Effects of insulin-like growth factor-I on final oocyte maturation and steroid production in Fundulus heteroclitus

Recent evidence has indicated the presence of IGF-I and IGF-I receptors in mammalian and teleost ovarian follicles. Since growth hormone (GH), which can be secreted from the pituitary concomitant with a gonadotropin as a response to gonadotropin-releasing hormone, generally acts to release IGF-I from tissues including the ovary, the effect of IGF-I itself on ovarian steroidogenesis and oocyte maturation was investigated in the model teleost, Fundulus heteroclitus. IGF-I was found to be without effect on ovarian follicle steroidogenesis, but initiated oocyte maturation in a dose-dependent manner even more rapidly and effectively than 17,20-dihydroxy-4-pregnene-3-one (DHP), the naturally occurring maturation-inducing steroid. IGF-II also induced oocyte maturation in a dose-dependent manner. IGF-I induction of oocyte maturation occurred in the absence of DHP production by the granulosa cells (which is normally stimulated by gonadotropin), and could be inhibited by cycloheximide but not actinomycin D, thus implicating the role of protein synthesis. These results suggest that GH-stimulated release of ovarian IGF-I may have an even more direct role than DHP on the reinitiation of oocyte maturation.     

Insulin and IGF-I receptors and tyrosine kinase activity in carp ovaries: changes with reproductive cycle

Insulin and insulin-like growth factor I (IGF-I) receptors from carp ovaries were semipurified with wheat germ agglutinin at different moments of the reproductive cycle and their binding characteristics and tyrosine kinase activity were studied. Specific receptors for insulin and IGF-I were found. IGF-I receptors presented higher binding (23.8 ± 1.5%), number of receptors (965 ± 20fm/mg) and affinity (K_D 0.24 ± 0.03nM) than those shown for insulin receptors (4.1 ± 1%, 530 ± 85fm/mg and 0.85 ± 0.1nM, respectively). Insulin and IGF-I receptors have a tyrosine kinase activity which is not different from that found in muscle of the same species. Seasonal changes were found in binding, with maximum values for insulin and IGF-I reached at the end of pre-spawning period (June). However, while IGF-I binding was observed in all stages, insulin binding decreased in autumn and disappeared in winter, which suggests a different role for the two peptides in ovarian physiology.     

The ovarian regulation of ovulation in teleost fish

Ovulation is the release of a mature oocyte from its follicle wall enclosure in the ovary. This process requires the separation of the oocyte from the granulosa layer, the rupture of the follicle wall and the active expulsion of the oocyte through the rupture site. Results of experiments on various vertebrates, including fish, have shown that the control of these processes may involve the cooperative action of a number of ovarian regulators including proteases, protease inhibitors, progestational steroids, eicosanoids, catecholamines and vasoactive peptides. We have used two teleost models, the brook trout (Salvelinus fontinalis) and the yellow perch (Perca flavescens) to study the mechanism and control of ovulation in fish. Using subtractive cDNA cloning, a family of ovarian and ovulation specific mRNAs (TOPs = trout ovulatory proteins) was isolated from the brook trout ovary. These mRNAs have not previously been observed in the ovary of any vertebrate species; however, the proteins they code for have significant sequence homology to a group of mammalian protease inhibitors called antileukoproteinases. These inhibitors have been isolated from several mammalian mucosal tissues and their function may be to protect the mucosal lining from nonspecific degradation by proteases released from infiltrating leukocytes. The ovarian proteins encoded by the TOP mRNAs have now been characterized by Western blotting using antibodies derived against recombinant TOPs. Given the similarity of TOPs to antileukoproteinases, one function of TOPs may be to regulate proteolysis at the time of ovulation. In yellow perch, the maturational steroid, 17,20-dihydroxy-4-pregnen-3-one (17,20-PG), stimulates both germinal vesicle breakdown and ovulation in vitro. The stimulation of ovulation can be blocked by indomethacin, a prostaglandin endoperoxide synthase inhibitor. Thus, it appears that 17,20-PG acts through the production of an eicosanoid that is most likely a primary prostaglandin. This hypothesis is further supported by the observations that (1) a direct correlation exists between indomethacin levels that block ovulation and those that block primary prostaglandin synthesis in the ovary; (2) ovulation can be restored in indomethacin-blocked incubates with primary prostaglandins; (3) PGF levels increase at the time of ovulation in incubations of yellow perch follicles stimulated with 17,20-PG; and (4) the stimulation of PGF by steroids in the ovary is specific for 17,20-PG. Finally, 17,20-PG-stimulated ovulation and follicular prostaglandin synthesis requires the close interaction of extrafollicular tissue and other follicle wall layers.     

