Biosynthesis of steroids in ovarian follicles of red seabream,Pagrus major (Sparidae,Teleostei) during final oocyte maturation and the relative effectiveness of steroid metabolites for germinal vesicle breakdown in vitro

The steroid synthesis pathway in the ovarian follicles of the red seabream during final oocyte maturation (FOM) was investigated by incubating intact follicles with different radioactively labeled steroid precursors. During FOM, the steroidogenic shift from estradiol-17beta to 20 beta-hydroxylated progestin production occurred mainly due to a combination of inactivation of C 1720-lyase and activation of 20 beta-hydroxysteroid dehydrogenase. Of the steroids produced, 1720 beta-dihydroxy-4-pregnen-3-one (1720 beta-P) and 1720 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) exhibited the greatest effect on germinal vesicle breakdown (GVBD) in vitro. 1720 beta-P was further converted to its 5 beta-reduced form, 1720 beta-dihydroxy-5 beta-pregnan-3-one (1720 beta-P-5 beta), which had lower GVBD activity, suggesting that 5 beta-reduction plays a role in the inactivation of the maturation-inducing ability of 1720 beta-P. In contrast, no 5 beta-reduced metabolite of 20 beta-S was found. Serum levels of 1720 beta-P and 20 beta-S, measured by ELISA, showed that circulating levels of both progestins increased during FOM, and 20 beta-S levels were approximately twice as high as 1720 beta-P levels. This study clarified the complete steroidogenesis pathway during FOM in red seabream ovarian follicles and showed that two 20 beta-hydroxylated progestins, 1720 beta-P and 20 beta-S, act as maturation-inducing hormones in this species. The catabolites of these two progestins and their physiological roles in reproduction are also discussed.     

Correlation between plasma steroid hormones and vitellogenin profiles and lunar periodicity in the female golden rabbitfish, Siganus guttatus (Bloch)

Characteristics of the lunar reproductive cycle in the golden rabbitfish, Siganus guttatus, were determined by histological observations of ovarian development, and immunological measurements of plasma steroid hormones, estradiol-17beta (E2), testosterone (T), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), and vitellogenin (VTG). Ovarian and plasma samples were collected every week according to the lunar phases from May to July. Weekly change of gonadosomatic index (GSI) showed two peaks at the first lunar quarter in June and July. Yolky oocytes were also observed around this time. Histological observations revealed that the vitellogenic oocytes appeared again 1 week after spawning and developed synchronously. These results suggest that this species is a multiple spawner and the oocyte development is in a group-synchronous manner. Plasma steroid hormones (E2, T, DHP and 20beta-S) and VTG levels changed in parallel with changes in GSI. The peak of plasma VTG level occurred prior to spawning. These cyclic changes of plasma steroid hormones and VTG support the hypothesis that lunar periodicity is the major factor in stimulating reproductive activity of S. guttatus.     

Steroid metabolism in vitro during final oocyte maturation in white croaker Micropogonias furnieri (Pisces: Sciaenidae).

Final oocyte maturation (FOM) is a process involving a complex set of genetical, biochemical, and morphological mechanisms. FOM involves the shift of a post-vitellogenic follicle to a pre-ovulated oocyte, which is necessary for fertilization by spermatozoan to occur. This process is regulated by a maturation-inducing steroid (MIS) at the follicular level. In other species of scienids fish the MIS, a hydroxilated derivatives of progestagen 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S), was identified. Although Micropogonias furnieri is the second fishery resource of Uruguay, basic knowledge about its endocrine process is very scarce. The aim of this work was to investigate what steroids are synthesized in vitro by the oocyte follicle of M. furnieri during the maturation process. Fragments of ovary (1 g) in three stages: post-vitellogenic (PV), maturing (Mtg), and mature (M) were incubated with 1 microg x g(-1) of tritiated progesterone (P) at 30, 60, and 180 min. After extraction with ethanol and dichloromethane, steroid metabolites were purified by TLC and rpHPLC. Two progesterone derivatives with identical chromatographic properties of 20beta-S and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were purified. In other Teleost fish these steroids are biologically active as MIS. The 17,20beta-P was clearly detected in Mtg and M stages and confirmed by enzymatic oxidation with enzyme 20beta-HSD. The 20beta-S was strongly detected in all Mtg oocytes. The results do not corroborate 20beta-S as a major hormone synthesized in the ovary in FOM as occurs in other scienid fish. A differential steroid synthesis in the advanced oocyte stages suggests that the 20beta-S is acting as a MIS in M. furnieri.     

