Plasma levels of Gonadotropin-II and gonadal sex steroids in triploid catfish, Heteropneustes fossilis (Bloch)

Triploid fish have under-developed gonads due to altered reproductive endocrinology. Triploids of Indian catfish (H. fossilis) showed significantly reduced plasma levels of gonadotropin (GtH-II), testosterone (T) and estradiol-17 (E₂) than that of diploids throughout the year, except for the resting phase, irrespective of sex. Plasma levels of GtH-II were significantly different (p<0.001) between diploid and triploid fish during preparatory, prespawning and spawning phase. The plasma testosterone contents in triploids were significantly less (p<0.001) than that of diploids, except during the resting phase. Triploid females showed very low titres of estradiol-17 (<1 ng ml^–1) throughout the annual reproductive cycle in contrast to highly fluctuating levels in diploid females. Thus, this study for the first time provides information on reduced levels of GtH-II and sex steroids in plasma of male triploid fish and additional information on species-specific alteration of sex hormones in female triploids.     

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.     

Gonadotropin-induced changes in steroid production by ovaries of the common careCyprinus carnio L. around the time of ovulation

Ovarian fragments from both primed (gonadotrophin treated) and unprimed female carp were incubated either with or without carp hypophysial homogenate and steroid hormone production measured. In incubations without hypophysial homogenate, production of all the steroids measured was either very low or nondetectable and there was no significant difference between tissue from primed and unprimed fish. In the presence of carp hypophysial homogenate a very significant increase in production of testosterone, 17-hydroxyprogesterone and testosterone glucuronide was observed, but there was no significant difference between primed and unprimed fish. 17,20-Dihydroxy-4-pregnen-3-one (17,20P) was not stimulated by carp hypophysial homogenatein vitro in ovaries from unprimed fish, but a very significant increase in production of this hormone was observed in tissue from fish which had received a priming dose of pituitary hormone. It is suggested that the priming dose of pituitary extract used in the normal hypophysation procedure to induce ovulation in teleosts initiates the potential for synthesis of 17,20P in response to later gonadotrophin challenge, and that this initiation may be related to the migration of the germinal vesicle.A preliminary account of these results was presented at the Fish Culture Conference, Barcelona, August 1985 (Kime and Bieniarz 1985).     

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.     

Effects of steroid hormones on immunoglobulin M (IgM) in rainbow trout, Oncorhynchus mykiss

The immunosuppressive effects of steroid hormones were evaluated as the response against implanted steroid hormones, cortisol (F), testosterone (T), estradiol-17 (E2), and 11- ketotestosterone (11-KT), in juvenile rainbow trout. In long term experiments (5 weeks), fish were given a single intraperitoneal implant of F or T. A clear suppressive effect of plasma IgM levels with F and T was not necessarily obtained, although mucus IgM levels were reduced corresponding to the elevated plasma steroid hormone levels. In short term experiments (1 week), intraperitoneal implantation of T, 11-KT and E2 suppressed plasma and mucus IgM levels, although the effects were not dose-dependent. When administered through diet, F and T caused a suppression of plasma IgM levels; F administration at both high and low dosages caused a significant decrease in plasma IgM levels, while only a high dose of T caused the suppression. These results suggest that sex steroid hormones, as well as F, have immunosuppressive functions in rainbow trout.     

In vitro steroidogenesis by testicular fragments and ovarian follicles in a hybrid sturgeon, Bester