Biochemical characterization of growth hormone receptor in rainbow trout (Oncorhynchus mykiss) before and after purification

The aim of this work was to verify if, compared to mammals, the lower molecular weight of GH-R previously reported in salmonid is real or due to the experimental process. For this purpose, we compared the apparent molecular weight of GH-R, obtained by SDS-PAGE after cross-linking with ⁵⁰ I-rtGH, obtained from rainbow trout crude liver membrane preparation, incubated in different buffers with those obtained after purification with affinity chromatography.Using crude liver membrane preparation, two specific bands of ⁵⁰ I-rtGH-protein complex were observed: the major one corresponds to a MW of 70 kDa and the minor one to 45 kDa. However, the pattern of electrophoresis varied according to the different incubation buffers tested. Digestion of the cross-linked complex with -galactosidase and phospholipase did not significantly modify the position of bands, whilst N-glycosidase F induced a large smear including 4 more pronounced bands (50, 65, 97 and > 130 kDa), the heavier band corresponding to the most intensive signal.GH receptors were purified using solubilisation and affinity chromatography. The yield of the liver GH-R from crude liver membrane preparation by the solubilization technique was optimized (48%) using Triton 1% for 1 h (12°C ). Specific binding sites in the solubilized membrane proteins were saturable when incubated with increasing ⁵⁰ I-rtGH concentrations, and revealed a high affinity constant (Ka=0.7×10⁹ M^–1). After affinity chromatography, specific binding activity was increased 64,000 fold. However, the purity of the preparation was partial and the purification yield was very low (about 0.3%). This enriched fraction, analysed by SDS-PAGE after cross-linking, showed a very intense band (about 63 kDa) which disappeared with an excess of cold rtGH.These results suggest that the lower molecular weight observed in salmonid (41 kDa), compared to mamals, is not due to the experimental process. The significance of GH-R size difference between salmonids and mammals is discussed.     

Isolation and expression of rainbow trout (Oncorhynchus mykiss) ovarian cDNA encoding 3β-hydroxysteroid dehydrogenase/Δ5−4-isomerase

A distinct shift in steroidogenesis from testosterone to 17α-hydroxyprogesterone occurs in the salmonid ovarian thecal cell layers immediately prior to oocyte maturation, and is a prerequisite for the production of 17α,20β-dihydroxy-4-pregnen-3-one (maturation-inducing hormone of salmonid fishes) by granulosa cells during oocyte maturation. 17α-Hydroxylase/17,20-lyase cytochrome P-450 (P-450_17α) and 3β-hydroxysteroid dehydrogenase/Δ^5?4-isomerase (3β-HSD) are the two major steroidogenic enzymes involved in the production of 17α-hydroxyprogesterone and testosterone. Using mammalian cDNA probes, we isolated and characterized full-length cDNAs encoding these two enzymes from a rainbow trout (Oncorhynchus mykiss) ovarian thecal cell cDNA library. The cloning of 2.4-kilobase cDNA encoding P-450_17α and transient expression of this clone in nonsteroidogenic monkey kidney tumor COS-1 cells have recently been reported (Sakai et al. 1992). We have isolated a 1.4-kilobase cDNA which is hybridized to the mammalian 3β-HSD cDNAs. Expression of this cDNA in COS-1 cells led to the production of an enzyme which is capable of converting dehydroepiandrosterone to androstenedione. In this study, enzymatic activities and expression of rainbow trout ovarian P-450_17α and 3β-HSD are discussed in relation of the steroidogenic shift occurring in the ovarian follicle layers.     

