GTHs with Fadrozole. In Experiment 3, groups of 4 follicles per well were incubated in 1 mL medium in the presence of 100 ng of GTH I, GTH II, or the steroid precursors testosterone (17p-estradiol precursor), and 17cc-hydroxyprogesterone (17a-20p-P precursor)/mL, or a combination of them. Six replicate incubations were made for each treatment and for the control in Experiments 1, 2 and 3. 6.3. Results 147 6.3.1. Experiment 1. The effect of the aromatase inhibitor Fadrozole on 17p-estradiol secretion by ovarian follicles of coho salmon. Follicles incubated with Fadrozole secreted significantly less 17P-estradiol (p<0.05) than the control follicles, and with the two concentrations tested (10 uM and 100 uM) there was no dose-dependent effect (Figure 41). The addition of exogenous testosterone to the medium in the absence of Fadrozole significantly increased 17P-estradiol secretion (p<0.05) in a concentration-dependent manner (Figure 42). All treatments in which testosterone was combined with Fadrozole resulted in an almost complete inhibition of 17P-estradiol secretion (Figure 43). 6.3.2. Experiment 2. The effects of the aromatase inhibitor Fadrozole, G T H I and G T H II on sex steroid secretion by ovarian follicles of coho salmon in vitro throughout the periovulatory period. The plasma sex steroid hormone levels and the oocyte developmental stages of the fish used in the next two in vitro experiments can be seen in Table 4. Fadrozole was able to significantly inhibit 17P-estradiol secretion (p<0.05) below control levels throughout the periovulatory period (Figure 44). GTH I and GTH II stimulated 17p-estradiol secretion significantly (p<0.05) above basal levels in a similar manner in follicles in the immature stage and in follicles with a central germinal vesicle. In follicles in the peripheral germinal vesicle stage, when basal 17P-estradiol secretion decreased (graph unit picogram/mL), GTH I and GTH II inhibited 17P-estradiol secretion significantly (p<0.05). Throughout the periovulatory period, the combination of 148 Figure 41. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 h period in the presence of Fadrozole (10 or lOOuM). Treatments which are similar (p>0.05) as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. 149 0 10uM 100uM Fadrozole concentration (uM) 150 Figure 42. 17f}-Estradiol levels secreted by cultured ovarian follicles over an 18 h period in the presence of exogenous testosterone (0.10, 0.20, or 0.40 LIM). Treatments which are different (p<0.05) as determined by Student-Newman-Keuls test, are identified by different superscript letters. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. Testosterone concentration (uM) 152 Figure 43. 17P-Estradiol levels secreted by cultured ovarian follicles over an 18 h period in the presence of Fadrozole (10 or IOOLXM) and with or without exogenous testosterone (0.10, 0.20, or 0.40 uM). Treatments which are similar (p>0.05) as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. 153 Fadrozole + Testosterone concentration (uM) 154 Table 4. Plasma hormone levels of 17fi-estradiol, 17a-20P-dihydroxy-4-pregnen-3-one (17a-203-P), and testosterone in individual female coho salmon at different times during sexual maturation. Plasma hormone levels (ng/ml) •Oocyte developmental 17P-estradiol 17a-20p-P testosterone stage Aug. 8, 1996 Immature3 20.59 1.80 140.40 Nov. 4, 1996 Central 13.25 4.80 210.70 Nov. 27, 1996 Peripheral 4.25 29.90 335.30 •Position of the germinal vesicle. anot visible 155 Figure 44. 17f3-Estradiol levels secreted by cultured ovarian follicles over an 18 h period in the presence of Fadrozole (10 uM) and with or without GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. Treatments which are similar (p>0.05), at each stage, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. C, control; F, Fadrozole. Note that there are different scales for the same hormone among the stages. 156 GTH II F F GTH I GTH II 18 16 14 12 10 8 6 4 2 0 1000 800 Central germinal vesicle stage a n GTH I GTH F F GTHI GTH 600 H 400 200 0 Peripheral germinal vesicle stage GTH I GTH F F GTH I GTH 157 Fadrozole with GTH I and GTH II inhibited 17f}-estradiol secretion below the levels when follicles were incubated with Fadrozole alone. 17oc-20P-P secretion was undetectable in the control incubations of follicles in the immature stage and in follicles in the peripheral germinal vesicle stage, but was detectable at the central germinal vesicle stage (Figure 45). Fadrozole alone did not affect 17a-20f3-P secretion throughout the periovulatory period. Follicles in the immature stage did not secrete significant amounts of 17a-20P-P in any of the treatments. The ability of the ovarian follicles to secrete 17a-20P-P in response to GTH I and GTH II increased during the periovulatory period. This is illustrated by the different scales used to represent 17a-20P-P secretion during different ovarian follicular stages. In follicles with central germinal vesicle GTH I and GTH II stimulated 17cc-20P-P secretion, and GTH II was more potent than GTH I. In follicles with peripheral germinal vesicle, both GTH I and GTH II stimulated 17a-20P-P secretion, but GTH II was 9 times more potent than GTH I. At the central germinal vesicle stage, the association of Fadrozole with GTH II acted synergistically, increasing 17a-20P-P secretion significantly (p<0.05) above the levels observed when follicles were incubated with GTH II alone. At the peripheral germinal vesicle stage both GTH 1 and GTH II acted synergistically with Fadrozole increasing 17a-20P-secretion, but the synergism was more potent between Fadrozole and GTH II. Basal testosterone secretion was undetectable in follicles in the immature stage and in follicles in the central germinal vesicle stage, but slightly detected at the peripheral germinal vesicle stage (Figure 46). Fadrozole stimulated testosterone secretion slightly but significantly (p<0.05) in follicles in the immature and in the central germinal vesicle stage. In the immature stage and in the central germinal vesicle stage, GTH I and GTH II were equally potent in slightly stimulating testosterone secretion. In follicles in the peripheral germinal vesicle stage both GTH I and GTH II stimulated testosterone secretion and GTH II was more potent than GTH I. GTH I and GTH II 158 Figure 45. 17a-203-dihydroxy-4-pregnen-3-one levels (17a-20P-P) secreted by cultured ovarian follicles over an 18 h period in the presence of Fadrozole (10 uM) and with or without GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. Treatments which are similar (p>0.05), at each stage, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. C, control; F, Fadrozole. Note that there are different scales for the same hormone among the stages. 159 12 10 8 6 4 2 0 70 60 50 40 30 20 10 0 Central germinal vesicle stage b MM Mm vw/m GTH I GTH Peripheral germinal vesicle stage e T c ....[ . 11111  iilSil lllllll ^^^^^ d b \ ~1 a a l i l l l GTH I GTH II F F GTHI GTH 160 Figure 46. Testosterone levels secreted by cultured ovarian follicles over an 18 h period in the presence of Fadrozole (10 uM) and with or without GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. Treatments which are similar (p>0.05), at each stage, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. C, control; F, Fadrozole. Note that there are different scales for the same hormone among the stages. 161 GTH I GTH II 10 8 8 4H 0 Central germinal vesicle stage Wm GTH I GTH I m m GTHI GTH 70 60 50 H 40 30 20 -10 -0 Peripheral germinal vesicle stage 3 c_ JL i i i ! GTH I GTH II F F GTHI GTH 162 significantly (p<0.05) stimulated testosterone secretion 8 and 16 times, respectively, above the basal levels. In follicles in the immature stage, the association of Fadrozole with GTH I and GTH II increased testosterone secretion significantly (p<0.05), and this synergistic effect was 10 times more potent in stimulating testosterone secretion than Fadrozole, GTH I and GTH II alone. In follicles with central germinal vesicle, the association of Fadrozole with GTH I and GTH II was more potent in stimulating testosterone secretion than Fadrozole, GTH I and GTH II alone. The combination of GTH II with Fadrozole was 8 times more potent in stimulating testosterone secretion than GTH I and GTH II alone and 2 times more potent than Fadrozole with GTH I. In follicles with peripheral germinal vesicle, no significant synergistic effect (p>0.05) was observed with Fadrozole and GTH I and GTH II. 6.3.3. Experiment 3. The effects of GTH I, GTH II, testosterone, and 17a-hydroxyprogesterone on sex steroid secretion by ovarian follicles of coho salmon in vitro throughout the periovulatory period. In this experiment indirect measures of aromatase, and 20P-hydroxysteroid dehydrogenase were obtained by incubating the ovarian follicles with exogenous substrate (100 ng/mL): conversion of exogenous testosterone to 17P-estradiol was used as an indicator of aromatase activity, and conversion of 17a-hydroxyprogesterone to 17a-20p-P was used as an indicator of 20P-hydroxysteroid dehydrogenase. In follicles at the immature stage, testosterone, 17a-hydroxyprogesterone, GTH I and GTH II stimulated 17P-estradiol secretion significantly (p<0.05) above basal levels (Figure 47). However, testosterone, GTH I, and GTH II, which stimulated 17P-estradiol secretion similarly, were more potent than 17a-hydroxyprogesterone. The combination of testosterone and 17-hydroxyprogesterone with 163 Figure 47. 17|3-Estradiol levels secreted by cultured ovarian follicles over an 18 h period in the presence or absence of exogenous testosterone (100 ng/mL), 17a-hydroxyprogesterone (100 ng/mL) and GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. Treatments which are similar (p>0.05), at each stage, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. CI, control with incubation medium; C2, control with incubation medium and ethanol (used to dissolve the steroids, see Chapter 2); P, 17oc-hydroxyprogesterone; T, testosterone. Note that there are different scales for the same hormone among the stages. 164 16 14 1 1 2 ]3> 10 5 8 TJ 2 6 </) L U 4 ca £ 2 0 Immature stage CI C2 b d X H I I-o I-+ I J3_ Q_ + I 1000 800 H 600 400 200 0 Peripheral germinal vesicle stage a J L C1 C2 a J L a J U b x t— J2_ x i -J2_ I— + x \— CD b 0. + X H o a. + X t— o 165 GTH I and GTH II was more potent in stimulating 17f3-estradiol secretion than GTH I, GTH II, testosterone, and 17a-hydroxyprogesterone alone In follicles with central germinal vesicle, testosterone, GTH I, and GTH II stimulated significantly (p<0.05), and in a similar manner, 17(3-estradiol production above basal levels. At this point 17a-hydroxyprogesterone did not stimulate 17P-estradiol secretion above basal levels. The combination of testosterone with GTH I and GTH II further increased 17p-estradiol secretion, and the association of testosterone with GTH II resulted in secretion of 17P-estradiol significantly higher (p<0.05) than testosterone with GTH I. In this stage, the combination of 17a-hydroxyprogesterone with GTH I and GTH II, significantly inhibited 17P-estradiol secretion (p<0.05) below basal levels. In follicles with peripheral germinal vesicle, testosterone and 17a-hydroxyprogesterone did not stimulate 17P-estradiol secretion above basal levels. On the other hand, GTH I and GTH II alone or in association with testosterone or 17a-hydroxyprogesterone inhibited 17P-estradiol secretion significantly (p<0.05) below basal levels.. In follicles at the immature stage, only the combination of GTH II with 17a-hydroxyprogesterone resulted in significant 17a-20P-P secretion (Figure 48). In follicles with central germinal vesicle, 17a-hydroxyprogesterone, GTH I and GTH II increased 17a-20P-P secretion significantly (p<0.05) above basal levels. Whereas 17a-hydroxyprogesterone slightly increased 17a-20P-P, GTH I and GTH II were 5 and 9 times more potent than 17a-hydroxyprogesterone in stimulating 17a-20P-P secretion, respectively. Combination of GTH I with testosterone or 17a-hydroxyprogesterone resulted in 17a-20P-P secretion similar to that seen with GTH I alone. The same effect was observed when GTH II was combined with testosterone. However, there was a significant (p<0.05) synergism between GTH II and 17a-hydroxyprogesterone, which caused an increase of 21 times in 17a-20P-P secretion above basal levels. In follicles with peripheral germinal vesicle, GTH II 166 Figure 48. 