Occurrence and in vitro biosynthesis of 11-ketotestosterone in Siberian sturgeon,Acipenser baeri Brandt maturing females

High levels of 11-ketotestosterone (11KT) were found (49 to 160 ng ml^–1) in plasma of Siberian sturgeon females during the end of their reproductive cycle. These levels were measured either by specific radioimmunoassay, or both by specific radioimmunoassay and by UV absorption after HPLC (isocratic conditions, 33% methanol, 26% acetonitrile, 41% water). In order to find the origin of 11KT synthesis, ovaries were incubated (30 min and 2h at 20°C) with tritiated 17-hydroxyprogesterone (17OHP) or with tritiated androstenedione (A4). Testosterone (conversion rate from tritiated 17OHP: 4%) and 11-ketotestosterone (conversion rate from tritiated A4: 1.6%) were identified as metabolites of respectively 17OHP and A4 (TLC, HPLC and crystallization). 11-hydroxyandrostenedione (11OHA4) and 11-hydroxytestosterone (11OHT) were suggested to be intermediate metabolites. Besides interrenal and blood cells were incubated respectively with tritiated cortisol and tritiated A4. 11OHA4 was identified in interrenal incubation (yield from tritiated cortisol: 1.2%). 11KT in interrenal (yield from tritiated cortisol: 0.14%), and 11OHA4 and 11KT in blood cells (yield from tritiated A4: 1.6%), were suspected to be synthesized (TLC, HPLC, acetylation). No significant metabolization of tritiated cortisol could be found in liver. The possible contribution of each of these tissues to high 11KT levels found in plasma is discussed.To whom correspondence should be sent     

Sex steroids in intersexual fishes

Studies of thein vitro gonadal steroidogenesis in intersexual fishes, using labelled testosterone as precursor, showed large species variation. The protogynousMonopterus albus produced predominantly 5α-reduced metabolites while the protandrousRhabdosargus sarba produced mainly 5β-reduced products. Both fishes synthesized 11-oxotestosterone; the synthesis of which appeared to mediate mainly through adrenosterone inM. albus butvia 11β-hydroxytestosterone inR. sarba. When the plasma levels of androstenedione, testosterone, 11-oxotestosterone, 11β-hydroxytestosterone, estrone and 17β-estradiol among the male, intersexual and female phase of the same species were compared, available data showed that either there was no obvious difference among the different sexual phases or the differences could be accounted for by the seasonal reproductive activities of the animal. Except for androstenedione, there are no marked changes in plasma testosterone, 11-oxotestosterone, 11β-hydroxytestosterone, estrone and 17β-estradiol levels in the intersexual phase compared with the female and male, it is unlikely that these classical sex steroids act as a primary trigger of natural sex reversal in these fishes; the role of androstenedione awaits further elucidation. Department of Zoology, University of Hong Kong     

Sperm characteristics and androgens in Acipenser ruthenus after induction of spermiation by carp pituitary extract or GnRHa implants

Spermiation and changes in androgen (testosterone, T and 11-ketotestosterone, 11-KT) levels were studied in sterlet (Acipenser ruthenus) treated with GnRH agonist implants (dAla⁶-Pro⁹-LHRHa) at 25 and 75?μg?kg^?1 b.w. and compared with those males treated with 4?mg?kg^?1 b.w. of carp pituitary extract (CPE) and 3 pellets of Ovopel kg^?1 b.w., which contains dAla⁶-Pro⁹NEt-mGnRH and metoclopramide. Sperm quality (sperm mass, spermatozoa concentration and sperm motility and velocity) was evaluated 24, 48 and 72?h after hormonal treatments. Males did not release sperm in the control group injected with physiological solution, while sperm could not be collected 7?days after treatments in all hormonally treated groups. Spermiation rates were 100?% in the CPE and Ovopel groups and 25–50?% in the GnRHa-treated groups. Sperm production was significantly lower in the GnRHa-treated groups than in the CPE and Ovopel groups and decreased 72?h after hormonal treatment. Sperm motility and velocity were higher in the Ovopel and GnRHa (75?μg) groups compared to the CPE and GnRHa (25?μg) groups and decreased 72?h after hormonal treatment. Androgens were only affected in spermiating males and changed in the Ovopel and GnRHa (75?μg) after hormonal treatment. Significant correlations were observed between sperm production, sperm motility and sperm velocity, but not androgens. The present study suggests involvement of dopamine in sturgeon spawning. Additionally, better sperm quality observed in the Ovopel group and particularly sperm motility in the GnRHa (75?μg) suggests enhancement of sperm quality in sturgeon treated with GnRHa. Therefore, further study is needed to induce fully spermiation using GnRHa implants in combination with a dopamine inhibitor.     

