Effects of steroid hormones on in vitro oocyte maturation in white sturgeon (Acipenser transmontanus)

The in vitro effects of several steroids on the maturation of intact white sturgeon (Acipenser transmontanus) ovarian follicles were investigated. At the highest concentration (1024 ng ml^–1 for the C21 steroids and 1139 ng ml^–1 for the C19 steroids), all of the C21 steroids tested, progesterone (P4), 17-hydroxyprogesterone (17OHP), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,(20,21-trihydroxy-4-pregnen-3-one 20-S), 11-deoxycortisol (S) and cortisol (F), as well as testosterone (T) induced germinal vesicle breakdown (GVBD) at 14 and 22 h. At 6 h, only P4 and 17,20-P induced maturation at the highest concentration (1024 ng ml^–1). At 14 and 22 h, 11-deoxycortisol was the most potent steroid inducer of GVBD followed by P4, 17OHP, 17,20-P, and 20-S. The steroid 11-hydroxytestosterone (11OHT) was completely ineffective at all concentrations and exposure times. The C21 steroids induced oocyte maturation at concentrations ranging from 4 to 1024 ng ml^–1, whereas T induced GVBD at 225 to 1139 ng ml^–1. Calculation of the mean effective concentration that induced 50% GVBD (EC50) from the 22 h incubations revealed the following order of potencies: S > P4 > 17OHP > 17,20-P > 20-S >> F > T. These bioassay results, together with previous findings on the endogenous production of steroids by ovarian follicles from gonadotropin-primed females, indicate that more than one steroid has a biological role in the resumption of meiosis in sturgeon oocytes and provides empirical evidence for P4, 17OHP, S, 20-S, and 17,20-P as maturation-inducing steroids in white sturgeon.     

Steroidogenesis in rainbow trout (Salmo gairdneri) at various preovulatory stages: changes in plasma hormone levels andin vivo andin vitro responses of the ovary to salmon gonadotropin

In order to specify the timing of some changes in ovarian steroid production during the transition from vitellogenesis to ovulation, plasma hormones levels andin vivo andin vitro responses of the ovary to salmon gonadotropin (s-GtH) or dibutyryl-cyclic adenosine mono-phosphate (db-cAMP) were recorded in relationship with the state of germinal vesicle migration in the oocyte.In vivo, a small, but significant, increase of plasma 17-hydroxy-20-dihydroprogesterone (17, 20-OH-P) level was detected earlier (at the subperipheral germinal vesicle stage) than the increase of GtH level (detectable at the peripheral germinal vesicle stage) and the decline of oestradiol-17 (E2–17) (also detectable at the peripheral germinal vesicle stage). Negative correlations were established between E2–17 levels and GtH (=–0.53) or 17,20-OH-P (=–0,43) levels while a positive correlation occurred between 17,20-OH-P and GtH levels (=+0,54).In vivo no action of GtH on the decline of E2–17 levels was detected GtH did not stimulate 17,20-OH-P production, within 72h, in females at the end of vitellogenesis stage. It had significant effect in females at other stages closer to ovulation, but the pattern of responses changed according to the stage.In vitro db-cAMP like GtH was able to stimulate 17,20-OH-P output from ovarian follicles. The greatest response was observed at the later stage. (GVBD). Testosterone output was also increased by GtH, but the lowest response was observed at the later stage (GVBD). Androstenedione output was lower than testosterone output.In vitro, a small but significant decline of E2–17 output was induced by GtH. We conclude that substantial changes occur during the very last stages prior to ovulation, both in the steroidogenic potential of the ovary and in the ovarian sensitivity to GtH. 20-oxydoreductase is probably progressively induced during GV migration when GtH basal levels are increasing but still relatively low. Without minimizing the role of discrete pulses of GtH on this induction, we could expect synergic actions of other hormones. Thus a high testosterone/oestradiol ratio in the follicle environment favours 17,20-OH-P secretion.     