17a-20f3-dihydroxy-4-pregnen-3-one levels (17a-20p-P) secreted by cultured ovarian follicles over an 18 h period in the presence or absence of exogenous testosterone (100 ng/mL), 17a-hydroxyprogesterone (100 ng/mL) and GTH I or GTH II (100 ng/mL) during different stages of the reproductive development. Treatments which are similar (p>0.05), at each stage, as determined by Student-Newman-Keuls test, are identified by the same superscript letter Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. C1, control with incubation medium; C2, control with incubation medium and ethanol (used to dissolve the steroids, see Chapter 2); P, 17cc-hydroxyprogesterone; T, testosterone. Note that there are different scales for the same hormone among the stages. 167 1400 1200 H |~ 1000 2 800 600 H 400 H 200 0 Immature stage a C1 a C2 a T X __J3L_ 1 o 28 24 -| 2 0 -C D . „ 16 -CL ca 12 o ^ 8 4 H 0 Central germinal vesicle stage a C1 a 02 a T b p c ± d c £ X T 36 32 _ 28 H 1 24 S 20 ^ 16 H ca 8 1 2 H £ 8 4 0 Peripheral germinal vesicle stage a CI a C2 a p c X z C d a. + 168 was strongly more potent than GTH I in stimulating 17a-20f3-P secretion. At this time there was no synergistic effect between GTH II and 17cc-hydroxyprogesterone. In follicles throughout the periovulatory period, 17cc-hydroxyprogesterone alone did not stimulate testosterone secretion (Figure 49). In follicles at the immature stage, GTH I and GTH II slightly increased testosterone secretion, and combination of GTH I and GTH II with 17a-OH-progesterone caused even further significant (p<0.05) increase in testosterone secretion. In follicles with central germinal vesicle, GTH I and GTH II were able to slightly stimulate testosterone secretion. The association of GTH I and GTH II with 17a-hydroxyprogesterone was even more potent in stimulating testosterone secretion, and the synergistic effect was significantly greater with GTH II (p<0.05). In follicles with peripheral germinal vesicle, both GTH I and GTH II stimulated testosterone secretion, although GTH II was more potent. The association of GTH I with 17a-hydroxyprogesterone increased testosterone secretion significantly (p<0.05) above levels observed when follicles were incubated with GTH I alone. On the other hand, testosterone secretion by follicles incubated with GTH II and 17a-OH-progesterone was similar to that when follicles were incubated with GTH II alone. 169 Figure 49. Testosterone levels secreted by cultured ovarian follicles over an 18 h period in the presence or absence of exogenous testosterone (100 ng/mL), 17a-hydroxyprogesterone (100 ng/mL) and GTH I or GTH II (100 ng/mL) during different stages of the reproductive development.. Treatments which are similar (p>0.05), at each stage, as determined by Student-Newman-Keuls test, are identified by the same superscript letter. Each treatment group consisted of 6 replicates containing 4 ovarian follicles in each. CI, control with incubation medium; C2, control with incubation medium and ethanol (used to dissolve the steroids, see Chapter 2); P, 17a-hydroxyprogesterone. Note that there are different scales for the same hormone among the stages. 170 Central germinal vesicle stage d JL a C1 a C2 a P b I i -O I H o C Q. + X o a. + x o Peripheral germinal vesicle stage b a C1 a C2 a P x o c T T + + X X X H 1— o o o 171 6.4. Discussion These results provide direct evidence that 17P-estradiol synthesis in teleost ovarian follicles can be inhibited when the action of the enzyme aromatase responsible for its synthesis is blocked by Fadrozole These results are similar to studies conducted in mammals which have also shown that Fadrozole is able to reduce estrogen synthesis in the ovary (Steele et al., 1987). In addition, Fadrozole has also been shown to reduce estrogen formation in the avian brain (Wade et al., 1994) and in the fish brain (Antonopouplou et al., 1995). These results further demonstrate the ability of intact salmon ovarian follicles to produce increased amounts of 17f3-estradiol in response to increasing amounts of exogenous testosterone and indicates the elevated availability of aromatase in the ovarian follicles during vitellogenesis. Similar increases in 17(3-estradiol production by ovarian follicular layers incubated with increasing amounts of testosterone has been found in studies on amago salmon (Young et al., 1983a) and goldfish (Kagawa et al., 1984). Fadrozole has been described as a reversible competitive inhibitor (Steele et al., 1987). Since in competitive inhibition the binding of both substrate and inhibitor involves the same site, the effect of a competitive inhibitor can be overcome by increasing the substrate concentration . However, in this case the combination of Fadrozole with increasing substrate concentration appeared to increase the potency of the aromatase inhibitor Fadrozole. Supporting these results are those obtained by Wade et al. (1994), where increasing amounts of Fadrozole and testosterone further inhibited aromatase activity in the zebra finch bird brain. Furthermore in Wade et al. (1994) the inhibition was not overcome by increasing the concentration of substrate. Granulosa cells of rats incubated for 48 h in the presence of the non steroidal inhibitor R151885 and 0.1 M of testosterone (one of the doses tested in this study) also had estrogen secretion suppressed (Wickings et al. 1987). It may be possible that the failure of 172 testosterone to overcome the inhibition is because Fadrozole and testosterone are binding to different sites of the enzyme. This is referred to as a non-competitive inhibition, and since this type of inhibition involves different sites on the enzyme, the inhibition cannot be overcome by increasing the substrate concentration (Wilson and Goulding, 1992). Another possibility to explain the further decrease in 17(3-estradiol secretion when Fadrozole was combined with testosterone, which needs further study, is that testosterone, which is not been aromatized to estrogens due to Fadrozole, is being converted to 5a-dihydrotestosterone, which in turn has been shown to inhibit aromatase activity in vitro in mammals (Siiteri and Thompson, 1975; Hillier et al., 1980; and Wickings, et al., 1987). The enzyme responsible for the conversion of testosterone to 5a-dihydrotestosterone, 5a-reductase, has been detected in the ovary of rainbow trout (Sire and Depeche, 1981). The results from Experiment 2 demonstrate that both the basal and gonadotropin-stimulated secretion of 17P-estradiol by coho salmon ovarian follicles changed throughout the stages examined, and the most notable difference is the decrease in 17p-estradiol secretion at the peripheral germinal vesicle stage. Since in the first.two stages the follicles are vitellogenic, the increased capacity of secretion correlates well with the involvement of 17P-estradiol with vitellogenesis. On the other hand, the decline in 17P-estradiol secretion by ovarian follicles with peripheral germinal vesicle correlates well with the fact they are late in vitellogenesis and close to final maturation. This pattern has been observed in other in vitro studies involving salmonid fish ( Kagawa, et al., 1983; Young et al., 1983a; Van Der Kraak et al., 1986, Fostier and Jalabert, 1986, Kanamory, et al, 1988). The pattern of 17p-estradiol secretion by ovarian follicles in vitro also correlates well with the plasma hormone levels of the fish used in this experiment as well as with the plasma hormone profile observed in vivo in several salmonids, where a decline in plasma 17P-estradiol concentration occurs late in vitellogenesis and close to final maturation (Scott et al., 1982; Van Der Kraak et al., 1984; Fitzpatrick et al., 1986; 173 Yamauchi et al., 1984; Slater et al., 1994; and Dye et al., 1986). Young et al. (1983a), who quantified aromatase activity indirectly by measuring the secretion of 17f3-estradiol by amago salmon ovarian follicles incubated with testosterone, postulated that the decline in 17P-estradiol secretion was due to a progressive decline in aromatase activity. The results in this study support this hypothesis and furthermore contribute to clarify it by demonstrating that the aromatase inhibitor Fadrozole inhibited aromatase activity throughout the different stages. Therefore, the combined results from the in vitro and in vivo studies in this thesis, suggests that the patterns of 17P-estradiol secretion, in particular the declines observed in the stages close to final maturation, are probably due to a decrease in aromatase activity at the ovarian level instead of an increased metabolic clearance of 17p-estradiol as proposed by Baroiller et al. (1987). This study demonstrated that chum salmon GTH I and GTH II were equally potent in stimulating 17p-estradiol secretion in vitro by coho salmon ovarian follicles during vitellogenesis. These results are supported by those described by Swanson et al. (1989 and 1991) for juvenile coho salmon ovarian tissue using coho salmon GTH I and GTH II, by Suzuki et al. (1988c) for midvitellogenic amago salmon ovarian follicles using chum salmon GTH I and GTH II, by Van Der Kraak et al. (1992) for vitellogenic goldfish ovarian follicles using common carp GTH I and GTH II, and by Planas (1993) for pre-germinal vesicle breakdown brook trout ovarian follicles using coho salmon GTH I and GTH II. These findings related above, contrast, however with the situation in rainbow trout, where Sumpter et al. (1991) found that only chum salmon GTH I was able to stimulate 17P-estradiol secretion in vitro during vitellogenesis. In view of the fact that in most of the species above both GTH I and GTH JJ are equally potent in stimulating 17P-estradiol secretion in vitro, it would be interesting to conduct more studies with rainbow trout to further understand the effects of GTH I and GTH II on 17p-estradiol secretion in this species. The results 174 found by Sumpter et al. (1991) are coherent with the findings that during vitellogenesis GTH I is the dominant gonadotropin circulating in the plasma (Suzuki et al., 1988d, and Swanson, 1991; Prat et al., 1996). Also, Tyler et al. (1991) provided the first evidence in vivo and in vitro that in rainbow trout chum salmon GTH I but not GTH II stimulated vitellogenin uptake by vitellogenic follicles. It is interesting to notice that although GTH I and GTH II are equally potent in stimulating 17P-estradiol secretion in vitro, in vivo studies showed that plasma GTH I levels are high during vitellogenesis declining close to final maturation, the time when plasma GTH II levels increase (Suzuki et al., 1988d, and Swanson, 1991; Prat et al., 1996). This raises the question of whether the observed action of GTH II in vitro during vitellogenesis is physiologically important. The stimulatory effects of GTH II on 17f3-estradiol secretion in vitro during vitellogenesis can be explained by the existence of gonadotropin receptors in the membranes from thecal layers and granulosa cells of coho salmon ovaries (Yan et al., 1992). In this study they proposed a two-receptor model for salmon gonadotropins I and II, where the type I receptor, present in the thecal layers and granulosa cells binds both GTH I and GTH II, but with higher affinity for GTH I, whereas the type II receptor, present in the granulosa cell, binds GTH II specifically. This study was further extended in male and female coho salmon at different stages of sexual maturation by Miwa et al. (1994). This study demonstrated that in both males and females the type I receptor, which binds GTH I and GTH II is present at all stages of gametogenesis, whereas the type II receptor, which binds GTH II specifically appears in the gonads of salmon which are close to spawning. This study further noted that in vitellogenic females the type I receptor was localized on the granulosa cells, and to a lesser extent on the thecal layer, most probably on the special thecal cells. On the other hand in postvitellogenic/ preovulatory females the type I receptor was only detected on the thecal layer, and in the granulosa cells only the type II receptor was found. 