In this study, developmental changes in the steroidogenic capacity of testicular fragments and isolated ovarian follicles of a hybrid sturgeon, Bester, at a variety stage of developments were examined. Testicular fragments or isolated ovarian follicles were incubated in L-15 medium in the presence or absence of different concentrations of five preparations; forskolin, human chorionic gonadotropin (HCG), pregnenolone (P₅), 17-hydroxyprogesterone (17OHP) and testosterone (T) for 18 h at 15 °C. After incubation, concentrations of 11-ketotestosterone (11 KT) (testis) and, 17-estradiol (E₂) (ovarian follicles) and 17,20-dihydroxy-4-pregnen-3-one (DHP) (testis and ovarian follicles) were measured. 11KT was detected in the media following incubation with P₅, 17OHP and T. Its concentration was higher during late spermatogenesis and prespermiation and lower at the degeneration stage. Both P₅ and 17OHP were converted to DHP during the prespermiation stage. Forskolin had little stimulatory effect on the synthesis of 11KT and DHP and HCG did not induce the production of these steroids.E₂ was detected in the medium following incubation of follicles with P₅, 17OHP and T at all stages of oocyte development. The concentration of E₂ in the medium increased during vitellogenesis with the peak production occurring at the tertiary yolk stage. In contrast, the potencies of follicles to produce steroids shifted to the production of DHP during migratory nucleus stage. Forskolin and HCG had little effect on the synthesis of E₂ and DHP. These results demonstrated that the failure of spontaneous spermiation or ovulation is not due to the insufficient synthesis of DHP, but may due to the lack of availability of precursors.     

Melatonin, but not somatostatin-14 influences in vitro steroidogenesis by ovarian follicles of rainbow trout

Ovarian follicles taken from sexually maturing rainbow trout at the mid-vitellogenic stage of ovarian development were incubated in vitro in the presence or absence of melatonin or somatostatin-14 (SRIF-14) to determine whether there is evidence of a direct action of these factors on gonadal steroidogenesis in fishes. The steroidogenic capacity of the ovarian follicles was assessed by measuring testosterone (T) and 17-estradiol (E₂) release into the incubation medium, and by examining the steroid metabolites produced following incubation of follicles with radiolabelled steroid precursors.Melatonin appears to elicit a biphasic effect on steroidogenesis by in vitro rainbow trout ovarian follicles; at a concentration of 1 × 10^–3 M, melatonin stimulated basal T and E₂ production, but at a concentration of 1 × 10^–2 M there was an inhibition of basal and sGtH-stimulated T and E₂ Melatonin may act to reduce the activity of specific steroidogenic enzymes, since there was evidence of melatonin at 1 × 10^–2 M enhancing the accumulation of [³H]17-hydroxyprogesterone in the medium following incubation with [³H]pregnenolone, possibly suggesting the inhibition of C_17,20-lyase activity. In contrast, SRIF-14, used at concentrations of 1 × 10^–8 M and 1 × 10^–6 M, had no effect on basal or sGtH-stimulated E₂ or T production by ovarian follicles, incubated in vitro.     

Reproduction related immunoglobulin changes in rainbow trout

Annual changes in plasma immunoglobulin (IgM) levels were investigated in three strains of rainbow trout Oncorhynchus mykiss which have different spawning periods, i.e., September–October, November–December, and January, reared under constant water temperature and natural day length. Plasma IgM levels decreased during the spawning season in all strains tested. The IgM changes became reversed in response to significant increases in plasma testosterone (T) and estradiol-17 in females and T and 11-ketotestosterone in males. Though the IgM decline showed a connection with suppressed immunocompetence, since many mature fish caught fungal diseases, no clear differences were observed in the plasma IgM levels between infected and noninfected fish during the spawning season. Incidentally, plasma IgM levels in infection prone fish were higher than in noninfection prone fish prior to the spawning season, whereas coincident differences in the plasma steroid levels were observed. Immature fish reared under lower water temperatures showed lower IgM levels. The effect of water temperature may have to be considered when analyzing the defense mechanism during the spawning season in rainbow trout.     