Steroidogenesis by ovaries and testes of the European catfish,the wels (Silurus glanis), in vitro

Testosterone, 3,17-dihydroxy-5-pregnen-20-one, 17,20-dihydroxy-4-pregnen-3-one (17,20P) and 5-pregnane-3,17,20-triol were identified as the major metabolites of [³H] 17-hydroxyprogesterone in ovarian incubations of the European catfish Silurus glanis. 17,20P and the reduced triol were present only in ovaries from fish primed with carp hypophysial homogenate (chh) while testosterone yields were significantly higher in controls than in treated fish. 11-Ketotestosterone, 11-hydroxytestosterone and 17,20-dihydroxy-4-pregnen-3-one (17,20P) were identified as the major metabolites of [³H]17-hydroxyprogesterone in in vitro incubations of testes of a spermiating catfish. There was no significant production of conjugates or other water soluble metabolites by either sex. The stimulation of plasma 17,20P, 17,20P and 11-hydroxytestosterone by chh in primed but not control males suggests that the role of these steroids in spermiation should be further examined.     

Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus)

In two separate spawning seasons, spermiating male Atlantic halibut were implanted with pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Males were bled repeatedly, and milt samples were collected. Blood samples were assayed for free and conjugated steroids: testosterone, 11-ketotestosterone, 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20,21-trihydroxy-4-pregnen-3-one and steroids with a 17,20 configuration. Towards the end of the first season, pellets were implanted into three wild-caught and three hatchery-reared males. No control fish were available. The major progestogen in plasma was identified as sulphated 5-pregnane-3,17,20-triol (3,17,20-P-5-S). Concentrations of this steroid were stimulated by the GnRHa. Sulphated 17,20-P was also identified in the plasma, but at 10-fold lower concentrations than 3,17,20-P-5-S. In the middle of the second season, pellets were implanted into five hatchery-reared males; five unimplanted males were used as controls. Levels of androgens fell following GnRHa treatment, levels of progestogens rose briefly, and there was a significant increase in the fluidity of the milt. Of all the measured steroids, free and sulphated 17,20-P showed the best correlation with milt fluidity.     

Hormonal changes during meiotic maturation and ovulation in the brook trout (Salvelinus fontinalis)

The plasma levels of estradiol-17 (E2), 17, 20-dihydroxy-4-pregnen-3-one (17,20-P) and gonadotropin (GTH) were measured in brook trout (Salvelinus fontinalis) during the period from the end of vitellogenesis to postovulation. Blood samples were taken according to specific stages of maturation, including germinal vesicle breakdown (GVBD) and ovulation. E2 levels were quite high (45 ng/ml) at the end of vitellogenesis (and prior to GVBD) and dropped precipitously by GVBD (2 ng/ml). They remained low through ovulation and postovulation. 17,20-P levels were low prior to GVBD (0.7 ng/ml) and increased dramatically at GVBD (148 ng/ml). The levels of 17,20-P remained high at ovulation (142 ng/ml) and then dropped significantly within 24 h to approximately half of the ovulatory values. They decreased even further by 7 days postovulation. GTH levels rose gradually through GVBD and ovulation from a postvitellogenic level of approximately 3 ng/ml to a 7 day postovulatory value of approximately 10 ng/ml. The overall results; 1) decrease in estradiol prior to GVBD, 2) increase in 17,20-P at GVBD and 3) gradual GTH rise through GVBD and ovulation, are similar to those reported for other salmonids.     