175 Therefore, based on this two-receptor model (Yan et al., 1992, and Miwa et al., 1994) it can be suggested that the ability of GTH II to stimulate 17p-estradiol secretion during early vitellogenesis is related to the capacity of the type I receptor to binding both GTH I and GTH II. Both GTH I and GTH II inhibited 17(3-estradiol secretion in ovarian follicles at the peripheral germinal vesicle stage, a period when basal secretion of 17P-estradiol was reduced (refer to 17|3-estradiol levels in the control groups). It can be seen from the measurement of testosterone secretion that 17p-estradiol was not synthesized due to lack of substrate, since testosterone was being secreted. Inhibition of 17P-estradiol secretion by gonadotropins has been observed in rainbow trout by Sire and Depeche (1981), GTH II inhibited 17p-estradiol secretion in midvitellogenic amago salmon ovarian follicles (Suzuki et al., 1988c), in peripheral germinal vesicle coho salmon ovarian follicles (Planas et al., 1995). In vitellogenic rainbow trout ovarian follicles (Fostier, 1995) demonstrated that both GTH I and GTH II had an inhibitory effect on 17p-estradiol secretion, but the effect of GTH II was stronger than GTH I. Assuming that the two-receptor model is valid in amago salmon and rainbow trout, the finding that GTH II inhibited 17P-estradiol secretion during midvitellogenesis in amago salmon (Suzuki et al. 1988c) and in vitellogenic rainbow trout (Fostier, 1995), is possible. However, it is questionable whether this is normal physiological response, since plasma and pituitary content of GTH II increase only close ovulation (Suzuki et al., 1988c). On the other hand, the results above described by Planas et al. (1995) and by Fostier (1995) that GTH II inhibited 17p-estradiol secretion close to maturation, as observed in the present study, fit well with the two-receptor model where at this developmental stage there is a specific GTH II receptor on the granulosa cells and also because at this time plasma GTH II is elevated (Swanson, 1991). The inhibitory effect of GTH I observed in this study in follicles close to maturation is more difficult to explain in terms of a normal physiological 176 response, since it has been shown that in mature coho salmon plasma GTH I levels have declined (Swanson, 1991). Also, considering the two-receptor model (Yan et al., 1992, and Miwa et al., 1994) close to maturation there are GTH I receptors only at the thecal layers, which in accordance with the two-cell type model proposed by Nagahama (1987a) to explain steroidogenesis by the ovarian follicles produces the aromatizable androgens, which then diffuses to the granulosa cells where aromatase is present. Therefore if GTH I has an inhibitory action on 17(3-estradiol secretion, it would be by stimulating production of a substance primarily secreted by the thecal layers. Although these in vitro studies demonstrated that GTH II inhibited 17{3-estradiol secretion close to final maturation, they revealed that basal secretion of 17p-estradiol was already low, and the question of whether the decrease on aromatase activity is due to GTH II still remains to be elucidated. Considering the fact that in vitro experiments, using amago salmon ovarian follicles at different stages of reproductive development, demonstrated that partially purified chinook salmon glycoprotein gonadotropin (SG-G100) or partially purified gonadotropin (SGA) did not stimulate aromatase activity (Young et al., 1983a; Kanamori et al., 1988), it is possible to speculate that the inhibitory effect of GTH II is not directly through inhibition of aromatase activity. It could be possible that GTH II is stimulating the synthesis of another substance, which might be inhibiting aromatase activity at a stage when the activity is already low. This hypothesis, however needs to be investigated. The association of Fadrozole with both GTH I and GTH II acted synergistically to further inhibit 17p-estradiol secretion, independently of the reproductive stage. Supporting these findings Khalil et al. (1989) found that incubation of porcine granulosa cells in vitro with the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A-dione) inhibited estradiol secretion below basal 177 levels. Similarly, immature female rats primed with Fadrozole and pregnant mare serum gonadotropin (PMSG) presented significantly lower intrafollicular estradiol levels than the group treated with PMSG only. However intrafollicular estradiol levels in the group treated with Fadrozole and PMSG were comparable to the control group, which was not primed with PMSG and Fadrozole (Selvaraj, et al., 1994). These authors also verified that the intrafollicular testosterone and progesterone levels increased significantly above basal levels in the group primed with Fadrozole and PMSG. Comparing the inhibitory action of Fadrozole and gonadotropins on 17(3-estradiol secretion observed in this experiment with the results obtained in the first in vitro experiment, where association of Fadrozole with testosterone further inhibited 17f3-estradiol secretion, suggests that the inhibitory action on 17P-estradiol secretion is related to testosterone. This idea can be supported by the data on testosterone secretion in this experiment. Incubation of ovarian follicles with Fadrozole alone, caused a discrete accumulation of testosterone. On the other hand, the association of Fadrozole with GTH I and GTH II caused a significant increase in testosterone secretion. The mechanism involved in this inhibitory effect is not known. These findings further support the idea that the testosterone produced via GTH I and GTH II stimulation, which is not being aromatized to estrogens due to Fadrozole, might be converted to 5a-dihydrotestosterone, which in turn has been shown to inhibit aromatase activity in vitro in mammals (Siiteri and Thompson, 1975, Hillier et al., 1980; and Wickings, et al., 1987). In this study /// vitro basal secretion of 17cc-20|3-P was very low or undetectable in the three different ovarian developmental stages. Similar results were obtained by Van Der Kraak et al. (1986) in coho salmon and Young et al. (1983b) and Kanamory et al. (1988) in amago salmon Similarly, incubation of ovarian follicles with the aromatase inhibitor Fadrozole at different reproductive stages did not increase the secretion of 17a-20P-P by coho salmon ovarian 178 follicles. This result clearly demonstrated that inhibition of 17(3-estradiol per se did not cause an increase in 17a-20p-P secretion. Correlating this result with the normal plasma reproductive steroid hormone profiles during sexual maturation and with the in vivo experiments in females in this thesis, where the switch from 17P-estradiol to 17a-20P-P production has been demonstrated to occur either naturally or after injection with the aromatase inhibitor Fadrozole, the increase in 17a-20P-P must be dependent on an extragonadal factor. Similar to this study, several in vitro studies have demonstrated that gonadotropins stimulate 17tx-20p-P secretion (Young et al., 1983b; Fostier and Jalabert, 1986; Van Der Kraak et al., 1986; Kanamori et al., 1988) Also supporting this study Young et al. (1983b), Van Der Kraak et al. (1986) and Kanamori et al. (1988) demonstrated that in early stages of ovarian development gonadotropins did not stimulate 17a-20p-P secretion. Furthermore, this study is in agreement with those studies which used purified GTH I and GTH II, and observed that GTH II was more potent than GTH I in stimulating in vitro 17a-20P-P secretion by ovarian follicles (Suzuki et al., 1988c; Planas et al., 1995), and that in later reproductive stages the stimulatory capacity of GTH I on 17a-20p-P secretion decreases (Planas et al., 1995). The stimulation of 17a-20p-P secretion by both GTH I and GTH II during vitellogenesis in vitro, can be explained by the two-receptor model (Yan et al., 1992, and Miwa et al., 1994), which states that during vitellogenesis the type I receptor can bind both GTH I and GTH II, although in vivo studies showed that GTH I is the dominant gonadotropin in salmon plasma during this period (Suzuki et al., 1988d; Swanson 1991; Prat et al., 1996). Basal testosterone secretion was only detected in coho salmon ovarian follicles at the peripheral germinal vesicle stage. Similarly, Van Der Kraak et al. (1986) working with coho salmon and Kanamori et al. (1988) working with amago salmon found that basal testosterone 179 secretion was undetectable or low in the early developmental stages and increased just prior to oocyte maturation. Incubation of the ovarian follicles with Fadrozole caused a slight increase in testosterone secretion at the immature and at the central germinal vesicle stage, but not at the peripheral germinal vesicle stage. Analyzing the results of testosterone secretion in the control and in the group treated with Fadrozole, it can be postulated that an extragonadal factor is also responsible for testosterone stimulation. If this factor was present at the ovarian level it would be expected that inhibition of 173-estradiol synthesis by Fadrozole, would cause an increase in testosterone secretion. During the period where 17p-estradiol was been actively secreted by the ovarian follicles (immature and central germinal vesicle stage) GTH I and GTH II did not stimulate or slightly stimulated testosterone accumulation. The low testosterone accumulation in the groups treated with GTH I or GTH II can be explained by the fact that testosterone was being actively converted to 17fJ-estradiol . This can be further confirmed by the fact that association of the aromatase inhibitor Fadrozole with GTH I and GTH II synergistically caused a drastic increase in testosterone production, suggesting that GTH I and GTH II acted to stimulate the pathway involved in testosterone production. Sire and Depeche (1981) examined the conversion rate of androstenedione to testosterone in rainbow trout ovarian follicles in vitro and demonstrated that the rate increased when highly purified salmon gonadotropin (mainly GTH II) was added to the incubation medium, indicating a stimulation of the 17P-hydroxysteroid dehydrogenase activity (enzyme which converts androstenedione to testosterone). Another interesting point was that the association between Fadrozole and GTH I or GTH II revealed differences in the potency of GTH I and GTH II. At the immature stage both GTH I and GTH II were equally potent. On the other hand at the central germinal vesicle stage, the combination of Fadrozole with GTH II was more potent in stimulating testosterone secretion. Even though there 180 was no synergism between Fadrozole and GTH I or GTH II, at the peripheral germinal vesicle stage GTH II was more potent than GTH I. A similar result in terms of GTH II potency at the peripheral