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.     

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.     

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.     

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.     

Biochemical characterization of the Arctic char (<Emphasis Type="Italic">Salvelinus alpinus</Emphasis>) ovarian progestin membrane receptor

Membrane progestin receptors are involved in oocyte maturation in teleosts. However, the maturation-inducing steroid (MIS) does not appear to be conserved among species and several progestins may fulfill this function. So far, complete biochemical characterization has only been performed on a few species. In the present study we have characterized the membrane progestin receptor in Arctic char (Salvelinus alpinus) and show that the 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) receptor also binds several xenobiotics, thus rendering oocyte maturation sensitive to environmental pollutants. We identified a single class of high affinity (K_d, 13.8 ± 1.1 nM), low capacity (B_max, 1.6 ± 0.6 pmol/g ovary) binding sites by saturation and Scatchard analyses. Receptor binding displayed rapid association and dissociation kinetics typical of steroid membrane receptors, with t_1/2 s of less than 1 minute. The 17,20beta-P binding also displayed tissue specificity with high, saturable, and specific 17,20beta-P binding detected in ovaries, heart and gills while no specific binding was observed in muscle, brain or liver. Changes in 17,20beta-P binding during oocyte maturation were consistent with its identity as the oocyte MIS membrane receptor. Incubation of fully-grown ovarian follicles with gonadotropin induced oocyte maturation, which was accompanied by a five-fold increase in 17,20beta-P receptor binding. In addition, competition studies with a variety of steroids revealed that receptor binding is highly specific for 17,20beta-P, the likely maturation-inducing steroid (MIS) in Arctic char. The relative-binding affinities of all the other progestogens and steroids tested were less than 5% of that of 17,20beta-P for the receptor. Several ortho, para derivatives of DDT also showed weak binding affinity for the 17,20beta-P receptor supporting the hypothesis that xenobiotics may bind steroid receptors on the oocyte's surface and might thereby interfere with oocyte growth and maturation.     

Identification of 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as the major ovarian steroid produced by the teleost Micropogonias undulatus during final oocyte maturation

This study describes the identification of 17 alpha,20 beta, 21-trihydroxy-4-pregnen-3-one (20 beta-dihydro-11-deoxycortisol, 20 beta-S) as a major steroid product of the ovary of Atlantic croaker (Micropogonias undulatus) incubated in vitro. This is the first report which describes 17 alpha,20 beta,21-trihydroxy-4-pregnen-3-one as a major product of teleost steroidogenic tissue. The steroid was identified by a variety of methods, including HPLC, TLC, GC-MS, UV absorbance, and reactions with specific enzymes. Full grown oocytes, prior to final oocyte maturation (FOM), accumulated only small amounts of 20 beta-S. However, a substantial increase in 20 beta-S production occurred at the time of FOM. These results suggest that 20 beta-S is a maturational steroid in this species.     

Binding characteristics, hormonal regulation and identity of the sperm membrane progestin receptor in Atlantic croaker

Recently a novel cDNA was discovered in spotted seatrout ovaries encoding a protein with seven transmembrane domains that has the characteristics of the membrane progestin receptor (mPR) mediating maturation-inducing steroid (MIS) induction of oocyte maturation in this species. Preliminary results suggested the MIS also activates an mPR on the spermatozoa of spotted seatrout and a closely related species, Atlantic croaker, to stimulate sperm motility. We show here that plasma membranes of croaker sperm demonstrate high affinity (Kd approximately 20 nM), limited capacity (Bmax 0.08 nM), specific and displaceable binding for progestins that is characteristic of mPRs. The MIS (17,20beta,21-trihydroxy-4-pregnen-3-one, 20beta-S) displayed the greatest binding affinity for the sperm mPR among the steroids tested. Treatment of croaker testicular tissue in vitro with gonadotropin caused a several-fold increase in sperm mPR receptor concentrations that was partially blocked in the presence of cyanoketone, which suggests this action of gonadotropin is partially mediated by stimulation of steroidogenesis. Protein bands of the predicted sizes for the mPR and its dimer (40 and 80 kDa) were detected by Western blotting of croaker sperm membranes using a specific antibody to the novel seatrout mPR (mPRalpha). Immunocytochemistry of whole croaker spermatozoa with the mPRalpha antibody showed that staining was primarily localized on the midpiece, consistent with a role of the mPRalpha in mediating MIS stimulation of sperm motility. The results suggest that the mechanism of progestin action on fish sperm involving mPRs is basically similar to that in mammals and has been evolutionarily conserved amongst vertebrates.     