Morphology,sex steroid level and gene expression analysis in gonadal sex reversal of triploid female (XXX) rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>)

In non-mammalian vertebrates, estrogens and expressions of cyp19a1 and foxl2 play critical roles in maintaining ovary differentiation and development, while dmrt1 and sox9 are male-specific genes in testicular differentiation and are highly conserved. In order to deeply understand the morphological change, sex steroids level and molecular mechanism of triploid female gonadal reversal in rainbow trout, we studied the ovary morphology, tendency of estradiol-17β (E2) and testosterone (T) levels and the relative expressions of dmrt1, cyp19a1, sox9 and foxl2 in juvenile and adult fish. Our results demonstrated that the development of triploid female gonads in rainbow trout went through arrested development, oocytes dedifferentiation, ovary reconstruction and sex reversal finally. During early gonadal development (154–334 days post-fertilization), the expressions of foxl2 and cyp19a1 increased linearly, while expressions of dmrt1 and sox9 were extremely suppressed, and E2 level was higher, while T level was lower. During the mid-to-late period of triploid female gonadal development (574–964 days post-fertilization), the expressions of dmrt1 and sox9 remained high and were very close to the quantity of diploid male genes, and T levels were even reaching diploid male plasma concentrations, while expressions of cyp19a1 and foxl2 were decreased, leading to decrease in E2 level. We realized that the development model of rainbow trout triploid female gonads was extremely rare, and the regulatory mechanism was very special. Genes involved in gonadal development and endogenous estrogens are pivotal factors in fish natural sex reversal.     

Gonadotropic hormone (GtH) receptors in the testis of the troutSalmo gairdneri: in vitro studies

A particulate fraction obtained from trout testis at the time of spermiation shows saturable binding sites for¹²⁵I-labeled salmon gonadotropin (¹²⁵I-GtH). Non-gonadal tissues (liver, muscle and spleen) did not demonstrate specific¹²⁵I-GtH binding. The tracer's specific activity was determined by the self-displacement method (18 to 30 Ci/g). Maximal specific binding ability of¹²⁵I-GtH varied from 20 to 30% of the labelled ligand added, depending on the hormone preparation. Specific binding of¹²⁵I-GtH to 20 mg of the testis membrane varied from 40 to 85% of the total binding depending on the method of membrane prepratation, and was competitively inhibited by concentrations of unlabelled GtH ranging from ca 1 to 1000 ng/ml of incubate. Gonadotropin of mammalian origin, ovine TSH or salmon prolactin competed only weakly, or not at all, for testicular gonadotropin binding sites (relative potencies s-GtH>>FSH=hCG>s-PRL>bTSH). Scatchard analysis of equilibrium binding studies shows that saturable gonadotropin binding was due to a class of high affinity binding sites (sites I Ka3×10¹⁰ M^–1) and possibly to a second class of lower affinity binding sites (sites II Ka=5 to 14×10⁸ M^–1). The binding capacity of sites I, as measured in enriched membrane preparations, was 45±18 fmoles/g of testis during the period of spermiation. The concentration of GtH required to obtain half maximal displacement of¹²⁵I-GtH in the binding studies was of the same order of magnitude as the apparent ED50 for GtH stimulation of 11-Cetotestosterone (11KT) secretion by trout testesin vitro. Mammalian LH and FSH were 100 to 1000 folds less potent than salmor GtH to increase 11 KT secretion.     

Extrapituitary gonadotropin-releasing hormone (GnRH) binding sites in goldfish

In teleosts, as in other vertebrates, the secretion of pituitary gonadotropin (GTH) is mediated by the hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH). Recent findings in teleosts indicate that GnRH receptors are not restricted to the pituitary gonadotropes and are also associated with somatotropes as well as being present in a number of other tissues. In the present study, we provide novel information on GnRH binding in a number of extrapituitary tissues in goldfish. However, we do not intend to provide full characterization of GnRH binding sites in various extrapituitary tissues in goldfish as this would clearly be outside the scope of this paper. In this study we examined GnRH binding in a number of extrapituitary tissues in goldfish and observed specific binding in ovary, testis, brain, liver and kidney. No specific GnRH binding was observed in muscle, skin, gut, gill and heart. In general, the present findings together with the results of other studies carried out in our laboratory demonstrate that mature goldfish ovary and testis contain two classes of GnRH binding sites, high affinity/low capacity and low affinity/high capacity sites with binding characteristics similar to those of the pituitary GnRH receptors. The brain of goldfish was also found to contain two classes of GnRH binding sites, a super-high affinity/low capacity and a low affinity/high capacity sites. Furthermore, study of goldfish liver and kidney demonstrated the presence of a single class of GnRH binding sites with characteristics different from those of pituitary, ovary, testis and brain. Overall, it is evident that goldfish contains a family of GnRH binding sites which can be classified into four groups based on binding affinities: 1) A class of high affinity binding sites present in the pituitary, ovary and testis, 2) a class of super high affinity sites so far only detected in the brain, 3) a class of intermediate-affinity GnRH binding sites in the liver and kidney, and 4) a class of low affinity binding sites present in all the tissues containing specific GnRH binding sites except for liver and kidney.     