Induction of maturation of Atlantic croaker oocytes by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one in vitro: consideration of some biological and experimental variables

We characterized the in vitro control of germinal vesicle breakdown (GVBD) by 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one (20 beta-S) in intact ovarian follicles of gonadotropin-primed Atlantic croaker. 20 beta-S-induced GVBD was determined in relation to ovarian (oocyte) morphology, duration of incubation, steroid metabolism, and interaction with other steroids. The rate of GVBD in vitro in the absence of exogenous steroid was positively correlated with initial stage of ovarian morphological development. Maximal responsiveness to 20 beta-S was seen in ovaries with oocytes showing the first signs of morphological maturation. Dose-response experiments with 20 beta-S and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-P) over a range of incubation times yielded similar results for both steroids, suggesting that conversion of 17 alpha,20 beta-P to 20 beta-S is not required for 17 alpha,20 beta-P-induced GVBD. The ED50 of these steroids markedly decreased with increasing incubation times. Comparisons between patterns of follicular transformation of various radiolabelled steroids to 20 beta-S and their respective activities (using unlabelled steroids) in the GVBD bioassay suggested that, in addition to 17 alpha,20 beta-P, progesterone has some intrinsic maturational activity. However, the maturational effects of 11-deoxycortisol and pregnenolone may be explained by their conversion to 20 beta-S. For the first time in any vertebrate, we showed that the proposed maturation-inducing steroid (20 beta-S) is not significantly transformed to any extractable, potentially active metabolite by intact, maturing ovarian follicles. These findings strongly suggest that 20 beta-S is the terminal product of the MIS biosynthetic pathway in Atlantic croaker ovaries. Estradiol had no acute effects on 20 beta-S-induced GVBD. However, testosterone decreased and cortisol augmented the maturational activity of 20 beta-S. Excess progesterone reduced the activity of a maximally effective dose of 20 beta-S, but pregnenolone was without effect. The effects of these steroids on 20 beta-S-induced GVBD are discussed in relation to their possible interactions with 20 beta-S at the MIS receptor level.     

Steroidogenesis in ovarian follicles of chub mackerel, Scomber japonicus

We incubated different radiolabeled steroid precursors with intact chub mackerel ovarian follicles to clarify the synthetic pathways of steroid hormones during vitellogenesis and following final oocyte maturation (FOM). During vitellogenesis, estradiol-17beta (E2) was synthesized from pregnenolone via 17-hydroxypregnenolone, 17-hydroxyprogesterone, androstenedione, and testosterone. The physiological significance of the intermediate metabolites of E2 in the ovarian follicles was examined by comparing follicular steroidogenesis between gonochoric and hermaphroditic fish species. After vitellogenesis, the steroidogenic pathway shifted from E2 to maturation-inducing hormone (MIH) production owing to the inactivation of 17,20-lyase and the activation of 20 beta-hydroxysteroid dehydrogenase. Of the new steroids produced during FOM, 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) was most effective at inducing germinal vesicle breakdown in vitro. Circulating levels of 17,20beta-P increased specifically around the time of germinal vesicle migration, while another FOM-specific 20beta-hydroxylated progestin, 17,20beta,21-trihydroxy-4-pregnen-3-one, was present at consistently low levels during FOM. These results indicate that 17,20beta-P is the MIH of chub mackerel.     

Oocyte maturation-inducing steroids in protandrous black porgy, Acanthopagrus schlegeli

The study objectives aimed to investigate the maturation-inducing steroid (MIS) in marine protandrous black porgy, Acanthopagrus schlegeli. The characteristics of oocyte maturation were also described. Females were injected with two successive doses of LHRH analog (LHRH-A, 10 and 50 microg/kg of fish). The ovarian tissue was obtained at 6-h intervals for in vitro oocyte maturation. Both 17,20 beta-dihydroxy-4-pregnen-3-one (DHP) and 17,20 beta,21-trihydorxy-4-pregnen-3-one (20 beta-S) were the most effective steroids to induce in vitro maturation (e.g. germinal vesicle breakdown, GVBD) in oocytes cultured for either 24 h or 1 min. 20 beta-S had a better potency than DHP in inducing oocyte maturation. 17-hydroxyprogesterone, 11-deoxycortisol, and 20 beta-21-dihydroxy-4-pregnen-3-one also significantly induced oocyte maturation at high concentrations. The process of oocyte maturation (after the injection of LHRH analog) was founded to be divided into four stages: hormone-insensitive stage (insensitive to gonadotropin and MIS); MIS-insensitive (respond to gonadotropin, but not MIS); MIS-sensitive (respond to MIS); and spontaneous stage (GVBD in the hormone-free condition), respectively. Cycloheximide blocked GVBD at the MIS-insensitive stage, control (hormone-free), and hormone-induced GVBD at the MIS-sensitive stage in a dose-dependent effect.     