Dimorphic expression of sex-related genes in different gonadal development stages of sterlet, <Emphasis Type="Italic">Acipenser ruthenus</Emphasis>, a primitive fish species

Molecular mechanism of sex determination and differentiation of sturgeon, a primitive fish species, is extraordinarily important due to the valuable caviar; however, it is still poorly known. The present work aimed to identify the major genes involved in regulating gonadal development of sterlet, a small species of sturgeon, from 13 candidate genes which have been shown to relate to gonadal differentiation and development in other teleost fish. The sex and gonadal development of sterlets were determined by histological observation and levels of sex steroids testosterone (T), 11-ketotestosterone (11-KT), and 17β-estradiol (E2) in serum. Sexually dimorphic gene expressions were investigated. The results revealed that gonadal development were asynchronous in 2-year-old male and female sterlets with the testes in early or mid-spermatogenesis and the ovaries in chromatin nucleolus stage or perinucleolus stage, respectively. The levels of T and E2 were not significantly different between sexes or different gonadal development stages while 11-KT had the higher level in mid-spermatogenesis testis stage. In all the investigated gonadal development stages, gene dmrt1 and hsd11b2 were expressed higher in male whereas foxl2 and cyp19a1 were expressed higher in female. Thus, these genes provided the promising markers for sex identification of sterlet. It was unexpected that dkk1 and dax1 had significantly higher expression in ovarian perinucleolus stage than in ovarian chromatin nucleolus stage and in the testis, suggesting that these two genes had more correlation with ovarian development than with the testis, contrary to the previous reports in other vertebrates. Testicular development-related genes (gsdf and amh) and estrogen receptor genes (era and erb) differentially expressed at different testis or ovary development stages, but their expressions were not absolutely significantly different in male and female, depending on the gonadal development stage. Expression of androgen receptor gene ar or rspo, which was supposed to be related to ovarian development, presented no difference between gonadal development stages investigated in this study whenever in male or female.     

Gonad histology and plasma steroid profiles in wild New Zealand freshwater eels (Anguilla dieffenbachii and A. australis) before and at the onset of the natural spawning migration. I. Females*

To determine steroid profiles in immature and maturing female eels from the wild, non-migratory and migratory New Zealand longfinned (Anguilla dieffenbachii) and shortfinned (A. australis) eels were caught and blood and ovarian samples collected. Plasma steroid levels were determined and related to the developmental stage of the ovary. Ovaries of non-migrants contained oogonia and previtellogenic oocytes. Vitellogenic oocytes were never observed in these groups, but instead were very common among migrants (up to 88% of oocytes). Concentrations of both androgens (androstenedione (AD), testosterone (T)) and estradiol-17 (E2) were higher in migrants than in non-migrants. Among migrants, T levels were higher in shortfins (2.27 ± 0.14 ng ml–1) than in longfins (0.82 ± 0.10 ng ml–1), whereas E2 levels were higher in longfins (mean 2.46 ng ml–1) than in shortfins. Levels of sex steroids were generally low in non-migrants. In contrast, plasma levels of 17-hydroxyprogesterone were significantly higher in non-migrants than in migrants. Similarly, cortisol levels were higher in non-migrating than in migrating shortfinned, but not longfinned, females. 17,20-Dihydroxy-4-pregnen-3-one, the putative maturation-inducing steroid in anguillids, was near minimum-detectable levels for all animals examined. Surprisingly, very high levels of 11-ketotestosterone (KT) were found in migrants, averaging nearly 3 ng ml–1 in longfins and over 20 ng ml–1 in shortfins. The identity of KT and several 5-reduced androgens was confirmed using gas chromatography - mass spectrometry. The function of KT in females is not known, but we suggest that this steroid hormone may play a role in preparing maturing animals for their spawning migration.     