germinal vesicle stage was reported by Planas (1993) who measured testosterone secretion by isolated coho salmon thecal cell layers. In terms of the two-receptor model it is reasonable to assume that the ability of GTH II to stimulate testosterone secretion when in association with Fadrozole during the immature and central stages is due to the presence of the type I receptor in the thecal cells follicles which binds both GTH I and GTH II, however they should be equally potent, and that was not the case at the central germinal vesicle stage. It seems that even though at the central germinal vesicle stage GTH I is the gonadotropin dominant in the plasma, the type I receptor has increased its capacity to bind to GTH II. In accordance with the two-cell type model proposed to explain the sex steroid synthesis in the ovary, testosterone is synthesized in the thecal cells (Nagahama 1987a). It is difficult to explain, however the situation that GTH II stimulates testosterone in ovarian follicles at the peripheral germinal vesicle stage, if the two receptor model proposed by Yan et al. (1992) and Miwa et al. (1994) states that close to final maturation the type two receptor which binds GTH II specifically is present only in the granulosa. Therefore it can be speculated that other types of gonadotropin receptor could be present at the ovarian level in coho salmon. Intracellular mechanisms can also be involved in regulating the steroidogenic action of gonadotropin (Kanamori and Nagahama, 1988). The results from Experiment 3 revealed that the substrate testosterone was capable of increasing 170-estradiol secretion by vitellogenic follicles above basal levels at the immature stage and at the central germinal vesicle stage. This result is supported by those obtained in experiment 1 discussed earlier, where follicles were incubated with increasing concentrations of testosterone, and by the results obtained by Young et al. (1983a), and Kagawa et al. (1984) . Similarly, GTH I and GTH II were equipotent to testosterone in stimulating 170-estradiol 181 secretion at the same stages. These findings demonstrated that 17P-estradiol production is not dependent on the direct action of gonadotropins on aromatase activity, since testosterone alone is able to stimulate 17f}-estradiol secretion. The possible lack of action of the gonadotropins on aromatase activity is further supported by the results in Experiment 2, which showed that GTH I and GTH II stimulated testosterone secretion when aromatase activity was blocked. Therefore the combination of these results further suggests that the gonadotropins are acting through the steroidogenic pathways involved in testosterone production. These results, however, contrast with the situation in mammals, where the major role of gonadotropins is to stimulate aromatase activity (Dorrington and Armstrong, 1979). It has been proposed by Nagahama et al. (1985) and Kanamori and Nagahama (1988) that the gonadotropins act on both granulosa and thecal cells to stimulate steroidogenesis via a mechanism involving adenylate cyclase-cAMP-dependent steps. Kanamori and Nagahama (1988) demonstrated that in amago salmon partially purified salmon gonadotropin (SGA) and several agents that increase intracellular cAMP level failed to increase aromatase activity in granulosa layers incubated with the substrate testosterone in different oocyte developmental stages. On the other hand the same authors showed that the same agents were able to stimulate testosterone production by isolated thecal layers. Contrasting with the above results were those obtained by Planas (1993) with brook trout species ovarian follicles. In a series of experiments using agents which stimulate intracellular cAMP, Planas found that these agents stimulated 17P-estradiol production. However it is difficult to conclude that these agents stimulated aromatase activity directly since he worked with intact ovarian follicles, whereas Kanamori and Nagahama (1988) carried out the experiments in isolated cell layers. Therefore the results obtained by Planas (1993) could have resulted from the action of those agents on the thecal cells, as was observed by Kanamori and Nagahama (1988). 182 The stimulation of 17P-estradiol secretion at the immature stage due to the synergistic effect of the gonadotropins with both substrates (testosterone and 17a-hydroxyprogesterone) can be attributed to the capacity of GTH I and GTH II to stimulate testosterone secretion, therefore producing more substrate which will be available for the aromatase reaction. On the other hand at the central germinal vesicle stage, the association of the gonadotropins with 17a-hydroxyprogesterone inhibited 17p-estradiol secretion. It is difficult to explain why this inhibitory action happened. It may be possible that the addition of the substrate combined with the gonadotropins caused a shift in the steroidogenic pathway. Indeed, the association of GTH II and 17a-hydroxyprogesterone caused an acute increase in 17a-20p-P and testosterone secretion. On the other hand, the association of GTH I and 17a-hydroxyprogesterone, which also inhibited 17p-estradiol secretion, did not synergistically stimulate 17a-20p-secretion, but stimulated testosterone secretion. It is clear that the results of this experiment are not enough to explain this inhibitory effect. The fact that GTH I and 17a-hydroxyprogesterone did not stimulate 17a-20p-P above the level secreted by the ovarian follicles incubated with GTH I alone, as did GTH II and 17a-hydroxyprogesterone, can be explained by the fact that GTH II is more potent than GTH I in stimulating 17a-20P-P in the presence of 17a-hydroxyprogesterone, (Suzuki et al., 1988c). It has also been demonstrated that gonadotropin stimulates 17a-20p-P by increasing 20P-hydroxysteroid dehydrogenase activity (Nagahama et al., 1985; Kanamori et al., 1988). Both GTH I and GTH II are also able to stimulate 17a-hydroxyprogesterone secretion in vitro by amago salmon ovarian follicles, and GTH II is more potent than GTH I (Suzuki et al., 1988c). Since 17a-hydroxyprogesterone alone did not alter 17p-estradiol secretion, and GTH I, and GTH II alone stimulated 17p-estradiol secretion, and considering the fact that GTH I and GTH II stimulate the pathways involved in the metabolism of 17a-hydroxyprogesterone (text 183 above), it is possible that the GTHs stimulated the conversion of 17a-hydroxyprogesterone to a substance which cause aromatase inhibition. However this experiment per se does not elucidate this mechanism. Both GTH I and GTH II or the combination of them with the substrates inhibited 17(3-estradiol secretion in ovarian follicles at the peripheral germinal vesicle stage, a period when basal secretion of 17(3-estradiol was reduced. It can be seen from the measurement of testosterone secretion that 17(3-estradiol was not synthesized due to lack of substrate, since testosterone was being secreted. Inhibition of 17(3-estradiol secretion by gonadotropins has been observed in rainbow trout by Sire and Depeche (1981), GTH II inhibited 173-estradiol secretion in midvitellogenic amago salmon ovarian follicles (Suzuki et al., 1988c), and in peripheral germinal vesicle coho salmon ovarian follicles (Planas et al., 1995). In vitellogenic rainbow trout ovarian follicles (Fostier, 1995) demonstrated that both GTH I and GTH II had an inhibitory effect on 173-estradiol secretion, but the effect of GTH II was stronger than GTH I. Assuming that the two-receptor model is valid in amago salmon and rainbow trout, the findings that GTH II inhibited 173-estradiol secretion during midvitellogenesis in amago salmon (Suzuki et al. 1988c) and in vitellogenic rainbow trout (Fostier, 1995) are possible, however, whether this a normal physiological response is questionable, since plasma and pituitary content of GTH II only increase close to ovulation (Swanson, 1991, Suzuki et al., 1988c; Prat et al., 1996 ). On the other hand, the results above described by Planas et al. (1995) and by Fostier (1995) that GTH II inhibited 17P-estradiol secretion close to maturation, as it was observed in this study, fit well with the two-receptor model where at this stage there is a specific GTH II receptor on the granulosa cells and also because at this time plasma GTH II is elevated (Swanson, 1991, Suzuki et al., 1988c, Prat et al., 1996). The inhibitory effect of GTH I observed in this study in follicles close to maturation is 184 more difficult to explain in terms of a normal physiological response, since it has been shown that in coho salmon plasma GTH I levels have declined at this time (Swanson, 1991). Also, considering the two-receptor model (Yan et al., 1992, and Miwa et al., 1994) in oocytes close to maturation there are GTH I receptors only in the thecal layers, which in accordance with the two-cell type model proposed by Nagahama (1987a) to explain steroidogenesis by the ovarian follicles produce the aromatizable androgens, which then diffuses to the granulosa cells where aromatase is present. Therefore if GTH I has an inhibitory action on 17P-estradiol secretion, it would be by stimulating production of a substance primarily secreted by the thecal layers. Although these in vitro studies demonstrate that GTH II inhibited 17p-estradiol secretion close to final maturation, they revealed that basal secretion of 17P-estradiol was already low, and the question whether the decrease in aromatase activity and consequently 17P-estradiol secretion is due to GTH II still remain to be elucidated. Considering the fact that in vitro experiments using amago salmon ovarian follicles at different stages of reproductive development demonstrated that partially purified chinook salmon gonadotropin (SG-G100) or partially purified salmon gonadotropin (SGA) did not stimulate aromatase activity (Young et al., 1983a; Kanamori et al., 1988d), it is possible to speculate that the inhibitory effect of GTH II is not direct through inhibition of aromatase activity. It could be possible that GTH II is stimulating the synthesis of another substance, which might be inhibiting aromatase activity at a stage where the activity is already low. This hypothesis, however needs to be investigated In contrast to 17p-estradiol, which can be directly synthesized when testosterone is added to the medium, 17a-20P-P secretion is low or undetectable in incubation containing no substrate (precursor) or when the precursor 17a-hydroxyprogesterone is added to the medium. On the other hand GTH I and GTH II or 17a-hydroxyprogesterone in the presence of gonadotropins can 185 stimulate 17a-20f3-P secretion, and GTH II is more potent than GTH I in stimulating 17a-20f3-P secretion. The results obtained in this experiment are supported by those of Suzuki et al. (1988c) that GTH II is more potent than GTH I in stimulating 17a-20fJ-P in the presence of 17a-hydroxyprogesterone. Also this experiment further confirms that gonadotropin stimulates 17a-20P-P by increasing 20p-hydroxysteroid dehydrogenase activity (Nagahama et al., 1985, Kanamori et al., 1988), since the levels of 17a-20P-P were low or undetectable when the follicles were incubated with the precursor 17a-hydroxyprogesterone alone. This series of in vitro experiments demonstrated that Fadrozole is capable of inhibiting 17P-estradiol secretion throughout the periovulatory period, and that inhibition of 17p-estradiol per se does not elicit 17a-20P-P secretion, showing that gonadotropin is necessary for its secretion. It was also evident that 17P-estradiol secretion is dependent on availability of the substrate testosterone, which in turn is secreted in response to the gonadotropins. On the other hand 17a-20p-P secretion is directly dependent on stimulation of 20P-hydroxysteroid dehydrogenase by