Analysis of steroid metabolites produced by theca cells from the adult domestic hen

In a previous study on steroid metabolism by hen ovarian cells we reported on the production of 17-hydroxyprogesterone (17OH), androstenedione (A), testosterone (T), and oestrogens from [3H]progesterone (P) by theca cells. The present study examines further the metabolism of P by theca cells from the preovulatory follicles of the hen. The results show that the major metabolite of P is 20 beta-hydroxy-4-pregnen-3-one (20 beta-DHP), representing up to 40% of the recovered radioactivity. In addition, 3 alpha-hydroxy-4-pregnen-20-one (3 alpha-DHP) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta) were identified as metabolites of P, comprising 1 and 3% of the recovered radioactivity, respectively. This is the first evidence that the allylic steroid, 3 alpha-DHP, can be produced by avian ovaries.     

Endocrine control of diurnal oocyte maturation in the kyusen wrasse,Halichoeres poecilopterus

The present study examined diurnal cycles of oocyte development and maturation in the kyusen wrasse, Halichoeres poecilopterus, and investigated the sensitivity of oocytes to maturation-inducing hormone (MIH) and gonadotropic hormone (GTH). Female fish were sampled at fixed intervals throughout the day, revealing that final oocyte maturation and ovulation were completed by 6:00 hr, and that spawning occurred daily between 6:00 and 9:00 hr. In vitro experiments showed that the steroids 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) and 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S) were equally potent and highly effective inducers of germinal vesicle breakdown (GVBD) in kyusen wrasse oocytes. Additionally, circulating levels of 17,20beta-P and 20beta-S increased around the time of GVBD and ovulation, suggesting that 17,20beta-P and 20beta-S act as MIHs in the kyusen wrasse. Moreover, in vitro experiments clearly showed that kyusen wrasse oocytes had a daily developmental cycle of GTH and MIH sensitivity, and that oocytes that completed vitellogenesis acquired GTH-induced maturational competence. An endogenous GTH surge likely occurs between 12:00 and 15:00 hr, and this daily pre-maturational GTH surge probably controls the diurnal maturation cycles of kyusen wrasse oocytes.     

Role of the tidal cycle in the gonadal development and spawning of the tropical wrasse Halichoeres trimaculatus

Many wrasses on coral reefs exhibit daily spawning that peaks around daytime high tides. In this study, we examined tidal-related ovarian development in the threespot wrasse, Halichoeres trimaculatus, a species common on coral reefs in the Indo-Pacific Ocean. When the fish were collected in the morning at different tidal phases, the gonadosomatic index (GSI) and ovarian histology changed; concomitant with increases in GSI towards high tide, a clutch of the most advanced oocytes developed from vitellogenic to maturation stages. Ovulated eggs and post-ovulated follicles (POF) existed in most ovaries around high tide, but only POF remained around ebb tide, suggesting that spawning occurred during or after high tide. We noticed that tidal-related spawning was considerable in the morning and that most ovaries collected on the afternoon high tide exhibited post-spawning features. This suggests that certain labrid species possess plasticity with regard to their spawning time and utilize potent environmental cues to ensure their reproductive success. When pieces of ovary were incubated with precursor steroids, high conversion of testosterone to 17beta-estradiol occurred during high and ebb tides, while that of 17alpha-hydroxyprogesterone to 17alpha,20beta-dihydroxy-4-pregnen-3-one and 17alpha,20beta,21-trihydroxy-4-pregnen-3-one was observed during low and flood tides. Incubation of pieces of ovary with human chorionic gonadotropin resulted in similar fluctuations in the steroid hormones with tidal phase. Production of these steroid hormones correlated with oocyte development in the ovaries and was probably regulated by gonadotropin. These results demonstrate that the daily cycle is fundamental for oocyte development, and that the tidal cycle is superimposed on this process.     