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.     

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.     

Gonadal Stage and Sex Steroid Correlations in Siberian Sturgeon,Acipenser baeri,Habituated to a Semitropical Environment

Stages of gonadal development, in association with plasma concentrations of the sex steroids 17β‐estradiol (E2), progesterone (P4), testosterone (T), and 11‐ketotestosterone (11‐KT), were investigated for a single time point during a natural breeding season in 7‐yr‐old Siberian sturgeon, Acipenser baeri Brandt, exposed lifelong to a warmwater environment. Among females, examination of gonadal tissue showed variation in ovarian stage, with 12.5, 47.5, 22.5, and 17.5% of females found at Stages 2 (previtellogenic), 3 (early vitellogenic), 4 (mid‐vitellogenic), and 5 (migratory nucleus), respectively. Although patterns varied among the hormones, plasma concentrations of E2, T, and 11‐KT became increasingly elevated in females as maturation progressed. On the basis of histological criteria, males were classified as either premeiotic (quiescent) or meiotic and 50% of the males sampled were found at each stage. Significant elevations in circulating concentrations of plasma E2 and T were observed in meiotic versus premeiotic males, and there was a rise in plasma 11‐KT concentration that approached significance (P = 0.056).     

Effects of dietary cottonseed meal protein level on growth, gonad development and plasma sex steroid hormones of tropical fish tilapia Oreochromis sp

Five experimental diets containing increasing proportions of cottonseedmeal (CSM) protein (0, 25, 50, 75 and 100%; diets 1 to 5, respectively) toreplace fish meal (FM) protein were formulated for intensive culture of tilapiaOreochromis sp. Each diet was fed to three replicategroupsof fish (mean weight ± SE = 11.3 ± 3.9 g) in30L aquaria connected as a closed recirculating-water system andmaintained at 27 ± 1 °C. Fish were fed three times adayby hand at a rate of 3% of body weight during four weeks, after which thefeeding rate was gradually decreased to reach 1.5% at 16 weeks. Thesubstitutionof 75 and 100% of FM proteins by CSM proteins resulted in significantly lowerbody weights in both sexes. In both sexes, gonadosomatic indexes and plasmaconcentrations of sex steroids (testosterone, 11-ketotestoterone,estradiol-17 and 17,20-dihydroxy-4-pregnen-3-one) were notsignificantly different among dietary treatments. The concentration ofgossypol,an antifertility agent contained in CSM, was measured in reproductive tissues.The total gossypol concentration in the testis was consistently lower than thatmeasured in the ovaries of the same group. Moreover, in both sexes, theconcentration of the (+)isomer of gossypol was always higher than that of(–)isomer. The total gossypol concentrations in testes increasedsignificantly with the increase of CSM in the diet. The highest levels of the(+)isomer (7.64 ± 0.62 g g^–1)were found in the testes of fish fed diet 4, whereas the (–)isomerreached its highest values in the testes of fish fed diet 5. The highest levelsof both enantiomers of gossypol were found in the ovaries of fish fed diet 4(14.2 ± 2.7 and 5.6 ± 1.5 g g^–1for (+) and (–)isomers, respectively). In both sexes, thehistological analysis of the gonads did not reveal differences among the fishfed different levels of CSM. Although CSM at any levels did not affect thereproductive parameters examined in this study, it cannot be used to substitutemore than 50% of FM since at higher levels growth of tilapia was compromised.     