Effects of cortisol and triiodo-L-thyronine on the steroidogenic capacity of rainbow trout ovarian follicles at two stages of oocyte maturation

The in vitro basal and salmon gonadotropin (sGTH)-stimulated steroidogenic capacity of rainbow trout follicles was examined at four stages [early (EV)-, mid (MV)- and peak-vitellogenic (PV), and pre-ovulatory, post-vitellogenic (PO)] of gonadal recrudescence using radioimmunoassays (RIAs) to measure 17-estradiol (E₂) and testosterone (T) production. In addition, follicles were incubated in the presence of [³H]pregnenolone ([³H]P⁵) and the radiolabelled steroid metabolites produced were separated using high performance liquid chromatography (HPLC). Peak basal and sGTH-stimulated E₂ and T production was found in PV stage follicles and lowest in PO stage follicles, and there were marked differences in the HPLC profiles of steroid metabolites. For EV stage follicles the major metabolite eluted as a peak that co-eluted with the androstenedione (A₄) and 17-hydroxyprogesterone (17-OHP) standards. A smaller peak that co-eluted with 11-hydroxyandrostenedione (11-OHA₄) and very small peaks co-eluting with 20-dihydroprogesterone (20-DHP) and E₂ were also seen. MV and PV stage follicles produced predominantly E₂, together with a small combined A₄ + 17-DHP peak, traces of 11-OHA₄ and two peaks that did not co-elute with any of the reference standards. The PO stage follicles produced only 17, 20-dihydroxy-4-pregnene-3-one (17,20-P).In addition, the effects of cortisol and triiodothyronine (T₃) on steroidogeneis were investigated in PV and PO stage ovarian follicles. For PV stage follicles, cortisol at 100 ng ml^–1 in the incubation medium significantly suppressed both basal and sGtH- stimulated T and E₂ production relative to control treatments. T₃ at 10 ng ml^–1 in the medium had no significant effect on either basal or sGtH-stimulated T or E₂ production compared to the controls, nor did it have any beneficial effect over the suppressive effect of cortisol. PO phase follicles taken 1 to 2 weeks prior to anticipated spawning had very low E₂ and T production, and there was no effect of cortisol or T₃, alone or in combination, on E₂ or T production. For PV stage follicles incubated in the presence of [³H]P⁵, cortisol suppressed T and E₂ production, but did not block the steroid pathway at any specific level; T₃ had no apparent affect on the metabolism of [³H]P⁵. The PO stage follicles produced little or no E₂; the major metabolite was 17,20-P. Cortisol and T₃ had no apparent effect on either basal or sGtH-stimulated 17,20-P production by the follicles at this stage of maturation.     

Endocrine changes during the annual reproductive cycle of the red porgy, Pagrus pagrus (Teleostei, Sparidae)

Changes in 17-estradiol (E2), estrone (E1), testosterone (T), 11-ketotestosterone (11KT), and 17,20-dihydroxy-4-pregnen-3-one (17,20-P) levels were correlated to changes in gonadosomatic index (GSI), vitellogenin concentration (Vg), ovarian and testicular histology during the annual reproductive cycle of the red porgy, Pagrus pagrus. The production of E2, E1, T and 17,20-P was confirmed by analysis of the steroidogenic activity of ovaries. In females, the average concentration of E2 was lower than 2 ng ml^–1. E2 values first increased significantly at the stage of endogenous vitellogenesis and remained high during exogenous vitellogenesis. E1 levels were lower values than E2 (less than 300 pg ml^–1), but they increased at the beginning of exogenous vitellogenesis. Estrogens concentrations followed similar pattern to Vg and were significantly correlated. Mean levels of T were mostly lower than 1 ng ml^–1. They followed a pattern similar to that of E2 except for a further increase observed at the stage of final maturation. T and E2 levels were significantly correlated. The concentration of 11KT did not change significantly. The levels of 17,20-P ranged between 0.22 and 1.22 ng ml^–1 but changes were not related to gametogenesis. In males, the concentrations of T and 11KT fluctuated significantly during the sexual maturity stages, showing a similar pattern and were significantly correlated to GSI changes. T levels increased during spermiogenesis and spermiation stages to reach about 3 ng ml^–1. 11KT levels stayed about half those of T. The levels of estrogens showed no significant changes. Level of 17,20-P showed no significant variation related to male maturity. Results are discussed in relation to changes in plasma steroid levels during gametogenesis of other multiple spawner species.     