Induction and inhibition of in vitro oocyte maturation and production of steroids in fish follicles by forskolin

The effects of forskolin (FK) on in vitro oocyte maturation and production of steroids were examined in Oryzias latipes. When oocytes within preovulatory follicles were preincubated in the presence of FK for 2-10 hr, they matured normally after additional incubation for 10-20 hr in plain culture medium. Naked (follicle cell-free) oocytes did not mature under these conditions. FK stimulated dose-dependent production of steroids (estradiol-17 beta, E2, and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one, 17 alpha,20 beta-diOHprog) and cAMP in follicle (granulosa) cells. On the other hand, exposure to FK within 2 hr after 17 alpha,20 beta-diOH prog stimulation caused reversible inhibition of gonadotropin (PMS)- or 17 alpha,20 beta-diOH prog-induced maturation of the intrafollicular oocytes in vitro. FK also significantly inhibited the 17 alpha,20 beta-diOHprog-induced maturation of naked oocytes, suggesting the existence of adenylate cyclase in fish oocytes. These data indicate that in Oryzias latipes, FK induces oocyte maturation by stimulating follicular production of maturation-inducing steroid (MIS), probably 17 alpha,20 beta-diOH prog, via an increase in cAMP, and that it may inhibit oocyte maturation by increasing ooplasmic cAMP and some inhibitory interaction between the granulosa cells and the oocyte through intercellular communication.     

In-vitro Production of Meiosis Inducing Substance (MIS) by Isolated Amphibian (Rana pipiens) Follicle Cells*

Previous studies indicated that pituitary hormone induced oocyte maturation in preovulatory amphibian ovarian follicles is mediated by somatic elements of the follicle. In this study procedures were developed for isolating and culturing follicle cells and their ability to produce meiosis inducing substance (MIS) was assessed. Defolliculated oocytes surrounded by a single layer of follicle cells but not denuded oocytes matured in response to frog pituitary hormone (FPH) stimulation. Cultured follicle cells secreted MIS following stimulation with FPH. The amount of MIS activity produced was related to the number of follicle cells cultured and the dose of FPH utilized. Radioimmunoassay (RIA) analysis of medium from follicle cell cultures demonstrated that FPH stimulated steroid (progesterone) secretion from these cells. Addition of cAMP to follicle cell cultures enhanced FPH stimulated steroid production. The results indicate that follicle cells retain FPH responsiveness when uncoupled from the immature oocyte and exhibit both MIS and steroid secretory functions.     

Progestin membrane receptors involved in the meiotic maturation of teleost oocytes: a review with some new findings

The endocrine control of oocyte maturation in fish has proven to be a valuable model for investigating rapid, nongenomic steroid actions at the cell surface. Considerable progress has been made over the last decade in identifying and characterizing progestin membrane receptors mediating these actions in fish, in understanding the hormonal regulation and physiological roles of these receptors in oocyte maturation, in elucidating the signal transduction pathways they activate, and in determining their nature. Recent advances on these topics are briefly reviewed. New data demonstrating the involvement of pertussis toxin-sensitive inhibitory G-proteins in induction of oocyte maturation by the maturation-inducing steroid (MIS) in teleosts is also presented. In addition, the cloning strategy to isolate the MIS receptor gene from spotted seatrout ovaries and the characteristics of a novel gene and protein discovered by this approach are discussed. Current evidence suggests this G-protein-coupled receptor-like protein is the long sought after MIS receptor mediating meiotic maturation of teleost oocytes.     

Extrafollicular mediation of oocyte maturation by radial nerve factor in starfish Pisaster ochraceus

In starfish ovaries follicle cells that envelop each oocyte are thought to mediate the production of a maturation inducing substance (MIS), identified as 1-methyladenine, that induces maturation and spawning of oocytes after exposure to a gonadotropic substance secreted by the radial nerve (RNF). Studies were carried out to assess the possible role of extrafollicular cells within the ovarian wall in mediating this signal transduction process in the ovary of Pisaster ochraceus. Oocyte maturation and spawning occurred following the addition of RNF to intact ovarian tissue in vitro whereas no maturation occurred following the addition of RNF to germinal vesicle (GV) oocytes or GV oocytes surrounded by follicle cells. In contrast, oocyte maturation occurred when small ovarian wall fragments, lacking mature follicles, were incubated with GV oocytes and RNF. Neither actinomycin D nor cycloheximide altered RNF induction of oocyte maturation in the presence of the ovarian wall tissue whereas preheating (boiling water for 5 min) the tissue obliterated its response to RNF. Non-ovarian tissues failed to produce MIS in response to RNF. Results suggest that ovarian components other than the follicle cells that envelop fully grown immature oocyte are responsive to RNF and represent a significant and previously unrecognised intra-ovarian source of MIS.     

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.     