Steroid-induced oocyte maturation in Atlantic croaker (Micropogonias undulatus) is dependent on activation of the phosphatidylinositol 3-kinase/Akt signal transduction pathway

Exposure of fully grown fish and amphibian oocytes to a maturation-inducing steroid (MIS) activates numerous signal transduction pathways to initiate the final stage of oocyte maturation. These events culminate in the activation of maturation-promoting factor and germinal vesicle breakdown (GVBD). In most species, exposure to MIS causes a transient decrease in oocyte cAMP levels. Whether this reduction in oocyte cAMP concentration is sufficient to induce GVBD is unclear. The current study tested the hypothesis that activation of cAMP-independent signal transduction pathways by the naturally occurring MIS, 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S), is necessary for GVBD in Atlantic croaker (Micropogonias undulatus) oocytes. Results indicate that although 20beta-S treatment of oocyte membranes significantly reduced cAMP production, incubation of follicles with the cell-permeable cAMP-dependent protein kinase (Prka) inhibitors Rp-cAMP or KT5720 did not promote GVBD in the absence of 20beta-S. Additionally, treatment of follicles with the phosphodiesterase (Pde) inhibitors Cilostamide (Pde3) or Rolipram (Pde4) significantly reduced GVBD, but they were not able to completely block it. In contrast, pharmacologic inhibition of the cAMP-independent phosphatidylinositol 3-kinase (Pik3)/Akt signal transduction pathway using the Pik3 inhibitors Wortmannin or LY294002, or the Akt inhibitor ML-9, blocked 20beta-S-induced GVBD. Finally, mitogen-activated protein kinase (Mapk1/3) activity increased after treatment with 20beta-S; however, inhibition of Mapk1/3 activity using PD98059 or U0126 had no effect on GVBD. These results demonstrate that activation of cAMP-independent signaling pathways, especially the Pik3/Akt pathway, is necessary for 20beta-S-induced GVBD in Atlantic croaker oocytes.     

Evaluation of the antiviral activity of natural sulfated polyhydroxysteroids and their synthetic derivatives and analogs.

Disodium 3beta,21-dihydroxypregn-5-en-20-one disulfate (2), sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3), sodium 3beta,21-dihydroxypregn-5-en-20-one 21-sulfate (4), and disodium 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one disulfate (6) have been synthesized and completely characterized for the first time from readily available materials. Sulfation was performed using triethylamine-sulfur trioxide complex in dimethylformamide as the sulfating agent. Selective sulfation of 3beta,21-dihydroxypregn-5-en-20-one rendered sodium 3beta,21-dihydroxypregn-5-en-20-one 3-sulfate (3) as the major compound. The synthetic sulfated steroids as well as natural disulfated polyhydroxysteroids (7-9) isolated by us from the antarctic ophiuroid Astrotoma agassizii and the synthetic derivatives disodium 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene 3-acetate, 2,21-disulfate (7a) and 2beta,3alpha,21-trihydroxy-(20R)-cholesta-5,24-diene (7b) were comparatively evaluated for their inhibitory effect on the replication of one DNA (HSV-2) and two RNA (PV-3, JV) viruses. In general, steroids with sulfate groups at C-21 and C-2 or C-3 were the most effective in their inhibitory action against HSV-2 and also proved to be active against PV-3 and JV.     

Characterization of the major steroids present in amniotic fluid obtained between the 15th and 17th weeks of gestation

In pooled amniotic fluid obtained between the 15th and 17th weeks of gestation the concentration of free steroids and steroid glucuronides was found to be 40 micrograms/dl. The concentration of steroid monosulfates and disulfates was 19 micrograms/dl. About half of the characterized steroids are progesterone metabolites. The "fetal type" 3 beta-hydroxy-5-ene steroids were found exclusively in the sulfoconjugated form. Their concentration represents 20% of the total steroid content. The identification of two 15 beta-hydroxylated C21 steroids, 3 beta,15 beta,17 alpha-tridoxy-5-pregnen-20-one and 5-pregnene-3 beta,15 beta,17 alpha,20 alpha-tetrol isolated from mid-pregnancy amniotic fluid is reported here. Metabolites of cortisol and 17-deoxycorticosteroid metabolites had similar quantitative importance, 8.6 and 9.4%, respectively.     