Specific binding of 11-ketotestosterone in an androgen target organ,the kidney of the male three-spined stickleback,Gasterosteus aculeatus

The kidney of male three-spined stickleback,Gasterosteus aculeatus, hypertrophies during the breeding season and produces a glue which is used in the building of the nest. This hypertrophy is androgen dependent, with 11-ketotestosterone (11KT) being more effective than other tested steroids in stimulating this secondary sexual character. In the present study kidneys were excised from stickleback males that had been castrated two days earlier. The purpose of this gonadectomy was to reduce the endogenous levels of androgens without allowing time for the kidney to regress. Tissue fragments were incubated with tritiated 11KT with and without unlabelled steroids at increasing concentrations. Displaceable specific 11KT binding was found in kidney tissue fragments whereas only non-specific binding was observed when liver and muscle were investigated in a similar way. Unlabelled 11KT displaced specifically bound, tritiated 11KT with an ED50-value (50% of displaceable binding) of 28 nM. Similar ED50 values were found for 17\-hydroxy-5-androstane-3,11-dione (29 nM) and 5-dihydrotestosterone (20 nM), whereas higher ED50 concentrations were estimated for testosterone (T; 203 nM) and progesterone (69 nM). No displacement of tritiated 11KT was found for the other investigated substances tested; estradiol, 17,20-dihydroxy-4-pregnen-3-one, flutamide or cyproterone acetate. No specific binding to kidney tissue fragments could be detected when labelled T was used instead of labelled 11KT. Specific binding of 11KT or T was not found either in the kidney cytosol or nuclear extracts. However, using the kidney membrane fraction a displacement of tritiated 11KT with unlabelled 11KT (10^–6M) was observed. In conclusion there is a specific binding of 11KT in the stickleback kidney. The absence of binding in liver and muscle, the ED50 value observed and the displacement with some, but not all steroids are consistent with a receptor function. The presence of binding in membrane fractions, but not in cytosol or nuclear extracts suggests that the binding is not related to classic steroid receptors.     

Whole-body concentrations of cortisol and sex steroids in white sturgeon (Acipenser transmontanus, Richardson 1836) during early development and stress response

In general little is known about hormones and the ontogeny of the stress response in the early developmental stages of chondrostean fishes and in particular of white sturgeon (Acipenser transmontanus, Richardson 1836). In this study, we measured for the first time cortisol and sex steroids (testosterone and estradiol) in eggs, larvae, post-larvae, and fry of white sturgeon by radioimmunoassay (RIA), to elucidate some endocrine aspects of its development. The cortisol, testosterone, and 17β-estradiol of maternal origin found in unfertilized eggs of white sturgeon probably regulate both growth and development of the embryo. Cortisol decreased after fertilization, whereas testosterone and 17β-estradiol did not significantly change. During the late stages of embryo development and immediately after hatching, endogenous production of cortisol and sexual steroids, respectively, occurred. Sex steroids may be physiological inducers of gonad sex differentiation in sturgeon. All steroids showed an increase 10 days post-hatch (dph), near the transition from an endogenous to an exogenous energy source. Cortisol maintained the same basal levels even after metamorphosis, whereas testosterone and 17β-estradiol declined significantly in post-larvae at 35 and 45 days post-hatch. In addition, to evaluate the ontogeny of a functional hypothalamic-pituitary-interrenal (HPI) axis, larvae and fry were submitted to acute stress. The HPI axis did not seem to be functional on the first day post-hatch, but became so from the third day post-hatch onward.     