the gonadotropins, especially GTH II. 186 C H A P T E R 7 - General Summary and Conclusions The first set of experiments demonstrated that inhibition of 17p-estradiol close to final maturation caused a premature increase in the 17a-20P-P, which is the maturational hormone in salmonids. The switch in the steroid profile that was observed in this study after injection with Fadrozole was very similar to that occurring naturally in fish close to maturation, in that a decline in 17p-estradiol was followed by an increase in plasma 17a-20P-P. Fadrozole was able to mimic a situation which would later occur naturally, and showed that the increase in the maturation hormone is dependent upon a decrease in 17p-estradiol levels, which normally occurs close to final maturation due to a progressive decrease in aromatase activity. As discussed above it has been shown by neuroendocrine studies in several species of fish that 17p-estradiol has an indirect negative feedback effect at the central nervous system (CNS) level by decreasing dopamine degradation, and thus increasing its inhibitory effect on G T H n release from the pituitary (Peter et al., 1991; Linard et al., 1995; Peng, 1994). G T H U on the other hand has been shown to stimulate 17a-20p-P production in vitro. In vivo studies also have shown that there is a correlation between the increase in plasma G T H II levels and the increase in plasma 17a-20p-P. Therefore there is substantial evidence of the negative feedback effects of 17p-estradiol on 17a-20P-P biosynthesis, which gives support to the idea that the timing of the increase in 17a-20P-P close to final maturation is in part under the control of 17P-estradiol. This has been further confirmed by the results obtained after injecting Fadrozole in females during vitellogenesis and males during spermatogenesis, where it has been shown that plasma 17ct-20p-P levels also increase close to spermiation. In both experiments, injection of Fadrozole, and the consequent inhibition of aromatase activity caused a premature increase in 17a-20p-P, and in the case of the males they started to spermiate. The results 187 with males were interesting in that they suggest that spermiation is probably preceded by a decrease in estrogen synthesis from androgens in the CNS, probably due to a decrease in aromatase activity at the brain level, which presumably would abolish the feedback inhibition of 17P-estradiol over GnRH and GTH II release, which has also been shown to stimulate 17a-20p-P production by the testis. Although plasma GTH I and GTH II were not measured in the studies presented in this thesis, some insights about their actions can be obtained from several studies reporting plasma GTH I and GTH II levels in coho salmon and other salmonids. The results demonstrate that during vitellogenesis and spermatogenesis plasma GTH I exceeds GTH II, whereas close to maturation and spermiation the opposite is observed. Therefore the permanent increase in 17cc-20P-P observed in females treated with the highest dose of Fadrozole and subsequent ovulation might have been preceded by an increase in plasma GTH II levels which then stimulated 17a-20P-P biosynthesis by the ovary leading to maturation and ovulation. The same sequence of events in terms of plasma gonadotropin may also have occurred in the males. In the vitellogenic females, it would be expected that at that time GTH I would be the dominant gonadotropin in the plasma. Therefore the premature and transient increase in 17a-20p-P observed in the groups injected with 1.0 and 10 mg Fadrozole/kg was due to either stimulation by GTH I or induction of premature GTH II secretion. In vitro experiments have shown that GTH I is able to stimulate 17a-20p-P secretion by vitellogenic ovarian follicles. On the other hand, the increase in 17a-20P-P in the group injected weekly with Fadrozole, which caused a permanent decline in plasma 17p-estradiol may have been due to a switch in gonadotropin production from GTH I to GTH fl", but this remains to be investigated. In this same group the arrested growth of the ovary, shown by the gonadosomatic index and egg diameter combined with the atretic oocytes provided further evidence for the importance of 17P-estradiol during vitellogenesis both at the ovarian and hepatic level. At the same time this result shows that Fadrozole can potentially be used to control 188 sexual development in females, by arresting ovarian growth. However, before Fadrozole or similar compounds could be used for this purpose further studies would be necessary to fully understand its effects in fish. The in vitro studies using coho salmon ovarian follicles in this thesis helped to answer some questions raised by the in vivo studies. First of all, the in vitro study carried out with the ovarian follicles of vitellogenic females injected with Fadrozole, showed that the inhibitor acted at the brain and ovarian levels decreasing 17p-estradiol secretion. This result was further corLfirmed by the next experiment in vitro which showed that ovarian follicles incubated in the presence of Fadrozole secreted less 17P-estradiol than the control group. This same experiment showed that 17p-estradiol secretion increased dramatically and in a dose dependent manner, when the amount of the substrate testosterone was increased in the incubation medium. Similar results have already been observed with other salmonids, and suggest that the formation of 17P-estradiol is only substrate dependent. This experiment also revealed that a combination of Fadrozole with testosterone actually inhibited 17f3-estradiol secretion more than Fadrozole alone, suggesting that a interacting mechanism not known at the present time could be occurring at the ovarian level to decrease 17p-estradiol secretion. The next in vitro experiment where Fadrozole was combined with purified chum salmon gonadotropins GTH I and GTH n, helped to further understand the in vivo effects of Fadrozole. This experiment was carried out with ovarian follicles at three different stages of development, which included early and mid vitellogenic and close to maturing ovarian follicles. First of all most of the results in terms of the steroidogenic capacity of GTH I and GTH II alone were similar to those already available in the literature for salmonids, thereby further supporting the previous findings. In terms of helping to understand what happened in vivo, the in vitro experiment showed that inhibition of 17p-estradiol secretion with Fadrozole per se did not cause an increase in 17a-20P-P. 189 Therefore this result demonstrated that an internal stimulation factor was missing, and that ovarian follicles alone, are not able to increase 17a-20P-P secretion. On the other hand, when Fadrozole was combined with the gonadotropins, both of them were able to stimulate 17o>20p-P secretion, but GTH II was more potent than GTH I in both vitellogenic and in ovarian follicles close to maturation. Therefore since 17a-20P-P is produced by the ovary, the increase in plasma 17a-20p-P levels observed after injection of Fadrozole in vivo was most probably due to the effect of gonadotropin at the ovarian level. Again it cannot be stated unequivocally which of the two gonadotropins stimulated 17a-20P-P since they were not measured in the plasma. Another interesting point was that even though GTH I and GTH II were able to stimulate 17a-20P-P secretion when combined with Fadrozole, the GTH I stimulating capacity was not greater than GTH I was alone. On the other hand GTH II with Fadrozole synergized to increase 17a-20P-P secretion. This suggests that GTH I has a limited capacity for stimulating 17a-20P-P secretion and this may be linked to the fact that GTH II is more potent in stimulating 20p-hydroxysteroid dehydrogenase than GTH I. The present study showed that inhibition of 17P-estradiol secretion caused a slight increase in testosterone secretion. On the other hand coadministration of Fadrozole with the gonadotropins resulted in an eight fold increase in testosterone secretion at the immature stage (vitellogenic follicles) and between four and eight fold at the central germinal vesicle stage (vitellogenic follicles). Comparing the secretion of testosterone by follicles when 17P-estradiol secretion was inhibited by Fadrozole alone with that when Fadrozole was coadministered with the gonadotropins, it is clear that the gonadotropins are stimulating testosterone secretion. This evidence that gonadotropins stimulate testosterone secretion was further clarified at the peripheral germinal vesicle stage (close to maturation), when basal 17P-estradiol secretion is reduced This suggests that the gonadotropins stimulate 17P-estradiol production indirectly by stimulating production of the substrate testosterone. This is further supported 190 by the results of the in vitro experiment which showed that 17p-estradiol secretion was substrate dependent. The finding that gonadotropin associated with Fadrozole further inhibited 17P-estradiol secretion and stimulated testosterone secretion, correlates well with the in vitro experiment where testosterone in combination with Fadrozole further inhibited 17p-estradiol secretion. Therefore the inhibitory effect of Fadrozole and the gonadotropins on 17p-estradiol secretion can be explained by the dramatic increase in testosterone due to gonadotropin stimulation. A possible explanation of the increase in testosterone that occurred in vivo after injection of Fadrozole, may be that the increase was due to the stimulatory effect of the gonadotropins on testosterone. Another interesting finding is that GTH II was more potent than GTH I in stimulating testosterone secretion at the peripheral germinal vesicle stage (Close to final maturation), where in accordance with the two-receptor model for gonadotropin binding, GTH II receptors are only present in the granulosa cells. This does not fit with the two-cell type model that explains steroid production by the ovarian follicles, which states that testosterone is produced by the theca cells and not by the granulosa cells. One possible explanation for this effect would be that other gonadotropin receptors not known at the moment are present in the ovarian follicles. The next in vitro experiment, where the substrates testosterone and 17a-hydroxyprogesterone were combined with the GTHs, further confirmed that 17p-estradiol biosynthesis is dependent on substrate concentration. This study also showed that 17a-20P-P secretion is clearly dependent on GTH stimulation of 20P-hydroxysteroid dehydrogenase activity, since the substrate for 17a-20p-P production (17a-hydroxyprogesterone) alone did not stimulate 17a-20P-P as much as when substrate was combined with the gonadotropins, and GTH II was far more potent in this regard than GTH I. 191 In summary these experiments demonstrated that the aromatase inhibitor Fadrozole can be used as tool to study mechanisms involved in the regulation of sexual development in fish. A new method to induce ovulation and spermiation in maturing coho salmon emerged, different from the traditional ones which use pituitary hormones or gonadotropin releasing hormones. Also aromatase inhibitors such as Fadrozole can be used during vitellogenesis to inhibit reproductive development. Future research should look at ways to explore the practical applications of these findings. At a basic level the in vivo experiments in females demonstrated that the switch from 17p-estradiol to 17a-20P-P production is dependent upon a decrease in 17P-estradiol production. The in vitro experiments showed that 17p-estradiol secretion is not dependent on the direct action of the gonadotropins on aromatase activity, but that 17P-estradiol secretion is regulated by the direct action of the gonadotropins on testosterone production. The in vitro experiments also demonstrated that 17a-20p-P production is directly dependent on stimulation of 20P-hydroxysteroid dehydrogenase activity by the GTHs. Together the in vivo and in vitro experiments provided further evidence that the increase in 17a-20p-P production is dependent on GTH II. Future in vitro studies should further investigate how the gonadotropins influence testosterone production, which could further elucidate the function of GTH I during vitellogenesis. 