Gene Expression in Black Tiger Prawns Following Intramuscular Injection of β-gal Plasmid

Gene expression in black tiger prawns (Penaeus monodon) was studied following intra-muscular injection of CMVGal plasmid into the second abdominal segment. We used an in situ staining technique to detect -gal expression in one- and three-month-old injected prawns. We found that only one of the three-month-old prawns expressed the marker gene (2 days after injection), and the site of expression was confined to the sixth abdominal segment away from the injection site. We repeated the experiment on a new batch of three-month-old prawns, but using fluorometric determination technique. This time we found that -gal expression was detected (6/42) at the site of injection after 2, 7, and 14 days. In two other test samples, transgene expression was detected in the sixth abdominal segment only, further confirming the possibility of injected DNA dispersal. The results of the study also suggest that direct gene transfer is a feasible technique in black tiger prawns.     

The presence of 17α,20β-dihydroxy-4-pregnen-3-one receptor activity in the ovary of the brook trout,Salvelinus fontinalis,during terminal stages of oocyte maturation

The presence of 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP) oocyte receptor activity has been demonstrated in brook troutSalvelinus fontinalis. Scatchard analyses of the cytosol fraction during various terminal stages of oocyte maturation gave a high equilibrium association constant (K_a) value of 1.394±0.669 10⁸M^–1 (n=7) and low maximum binding capacities (N_max). The association kinetics of the receptor was second order k₊₁=2.292×10⁶M^–1 sec^–1. The dissociation rate constant k_a was 1.502×10^–2 sec^–1 for the first order dissociation reaction. The K_a=1.526×10⁸M^–1, when it was determined from k₊₁/k_–1 a value close to that found from the Scatchard analysis. Competition studies showed the following binding affinities testosterone > 17-HP > 17,20-DHP > Promegestone > progesterone > estradiol > pregnenolone; cortisol showed no competitive inhibition. Cytosolic extracts when pre-equilibrated with various labelled steroids and eluted from a Sephacryl S-300 column gave multiple specific binding peaks. On sucrose density gradient centrifugation specific binding was observed at 3.05 S in cytosol containing 0.15M sodium chloride buffer. The receptor lost binding activity when incubated with various proteases, but DNase and RNase had no effect. Blood plasma without heparin at (110) dilution also bound [³H]17,20-DHP, Ka was 8.04×10⁷ M^–1.The nuclear pellet extract (750×g) gave very little specific binding activity even at high radiolabelled steroid concentrations and a linear Scatchard plot was not obtained. Nevertheless the nuclear extract, after dextran-charcoal treatment, pre-equilibrated with [³H]17,20-DHP, bound specifically to DNA cellulose, and cytosol from the same oocytes also bound to DNA cellulose under similar conditions. Although specific binding to DNA cellulose was obtained the salt concentrations at which the steroid-receptor complex elution took place was not reproducible in both nuclear extracts and cytosol samples. Also binding activity was extremely small compared to the total cytosolic binding. The nuclear extract when pre-equilibrated with high concentrations (20 nM) of the labelled steroid and then chromatographed on Sephacryl S-300 column gave a specific binding peak which was similar to that of the cytosolic preparation.The receptor levels in cytosol decreased progressively during final maturation (Stages 1–7). There is preliminary evidence for the presence of 17,20-DHP receptor activity in cytosol of landlocked Atlantic salmonSalmo salar ouananiche, and rainbow troutSalmo gairdneri. The zona radiata fraction from late stages oocyes 5, 6, and 7 in brook and rainbow trout oocytes were isolated by ultracentrifugation; from this fraction a protein was characterized which covalently bound [³H]R5020 after photoaffinity labelling. The same protein also bound [³H]17,20-DHP after solubilization in Brig 35 buffer. The SDS gel electrophoresis subunit composition of the above protein was similar to the cytosol counterpart binding [³H]17,20-DHP, although the molecular weights were different. The blood sample [³H]R5020 binding component subunit composition was different from that of the membrane extracted protein. These results demonstrate the presence of 17,20-DHP receptor activity in the cytosol and zona radiata membranes of the oocytes during final maturation.A. Maneckjee is presently NSERC postgraduate scholar at MSRL and Ph.D. candidate at Department of Biochemistry, Memorial University of Newfoundland.     