Effects of unilateral ovariectomy on recruitment and growth of follicles in the rainbow trout,Oncorhynchus mykiss

Virgin female rainbow trout, Oncorhynchus mykiss, were unilaterally ovariectomised at various stages of ovarian development to investigate the effect of the removal of one ovary on subsequent recruitment and growth of follicles in the remaining ovary. The right ovary was removed from groups of 12–15 fish, 12, 7 and 4 months before they were due to ovulate, and the gonadosomatic index and follicle number and size determined just prior to ovulation. There were no differences in fecundity or follicle size in fish unilaterally ovariectomised at 12 and 7 months prior to ovulation compared to the controls. However, in the females unilaterally ovariectomised 4 months prior to ovulation, the remaining ovary either had the normal number of follicles for a single ovary but of a significantly larger size than follicles in the controls, or alternatively had almost 70% more than the normal number of vitellogenic follicles but comprising two distinctly different size populations. Differences in plasma oestradiol-17β concentrations at the final sample were seen only in females unilaterally ovariectomised 4 months prior to ovulation, where the levels were significantly lower than both the sham operated and control fish (p < 0.05). These data show that in the rainbow trout, complete compensatory ovarian hypertrophy following unilateral ovariectomy can occur throughout a major part of ovarian development, but that follicle recruitment is limited to stages up to (and therefore fecundity is determined by) mid-vitellogenesis.     

Insulin binding to isolated hepatocytes of Atlantic salmon and rainbow trout

The questions addressed in this study were: 1) whether insulin added to the incubation medium can down-regulate ¹²⁵I insulin binding to isolated hepatocytes of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss); 2) whether quantitative assessment of insulin processing can be made on isolated fish liver cells; 3) how ambient temperatures can affect insulin binding, and down-regulation of insulin receptors. After isolation and a short (up to 4h) “metabolic recovery period”, liver cells were used either directly in ¹²⁵I insulin binding assay or first preincubated for 18h at 4°C or for 3h at 15°C, with or without mammalian or salmon insulin in concentrations ranging from 1 to 1000 nM. Preincubation at 15°C, decreased binding capacity (number of binding sites per liver cell) in all five independent hepatocyte preparations treated with 1000 nM insulin and in four out of five preparations treated with 100 nM insulin. At 4°C insulin binding sites were down-regulated in less than 50% of all hepatocyte preparations and only in the presence of 1000 nM insulin. Differential quantitive assessment was made of a) intact free insulin; b) insulin degraded; c) intact insulin bound to the cell membrane; d) internalized but degraded insulin, and e) intact insulin internalized by liver cells. Hepatocytes preincubated with 100 – 1000 nM insulin at 15°C bound and internalized less ¹²⁵I insulin. We hypothesize that in vivo, at water temperatures of 15°C and higher, extreme physiological levels of plasma insulin may regulate the numbers of insulin receptors in the salmonid liver. In contrast, in fish inhabiting cold waters the regulation of insulin receptors by circulating plasma insulin seems to be of little physiological importance. Presented in part at the Western Regional Conference on Comparative Endrocrinology, Tempe, Arizona, U.S.A., 1991 and at the Meeting of Italian Society of Experimental Biology, Sorrento, Italy, 1991. Supported by grants from NSF of the USA#DCB 8915935 to E.M.P., NSERC of Canada OPGA 6944 to T.W.M., North Atlantic Treaty Organization (NATO) grant #0926/87 to C.O. and E.M.P., and CYCIT grant of Spain to J.G.     

Responses to prolonged hypoxia by rainbow trout (Salmo gairdneri) I. Free amino acids and proteins in plasma,liver and white muscle

In order to estimate the mobilization of nitrogen compounds for energetic purposes in trout under hypoxic conditions, commercial-size rainbow trout, acclimated to 15°C, were maintained for 10 weeks at an oxygen level of 5.3 ± 0.5 mg/l (hypoxic group) or 8.4 ± 0.4 mg/I (control group), and the changes in tissue concentrations of free amino acids and proteins studied. In animals subjected to hypoxia, there was a decrease in plasma free amino acids involved in gluconeogenesis, liver alanine and aspartic acid, plasma and liver protein concentrations, and muscle free histidine. These results suggest a trend of rainbow trout metabolic activity towards energy production at the expense of anabolism when oxygen availability in water is limited over a long period of time.     