Plasma profiles of fourteen ovarian steroids before,during and after ovulation in African catfish,Clarias gariepinus,determined by gas chromatography and mass spectrometry

The plasma concentrations of fourteen ovarian steroids were measured in postvitellogenic African catfish,Clarias gariepinus, which had been injected with pimozide and LHRHa. Postvitellogenesis persisted for at least four hours after pimozide and LHRHa administration. During this stage a limited rise in the plasma gonadotropin (GTH) level was accompanied by an increase in the testosterone concentration. The estradiol level was high and remained high except for a passing drop during the stage of germinal vesicle migration. At the stage of germinal vesicle migration a strong increase in the plasma GTH level coincided with a maximum in the testosterone concentration and a concomitant increase in the levels of 17,20-dihydroxy-4-pregnen-3-one and of five 5-reduced pregnanes. During germinal vesicle breakdown the GTH concentration remained high, the plasma level of 17-hydroxyprogesterone tended to increase, and the levels of 5-pregnane-3, 17-diol-20-one, 5-pregnane-3,17,20-triol and 5-pregnane-3,17,20-triol reached a maximum. At pre-ovulation the GTH concentration did not change, and peak levels were reached of 17,20-dihydroxy-4-pregnen-3-one and 5-pregnane-3,6,17-triol-20-one. Shortly after ovulation the GTH concentration slightly decreased together with a sharp decline in the concentrations of 17,20-dihydroxy-4-pregnen-3-one and the 5-reduced steroids, with the exception of 5-pregnane-3,17,20-triol, 5-pregnane-3,6,17,20-tetrol and 5-dihydrotestosterone. The plasma concentrations of androstenedione, estrone, etiocholanolone and 5-androstane-3,17-diol showed marginal fluctuations during oocyte maturation and ovulation. Apart from 17,20-dihydroxy-4-pregnen-3-one, the 5-reduced pregnanes might be candidates for the function of oocyte maturation inducing hormone inC. gariepinus.     

Relativein vitro effectiveness of estradiol-17β, androgens,corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown in oocytes of Indian major carps,Labeo rohita,Cirrhinus mrigala andCatla catla

The relative effectiveness of estradiol-17, androgens, corticosteroids, progesterone and other pregnene derivatives on germinal vesicle breakdown (GVBD) was investigatedin vitro using folliculated oocytes of three carps,Labeo rohita, Cirrhinus mrigala, andCatla catla. In all three species progesterone and 17-hydroxyprogesterone could induce GVBD but relatively 17,20-dihydroxyprogesterone was consistently found to be the most potent maturation-inducing steroid. Both estradiol-17 and testosterone were ineffective in inducing GVBD. Androsterone and dehydroepiandrosterone were found to be effective inC. catla at all the concentrations used. Deoxycorticosterone (DOC), hydrocortisone (HC) and cortisone were effective inducer of GVBD inC. catla whereas inL. rohita andC. mrigala only cortisone was found to be effective. All 5-reduced pregnenes were effective in inducing GVBD inL. rohita but inC. catla, only 5-pregnane-17-01-3,20-dione and 5-pregnane-3,17,20-triol and inC. mrigala, 5-pregnane-3-ol-20- one could induce oocyte maturation.     

Circulating levels and in vitro production of two maturation-inducing hormones in teleost: 17α,20β-dihydroxy-4-pregnen-3-one and 17α,20β,21-trihydroxy-4-pregnen-3-one, in a daily spawning wrasse, Pseudolabrus japonicus

The female bambooleaf wrasse, Pseudolabrus japonicus, spawns daily during the spawning season, and exhibits a diurnal rhythm of ovarian development. In the present study, we have investigated: (1) circulating levels of 17a, 20-dihydroxy-4-pregnen- 17,20-P) and 17,20,21-trihydroxy-4-pregnen-3-one (20-S) in females sampled at different times of the day during spawning season in captivity, and (2) in vitro production of 17,20-P and 20-S by follicle-enclosed oocytes at seven different develo tal stages. In addition, we developed a microtiter plate enzyme-linked immunosorbent assay (ELISA) for 17,20-P. Serum levels of 17,20-P and 20-S showed similar diurnal changes; substantial increases in these levels occurred around the time of germinal vesicle breakdown (GVBD). In vitro experiments showed that massive production of 17,20-P and 20-S occurred in follicles collected just before or during GVBD. Further, acute decreases in 17,20-P and 20-S production were found in the ovarian follicles just prior to ovulation, suggesting inactivation of the maturation-inducing hormone (MIH). These results, taken together with our previous data on the occurrence of GVBD in vitro, suggest a role for both 17,20-P and 20b-S as MIHs in the bambooleaf wrasse.     