17α,20β,21-trihydroxy-4-pregnen-3-one and 17α,20β-dihydroxy-4-pregnen-3-one stimulated spermiation in protandrous black porgy, Acanthopagrus schlegeli

The objective of this study was to investigate the effects of sex steroids on spermiation in protandrous male black porgy, Acanthopagrus schlegeli. Experiments on common carp (Cyprinus carpio) were also conducted for comparison. Fifty male black porgy were divided into 5 groups and injected with a superactive analogue of mammalian luteinizing hormone releasing hormone (LHRH-A), 17,20,21-trihydroxy-4-pregnen-3-one (20-S), 17,20-dihydroxy-4-pregnen-3-one (17,20-DHP), 11-ketotestosterone (11-KT) or saline. The dosage of the sex steroids given on days 0, 2, 4 and 6 was 330, 330, 990 and 1980 µg kg^-1 body weight, respectively. Milt volume and sperm concentrations were measured on days 0, 2, 4, 6, 8 and 10. Similar treatments were also conducted in 45 male common carp. Milt volume was significantly increased in black porgy after treatment with 20-S and 17,20-DHP; 17,20-DHP had stimulatory effects on spermiation at a lower dose (900 µg kg^-1 body weight, p < 0.05) as compared to 20-S (1980 µg kg^-1 body weight, p < 0.01). In the common carp, milt volume was also increased after treatment with LHRH-A and 17,20-DHP but not with 20-S. 17,20-DHP stimulated spermiation at a lower dose in common carp (330 µg kg^-1 body weight) than in black porgy (990 µg kg^-1 body weight). However, 11-KT did not stimulate spermiation in black porgy or common carp. The concentrations of plasma 11-KT could immediately reflect to the administration of exogenous 11-KT in black porgy.     

Synthesis and regulation of 17α-hydroxy-20β-dihydroprogesterone in immature males of Oncorhynchus mykiss

Three experimental approaches were chosen to study the question if the progestin 17-hydroxy-20-dihydroprogesterone (1720OHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20-hydroxysteroid dehydrogenase activity (20HSD), testes homogenates were incubated with ³H-labeled 17OHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 1720OHP-³H, indicating that already immature testes contain 20HSD activity and are able to produce 20-reduced steroids. Second, 1720OHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 1720OHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20HSD is present in testicular cells other than spermatozoa. Furthermore, 1720OHP is indeed secreted at a very early stage of testicular development; 1720OHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.     

Methallibure-induced inhibition of hypothalamo-hypophyseal-ovarian activity in the catfish Heteropneustes fossilis involves changes in hypothalamic monoamine activity

Administration of Methallibure, a non-steroidal gonadotropin (GTH) inhibitor 20 g g–1 body weight; i.p., daily for 10 days, to prespawning phase female Heteropneustes fossilis inhibited the brain-pituitary-ovarian axis as indicated by significant reductions in plasma and pituitary levels of GTH-II, and plasma levels of 17-estradiol (E2) and testosterone. Concurrently, the treatment resulted in significant reductions in the hypothalamic content of serotonin, noradrenaline (and adrenaline) that stimulate, and a significant elevation of dopamine that inhibits GTH-II release in this species. Activities of the monoamine degrading enzymes, monoamine oxidase and catechol-O-methyltransferase were significantly increased, while that of the synthesizing enzymes, dopamine--hydroxylase and phenylethanolamine-N-methyltransferase were significantly decreased. These results suggest that the mechanism of inhibition of GTH-II secretion includes, among others, differential actions of the drug on hypothalamic monoamine metabolism.     

Effects of isotonic swelling on the intracellular Bohr factor and the oxygen affinity of trout and carp blood

Blood samples from carp and trout were exposed to overnight tonometry in the presence of noradrenaline to obtain isosmotic cell swelling. The intracellular fixed acid Bohr factor (_fa(i)) were then measured and compared to the values for unswollen cells (Holk and Lykkeboe 1995). An increase in oxygen affinity appeared for both carp and trout. Part of this increase could be explained by a lower content of organic phosphates, whereas the rest of the increase was ascribed to changes in the association constants of at least one of the phosphate-hemoglobin or oxygen-hemoglobin complexes. However, in spite of a marked swelling and a slightly lower content of organic phosphates, no change in the _fa(i) appeared.     