Activation of a pertussis toxin-sensitive, inhibitory G-protein is necessary for steroid-mediated oocyte maturation in spotted seatrout

Oocyte maturation (OM) is initiated in lower vertebrates and echinoderms when maturation-inducing substances (MIS) bind oocyte membrane receptors. This study tested the hypothesis that activation of a G_i protein is necessary for MIS-mediated OM in spotted seatrout. Addition of MIS significantly decreased adenylyl cyclase activity in a steroid specific, pertussis toxin (PTX)-sensitive manner in oocyte membranes and microinjection of PTX into oocytes inhibited MIS-induced OM, suggesting the steroid activates a G_i protein. MIS significantly increased [³⁵S]GTPγS binding to ovarian membranes, confirming that MIS receptor binding activates a G-protein, and immunoprecipitation studies showed the increased [³⁵S]GTPγS binding was associated with Gα_i1-3 proteins. Radioligand binding studies in ovarian membranes using GTPγS and PTX demonstrated that the MIS binds a receptor coupled to a PTX-sensitive G-protein. This study provides the first direct evidence in a vertebrate model that MIS-induced activation of a G_i protein is necessary for OM. These results support a mechanism of MIS action involving binding to a novel, G-protein coupled receptor and activation of an inhibitory G-protein, the most comprehensive and plausible model of MIS initiation of OM proposed to date.     

Identification of membrane progestin receptors (mPR) in goldfish oocytes as a key mediator of steroid non-genomic action

One of the most extensively investigated and well characterized models of non-genomic steroid actions initiated at the cell surface is the induction of oocyte maturation (OM) in fish and amphibians by progestin. Gonadotropin induces the final phase of oocyte maturation indirectly by inducing the synthesis of maturation inducing steroids (MIS) by the ovarian follicles via its membrane receptor, membrane progestin receptor (mPR). Three mPR subtypes (α, β and γ) have been identified by cDNA cloning or by in silico analysis of genome sequence databases. Previously, we described the cloning of the mPRα cDNA from a goldfish ovarian cDNA library and obtained experimental evidence that the mPRα protein is an intermediary in MIS induction of OM in goldfish. Then we cloned one β and two γ subtypes (hereafter referred to as γ-1 and γ-2) from a goldfish ovarian cDNA library. RT-PCR showed different tissue expression patterns of the mRNAs for these mPR subtypes. However, in addition to mPRα, the β, γ-1 and γ-2 subtypes were also expressed in follicle-enclosed oocytes. Microinjection of goldfish oocytes with a morpholino antisense oligonucleotide to mPRβ blocked the induction of oocyte maturational competence, whereas injection of antisense oligonucleotides to mPRγ-1 and γ-2 were ineffective. These results suggest that goldfish mPRβ protein acts as an intermediary during MIS induction of OM in goldfish, in a manner similar to mPRα. We are establishing mutant strains of Medaka fish to investigate the roles of mPR proteins in vivo produced by Targeting Induced Local Lesions in Genomes (Tilling) strategy. By the screening, we have selected three strains in which a point mutation was induced in each strain at the coding sequence of mPRα. In near future results of phenotypic analysis of mPRα defective fish will be introduced.     

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.     

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.     

Effects of gonadotropin on ovarian intrafollicular processes during the development of oocyte maturational competence in a teleost, the Atlantic croaker: evidence for two distinct stages of gonadotropin control of final oocyte maturation

Full-grown oocytes of Atlantic croaker are insensitive to maturation-inducing steroid (MIS) unless they are primed with gonadotropin (GtH). The objective of this study was to examine the mechanism of GtH-induced maturational competence in croaker oocytes. Specifically, we determined the in vitro secretion of steroids by intact ovarian follicles of unprimed or hCG-primed fish, the direct effects of steroids on maturational competence, and the effects of steroid (cyanoketone), protein (cycloheximide), and RNA (actinomycin D) synthesis inhibitors on hCG-induced maturational competence and steroidogenesis in vitro. The steroid content of the incubation medium after hCG treatment was measured by RIA. The effects of hCG or exogenous steroid treatment on maturational competence were determined by recording the incidence of germinal vesicle breakdown (GVBD) after MIS-induced GVBD in a standard bioassay. Our major findings were: (1) induction of maturational competence occurred after exposure of ovarian follicles to hCG either in vivo or in vitro; (2) MIS secretion was detected in follicles of hCG-primed fish but not unprimed fish, and no MIS secretion was observed during hCG induction of maturational competence in vitro; (3) treatment with cyanoketone blocked the hCG-dependent secretion of testosterone and estradiol but not the development of maturational competence; (4) treatment with MIS or various other exogenous steroids in the absence of hCG did not induce maturational competence; and (5) hCG-induced maturational competence was inhibited by cycloheximide and actinomycin D. Therefore, the mechanisms of GtH induction of oocyte maturation in Atlantic croaker can be described in two distinct stages: a delta-4 steroid-(including MIS) and estrogen-independent priming stage followed by a MIS-mediated GVBD stage. The priming stage may involve mechanisms requiring RNA as well as protein synthesis.     

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.