Presence of maturation-promoting factor in 17α,20β-dihydroxy-4-pregnen-3-one-induced oocytes of catfish,Clarias batrachus

Full-grown immature Clarias batrachus oocytes respond in vitro to exogenous 17,20-dihydroxy-4-preg-nen-3-one ( 17,20-DP) by undergoing germinal vesicle breakdown (GVBD). Cytosolic extract (CE) prepared from 17,20-DP-induced oocytes has been shown to produce similar effect when microinjected into unstimulated immature oocytes of the same fish. A dose of 50 nl is enough to cause 100% GVBD after 4 h. Maturation-promoting factor was investigated from 17,20-DP-induced, immature and cycloheximide treated oocytes incubated in presence of [³⁵S] methionine. When the proteins were extracted and analyzed on SDS-PAGE, two prominent bands corresponding to molecular weight 34- and 46-kDa were detected in the CE of mature oocytes. However, labelling of [³⁵S] methionine was observed mainly in the region of 46 kDa protein band indicating de novo synthesis of this particular protein during l7,20-DP-induction. Further, immunoblotting study by using rabbit anti-cyclin B₁ antibody has clearly demonstrated that the protein which is newly synthesized is highly homologous to Xenopus cyclin B₁ and goldfish cyclin B.     

Oocyte maturation inClarias batrachus. III. Purification and characterization of maturation-inducing steroid

Maturation-inducing steroid (MIS) in the Indian female catfish,Clarias batrachus, was purified and characterized from the incubation medium in which fully grown but immature folliculated oocytes were incubated with salmon gonadotropin (SG-G100) for 36 h. Maturation-inducing (MI) activity of residues obtained at various steps of extraction and purification was assessed byin vitro germinal vesicle breakdown (GVBD) assay using folliculated oocytes ofC. batrachus. The post incubation medium was extracted with diethyl ether. The ether phase was partitioned using 50% methanol plus n-hexane. The methanol phase which had MI activity was fractionated into 7 fractions using reverse-phase high-performance liquid chromatography. Of these 7 fractions, fraction 3 was found to be active in having MI ability and identified as 17 ,20-dihydroxy-4-pregnen-3-one (17,20-diOHprog). The authenticity of 17,20-diOHprog as the major follicular mediator of gonadotropin-induced oocyte maturation was further confirmed by thin-layer chromatography (TLC) in which fraction 3 was run along with authentic 17,20-diOHprog standard. This investigation gives a direct evidence that 17,20-diOHprog is the major naturally occurring MIS in Indian female catfish,C. batrachus.     

Regulation of oocyte maturation in the rainbow trout,Salmo gairdneri: role of cyclic AMP in the mechanism of action of the maturation inducing steroid (MIS), 17α-hydroxy, 20β-dihydroprogesterone

