Maturation-inducing Steroid Research Articles

Hormonal regulation of the steroidogenic acute regulatory protein (StAR) in gonadal tissues of the Atlantic croaker ( Micropogonias undulatus)

The steroidogenic acute regulatory protein (StAR), a member of the StAR-related lipid transfer domain (START) family, is critical to regulated steroidogenesis in vertebrates. We have isolated a cDNA encoding StAR from a well-studied model of teleost physiology, the Atlantic croaker Micropogonias undulatus. This cDNA (1204 nucleotides total length) contains an open reading frame of 858 nucleotides encoding a protein of 286 amino acids. Molecular phylogenetic analysis indicates the putative Atlantic croaker StAR protein is more closely related to StAR proteins (62–85% identity) than to the related START protein MLN-64 (28–31% identity). Green monkey kidney cells (COS-1) cotransfected with Atlantic croaker StAR and human cholesterol side chain cleavage (SCC) expression constructs are able to produce significantly more pregnenolone than cells transfected with SCC alone. StAR mRNA is detected in the Atlantic croaker head kidney by reverse transcriptase-polymerase chain reaction (RT-PCR) and in the kidney and hypothalamus in some individuals. Gonadal StAR gene expression is below the level of detection by RT-PCR in most individuals, but can be detected using fluorescent probes in quantitative RT-PCR. StAR mRNA is not detected in the Atlantic croaker brain. Six hour in vitro treatment of Atlantic croaker ovarian follicles with human chorionic gonadotropin (hCG) is insufficient to significantly alter StAR mRNA levels; however, 24 h hCG treatment induces StAR mRNA levels 17-fold over untreated controls. Neither 6 nor 24 h treatment with hCG significantly alters StAR mRNA levels in Atlantic croaker testicular minces. Likewise, 6 h in vitro treatment with estradiol, testosterone or the maturation-inducing steroid 17,20β,21-trihydroxy-4-pregnen-3-one is without effect on gonadal StAR mRNA levels.

Sex steroids and oocyte maturation in the sterlet (Acipenser ruthenus L.)

Summary Sturgeon oocytes, like those of other vertebrates, are arrested in early meiosis and require a steroid or steroids to trigger meiosis resumption or maturation. The identity of the maturation-inducing steroid in sturgeons has present study was designed to investigate in sterlet the follicular secretion and relative blood changes of free and conjugated 17,20βP, 20βS and of the androgens T, 11KT, in response to hormonal treatment and to determine the relative potencies of the progestagens, 17,20β-P, 20βS, P4 and S, in inducing oocyte final maturation in vitro.

Ovarian development and plasma sex steroid levels in cultured female Senegalese sole Solea senegalensis

Ovarian development was studied in cultured female Senegalese sole Solea senegalensis. Females with regressed ovaries, mainly occupied by perinucleolar oocytes, predominated throughout summer exhibiting low condition factor (K), gonadosomatic index (I(G)), and plasma 17beta-estradiol and testosterone levels. Throughout autumn and winter (ovaries at early and intermediate maturation), oocytes progressed to cortical alveoli and vitellogenic stages accompanied by increasing K, I(G), and plasma 17beta-estradiol and testosterone levels. At late winter/early spring, ovarian development reached its maximum with the predominance of females at intermediate and final maturation (the latter occupied by late vitellogenic oocytes and few early maturation oocytes) and peak values of K, I(G), and 17beta-estradiol and testosterone concentrations. Steroid levels were lower (especially testosterone) than those for naturally-spawning females, which might cause extensive atresia without final oocyte maturation (no spawning was observed). This degenerative process reduced de size of the ovary (initial and intermediate phases of regression) in association with declining K, I(G), and plasma 17beta-estradiol and testosterone levels and increasing proportions of perinucleolar oocytes. The circulating 17,20beta-dihydroxy-4-pregnen-3-one levels, the proposed maturation-inducing steroid, remained relatively constant throughout the experimental period, suggesting that oocytes were unable to respond adequately to its stimulation. We propose the inadequate seasonal thermal regime as the main cause of such dysfunction.