Sex steroids relative to alternative mating behaviors in the simultaneous hermaphrodite Serranus subligarius (Perciformes: Serranidae)

This study is the first investigation of reproductive endocrinology in a simultaneously hermaphroditic teleost, the belted sandfish (Serranus subligarius). We address two questions: (1) Do steroid hormone levels vary during the spawning season or during the daily spawning cycle of sandfish? (2) Do hormone levels vary relative to an individual's phenotype-size, frequency of spawning and aggressive behaviors, and proportion of testis in the gonad? We analyzed circulating estradiol-17beta (E2), testosterone (T), 11-ketotestosterone (11KT), 17alpha,20beta,21-trihydroxy-4-pregnen-3-one (20betaS), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP) concentrations in a field population. Only E2 levels were significantly higher at the new and full moon, suggesting peak periods of vitellogenesis at these times. Naturally spawning sandfish were sampled every 2 h during the photophase of a 25-h period (12 pm to 1 pm the following day) and gonadosomatic index, degree of oocyte hydration and ovulation, and plasma levels of E2, T, DHP, and 20betaS were analyzed. E2 and T levels did not vary during photophase, suggesting continuous recruitment of oocytes into vitellogenesis. The 20betaS levels peaked around the time of final oocyte maturation. Since frequency of spawning behaviors changes with body size, we captured individuals of various sizes throughout the spawning season and analyzed circulating levels of hormones. 11KT and 20betaS levels increased significantly with body size. In 1992, we quantified frequency of spawning and aggressive behaviors, circulating T and 11KT levels and testicular mass relative to ovotestis mass in focal animals. 11KT levels tended to be positively correlated with frequency of courting male behavior, but were unrelated to the frequency of aggressive behavior or testis mass. Because hormone levels increased with size and frequency of each spawning behavior changes with size, we propose that sex steroids influence growth-related changes in spawning tactics of individuals.     

Purification and properties of an NADPH-dependent 21-oxo-20-hydroxysteroid reductase (17beta-aldol reductase) from sheep liver. Isolation of the 20beta-glycol product.

This investigation was undertaken to test the hypothesis that steroidal 20-hydroxy-21-aldehydes are intermediates in an alternative pathway of corticosteroid metabolism leading to steroidal 20,21-diols. A NADPH-dependent 21-oxo-20-hydroxysteroid reductase which catalyzed the reduction of 11beta,17,20beta-trihydroxy-3-keto-4-pregnen-21-al (isocortisol) to 11beta,17,20beta,21-tetrahydroxy-4-pregnen-3-one (Reichstein's compound E) was prepared from sheep liver. Other steroidal 17-aldols were also good substrates. Some steroidal 17-oxoaldehydes, D-, and L-glyceraldehyde were reduced, but less effectively than the steroidal aldols. Steroidal ketols and 21-oic acids were not substrates. The enzyme contains--SH groups, has a pH optimum of 6.9 to 7.5 and a molecular weight of about 28,000. Reversibility of the enzymic reaction could not be demonstrated. The reduction product obtained from isocortisol was isolated and characterized. Other 17-glycols were derived from their respective steroid aldols. Reductase activity was also present in hamster and rat liver. From a comparison of 21-oxo-20-hydroxysteroid reductase and 21-hydroxysteroid dehydrogenase with respect to pH optima, substrate specificity, stability to heat, and kinetic constants, we conclude that the two enzymes are distinct.     

Formation of 5-[17beta-2H]androstene-3beta,17alpha-diol from 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one by the microsomal fraction of boar testis