In fish, oocyte maturation (resumption of meiosis after completion of vitellogenesis and before ovulation) is triggered by maturation inducing steroids (MIS) which generally appear to be secreted in the ovary in response to stimulation by a pituitary maturational gonadotropin. Converging data from different laboratories show that 17-hydroxy, 20-dihydroprogesterone (17, 20-OH-P) is the principal MIS in salmonoids; but clear identification remains to be done in other taxonomic groups.The experiments reported here in the rainbow troutSalmo gairdneri examine the possible involvement of oocyte cAMP on the mechanism of MIS action. The action of 17, 20-OH-P, on germinal vesicle breakdown (GVBD) in oocytes incubatedin vitro within the follicle, was inhibited by various substances expected to elevate the intraoocyte concentrations of cAMP: cAMP ( 1 mM) or dibutyril cAMP ( 2 mM), phosphodiesterase inhibitors such as theophylline ( 0.2 mM) or 3-isobutyl-1 methylxanthine (IBMX 0.1 mM), adenylate cyclase activators such as cholera toxin (> 100 nM) or forskolin ( 0.03 mM). In fact, the combined action of IBMX (1 mM) and forskolin (0.01 or 0.05 mM)in vitro was to promote accumulation of intraoocyte cAMP within 1 to 5 hours. Oocyte cAMP concentrations exhibited a large variability between different females, depending on the stage of oocyte development; a significant positive correlation between oocyte cAMP concentration and the follicular weight, and a significant negative correlation between oocyte cAMP concentration and the median efficient dose of 17, 20-OH-P for induction of GVBD, were observed. Finally, when intrafollicular oocytes were incubatedin vitro, the addition of a maturation-inducing concentration of 17, 20-OH-P (3×10^–6M) induced a significant decrease of oocyte cAMP within the first 10 hours of incubation. These results show that cAMP appears to play a central role in the regulation of oocyte sensitivity to 17, 20-OH-P and in the intraoocyte mechanisms leading to GVBD in trout.These data are discussed together with the few indications available in fish concerning the mechanism of MIS action which can be compared to some extent with the amphibian model.     

Sulfation and uptake of the maturation-inducing steroid, 17α,20β-dihydroxy-4-pregnen-3-one by rainbow trout ovarian follicles

Rainbow trout ovarian follicles were incubated in vitro with tritiated 17,20-dihydroxy-4-pregnen-3-one (17,20-P; maturation-inducing steroid). Within 18–24 h, 56–66% had been converted to tritiated 17,20-dihydroxy-4-pregnen-3-one 20-sulfate (identification confirmed by HPLC) and 27% had been taken up (absorbed) by the follicles. Addition of 125 ng of cold (non-tritiated) 17,20-P to the incubations caused a decrease in the percentage of [³H]-17,20-P which was sulfated (56% 10%) and an increase in the percentage that was taken up (27% 57%). Seven steroids were tested for their effectiveness in decreasing the sulfation and increasing the uptake of tritiated [³H]-17,20-P. The order of effectiveness was in both cases the same: 17,20-P > cortisol > 11-deoxycortisol > 17,20,21-trihydroxy-4-pregnen-3-one > 17-hydroxy-4-pregnene-3,20-dione > 17-estradiol > testosterone. This indicated that the processes of sulfation and uptake of [³H]-17,20-P were related to each other and led to the hypothesis that, when cold 17,20-P is added to the medium, it reduces the proportion of [³H]-17,20-P which is sulfated and thus allows more free [³H]-17,20-P to enter the ovarian follicles. This hypothesis was supported by the finding that each ovarian follicle had the capacity in vitro to sulfate only ca. 2 ng of [³H]-17,20-P per 18h but a capacity to take up > 500 ng per 18h.Gonadotropin I, Gonadotropin II, forskolin and phorbol-12-myristate-13-acetate (which all have an affect on steroid biosynthesis) did not affect the amount of 17,20-P which was sulfated. Sulfating activity was localized in the thecal cell layer of the follicle. The yolk fraction was shown to be responsible for absorbing the [³H]-17,20-P.     