Specific gene expression profiles are associated with follicular maturational competence acquisition in rainbow trout (Oncorhynchus mykiss)

The expression profiles of several mRNAs were characterized during follicular maturational competence (FMC) acquisition. Post-vitellogenic female rainbow trout (Oncorhynchus mykiss) were assayed in vitro for follicular maturational competence (FMC). Ovarian follicles were stimulated in vitro for 60 h at 12 °C with a range of concentrations of partially purified gonadotropin and the efficient concentration for 50% germinal vesicle breakdown was calculated and used as an indicator of follicular maturational competence. Before in vitro assay, ovarian tissue was sampled in order to quantify mRNA abundance in the ovarian follicle by real-time PCR. The mRNA expression of Luteinizing Hormone receptor (LH-r), Follicular Stimulating Hormone receptor (FSH-r), Insulin-like Growth Factor 1 (IGF1), Insulin-like Growth Factor 2 (IGF2), Insulin-like Growth Factor receptor 1a (IGF-r1a) and 20β-hydroxysteroid dehydrogenase (20β-HSD), that are putatively expressed in the preovulatory ovary, was thus studied in females of varying FMC.     

The incorporation and metabolism of polyunsaturated fatty acids in phospholipids of cultured cells from chum salmon (Oncorhynchus keta)

The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids were studied in a cell line derived from chum salmon heart (CHH-1). Supplementing media with 25 M fatty acid considerably altered the cellular fatty acid composition but did not affect the lipid class composition or cause the appearance of cytoplasmic lipid droplets. CHH-1 cells exhibited considerable -6-desaturase activity but showed no preference between (n-3) and (n-6)PUFA substrates. CHH-1 cells also possess -5-desaturase activity which showed preference towards (n-3)PUFA, but -4-desaturase activity was totally absent. Elongation of 20-carbon PUFA was especially active in CHH-1 cells with 22-carbon PUFA being specifically incorporated into PE and PS lipid classes. The fatty acid composition of PI indicated specific incorporation of 20-carbon PUFA into this lipid class. Supplementation with 22:6(n-3) generated fatty acid compositions more closely resembling those of intact salmonid hearts. Substantial chain shortening of 22:6(n-3) to 20:5(n-3) occurred.Abbreviations BHT butylated hydroxytoluene - BSA bovine serum albumin - CL cardiolipin - FCS fetal calf serum - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PUFA polyunsaturated fatty acid - SM sphingomyelin     

Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.     

Evidences for involvement of growth hormone and insulin-like growth factor in ovarian development of starry flounder (<Emphasis Type="Italic">Platichthys stellatus</Emphasis>)

Although gonadotrophins are major regulators of ovarian function in teleosts and other vertebrates, accumulating evidence indicates that the growth hormone (GH)-insulin-like growth factor (IGF) axis also plays an important role in fish reproduction. As a first step to understand the physiological role of the GH-IGF system in the ovarian development of starry flounder (Platichthys stellatus), the expression profiles of GH and IGF messenger RNAs (mRNAs) and plasma GH, IGF-I, estradiol-17β (E2), and testosterone (T) levels during the ovarian development were investigated. The developmental stages of ovaries were divided into five stages (II, III, IV, V, and VI) by histological analysis. The hepatosomatic index (HSI) and gonadosomatic index (GSI) values increased and peaked at stage IV and stage V, respectively, and then declined at stage VI. Pituitary GH mRNA levels decreased sharply at stage III and raised to top level at stage VI. The hepatic IGF-I mRNA levels ascended to maximum value at stage V and then declined significantly at stage VI. However, the hepatic IGF-II mRNA levels remained stable and increased significantly at stage VI. In contrast, the ovarian IGF-I mRNA levels increased gradually and peaked at stage VI. The ovarian IGF-II mRNA levels were initially stable and increased significantly at stage V until the top level at stage VI. Consistent with the pituitary GH mRNA levels, plasma GH levels reduced sharply at stage III and remained depressed until stage V and then raised remarkably at stage VI. Plasma IGF-I level peaked at stage V and then declined to initial level. Plasma E2 level peaked at stage IV and then dramatically descended to the basal level. Plasma T level peaked at stage V and then declined significantly back to the basal level. Based on statistical analysis, significant positive correlations between hepatic IGF-I mRNA and GSI, ovarian IGF-II mRNA and hepatic IGF-II mRNA, ovarian IGF-I mRNA and ovarian IGF-II mRNA, and plasma IGF-I and plasma T were observed, respectively. These results suggest that the GH-IGF system may be involved in the ovarian development of starry flounder; GH and IGFs appear to play distinct roles in the regulation of the ovarian development in paracrine/autocrine manners. These findings extend our knowledge of the roles of the GH-IGF axis on reproduction regulation in fish.     