Synthesis of 17,20α- and 17,20β-dihydroxy-4-pregnen-3-ones, 11-ketotestosterone and their conjugates by gills of teleost fish

Goldfish, carp and trout gills were incubated with ³H-17-hydroxyprogesterone (17P). With goldfish gills, the metabolites were 17,20-dihydroxy-4-pregnen-3-one (17,20P; 82%), 17,20-dihydroxy-4-pregnen-3-one (17,20P; 8%), 11-ketotestosterone (KT) glucuronide (5.4%) and 17,20P glucuronide (0.2%). Sulfates were not detected. Carp gills converted 17P into 17,20P (11.2%), 17,20P (9.6%), KT (8.4%), glucuronides of 17,20P (1.3%) and 17,20P (1.6%) and sulfates of 17,20P (5.1%) and 17,20P (7.2%). 17,20P (38% free, 1.8% glucuronide and 21.1% sulfate) was the sole metabolite of ³H-17P in trout gill incubations. In the presence of high (10; µg ml^-1) substrate concentration, cyprinid gills gave predominantly free 17,20P, while trout gills yielded only free 17,20P. Production of 17,20P, predominantly as its sulfate, from endogenous precursors was demonstrated in trout gills but was not stimulated by trout primary extract. Our results demonstrate for the first time the steroidogenic potential of teleost gills and suggest that they may play a role in secretion of pheromones in some species.     

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.     

The modulating effect of vitellogenin on the synthesis of 17β-estradiol by rainbow trout (Oncorhynchus mykiss) ovary

In vivo induction of vitellogenin (VTG) in response to the administration of 17-estradiol (E₂) was achieved and the protein was isolated by gel filtration column chromatography of plasma samples. Adult female trout were injected with the vitellogenic fraction every ten days from July to November and levels were measured by RIA from September to December. The results showed a significant decrease (p<0.01) in plasma E₂ levels in injected females compared with the controls. In December, after finishing the treatment, the plasma E₂ concentration increased, in injected females to reach a level similar to that of control females at vitellogenesis. The in vitro study showed that in early vitellogenic oocyte (from September) the presence of the vitellogenic fraction in the incubation medium causes a significant decrease (p<0.01) in the synthesis of E₂ by the oocytes. These data suggest that the concentration of the VTG into the oocyte can alter VTG production by the liver, moderating the production of E₂ by the ovary.     

Characterization of vitellogenin induced by estradiol-17β in protandrous black porgy,Acanthopagrus schlegeli

The objectives of this study were to isolate and characterize vitellogenin from plasma of estradiol-treated protandrous black porgy,Acanthopagrus schlegeli. Vitellogenin concentrations in plasma measured by a validated enzyme-linked immunosorbent assay (ELISA) were also compared. Two-year-old black porgy (n=20) were fed with estradiol-17 (4 mg kg^–1 of feed). Plasma was collected for purification of vitellogenin. Two forms of vitellogenin were found in plasma after chromatography on Sepharose CL-2B column and hydroxylapatite, and polyacrylamide gel electrophoresis. Black porgy vitellogenins are phospho-lipo-glycoproteins based on their chemical staining properties. The apparent molecular weights of the two forms of vitellogenin were 636 kDa and 321 kDa, respectively. The amino acid composition of purified vitellogenin was also analyzed after acid hydrolysis. The presence of immunoreactive vitellogenin was identified in the plasma and mucus extract from control and estradiol-induced females on the basis of Western blotting. Serial dilution of the plasma and mucus extract taken from estradiol-induced black porgy showed reactivity to an antiserum against lipovitellin in the ELISA, whereas mucus extract and plasma from male fish did not. Significantly higher concentrations of plasma vitellogenin were detected in estradiol-stimulated black porgy than in the control males.