Steroid release from separated theca and granulosa layers of Atlantic salmon (Salmo salar) ovarian follicles: the effects of a purified salmon gonadotrophin preparation

Theca and granulosa layers were removed from ovarian follicles of mature Atlantic salmon (Salmo salar) and were separately incubated under sterile conditions with and without a partially purified salmon gonadotrophin preparation (GTH). Aliquots of the incubation media were removed at intervals and analysed for the steroids 17, 20-dihydroxy-4-pregnen-3-one (1720P), 17-hydroxyprogesterone, progesterone, androstenedione, testosterone and oestradiol. The biosynthesis of C19 and C21 steroids was very largely restricted to the thecal tissue and was markedly stimulated in the presence of GTH. Androstenedione (max 65 ng/ml) and testosterone (max 14 ng/ml) were released from the earliest stages of incubation whereas the release of 17-hydroxyprogesterone (max 51 ng/ml) and progesterone (max 5.5 ng/ml) commenced only after a lengthy induction period. A trace (1.0 ng/ml) of 1720P was produced by the theca in the presence of GTH but oestradiol was not detected. The granulosa preparations released levels of 17-hydroxyprogesterone and androstenedione only marginally above the detection limits (ca 0.7 ng/ml) and there was little stimulation of output with GTH. Oestradiol (max 4 ng/ml) was released only in the presence of GTH. 1720P, progesterone and testosterone were not detected as products of this tissue. These results, together with those derived earlier from incubations of complete follicles support the view that the synthesis of 1720P is essentially a two-cell process in which 17-hydroxyprogesterone produced in the theca is subject to the action of steroid 20-hydroxysteroid dehydrogenase in the granulosa. The temporal pattern of release of steroids in these and earlier experiments is considered in relation to mechanisms of steroid biosynthesis and to their possible roles in oocyte final maturation.     

Steroids and steroid glucuronides in the ovarian fluid of the African catfish,Clarias gariepinus,between ovulation and oviposition

As part of a series of experiments concerning a possible pheromonal function of steroids and steroid glucuronides excreted by the sex organs of the African catfish,Clarias gariepinus, qualitative and quantitative studies, using GCMS, were carried out to examine the presence of the steroids, that can be synthesized by the ovary during oocyte maturation and ovulation, and of the corresponding steroid glucuronides, in the fluid surrounding the eggs in the ovarian cavity shortly after ovulation.Full mass spectra were obtained of 5-pregnane-3,17-diol-20-one, 5-pregnane-3,17,20-triol, 5-pregnane-3,6,17-triol-20-one, 5-pregnane-3,6,17,20-tetrol, 5-androstane-3,17-diol and 5-androstane-3,17-diol-11-one. After selected ion monitoring the following steroids could be detected by the presence of at least two characteristic ions at the expected retention time: 5-pregnane-3, 17,20-triol, etiocholanolone, 5-dihydrotestosterone, 5-androstane-3,11-diol-17-one, testosterone and estradiol. After treatment with -glucuronidase the following steroids could be determined in a similar way: 5-pregnane-3,17-diol-20-one, 5-pregnane-3,17,20-triol, 5-pregnane-3,17,20-triol, 5-pregnane-3,6,17-triol-20-one, 5-pregnane,3,6,17,20-tetrol, 5-androstane-3,17-diol, etiocholanolone, 5-dihydrotestosterone, testosterone and estradiol.The free steroids 5-pregnane-3,6,17,20-tetrol and 5-pregnane-3,6,17-triol-20-one and the steroid glucuronides of testosterone, 5-dihydrotestosterone and estradiol appeared to be the most abundant of these compounds. The results indicate that very polar steroids and steroid glucuronides, synthesized in the ovary, can be excreted via the ovarian fluid shortly before and during oviposition, and possibly function as sex attractants, inducing reproductive behaviour in male conspecifics.     