Prevalence and pathogenicity of infectious pancreatic necrosis virus (IPNV) associated with feral lake trout, Salvelinus namaycush (Walbaum)

Abstract. A study was undertaken in 1987 to determine the prevalence of infectious panercatic necrosis virus (1PNV) infection in the lake trout population of Cornwall Lake. Alberta, Canada, and its pathogenicity to cultivable salmonid fish. Virological examination indicated that 44.4% of the adult lake trout in the lake, which is situated in a remote northern region of Alberta, were infected with the virus, mainly in the pyloric caeca and intestine. Virus was not detected in kidney, leucocytes, liver or gonads. In experimental immersion infection of brook trout fry, the virus caused a cumulative mortality of up to 74% in 30 days, beginning at 10 days post-infection. Pyloric caeca, intestine and to some extent gills were found to be early sites of viral replication. The virus was less pathogenic to rainbow trout causing a cumulative mortality of 10% and the survivors were IPNV carriers for at least 3 months. The virus did not cause mortality in young lake trout, the natural host, but the infected fish carried the virus during the experimental period of 30 days.     

Effect of gonadotropin type II and 17-hydroxy-4-pregnene-3,20-dione on 17,20β-dihydroxy-4-pregnen-3-one production by rainbow trout testes at different developmental stages

Plasma levels of 17,20-dihydroxy-4-pregnen-3-one (17,20OHP), which is involved in the regulation of spermiation in male salmonid fish, increase dramatically at the time of spermiation. To advance the understanding of the regulation of 17,20OHP production during the spermatogenetic cycle in trout, we have studied the in vitro effect of gonadotropin type II (GtH II) and the precursor 17-hydroxy-4-pregnene-3,20-dione (17OHP) on the production of 17,20OHP. The sensitivity with which testes secreted 17,20OHP following stimulation with GtH II was maximum during spermatogenesis. The addition of 17OHP (10 to 1600 ng ml-1) to the culture medium of testes fragments induced a significant and dose-related increase in 17,20OHP secretion. Although the capacity to produce 17,20OHP was not saturated by the 17OHP concentrations used, the conversion rate was highest for tested at an immature stage. As to the regulation of 17OHP, in vivo, a single injection of partially purified salmon gonadotropin (50 ng g-1 body weight) induced a significant increase in the circulating levels of 17OHP of immature males. In conclusion, the maximum sensitivity to GtH II stimulation and the highest conversion rate of 17OHP to 17,20OHP in vitro, occurred before the dramatic increase in the 17,20OHP secretion observed in rainbow trout at the time of spermiation.     

Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

A recombinant carp growth hormone (rcGH) was used to develop for a GH radioreceptor binding assay in the goldfish (Carassius auratus). Specific binding of¹²⁵I-rcGH to goldfish liver membranes was a pH, time, temperature, and membrane protein dependent process. Scatchard and LIGAND analysis indicated a single class of high affinity and low capacity binding site, with an association constant (Ka) of 1.9×10¹⁰ M^–1 and a maximum binding capacity (B_max) of 9 fmol mg^–1 protein. Liver tissue displayed the highest¹²⁵I-rcGH binding of all the tissues examined. Displacement of¹²⁵I-rcGH with various unlabeled teleost and mammalian GHs and prolactins revealed that the goldfish hepatic binding site was highly specific for teleost GH. Intraperitoneal administration of 0.1, 1.0, and 10 g rcGH g^–1 body weight to hypophysectomized goldfish resulted in a 27, 52, and 68% decrease in total binding sites, respectively. Injection of a high dose of rat prolactin (rPRL) (5 g rPRL g^–1 body weight) also resulted in a 32% decrease in total binding sites. These results suggest that endogenous GH may have a role in the regulation of its own receptors in the goldfish.