192 L I T E R A T U R E CITED Alexandre, C , and Balthazar, J. (1987). Inhibition of testosterone metabolism in the brain and cloacal gland of the quail by specific inhibitors and antihormones. J. Endocrinol. 112, 189-195. Al-Gubory, K.H., Driancourt, M-A., Antoine, M , Martal, J., and Neimer, N (1994) Evidence that a non-steroidal factor from corpus luteum of pregnant sheep inhibits aromatase activity of ovarian follicles in vitro. Journal of Reproduction and Fertility 100, 51-56. Andersson, E., Borg, B , and Lambert, J.G.D (1988) Aromatase activity in brain and pituitary of immature and mature atlantic salmon {Salmo salar L.) parr. Gen. Comp. Endocrinol. 72, 394-401. Antonopoulou, E., Mayer, I., Berlung, I. and Borg, B. (1995). Effects of aromatase inhibitors on sexual maturation in Atlantic salmon, Salmo salar, male parr Fish Physiology and a. Biochemistry 14, 15-24. Arnold, A P., and Schlinger, B.A. (1993). Sexual differentiation of brain and behavior: the zebra finch is not just a flying rat. Brain Behav. Evol. 42, 231-241. Baroiller, J.F., Fostier, A., Zohar, Y , and Marcuzzi, O. (1987). The metabolic clearance rate of estradiol-17P in rainbow trout, Salmo gairdneri R., estimated by both single injection and constant infusion methods increase during oocyte maturation. Gen. Comp. Endocrinol. 66, 85-94. Barry, TP., Aida, K., Okumura, T , and Hanyu, I. (1990). The shifth from C-19 to C-21 steroid synthesis in spawning male common carp, Cyprinus carpio, is regulated by the inhibition of androgen production by progestogens produced by the spermatozoa. Biol. Reprod. 43, 105-112. Bhatnagar, AS., Batzl, C , Hausler, A., and Nogues, V. (1993). The role of estrogen in the feedback regulation of follicle-stimulating hormone secretion in the female rat. J. Steroid Biochem. Molec. Biol. 47, 161-166. Billard, R. (1978). Testicular feedback on the hypothalamo pituitary axis in rainbow trout (Salmo gairdneri R). Ann. Biol. Anim. Biochem. Biophys. 18, 813-818. Billard, R., Breton, B., Fostier, A., Jalabert, B , and Weil, C. (1978). Endocrine control of the teleost reproductive cycle and its relation to external factors: Salmonid and Cyprinid model. In "Comparative Endocrinology" (P.J. Gaillard and HH. Boer, eds), pp.37-48. Bohemen, Ch. G. Van, and Lambert, J.G.D. (1981). Estrogen synthesis in relation to estrone, estradiol, and vitellogenin plasma levels during the reproductive cycle of the female rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 45, 105-114. 193 Bonsall, R.W., Clancy, A.N., and Michael, R.P. 1992. Effects of the nonsteroidal aromatase inhibitor, fadrozole, on sexual behavior in male rats. Hormones and Behavior 26, 240-254. Borg, B, Timmers, R.J.M., and Lambert, J.G.D. (1987a). Aromatase activity in the brain of the three-spined stickleback, Gasterosteus aadeatus. I. Distribution and effects of season and photoperiod. Exp. Biol. 47, 63-68. Borg, B., Timmers, R.J.M., and Lambert, J.G.D. (1987b). Aromatase activity in the brain of the three-spined stickleback, Gasterosteus aculeatus. II. Effects of castration in winter. Exp. Biol. 47, 69-71. Callard, GV. (1983). Androgen and estrogen actions in the vertebrate brain. Amer. Zool. 23, 607-620. Callard, G.V., Petro, Z , and Ryan, K.J. (1978a). Conversion of androgen to estrogen and other steroids in the vertebrate brain. Amer. Zool. 18, 511-523. Callard, G.V., Petro, Z , and Ryan, K.J. (1978b). Phylogenetic distribution of aromatase and other androgen-converting enzymes in the central nervous system. Endocrinology 103, 2283-2290. Callard, G.V., Petro, Z , and Ryan, K.J. (1981). Estrogen synthesis in vitro and in vivo in the brain of a marine teleost {Myoxocephalus). Gen. Comp. Endocrinol. 43, 243-255. Callard, G.V., Specker, J.L., Knapp, J., Nishioka, R.S., and Bern, HA. (1988). Aromatase is concentrated in the proximal pars distalis of tilapia pituitary. Gen. Comp. Endocrinol. 71, 70-79. Campbell, P.M. (1992). The effects of stress on the reproductive endocrinology and gamete quality of trout. Ph.D. Thesis, Brunei University, 285 p. Carrol, R.S., and Baum, M.J. (1989). Evidence that oestrogen exerts an equivalent negative feedback action on LH secretion in male and female ferrets. J. Reprod. Fert. 86, 235-245. Carson, R.S., Findlay, J.K., Clarke, I.J., and Burger, H.G. (1981). Estradiol, testosterone, and androstenedione in ovine follicular fluid during growth and atresia of ovarian follicles. Biol. Reprod. 24, 105-113. Clearwater, S.J., and Pankhurst, NW. (1997). The response to capture and confinement stress of plasma Cortisol, plasma sex steroids and vitellogenic oocytes in the marine teleost, red gurnard. Journal of Fish Biology 50, 429-441. Colombo, L., Colombo Belvedere, P., and Arcarese, G. (1978). Gonadal steroidogenesis and gametogenesis in teleost fishes. A sutdy on the sea bass Dicentrarchus labrax L. Boll. Zool. 45, 89-101. De Leeuw, R., Smit-van Dijk, W , Zigterman, J.W.J., van der Loo, J.C.M., Lambert, J.G.D., and Goos, H.J.Th. (1985). Aromatase, estrogen 2-hydroxylase, and catechol-O-methyltransferase 194 activity in isolated, cultured gonadotropic cells of mature African catfish, Clarias gariepinus (Burcheli;. Gen. Comp. Endocrinol. 60, 171-177. De Leeuw, R, Goos, H.J.Th, and Van Oordt, P.G.W.J. (1986a).The dopaminergic inhibition of the gonadotropin-releasing hormone-induced gonadotrpin release: an //; vitro study with fragments and cell suspensions from pituitaries of the African catfish, Claria gariepinus (Burcheli). Gen. Comp. Endocrinol. 63, 171-177. De Leeuw, R, Wurth, Y.A., Zandbergen, M.A.,Peute, J., and Goos, H.J.Th. (1986b). The effects of aromatizable androgens, non-aromatizable androgens, and estrogens on gonadotropin release in castrated African catfish, Clarias gariepinus (Burcheli): A physiological and structural study. Cell Tissue Res. 243, 587-594. Depeche, J., and Sire, O (1982). In vitro metabolism of progesterone and 17a-hydroxy-progesterone in the testis of the rainbow trout, Salmon gairdneri R., at different stages of spermatogenesis. Reprod. Nutr. Develop. 22, 427-438. Donaldson, EM. (1996). Manipulation of reproduction in farmed fish. Animal Reproduction Science 42, 381-392. Donaldson, E M , Hunter, G.A., and Dye, H.M. (1981/1982). Induced ovulation in coho salmon (Oncorhynchus kisutch). III. Preliminary study on the use of the antiestrogen tamoxifen. Aquaculture 26, 143-154. Dorrington, J.H., and Armstrong, DT. (1979). Effects of FSH on gonadal function Recent Prog. Horm. Res. 35, 301-342. Dye, H M , Sumpter, J.P., Fagerlund, U.H.M. and Donaldson, EM. (1986). Changes in reproductive parameters during the spawning migration of pink salmon, Oncorhynchus gorbuscha (Walbaum). J. Fish Biol. 29, 167-176. Elbrecht, A. and Smith, R. (1992). Aromatase enzyme activity and sex determination in chickens. Science 255, 467-470. Fitzpatrick, M.S., Van Der Kraak, G. and Schreck, C. (1986). Profiles of plasma sex steroids and gonadotropin in coho salmon, Oncorhynchus kisutch, during final maturation. Gen. Comp. Endocrinol. 62,437-451. Fitzpatrick, M.S., Redding, J.M,, Ratt, F.D. and Schreck, C. (1987). Plasma testosterone concentration predicts the ovulatory response of coho salmon (Oncorhynchus kisutch) to gonadotropin-releasing hormone analog. Can. J. Fish. Aquat. Sci. 44, 1351-1357. Fostier, A. (1995). Regulation of aromatase activity in the rainbow trout, Oncorhynchus mykiss, ovary. In: Reproductive Physiology of Fish 5th International Symposium (F W. Goetz and P.Thomas eds), July 2-8 1995, p. 293-295. Univ. of Texas. 195 Fostier, A., and Jalabert, B. (1986). Steroidogenesis in rainbow trout {Salmo gairdneri) at various preovulatory stages: changes in plasma hormone levels and in vivo and in vitro responses of the ovary to salmon gonadotropin. Fish Physiol. Biochem. 2, 87-99. Fostier, A., and Baek, H. (1994). Inhibition of production of maturation inducing steroid in rainbow trout granulosa cells: effect of oestradiol on gonadotropin stimulated 20(3-hydroxysteroid dehydrogenase activity. Reprod. Nutr. Develop. 33, 81-82. Fostier, A., Jalabert, B., Billard, R., Breton, B., and Zohar, Y. (1983). Hormonal control of oocyte maturation and ovulation in fishes. In: Fish Physiology (W.S. Hoar, D.J. Randall and E.M. Donaldson, Eds.) IXA, 277-372. Academic Press, New York. Goudie, C.A. (1994). Production of monosex fish population: The channel catfish model. In: High Performance Fish: Proceedings of an International Fish Physiology Symposium, held at the University of British Columbia in Vancouver, Canada, July 16-21, 1994"(Mackinlay, D.D. Ed.), pp.150-155. Fish Physiology Association. Hamazaki, T., Nagahama, Y., Iuchi, I, and Yamagamai, K. 1989. A glycoprotein from the liver constitutes the inner layer of the egg envelope (zona pellucida) of the fish, Oryzia latipes. Dev. Biol. 133,101-110. Hillier, S.G., Van Den Boogaard, A.M.J., Reichert, L.E.Jr., and Van Hall, E.V. (1980). Alterations in granulosa cell aromatase activity accompanying preovulatory follicular development in the rat ovary with evidence that 5a-reduced C19 steroids inhibit aromatase reaction in vitro. J. Endocrinol. 84, 409-419. Hyllner, S.J. and Haux, C. (1995). Vitelline envelope proteins in teleost fish. In: Reproductive Physiology of Fish 5th International Symposium (F.W. Goetz and P.Thomas eds.), July 2-8 1995, p. 331-335. Univ. of Texas. Ho, S.M. (1987). Endocrinology of vitellogenesis. In: Hormones and Reproduction in Fishes, Amphibians, and Reptiles (D.O. Norris, and R.E. Jones eds). 145-169. Plenum Press New York. Hsueh, A.J.W., Billig, H., and Tsafriri, A. (1994). Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocrine reviews 15, 707-724. Idler, D.R., Fagerlund, U.H.M., and Ronald, A.P. (1960). Isolation of pregnen-4-ene-17a-20(3-diol-3-one from, plasma of Pacific salmon Oncorhynchus nerka. Biochem. Biophys. Res. Commum. 2, 133-137. Idler, D.R., Hwang, S.J., Crim, L.W., and Reddi, D. (1981). Determination of sexual maturation stages of atlantic salmon Salmo salar captured at sea. Can. J. Fish. Aquat. Sci. 38, 405-413. Jalabert, B., and Fostier, A. (1984). The modulatory effect in vitro of oestradiol-17(3, testosterone or Cortisol on the output of 17a-hydroxy-20(3-dihydroprogesterone by rainbow trout (Salmo 196 gairdneri) ovarian follicles stimulated by the maturational gonadotropin s-GtH. Reprod. Nutr. Develop 24, 127-136. Jalabert, B , Goetz, F.W., Breton, B , Fostier, A., and Donaldson, EM. (1978). Precocious induction of oocyte maturation and ovulation in coho salmon, Oncorhynchus kisutch. J. Fish. Res. Board Can 35, 1423-1429 Kagawa, H. (1994). Oogenesis. In: Biochemistry and molecular biology of fishes (P.W. Hochachkaand TP. Mommsen, Eds.)3, 291-304. Elsevier, Amsterdam. Kagawa, H., Young, G. and Nagahama, Y. (1983). Relationship between seasonal plasma 17p-estradiol and testosterone levels and in vitro production by ovarian follicles of amago salmon (Oncorhynchus rhodurus). Biol. Reprod. 29, 301-309. Kagawa, H., Young, G. and Nagahama, Y. (1984). In vitro estradiol-170 and testosterone production by ovarian follicles of Goldfish, Carassius auratus. Gen. and Comp. Endocrinol. 