Gonadotropins I and II do not stimulate thein vitro secretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation

Thein vitro secretion of 17,20-dihydroxy-4-pregnen-3-one 20-sulphate (17,20-P-sulphate) and the free steroid 17,20-dihydroxy-4-pregnen-3-one (17,20-P), by rainbow trout (Oncorhynchus mykiss) gonads, in response to gonadotropin (GTH) I and GTH II, were studied during the final stages of sexual maturation. Substantial amounts of 17,20-P-sulphate were produced, by both mature ovaries and testes, indicating considerable 20-hydroxysteroid sulphotransferase (20-HST) activity within these tissues. In the post-ovulatory ovary the level of 17,20-P-sulphate (36.6 ng ml^–1) greatly exceeded that of 17,20-P (8.59 ng ml^–1). The amount of 17,20-P-sulphate produced in incubations of both mature ovary and testes was unaffected by either GTH I or GTH II treatment at physiological concentrations up to 100 ng ml^–1. Similarly, incubations of maturing ovary and testes, treated with GTH I or GTH II, in the presence of added 17,20-P at 100 ng ml^–1 of medium, produced levels of 17,20-P-sulphate that were similar to those of the controls. In incubations of mature ovarian follicles at the stages of germinal vesicle breakdown and preovulation, both GTHs significantly stimulated secretion of 17,20-P, although GTH II was always more potent than GTH I. GTH II significantly elevated the levels of 17,20-P in testicular incubations from mature males more than 4-fold relative to GTH I and controls, which did not differ from one another.In conclusion, 20-HST, the enzyme responsible for the sulphate conjugation of 17,20-P, was found to be active in the ovaries and testes of rainbow troutin vitro. However, the levels of this enzyme do not appear to be regulated by either GTH I or GTH II.     

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17-estradiol, 17-20-dihydroxy-4-pregnen-3-one (17-20-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17-estradiol levels. Inversely, plasma 17-20-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17-20-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.     

Influence of growth hormone on the hepatic mixed function oxidase and transferase systems of rainbow trout

The effect of GH treatment on hepatic cytochrome P450 content, aryl hydrocarbon hydroxylase (AHH), aminopyrine-N-demethylase (AND), testosterone hydroxylase, testosterone 5- and 5-reductase, UDP-glucuronyl transferase (UDPGT) and glutathione S-transferase (GST) activities in immature rainbow trout were investigated. Hepatic cytochrome P450 content, AHH and GST activities were measured in both GH implanted and GH injected animals whereas other activities were assayed in GH implanted trout only.GH implants significantly decreased cytochrome P450 content at 15 days compared to the control but no significant effect was observed at 15 or 30 d when GH was injected biweekly. In both cases, AHH activity was significantly decreased by GH treatment compared to the control whereas GST remained unchanged. Compared to the control, GH implanted fish exhibited a pronounced inhibition of AND, a decreased 6 and 16-testosterone hydroxylation, an inhibition of UDPGT with testosterone as substrate and an enhanced 17-testosterone oxidation.     

Activin and its receptors in the goldfish

Activin (_AA, _AB and _BB) is a dimeric protein that belongs to the transforming growth factor- (TGF-) superfamily of growth factors and is involved in the regulation of many physiological and developmental processes. Recently, we have demonstrated that porcine activin stimulated goldfish gonadotropin-II (GTH-II) and growth hormone (GH) secretion from dispersed pituitary cells in static culture and pituitary fragments in perifusion. The action of activin in the goldfish is unique in that it has an acute stimulatory effect on the secretion of GTH-II and GH, whereas in mammals activin usually exhibits long-term stimulatory actions on FSH secretion. The action mechanism of activin is different from that of gonadotropin-releasing hormone (GnRH). Using domain-specific antibodies against mammalian activin subunits, we subsequently demonstrated the existence of immunoreactive activin subunits (_A and _B) in the goldfish ovary, testis, pituitary and brain, suggesting endocrine, paracrine and autocrine roles for activin in the regulation of goldfish reproduction. Both activin _A and _B subunits have been cloned from goldfish genome by polymerase chain reaction (PCR). Using the PCR fragments as probes, we have cloned a full length cDNA coding for activin _B subunit from the goldfish ovary. Both activin _A and _B subunits show high homology to those of other vertebrates with the _B subunit much more conserved (93 and 98% identity with human and zebrafish _B subunit, respectively). The identity of the cloned _B subunit was further confirmed by expression in the Chinese hamster ovary (CHO) cells and detection of the specific activity of activin in the culture medium. The messenger RNA of activin _B subunit is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish. We have also cloned a full length cDNA coding for the activin Type IIB receptor from the goldfish ovary, suggesting that activin may have paracrine or autocrine actions on the ovarian functions. The identity of the cloned receptor was confirmed by specific binding of¹²⁵ I-activin on COS-1 cells transfected with the cloned Type IIB receptor.     

