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.     

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.     

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.     

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 aromatase inhibitors on sexual maturation in Atlantic salmon,Salmo salar,male parr

Atlantic salmon, Salmo salar, male parr were implanted with Silastic capsules filled with different aromatase inhibitors: 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and the non-steroidal CGS16949 A, 4-benzonitrile monohydrochloride (CGS). Aromatization in brain homogenates were lower in salmon implanted with CGS and ATD than in controls. This was not the case for 4OH, but administration of 4OH to brain homogenates reduced the aromatase activity. All three aromatase inhibitors had effected gonadal weights in fish sampled in the summer, but the effects were markedly different among inhibitors. Plasma levels of the androgen 11-ketotestosterone (11KT) and the progestin 17, 20-dihydroxy-4-pregnen-3-one (17,20P) were measured by means of radioimmunoassay. CGS and ATD, but not 4OH, significantly decreased the plasma 17,20P levels in the autumn. Plasma levels of 11 KT were not influenced by ATD or CGS treatment, but 4OH had a lowering effect in one autumn sampling. ATD and 4OH (CGS not tested) increased the proportion of maturing males.These findings suggest that aromatization is of physiological importance in different mechanisms controlling reproduction in salmon.     

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.     

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.     

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.     

Inhibition of HPI axis response to stress in gilthead sea bream (Sparus aurata) with physiological plasma levels of cortisol

This study investigates the effect of corticosteroid (cortisol) administration on the stress response of the gilthead sea bream Sparus aurata subjected to a 48 h confinement. The effect of (in-vitro and in-vivo ) cortisol administration on the in-vitro ACTH sensitivity of the interrenal tissue; the plasma levels and tissue concentration of cortisol; and the plasma levels of ACTH, -MSH, -endorphin and glucose were determined. Confinement caused a transient and concomitant increase in plasma cortisol and ACTH levels. However, in cortisol-fed fish the plasma ACTH levels were lower, indicating a suppresion of the ACTH release from the corticotropes by cortisol. In contrast to the activation of the corticotropes, the levels of plasma melanotrope derived peptides were not affected. In spite of the fact that interrenal cells of cortisol-fed gilthead sea bream released less cortisol than controls, the interrenal sensitivity to ACTH was not affected by in-vivo and in-vitro cortisol administration. This suggests that the interrenal sensitivity to ACTH in stressed (confinement) sea bream is probably not regulated by -MSH, N-ac--END, or by cortisol. Thus, in gilthead sea bream the interrenal sensitivity to ACTH could be regulated at the hypothalamus and/or pituitary and communicated via circulating ACTH levels.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Structural and functional effects of early exposure to estradiol-17β and 17α-ethynylestradiol on the gonads of the gonochoristic teleost Dicentrarchus labrax