자연산 문치가자미 (Limanda yokohamae)의 난소 발달에 따른 혈중 성호르몬과 난황단백전구체 농도 변동

This study correlated changes in estradiol-l7<TEX>$\beta$</TEX> (<TEX>$E_2$</TEX>), testosterone (T), 17<TEX>$\alpha$</TEX>,20-dihydroxy-4-pregnen-3-one (DHP), and vitellogenin (VTG) levels with changes in the gonadosomatic index (GSI) and ovarian histology during the annual reproductive cycle of the wild marbled sole, Limanda yokohamae. Synchronous oocyte development occurs in this fish. Ovary maturity was classified into four periods, based on histological observations: the spawning (December to February), post-spawning (February to April), recovery (May to August), and vitellogenic (September to November) periods. Seasonal changes in the GSI were inversely correlated with water temperatures and reflected the degree of ovarian maturity. Plasma VTG levels were correlated with changes in the GSI, which increased from September to a peak in January, and levels remained comparatively high until February. Estradiol-17<TEX>$\beta$</TEX> was at baseline levels (<0.11 ng/mL) during the spring and summer, and peaked rapidly (1.55<TEX>$\pm$</TEX>0.445 ng/mL) from October to January. Plasma T and DHP levels had a similar profile; they rose markedly during the spawning period and remained low (or were not detectable) from spring through autumn. These data indicate that changes in plsama steroid hormones and VTG levels are correlated with the annual ovarian activity of the marbled sole. Based on these results and published reports, it appears that in this species DHP is the most important maturation-inducing steroid and that T is also related to final maturation.

Cell-surface expression, progestin binding, and rapid nongenomic signaling of zebrafish membrane progestin receptors α and β in transfected cells

Recently, a unique family of membrane progestin receptors (mPRalpha, mPRbeta, and mPRgamma) was identified, which may be responsible for mediating rapid, nongenomic actions of progestins in a variety of target tissues. In this study, the mPRalpha and mPRbeta isoforms from zebrafish were shown to be rapidly and specifically activated by the maturation-inducing steroid (MIS) of this species, 4-pregnen-17,20beta-diol-3-one (17,20beta-DHP). The zebrafish mPRalpha and a previously uncharacterized mPRbeta isoform were stably expressed in nuclear progesterone receptor-deficient mammalian breast cancer cells, MDA-MB-231. Expression and surface localization of the receptors were verified by flow cytometry, biotin surface labeling, and Western blotting. Plasma membrane proteins from mPRalpha- or mPRbeta-transfected cells showed high affinity (mPRalpha, K(d) 7 nM; mPRbeta, K(d) 12 nM), saturable, displaceable, single-binding sites specific for 17,20beta-DHP, whereas negligible specific 17,20beta-DHP binding was observed in nontransfected cells. Progestin treatment caused significant activation of mitogen-activated protein kinase (MAPK) within 5 min in cells transfected with either of the receptors as measured by western blotting and flow cytometry. The rank order of the potencies of several progestins in activating MAPK via mPRalpha and mPRbeta was the same (17,20beta-DHP>progesterone >4-pregnen-17,20beta,21-triol-3-one). Interestingly, the MIS in zebrafish, 17,20beta-DHP, was also the most potent inhibitor, among the progestins tested, of adenylyl cyclase activity in cells transfected with either of the receptors. This progestin significantly decreased cAMP levels in both mPRalpha- and mPRbeta-transfected cells in a dose-responsive and time-dependent manner. In addition, signaling of the zebrafish mPRalpha was blocked by pertussis toxin, implying activation of a G(i) protein, while sensitivity to pertussis or cholera toxin was not shown with mPRbeta-mediated signaling, possibly indicating that this receptor activates a different pertussis toxin-insensitive G protein. The results of this study suggest that zebrafish mPRalpha and mPRbeta signal similarly upon progestin binding resulting in rapid activation of MAPK and downregulation of adenylyl cyclase activity.