54, 139-143. Kah, O., Anglade, I., Linard, B , Bennani, S., Pakdel, F. and Saligaut, C. (1995). Neuro-endocrine regulation of gonadotropin release in fish: What does What? In " St. Andrews Meeting, 3-7 April 1995, Animal and Cell Abstracts", p 46. The Trouser Press. Kanamori, A., and Nagahama, Y. (1988). Involvement of 3',5'-cyclic adenosine monophosphate in the control of follicular steroidogenesis of amago salmon (Oncorhynchus rhodurus). Gen. Comp. Endocrinol 72, 39-53. Kanamori, A., Adachi, S., and Nagahama, Y. (1988). Developmental changes in steroidogenic responses of ovarian follicles of amago salmon (Oncorhynchus rhodurus) to chum salmon gonadotropin during oogenesis. Gen. Comp. Endocrinol. 72, 13-24. Kaplan, ME., and McGinnis, MY. (1989). Effects of ATD on male sexual behavior and androgen receptor binding: A reexamination of the aromatization hypothesis. Horm. Behav. 23, 10-26 Khalil, M.W., Morley, P., Glasier, M.A., Armstrong, D.T., and Lang, T (1989). Formation of 4-oestrene-3,17-one (19-norandrostenedione) by porcine granulosa cells in vitro is inhibited by the aromatase inhibitor 4-hydroxyandrostenedione and the cytochrome P-450 inhibitors aminoglutethimide phosphate and ketoconazole. J. Endocrinol. 120, 251-260. Lambert, J.G.D. (1967). Steroidproductie in het ovarium van Poecilia reticulata. Ph.D. Thesis. Universiteit te Utrecht, 97 p. Lambert, J.G.D , and van Bohemen, C.G (1980). Oestrogen synthesis in the female trout Salmo gairdneri. Gen. Comp. Endocrinol. 40, 323. Lambert, J.G.D., and van Oordt, P.G.W.J. (1982). Catecholoestrogens in the brain of the female rainbow trout Salmo gairdneri . Gen. Comp. Endocrinol. 46, 401. 197 Lambert, J.G.D., Goos, H.J.Th., and Van Oordt, P.G.W.J. (1984). Aromatase activity in the brain of the rainbow trout, Salmo gairdneri. Gen. Comp. Endocrinol. 53, 459. Lance, VA. and Bogart, M H (1992). Disruption of ovarian development in alligator embryos treated with aromatase inhibitor. Gen. Comp. Endocrinol. 86, 59-71. Linard, B , Bennani, S. and Saligaut, C. (1995a). Involvement of estradiol in a catechol-amine inhibitory tone of gonadotropin release in the rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 99,192-196. Linardi, B , Anglade, I., Bennani, S., Salbert, G , Navas, J.M., Bailhache, T , Pakdel, F., Jego, P., Valotaire, Y., Saligaut, C. and Kah, 0. (1995b). Some insights into the sex steroid feedback mechanism in trout. In: Reproductive Physiology of Fish 5th International Symposium (F.W. Goetz and P.Thomas eds), July 2-8 1995, p. 49-51. Univ. of Texas. Linardi, B., Anglade, I., Corio, M , Navas, J.M., Pakdel, F., Saligaut, C , and Kah, O. (1996). Estrogen receptors are expressed in a subset of tyrosine hydroxylase-positive neurons of the anterior preoptic region in the rainbow trout. Neuroendocrinology, 63, Loir, M. (1988). Trout Sertoli and Leydig cells: isolation, separation and culture. Gamete Res. 20, 437-458. Loir, M. (1990a). Trout steroidogenic testicular cells in primary culture. Changes in free and conjugated androgens and progestogen secretions: effects of gonadotropin, serum, and lipoproteins. Gen. Comp. Endocrinol. 78, 347-387. Loir, M. (1990b). Trout steroidogenic testicular cells in primary culture. II. Steroidogenic activity of interstitial cells, and spermatozoa. Gen. Comp. Endocrinol. 78, 388-398. Louvet, J.P., Harman, S.M., Schrieber, JR., and Ross, G.T. (1975). Evidence of a role of androgens in follicular maturation. Endocrinology 97, 366-372. Matsuyama, M., Adachi, S., Nagahama, Y., and Matsuura, S. (1988). Diurnal rhythm of oocyte development and plasma steroid hormone levels in the female red sea bream, Pagrus major, during the spawning season. Aquaculture 73, 357-372. Mayer, I., Borg, B., Berglung, I., and Lambert, J.G.D. (1991). Effects of castration and androgen treatment on aromatase activity in the brain of mature male atlantic salmon (salmo salar L.) parr Gen. Comp. Endocrinol. 82, 86-92. Mayer, I., Lundqvist, H., Berglung, I., Schmitz, M., Schulz, R, and Borg, B. (1990). Seasonal endocrine changes in Baltic salmon, Salmon salar, immature parr and mature male parr. I. Plasma levels of five androgens, 17a-hydroxy-20fi-dihydroprogesterone, and 17P-estradiol. Can. J. Zool. 68, 1360-1365. 198 McEwen, B.S., Davis, P.G., Parsons, B, and Pfaff, D.W. (1979). The brain as a target for steroid hormone action. Ann. Rev. Neurosci. 2, 65-112. McEwen, B S , Biegon, A., Davis, P.G., Krey, L C , Luine, V.N, McGinnis, M Y , Paden, C M , Parsons, B, and Rainbow, T.C. (1982). Steroid hormones, humoral signals which alter brain cell properties and functions. Rec. Prog. Horm. Res. 38, 41-92. Miura, T , Yamauchi, K, Takahashi, H , and Nagahama, Y. (1992). The role of hormones in the acquisition of sperm motility in salmonid fish. J. Exp. Zool 261, 359-363. Miwa, S, Yan, L , and Swanson, P. (1994). Localization of two gonadotropin receptors in the salmon gonad by in vitro Iigand autoradiography. Biol. Reprod 50, 629-642. Morrison, CM. (1990). Histology of the Atlantic cod, Gadus morhua: an atlas. Part three. Reproductive tract. Can. Spec. Publ. Fish. Aquat. Sci. 110, 177p. Naftolin, F , Ryan, K.J, Davies, I.J, Reddy, V V , Flores, F , Petro, Z , Kuhn, M , White, R.J, Takaoka, Y , and Wolin, L. (1975). The formation of estrogens by central neuroendocrine tissues. Rec. Prog. Horm. Res. 31, 295-319. Nagahama, Y. 1983. The functional morphology of teleost gonads. In: Fish Physiology, IXA, (Hoar, W.S, Randall, D.J and Donaldson, EM. eds), p.223-275. Academic Press, New York. Nagahama, Y. (1987a). Gonadotropin action on gametogenesis and steroidogenesis in teleost gonads. Zoological Science 4, 209-222. Nagahama, Y. (1987b). 17a-20p-dihydroxy-4-pregnen-3-one: A Teleost Maturation-Inducing Hormone. Develop. Growth and Differ. 29, 1-12. Nagahama, Y. (1993). Regulation of oocyte maturation in aquatic animals: the comparative and general aspects. Biology International 28, 27-32. Nagahama, Y , and Yamashita, M. (1989). Mechanisms of synthesis of 17cc-20P-dihy-droxy-4-pregnen-3-one, a teleost maturation-inducing substance. Fish Physiology and Biochemistry 7,193-200. Nagahama, Y , Adachi, S, Tashiro, F, and Grau, E.G. (1982). Some endocrine factors affecting the development of seawater tolerance during the parr-smolt transformation of the amago salmo, Oncorhynchus rhodorus. Aquaculture 28, 81-90. Nagahama, Y , Kagawa, H , and Young, G. (1985). Stimulation of 17a-20P-dihydroxy-4-pregnen-3-one production in the granulosa cells of amago salmon, Oncorhynchus rhodurus, by cyclic nucleotides J. Exp. Zool. 236, 371-375. Nagahama, Y , Yoshikuni, M , Yamashita M , Sakai, N, and Tanaka, M. (1993). Molecular endocrinology of oocyte growth and maturation in fish. Fish Physiology 199 and Biochemistry 11, 3-14. Ng, T.B., and Idler, D.R. (1983). Yolk formation and differentiation in teleost fishes. In: Fish Physiology, Vol.9A, (Hoar, W.S., Randall, D.J. and Donaldson, EM. eds), p.373-404. Academic Press, New York. Oppen-Berntsen, DO., Gram-Jensen, E., and Walther, B.T. 1992. Zona radiata proteins are synthesised by the rainbow trout (Oncorhynchus mykiss) hepatocytes in response to estradiol- 17p J . Endocrinol. 135,293-302. Pasmanik, M., and Callard, G.V. (1985). Aromatase and 5a-reductase in the teleost brain, spinal cord, and pituitary gland. Gen. Comp. Endocrinol. 60, 244-251. Patino, R., and Schreck, C.B. (1986). Sexual dimorphism of plasma sex steroid levels in juvenile coho salmon, Oncorhynchus kisutch, during smoltification. Gen. Comp. Endocrinol. 61, 127-133. Peng. C , Peter, R.E., Chang, J.P. 1994. Neuroendocrine control of gonadotropin secretion and ovulation in teleosts. In: In: High Performance Fish. Proceedings of an International Fish Physiology Symposium, held at the University of British Columbia in Vancouver, Canada, July 16-21, 1994 (DD. Mackinlay, Ed), pp. 131-136. Fish Physiology Association. Peter, R.E. (1983). The brain and neurohormones in teleost reproducttion. In "Fish Physiology" (W.S. Hoar, D.J. Randall and EM. Donaldson, Eds.) IXA, 97-135. Academic Press, New York. Peter, RE., Chang, J.P., Nahorniak, C.S., Omeljaniuk, R.J., Sokolowska, M., Shih, S.H. and Billard, R. (1986). Interactions of catecholamines and GnRH in regulation of gonadotropin secretion in teleost fish. Recent Progr. Horm. Res. 42, 513-548. Peter, RE., Trudeau, V.L., Sloley, B.D., Peng, C. and Nahorniak, CS. (1991). Actions of catecholamines, peptides and sex steroids in regulation of gonadotropin-II in the goldfish. ^"Reproductive Physiology of Fish 1991" (A.P. Scott, J.P. Sumpter, DE. Kime, M.S. Rolfe, Eds), pp.30-34. Fish Symp.91, Sheffield. Peter, R.E , Lin, H.R., Van Der Kraak, G , and Little, M. (1993). Releasing hormones, dopamine antogonists and induced spawning. In: Recent advance in aquaculture IV (J.M. Muir and R.J. Roberts eds), pp 25-30 Blackwell Scientific Publications, Ontario. Pickering, A D , and Pottinger, T.G. (1983). Seasonal and diel changes in plasma Cortisol levels of the brow trout. Gen. Comp. Endocrinol. 49, 232-239. Piferrer, F., Zanuy, S., Carrillo, M , Solar, 1.1., Devlin, RH. and Donaldson, EM. (1994). Brief treatment with an aromatase inhibitor during sex differentiation causes chromosomally female salmon to develop as normal, functional males The Journal of Experimental Zoology 270, 255-262 Piferrer, F., Baker, I.J. and Donaldson, EM. (1993). Effects of natural, synthetic, 200 aromatizable, and nonaromatizable androgens in inducing male sex differentiation in genotypic female chinook salmon (Oncorhynchus tshawytscha). Gen. Comp. Endocrinol. 91, 59-65. Planas, J.V. (1993). Hormonal regulation of gonadal steroidogenesis in salmonids. PhD Thesis, University of Washington, 168 p. Planas, J.V., and Swanson, P. (1995). Maturation-associated changes in the response of the salmon testis to the steroidogenic actions of gonadotropins (GTH I and GTH II) in vitro. Biology of Reproduction 52, 697-704. Planas, J.V., Athos, J., and Swanson, P. (1995). Regulation of ovarian steroidogenesis in vitro by gonadotropins during sexual maturation in coho salmon (Oncorhynchus kisutch). In: Reproductive Physiology of Fish 5 th International Symposium (F. W. Goetz and P.Thomas eds.), July 2-8 1995, p. 296-298. Univ. of Texas. Prat, F., Sumpter, J.P., and Tyler, C.R. (1996). Validation of radioimmunoassays for two salmon gonadotropins (GTH I and GTH II) and their plasma concentration throughout the reproductive cycle in male and female rainbow trout (Oncorhynchus mykiss). Biology of' Reproduction 54, 1375-1382. Richter, C.J.J., Eding, E.H., Goos, H.J.TH., De Leeuw, R, Scott, A.P. and Van Oordt, P.G.W.J. (1987). The effect of pimozide/LHRHa and 17a-hydroxyprogesterone on plasma steroid levels and ovulation in the african catfish, Clarias gariepinus. Aquaculture 63, 157-168. Sakai, N , Iwamatsu, T., Yamauchi, K., Suzuki, N , and Nagahama, Y. (1988). Influence of follicular development on steroid production in the medaka (Oryzias latipes) ovarian follicles in response to exogenous substrates. Gen. Comp. Endocrinol. 71, 516-523. Sakai, N , Ueda, H., Suzuki, N , and Nagahama, Y. (1989). Steroid production by amago salmon (Oncorhynchus rhodurus) testes at different developmental stages. Gen. Comp. Endocrinol. 75, 231-240. Sakai, N , Ueda, H., Suzuki, N., and Nagahama, Y. (1990). Involvement of sperm in the production of 17a-20P-dihydroxy-4-pregnen-3-one in the testis of spermiating rainbow trout, salmo gairdneri. Biomedical Res. 10, 131-138. Santen, R.J., Manni, A., Harvey, H. and Redmond, C. (1990). Endocrine treatment of breast cancer in women. Endocrine Reviews 11, 221-265. Schieweck, K , Bhatnagar, AS. and Matter, A. (1988). CGS 16949A, a new nonsteroidal aromatase inhibitor: effects on hormone-dependent and independent tumors in vivo. Cancer Research 48, 834-838. Schlinger, B.A., and Arnold, A.P. (1991). Brain is the major site of estrogen synthesis in a male songbird. Neurobiology 88, 4191-4194. 