中华鲟piwil1基因的克隆表达特征研究

Piwi是鱼类配子发生和性腺发育的重要调控因子。本研究从中华鲟(Acipenser sinensis)性腺中克隆得到了piwi基因的全长c DNA序列,该序列长3 393 bp,开放阅读框为2 583 bp,编码860个氨基酸。氨基酸序列多重对比发现,该序列具有PAZ和PIWI保守结构域,与其他鱼类piwil具有较高同源性;系统进化分析显示,中华鲟piwil聚于piwil1分支;因此,所得序列为piwi-like 1基因,命名为Aspiwil1。荧光定量PCR研究表明,Aspiwil1为母源表达,且其表达量随着胚胎发育逐渐降低;Aspiwil1在性腺中大量表达,在脑组织中也有较低水平的表达。性腺组织切片RNA原位杂交结果表明,Aspiwil1仅在生殖细胞表达,且其杂交信号随着卵子发生逐渐增强、精子发生逐渐减弱。本研究为中华鲟配子发生过程中Aspiwil1的功能研究提供了基础。     

Presence of 11-ketotestosterone in pre-differentiated male gonads of Odontesthes bonariensis

The involvement of androgens during sex differentiation period was investigated in the pejerrey Odontesthes bonariensis, by classic biochemical studies and gonadal histology. We studied in particular whether the enzyme activities involved in 11-oxygenated androgen production were active in a gonadal/peritoneum complex (GPC) of very small larvae exposed to masculinizing temperatures previous to morphological sex differentiation (5 weeks post-hatching). The GPC was incubated with 17-hydroxyprogesterone (³H-17P), and the presence of 11-KT as major metabolite in early gonads undergoing masculine pathway after temperature treatment exposure is reported. 11-KT was identified by thin-layer chromatography and high-pressure liquid chromatography. The present results show that 11-KT is produced at very early stages of testis development in pejerrey, being this androgen one of the main mediators of the masculinization induced by temperature treatment at the gonad level.     

Effects of acidic water,aluminum, and manganese on testicular steroidogenesis in <Emphasis Type="Italic">Astyanax altiparanae</Emphasis>

Metals can influence the gonadal steroidogenesis and endocrine systems of fish, thereby affecting their reproduction. The effects of aluminum and manganese in acidic water were investigated on steroidogenesis in sexually mature male Astyanax altiparanae. Whether mature male fish recover from the effects of metals in metal-free water was also assessed. The fish were exposed to 0.5 mg L^?1 of isolated or combined aluminum and manganese in acidic pH (5.5) to keep the metals bioavailable. The fish underwent 96 h of acute exposure, and samples were taken 24 and 96 h after the beginning of the experiment. The fish were then maintained in metal-free water for 96 h. Plasma levels of testosterone, 11-ketotestosterone, 17β-estradiol, and cortisol were measured. Acidic water increased the plasma concentration of testosterone and 11-ketotestosterone. Aluminum increased the testosterone levels after 96 h of exposure. Manganese increased the 17β-estradiol levels after 24 h of exposure and maintained at high levels until the end of the experiment. With the exception of acidic pH, which increased cortisol levels after 24 h of exposure, no changes were observed in this corticosteroid during the acute experiment. Aluminum and manganese together also altered steroid levels but without a standard variation. The fish recovered from the effects of most exposure conditions after 96 h in metal-free water. A. altiparanae could use reproductive tactics to trigger changes in testicular steroidogenesis by accelerating spermatogenesis and spermiogenesis, which may interfere with their reproductive dynamics.     

Relationships between circulating androgens,aggressive behaviour and breeding tubercles in males of the common bream <Emphasis Type="Italic">Abramis brama</Emphasis> L. in an aquarium environment

In this study, relationships between circulating androgens, aggressive behaviour and breeding tubercles in males of common bream Abramis brama were examined in an aquarium environment. The breeding tubercles of fish were counted, the number of attacks was quantified by male status and circulating rates of testosterone and 11-ketotestosterone from blood plasma were analysed using radioimmunoassay procedures. The results revealed that no significant differences were found between circulating testosterone and 11-ketotestosterone in territorial and nonterritorial males. Furthermore, no significant correlations were found between circulating androgens, androgens and aggression, androgens and tubercles and breeding tubercles and aggression in common bream by male status. However, territorial fish displayed a significantly higher level of aggressive behaviour and breeding tubercles than nonterritorial fish. In natural environments, the occurrence of breeding tubercles during the spawning season could contribute to identifying the behavioural status of common bream males.     