LH-β and FSH-β mRNA expression in nesting and post-breeding three-spined stickleback, Gasterosteus aculeatus, and effects of castration on expression of LH-β, FSH-β and spiggin mRNA

The mRNA expression of the LH- and FSH- subunits were measured in nesting and post-breeding male three-spined sticklebacks, Gasterosteus aculetaus, as well as in castrated and sham-operated nesting males. Furthermore, expression of an androgen induced kidney protein, spiggin, and 11-ketotestosterone (11KT) levels, were measured in the castrated and sham-operated males. Nesting males had significantly higher levels of both LH- and FSH- mRNA expression compared to post-breeding males. Furthermore, sham-operated males had significantly higher levels of LH- mRNA and spiggin mRNA expression than the castrated fish. Expression of FSH-, on the other hand, did not differ between castrated and sham-operated males. There were strong positive individual correlations between circulating levels of 11KT on the one hand and expressions of LH- and spiggin mRNA, whereas the correlation between 11KT levels and FSH- mRNA was weak. The negative effect of castration on -LH mRNA indicates that gonadal hormones stimulate this expression, whereas this was not the case for -FSH. The observed decline in -LH expression after the end of the breeding season may be the result of cessation of the gonadal stimulation of the pituitary. On the other hand, it is not likely that this can explain the decline in FSH- expression.     

Binding characteristics and regulation of the 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) receptor on testicular and sperm plasma membranes of spotted seatrout (Cynoscion nebulosus)

The binding characteristics of 17,20,21-trihydroxy-4-pregnen-3-one (20-S) to plasma membranes prepared from the testes and sperm of spotted seatrout (Cynoscion nebulosus) were investigated using a filtration method to retain the bound 20-S. A single class of high affinity (K_d = 17.9 nM), low capacity (B_max = 0.072 nM g^-1 testes) binding sites was identified by saturation and Scatchard analyses on testicular membranes of spermiating spotted seatrout. A corresponding receptor (K_d = 22.17 nM, B_max = 0.00261 nM ml^-1 milt) was also detected in spermatozoan membrane preparations. The rates of 20-S association and dissociation were rapid, both had T_halfs of less than 1 min. Competition studies indicated that the receptor was highly specific for 20-S. 17,20-dihydroxy-4-pregnen-3-one, which had the highest affinity of the other steroids tested, had a relative binding affinity (RBA) of 14.3%. Progesterone, 11-deoxycortisol and testosterone competed with an order of magnitude less affinity (RBA's of 7.4, 1.8 and 1.1%, respectively). Estradiol displayed low affinity for the receptor (RBA = 0.4%) and cortisol did not cause any displacement at 1000-fold excess concentration. Specific 20-S receptor binding was detected in plasma membranes from testes of both spermiating and non-spermiating seatrout and on spermatozoa. Prolonged incubation of testicular fragments from a spermiating fish with gonadotropin (15 IU ml^-1 human chorionic gonadotropin) or forskolin (10 µM) caused a 2–3 fold increase in membrane receptor binding. Previous studies have shown that gonadotropin-induced upregulation of the 20-S plasma membrane receptor in seatrout ovaries is required for the oocytes to become responsive to 20-S and undergo final maturation. The existence of a 20-S membrane receptor on sperm and its upregulation in the testes by gonadotropin raises the possibility that final maturation of spermatozoa in male seatrout may be regulated by a similar mechanism.