201 Schlinger, B.A, and Arnold, AP. (1993). Estrogen synthesis in vivo in the adult zebra finch: additional evidence that circulating estrogens can originate in brain. Endocrinology 133, 2610-2616. Schreck, C.B, and Hopwood, ML. (1974). Seasonal androgen and estrogen patterns in the goldfish, Carassius auratus. Trans. Am. Fish. Soc. 2, 375-378. Schreck, C.B, Lackey, R T , and Hopwood, ML. (1973). Plasma oestrogen levels in rainbow trout Salmon gairdneri Richardson. J. Fish Biol. 5, 227-230. Schulster, D, Burstein, S, and Cooke, B.A. (1976). Molecular Endocrinology of the steroid hormones. John Wiley & Sons, Toronto. 321 p. Schulz, R, and Blum, V. (1990). Steroid secretion of raibow trout testis in vitro, variation during the reproductive cycle. Gen. Comp. Endocrinol. 80, 189-198. Scott, A P , and Baynes, SM. (1982). Plasma levels of sex steroids in relation to ovulation and spermiation in rainbow trout Salmo gairdneri. In "Proceedings of the first International Symposium on Reproductive Physiology of Fish" (C.J.J. Richter, H.J. Goos, Eds), p. 103-106, Wageningen, Pudoc. Scott, AP. and Sumpter, J.P (1989). Seasonal variations in testicular germ cell stages and in plasma concentration of sex steroids in male rainbow trout (salmon gairdneri) maturing at two years old. Gen. Comp. Endocrinol. 73, 46-58. Scott, A P , Sheldrick, E L , and Flint, A.P.F. (1982). Measurement of 17a-20p-dihy-droxy-4-pregnen-3-one in plasma of trout (Salmo gairdneri Richardson): seasonal changes and response to salmon pituitary extract. Gen. Comp. Endocrinol. 46, 444-451. Scott, A P , Sumpter, J.P, and Hardiman, PA. (1983). Hormone changes during ovulation in the rainbow trout (Salmo gairdneri Richardson). Gen. Comp. Endocrinol. 49, 128-134. Selvaraj, N , Shetty, G , Vijayalaksmi, K, Bhatnagar, A S , and Moudgal, NR. (1994). Effect of blocking oestrogen synthesis with a new generation aromatase inhibitor CGS 16949A on follicular maturation induced by pregnant mare serum gonadotropin in the immature rat. J. Endocrinol. 142, 563-570. Siiteri, P.K, and Thompson, EA. (1975). Studies of human placental aromatase. J. Steroid Biochem. 6, 317-322. Sire, O, and Depeche, J. (1981). In vitro effect of a fish gonadotropinon aromatase and 170-hydroxysteroid dehydrogenase activities in the ovary of the rainbow trout (salmo gairdneri Rich). Reprod. Nutr. Develop. 21, 715-726. Slater, C H , Schreck, C.B, and Swanson, P. (1994) Plasma profiles of the sex steroids and gonadotropins in maturing female spring chinook salmon (Oncorhynchus Tshawytscha). Comp. Biochem. Physiol. 109A, 167-175. 202 Sower, S.A. and Schreck, C. (1982). Steroid and thyroid hormones during sexual matura-tion of coho salmon (Oncorhynchus kisutch) in seawater or fresh water. Gen. Comp. Endocrinol. 47, 42-53 Steele, R.E, Mellor, L.B, Sawyer, W.K, Wasvary, J.M. and Browne, L.J. (1987). In vitro and in vivo studies demonstrating potent and selective estrogen inhibition by the nonsteroidal aromatase inhibitor CGS 16949A. Steroids 50, 147-161. Sumpter, J.P, Tyler, C R , and Kawauchi, H. (1991). Actions of GTH I and GTH II on ovarian steroidogenesis in the rainbow trout, Oncorhynchus Mykiss. In: In: Proceedings of the fourth Internationl Symposium on the Reproductive Physiology of Fish (A.P. Scott, J.P. Sumpter, D.E. Kime, and M.S. Rolfe, Eds.), p.27. Fish Symp.91,Sheffield. Suzuki, K , Kawauchi, H. and Nagahama, Y. 1988a. Isolation of two distinct salmon gonadotropins from chum salmon pituitary glands. Gen. Com. Endocrinol. 71, 292-301. Suzuki, K , Kawauchi, H. and Nagahama, Y. 1988b. Isolation and characterization of subunits from two distinct salmon gonadotropins. Gen. Com. Endocrinol. 71, 302-306. Suzuki, K , Kawauchi, H. and Nagahama, Y. 1988c. Steroidogenic activity of of two distinct salmon gonadotropins. Gen. Com. Endocrinol. 71, 452-458. Suzuki, K, Kanamori, A , Nagahama, Y , and Kawauchi, H. (1988d). Development of salmon GTH I and GTH II radioimmunoassays. Gen. Comp. Endocrinol. 71, 459-467. Swanson, P. (1991). Salmon gonadotropins: reconciling old and new ideas. In: Proceedings of the fourth Internationl Symposium on the Reproductive Physiology of Fish (A.P. Scott, J.P. Sumpter, D.E. Kime, and M.S. Rolfe, Eds.), p. 2-7. Fish Symp.91,Sheffield. Swanson, P. (1994). Regulation of gametogenesis in fish by gonadotropins. In: High Performance Fish: Proceedings of an International Fish Physiology Symposium, held at the University of British Columbia in Vancouver, Canada, July 16-21, 1994 (D.D. Mackinlay, Ed.), pp. 131-136. Fish Physiology Association. Swanson, P, Bernard, M , Nozaki, M , Suzuki, K, Kawauchi, H , and Dickhoff, W.W. (1989). Gonadotropins I and II in juvenile coho salmon. Fish Physiol. Biochem. 7, 169-176. Swanson, P, Suzuki, K, Kawauchi, H. And Dickhoff, W.W. 1991. Isolation and characterization of two salmon gonadotropins, GTH I and GTH II. Biol. Reprod. 44, 29-38. Timmers, R.J.M. (1988). Gonadal steroids their metabolism and interaction with dopamine in the brain of the African catfish, Claria gariepinus. Ph.D thesis, Utrecht. 156 p. Timmers, R.J.M, and Lambert, J.G.D. (1987). Measurement of aromatase activity in the brain of the African catfish, Clarias gariepinus - A comparison of two assay methods. Comp. Biochem. Physiol. 88B, 453-456. 203 Timmers, R.J.M., Lambert, J.G.D., Peute, J., Vullings, H.G.B., and van Oordt, P.G.W.J. (1987). Localization of aromatase in the brain of the male African catfish, Clarias gariepinus Burcheli), by microdissection and biochemical identification. J. Comp. Neurol. 258, 368-377. Timmers, R.J.M., Granneman, J.C.M., Lambert, J.G.D., and van Oordt, P.G.W.J. (1988). Estrogen-2-hydroxylase in the brain of the male African catfish, Clarias gariepinus. Gen. Comp. Endocrinol. 72, 190-203. Tyler, C. (1991). Vitellogenesis in salmonids. In "Reproductive Physiology of Fish 1991" (A.P. Scott, J.P. Sumpter, D.E. Kime, M.S. Rolfe, Eds.), pp.295-302. Fish Symp.91, Sheffield. Tyler, C, and Sumpter, J.P. (1996). Oocyte growth and development in teleosts. Rev. Fish Biol. And Fish. 6, 287-318. Tyler, C.R., Sumpter, J.P. and Witthames, P.R. (1990). The dynamics of oocyte growth during vitellogenesis in the rainbow trout (Oncorhynchus mykiss). Biol. Reprod. 43, 202-209. Tyler, C.R., Sumpter, J.P., Kawauchi, H., and Swanson, P. (1991). Involvement of gonadotropin into vitellogenic oocytes of the rainbow trout, Oncorhynchus mykiss. Gen. Comp. Endocrinol. 84, 291-299. Truscott, B., Idler, D.R., So, Y.P., and Walsh, J.M. (1986). Maturational steroids and gonadotropin in upstream migratory sockeye salmon. Gen. Comp. Endocrinol. 62, 99-110. Ueda, H., Kambegawa, A., and Nagahama, Y. (1984). In vitro 11-ketotestosterone and 17a-20P-dihydroxy-4-pregnen-3-one production by testicular fragments and isolated sperm of raibow trout, Salmo gairdneri. J. Exp. Zool. 231, 435-439. Ueda, H., Kambegawa, A., and Nagahama, Y. (1985). Involvement of gonadotropin and steroid hormones in spermiation in the amago salmon, Oncorhynchus rhodurus, and goldfish, Carassius auratus. Gen. Comp. Endocrinol. 59, 24-30. Ueda, H., Young, G., Crim, L.W., Kambegawa, A., and Nagahama, Y. (1983). 17a-20p-dihydroxy-4-pregnen-3-one: plasma levels during sexual maturation and in vitro production by the testis of amago salmon (Oncorhynchus rhodurus) and rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 51, 106-112. Wade, J., Schlinger, B.A., Hodges, L. and Arnold, A.P. (1994). Fradazole: a potent and specific inhibitor of aromatase in the zebra finch brain. Gen. Comp. Endocrinol. 94, 53-61. Wallace, R.A. (1985). Vitellogenesis and oocyte growth in nonmammalian vertebrates. In "Developmental Biology"(L.W. Browder, Ed) 1, 127-177. Plenum Press, New York. Wennstrom, K.L. and Crews, D. (1995). Making males from females: The effects of aromatase inhibitors on a parthenogenetic species of whiptail lizard. Gen. Comp. 204 Endocrinol. 99, 316-322 Wickings, E.J, Middleton, M.C, and Hillier, S.G. (1987). Non-steroidal inhibition of granulosa cell aromatase activity /// vitro. J. Steroid Biochem. 26, 641-646. Wilson, K, and Goulding, K.H. (1992/ A Biologist's giude to principles and techniques of practical biochemistry. State Mutual Book and Periodical Service, USA, 396 p. Van Der Kraak, G. (1984). 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 gonadotropin binding sites. Ph.D. Thesis, The University of British Columbia, 178 p. Van Der Kraak, G , and Donaldson, EM. (1986). Steroidogenic capacity of coho salmon ovarian follicles throughout the periovulatory period. Fish Physiol. Biochem. 1, 179-186. Van Der Kraak, G , Dye, H.M. and Donaldson, EM. (1984). Effects of LH-RH and Des-Gly10[D-Ala6]LH-RH-Ethylamide on plasma sex steroid profiles in adult female coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 55, 36-45. Van Der Kraak, G , Lin, H-R, Donaldson, E M , Dye, H.M and Hunter, G A (1983). Effects of LH-RH and Des-Gly10[D-Ala6]LH-RH-Ethylamide on plasma gonadotropin levels and oocyte maturation in adult female coho salmon (Oncorhynchus kisutch). Gen. Comp. Endocrinol. 49, 470-476. Van Der Kraak, G , Dye, H.M, Donaldson, E M , and Hunter, G.A. (1985). Plasma gonadotropin, 17f3-estradiol, and 17c-20p-dihydroxy-4-pregnen-3-one levels luteinizing hormone-releasing hormone analogue and gonadotropin induced ovulation in coho salmon (Oncorhynchus kisutch). Can. J. Zool. 63, 824-833. Van Der Kraak, G , Suzuki, K , Peter, R E , Itoh, H , and Kawauchi, H. (1992). Properties of common carp gonadotropin I and gonadotropin II. Gen. Comp. Endocrinol. 85, 217-229. Vermeulen, G.J. 1994. Testicular steroids and catecholamines in the brain of the African catfish, Claria gariepinus, in relation to gonadotropin release. Ph.D. Thesis, Utrecht, 119 p. Vizziano, D, Le Gac, F, and Fostier, A. (1995). Synthesis and regulation of 17a-hydroxy-20P-dihydroxy-progesterone in immature males of Oncorhynchus mykiss. Fish Physiol. Biochem. 14, 289-299. Vizziano, D, Fostier, A, Le Gac, F , and Loir, M. (1996). 20P-Hydroxysteroid dehydrogenase activity in nonflagellated germ cells of rainbow trout testis. Biology of Reproduction 54, 1-7. Yamauchi, K, Kagawa, H, Ban, M , Kasahara, N. and Nagahama, Y. (1984). Changes in plasma estradiol-17P and 17oc-20P-dihydroxy-4-pregnen-3-one levels during final oocyte maturation of the masu salmon, Oncorhynchus rhodurus. Bull. Jap. Soc. Sci. Fish. 50: 2137 205 Yan, L , Swanson, P., and Dickhoff, WW. (1992). A two-receptor model for salmon gonadotropins (GTH I and GTH II) Biol. Reprod. 47, 418-427 Young, G , Kagawa, H , and Nagahama, Y. (1983a). Evidence for a decrease in aromatase activity in the ovarian granulosa cells of amago salmon (Oncorhynchus rhodurus) associated with final oocyte maturation. Biol. Reprod. 29, 310-315. Young, G , Crim, L.W., Kagawa, H , Kambegawa, A., and Nagahama, Y. (1983b). Plasma 17a-20P-dihydroxy-4-pregnen-3-one levels during sexual maturation of amago salmon (Oncorhynchus rhodurus). correlation with plasma gonadotropin and in vitro production by ovarian follicles. Gen. Comp. Endocrinol. 51, 96-105. Young, G , Adachi, S., and Nagahama, Y. (1986). Role of ovarian thecal and granulosa layers in gonadotropin-induced synthesis of a salmonid maturation-inducing substance (17a,20(J-dihydroxy-4-pregnen-3-one). Dev. Biol 118, 1-8. Zohar, Y., Breton, B , and Fostier, A. (1986). Short-term profiles of plasma gonadotropin and estradiol-170 levels in the female rainbow trout, from early recrudescence and throughout vitellogenesis. Gen. Comp. Endocrinol. 64, 172-188. Zumpe, D, Bonsall, R.W., and Michael, R.P. (1993). Effects of the nonsteroidal aromatase inhibitor, fadrozole, on the sexual behavior of male cynomolgus monkeys (Macaca fascicularis). Hormones and Behavior 27, 200-215. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0088288/manifest

Comment

Related Items