Effect of in vitro xenoestrogens on steroidogenesis in mature female fish, Chasmichthys dolichognathus

In this study, fully vitellogenic oocytes of the longchin goby (Chasmichthys dolichognathus) were exposed to in vitro xenoestrogens such as diethylstilbestrol (DES), bisphenol A (BPA) and nonylphenol (NP) at concentrations of 100 ng/ml in the presence of [³H]17α-hydroxyprogesterone as precursor. The major metabolites produced in vitro are progestogens [17α-hydroxy,20α-dihydroprogesterone (17α20αOHP) and 17α-hydroxy,20β-dihydroprogesterone (17α20βOHP)], androgens [androstenedione (A4) and testosterone (T)] and estrogens [estrone (E1) and estradiol-17β (E2)]. Comparative activities of these chemicals in oocyte steroid biosynthesis showed as follows: NP treatment resulted in clear stimulation of estrogen production, while the opposite occurred in DES treated incubation. Treatment with DES resulted in a higher synthesis of 17α20αOHP. BPA treatment increased the androgen, while estrogen synthesis was slightly inhibited. These results suggest that NP exhibited clearly estrogenic activity on the three chemicals.     

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.     

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.     

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.     

Investigation into the possible role of androgens in the induction of hepatic vitellogenesis in the European eel:in vivo andin vitro studies

Changes in the levels of plasma vitellogenin (Vg), estradiol (E₂) and testosterone (T) were examined following gonadal development induced by carp gonadotropin treatment (cGTH) of freshwater female yellow and silver eels (Anguilla anguilla L.). The animals received injections of cGTH (250 μg kg⁻¹ body weight) or saline vehicle three times a week, for 6 to 8 weeks. No effect of vehicle was observed. Steroidogenic activity of the ovary was stimulated by cGTH treatment as shown by the increase in circulating steroid levels in both stages. However, the responses of T, E₂ and Vg differed according to the stage of development of eels. At the yellow stage, the initial steroid plasma levels were undetectable (< 0.01 ng ml⁻¹) before treatment and ovarian steroidogenic activity was slightly stimulated following cGTH treatment; steroid levels reached their highest values after 3 weeks and 6 weeks of treatment for E₂ (0.62 ± 0.13 ng ml⁻¹ and T (0.33 ± 0.30 ng ml⁻¹), respectively. The cGTH treatment slightly increased plasma Vg levels (0.2–0.7 μg ml⁻¹ during the experiment compared with the initial values of the group. At the silver stage, the initial steroid levels were detectable (0.7 ng ml⁻¹ for E₂ and 0.1 ng ml⁻¹ for T); cGTH treatment did not significantly increase plasma E₂ level which remained at initial levels. Nevertheless, plasma T levels dramatically increased from 0.1 to 3 ng ml⁻¹ and peaked after 1 or 2 weeks of cGTH treatment; a rapid increase in plasma Vg levels occurred, reaching its highest value at 5 mg ml⁻¹ after 3 weeks of treatment. Thus, the steroid kinetic profiles in relation to the appearance of Vg in the plasma following cGTH treatment was closely related to androgen levels and there was a strong vitellogenic response induced by chronic cGTH treatment. In order to test if androgens could be implicated in the vitellogenic response, we evaluated the potencies of various androgens (testosterone and 5α-androstane-3β,17β-diol)in vivo andin vitro, associated with E₂ to induce the production of Vg.In vitro experiments showed that Vg synthesis was induced by high doses (10⁻⁶ to 10⁻⁵ M) of androgen in the eel. Tamoxifen totally inhibited the action of androgens suggesting that androgens were acting through binding to the E₂ receptor.In vivo, androgens given alone at 50 μg kg⁻¹ 3 times a week for 1 months had no significant effect on plasma Vg levels. In addition, E₂-androgen cotreatment showed that the presence of androgen did not modify the vitellogenic response induced by E₂.