This study investigated the effects of estrogens on sexual differentiation in sea bass (Dicentrarchus labrax L.), a gonochoristic marine teleost that under culture conditions has a histologically sexual undifferentiated period that covers most of the first year of life, after which most individuals develop as males. Sea bass that had no noticeable histological sign of sex differentiation were fed estrogens at two doses (5 or 10 mg kg^-1 food) and for different periods ranging from 48 to 426 days post fertilization (DPF). Exposure to the synthetic estrogen 17-ethynylestradiol (EE₂) at 10 mg kg^-1 food from 60 to 260 DPF, including the sensitive period to equivalent doses of synthetic androgens previously determined for this species (126-226 DPF), significantly (p < 0.05) more than doubled the number of juvenile females to 80%, compared to the control value of 33%, and completely suppressed gonadal development in the remaining 20% of the population. This suggests that the period during which sea bass gonads exhibit high sensitivity to androgens is also very sensitive to estrogens. A comparable exposure to the natural estrogen estradiol-17 (E₂) resulted in 13% of the fish having suppressed gonadal development, but induced 57% of the fish to develop gonads with germinal tissue of both sexes, suggesting a pivotal role for E₂ during this sensitive period. Earlier exposure to EE₂ at 10 mg kg^-1 food from 48-88 DPF, significantly (p < 0.05) increased the number of females to 62% from 36% in the control group, allowing for the normal testicular development in the remaining fish. In contrast, a later chronic exposure (226-426 DPF) to E₂, at either 5 or 10 mg kg^-1 food, starting when the gonads showed no sign of sexual differentiation but past the critical sensitive period, had no effect on the resulting overall sex ratios, indicating that after this period responsiveness of the gonads to estrogens decreases as gonadal sexual differentiation progresses. However, the consequences of this apparently innocuous exposure were later manifested in adults, exemplified by a significant dose-dependent reduction in the number of mature males at 626 DPF, coinciding with the second reproductive season, the time when males normally reach sexual maturation in cultured sea bass. This suggests that chronic exposure to E₂ past the critical sensitive period may not affect the sex ratio, but could result in alterations in the male reproductive organs. This was later verified by histological analysis which revealed a significant (p < 0.05) dose-dependent reduction of the surface of the testicular lobules in the remaining males that did not mature. Together, these experiments illustrate both readily observable and subtle effects of estrogens on sex proportions, gonadal morphology and maturation rates, providing evidence that estrogen exposure can have delayed action in a teleost in a manner similar to the effects described for mammalian species. The possible existence of effects of this latter type in adult fish could be considered when evaluating the consequences of deliberate or accidental exposure to estrogens or putative estrogenic chemicals, particularly if such exposure had taken place during sex differentiation.     

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.     

Thyroid hormones down-regulate thyrotropin β mRNA level in vivo in the turbot (Psetta maxima)

Thyroid stimulating hormone (TSH) is a vertebrate pituitary heterodimeric hormone that stimulates the thyroid gland to produce the thyroid hormones, T3 and T4. We report here the cloning, by PCR on reverse-transcribed pituitary RNAs, of a 180 bp fragment of the cDNA encoding TSH subunit in the turbot (Psetta maxima). The deduced amino acid sequence displayed 66 and 75% identity with the corresponding sequence from the European eel (Anguilla anguilla) and the rainbow trout (Oncorchyncus mykiss), respectively. This cDNA was then used as a probe for densitometric analysis of individual pituitary Northern blots. TSH mRNA levels were quantified in turbot where circulating thyroid hormones were modified by dietary treatments or hormone supplementation. Recombinant rainbow trout growth hormone had no effect on circulating thyroid hormone levels or on pituitary TSH mRNA level. In turbot fed heat-treated rapeseed meal, plasma T4 levels were lowered and TSH mRNA increased more than two fold. In contrast, when turbot were fed a standard fish-meal supplemented with T3, circulating T3 levels were elevated and there was a dramatic decrease in TSH mRNA level. It is concluded that both thyroid hormones are able to down-regulate TSH mRNA level in vivo in the turbot. These results are discussed in the context of the evolution of the TH feed-back on TSH production.     

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.     

Estradiol-17β suppresses testicular development and stimulates sex reversal in protandrous black porgy,Acanthopagrus schlegeli

Two year old black porgy (Acanthopagrus schlegeli) fed a diet containing 4.0 mg kg^–1 of estradiol-17 (E₂) for 5 months had significantly lower GSI than the control group during the spawning season. E₂ suppressed testicular development, spermiation and plasma testosterone (T) and 11-ketotestosterone (11-KT) and stimulated ovarian development, vitellogenesis and sex reversal. Spermiation in the control group occurred in January and February with the concentrations of 1.08–1.36 × 10¹⁰ sperm ml^–1 of milt. Higher plasma T and 11-KT, but lower E₂ levels were detected in the spermiating fish (control group). Higher plasma E₂ levels were detected in the sex reversing black porgy during the pre-spawning season. A sharp rise in plasma 11-KT and a drop in T levels were detected in spermiating fish (control group) from January to February. Plasma 11-KT levels correlated with the testicular development and spermiation. The data suggest that E₂ plays an important role in controlling the sex reversal of black porgy.to whom correspondence should be addressed.     

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.     

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.     

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).     

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.