After incubation of 3beta-hydroxy-5-[17,21,21,21-2H]-pregnen-20-one with the microsomal fraction of boar testis, the metabolites were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following metabolites were identified: 3beta,17alpha-dihydroxy-5-[21,21,21-3H]pregnen-20-one, 3beta-hydroxy-5-androsten-17-one, 5-androstene-3beta,17beta-diol, and 5-[17beta-2H]androstene-3beta,17alpha-diol. The presence of a 2H atom at the 17beta position of 5-androstene-3beta,17alpha-diol was confirmed by oxidizing the steroid with 3beta-hydroxy-steroid dehydrogenase of Pseudomonas testosteroni to obtain 17alpha-hydroxy-4-[2H]androsten-3-one and then by oxidizing the latter steroid with chromic acid to obtain nonlabeled 4-androstene-3,17-dione. Among these metabolites, the first three can be interpreted to be synthesized by a well documented pathway, including 17alpha-hydroxylation followed by side chain cleavage as follows: 3beta-hydroxy-5-[17,21,21,21-2H]pregnen-20-one leads to 3beta,17alpha-dihydroxy-2-[21,21,212H]-pregnen-20-one leads to 3beta-hydroxy-5-androsten-17-one leads to 5-androstene-3beta,17beta-diol. On the other hand, 5-androstene-3beta,17alpha-diol, which contained a 2H atom at the 17beta position, is not likely to be synthesized via above mentioned pathway in which nonlabeled 3beta-hydroxy-5-androsten-17-one is formed as the first C19-steroid. It seems that an alternate side chain cleavage mechanism leading from pregnenolone to 17alpha-hydroxy-C19-steroid exists in boar testis.     

Synthesis of deuterium-labeled tetrahydrocortisol and tetrahydrocortisone for study of cortisol metabolism in humans

A method is described for the preparation of multi-labeled tetrahydrocortisol (3alpha,11beta,17alpha,21-tetrahydroxy-5beta-[1, 2,3,4,5-2H5]pregnan-20-one, THF-d5), allo-tetrahydrocortisol (3alpha,11beta,17alpha,21-tetrahydroxy-5alpha-[1 ,2,3,4,5-2H5]pregnan-20-one, allo-THF-d5), and tetrahydrocortisone (3alpha,17alpha,21-trihydroxy-5beta-[1,2,3,4,5-2H5]pre gnane-11,20-dione, THE-d5) containing five non-exchangeable deuterium atoms in the steroid ring A. Reductive deuteration at C-1, C-2, C-3, C-4, and C-5 of prednisolone or prednisone was performed in CH3COOD with rhodium (5%) on alumina under the deuterium atmosphere. The isotopic purities of the labeled compounds as [2H5]-form were estimated to be 86.17 atom%D for THF-d5, 74.46 atom%D for allo-THF-d5 and 81.90 atom%D for THE-d5, based on the ion intensities in the region of the molecular ion of methoxime-trimethylsilyl (MO-TMS) derivatives measured by GC-MS.     

Microbial transformation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one by Trichoderma viride

Fermentation of 5alpha,6alpha-epoxy-3beta-hydroxy-16-pregnen-20-one (4) with Trichoderma viride under aerobic condition yielded 3beta,5alpha,6beta-trihydroxy-16-pregnen-20-one (5) and 3beta,5alpha,6beta,15beta-tetrahydroxy-16-pregnen-20-one (6). Each microbial metabolite was characterized by spectroscopic methods. Compounds 6 and 3beta,5alpha,15beta-trihydroxy-16-pregnen-6,20-dione (7) are reported for the first time.     

In vitro hormone induction of final oocyte maturation in striped bass (Morone saxatilis) follicles is inhibited by blockers of phosphatidylinositol 3-kinase activity

Oocyte germinal vesicle breakdown (GVBD) was induced in striped bass ovarian fragments when tissues were incubated with 100-nM recombinant human insulin-like growth factor-I (rhIGF-I), 25-IU human chorionic gonadotropin (hCG) ml(-1), or 290 nM of the maturation-inducing steroid (MIS), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S). Inhibitors of phosphatidylinositol 3-kinase (PI 3-K), wortmannin (100 nM) and LY 294002 (50 microM), inhibited GVBD induced by these hormones. Furthermore, the inhibitors attenuated hCG-induced steroid hormone synthesis. Previous studies report that gap junction uncouplers inhibit GVBD induced by hCG, but not by rhIGF-I, in striped bass. We show that 20beta-S-induced GVBD is also attenuated by 1 mM 1-heptanol or 1-octanol without being affected by incubation with 3 mM ethanol. Thus, the effects of inhibiting PI 3-K activity on GtH and MIS actions are similar to effects of uncoupling gap junctions. These data suggest that PI 3-K activity is required for GtH- MIS- and IGF-I induction of GVBD in striped bass. Our data are also consistent with the notion that a ligand that regulates PI 3-K activity, possibly an IGF, participates in maintenance of gap junctional communication required for maximal GtH and MIS action.