Hydration of rainbow trout oocyte during meiotic maturation andin vitroregulation by 17,20β-dihydroxy-4-pregnen-3-one and cortisol

Although oocytes of many teleost fish, especially marine species, are subjected to a hydration process during meiotic maturation, which leads to an important volume increase, no noticeable hydration of the preovulatory oocyte has ever been reported in rainbow trout (Oncorhynchus mykiss). In the present study, oocyte water content and dry mass were monitored using consecutive samples taken in vivo from the same female rainbow trout, from 4-5 days prior to ovulation to up to 7 days post-ovulation. In addition, yolk protein electrophoretic patterns were compared between oocytes sampled prior to germinal vesicle breakdown (GVBD) and unfertilized eggs. Furthermore, the effect of the maturation-inducing steroid (17,20beta-dihydroxy-4-pregnen-3-one, 17,20beta-P), cortisol and 11-deoxycorticosterone (DOC) on oocyte dry and wet masses, as well as GVBD occurrence was assessed in vitro. Finally, mRNA expression profiles of glucocorticoid and mineralocorticoid receptors as well as 11beta-hydroxysteroid dehydrogenase (11beta-HSD) were monitored in the periovulatory ovary by real-time PCR. Both in vivo and in vitro data showed, for the first time in rainbow trout, that a significant oocyte hydration occurs during oocyte maturation. In addition, an intra-oocyte dry matter increase was reported in vivo during the periovulatory period. However, yolk protein migration patterns were similar in preGVBD oocytes and unfertilized eggs, suggesting that no or little yolk proteolysis occurs during oocyte maturation. We also showed that oocyte hydration can be induced in vitro by 17,20beta-P and cortisol but not by DOC. In contrast, GVBD was only observed after 17,20beta-P stimulation. Finally, real-time PCR analysis showed an up-regulation of 11beta-HSD and glucocorticoid receptor 2 transcripts in the ovary at the time of oocyte maturation. Together, these results suggest that cortisol could participate in the control of oocyte hydration and possibly in other periovulatory ovarian functions.

In vitro effects of insulin-like growth factors and insulin on oocyte maturation and maturation-inducing steroid production in ovarian follicles of common carp, Cyprinus carpio

In vitro germinal vesicle breakdown (GVBD) in Cyprinus carpio oocytes was induced by recombinant human insulin-like growth factor-I and -II (IGF-I and IGF-II) and bovine insulin (b-insulin). Treatment of postvitellogenic ovarian follicles with IGF-I and b-insulin increased concentration of maturation-inducing hormone (MIH), 17α,20β-dihydroxy-4-pregnane-3-one (DHP) in the medium. IGF-I and IGF-II both and b-insulin induced GVBD in denuded oocytes. IGF-I analogue R3 IGF-I was more potent than IGF-I in inducing GVBD of postvitellogenic follicles suggesting that ovarian IGF binding proteins may inhibit IGF-I action. Vitellogenic follicles, which were immature for oocytes to complete GVBD in response to DHP or HCG, underwent GVBD by IGF-I, not by b-insulin. IGF-I was also able to stimulate DHP production in such follicles. Addition of DHP and HCG to the culture of vitellogenic follicles containing IGF-I or b-insulin did neither potentiate the stimulation of GVBD induced by IGF-I nor initiate the same in response to b-insulin. Incubation of postvitellogenic follicles with trilostane (3β-HSD inhibitor) had no inhibitory effects on IGF-I- and b-insulin-induced GVBD but attenuated the same under HCG stimulation. Trilostane, however, strongly inhibited DHP production induced by all these effectors. Induction of GVBD by IGF-I and b-insulin was not altered in the presence of actinomycin D. However, it significantly blocked the HCG-induced GVBD. Cycloheximide was shown to inhibit the induction of GVBD and DHP production by IGF-I, b-insulin and HCG. Both actinomycin D and cycloheximide were found to inhibit DHP production stimulated by all the three effectors. Collectively, these observations indicate that IGF-I and b-insulin can induce GVBD via MIH- and transcription-independent pathway. Incubation of the follicles with gap junction uncouplers, 1-heptanol or 1-octanol, had no effect on IGF-I- and b-insulin-induced GVBD, but attenuated the same induced by HCG. These uncouplers, however, inhibited DHP production induced by IGF-I, b-insulin and HCG. This result suggests that both IGF-I and b-insulin can induce oocyte maturation without coupled gap junction between oocytes and granulosa cells, while homologous gap junctions are required for DHP production. Inhibitors of phosphatidylinositol-3 kinase (PI-3 kinase), wortmannin and LY294002 inhibited GVBD by IGF-I and b-insulin. These two inhibitors also attenuated HCG-induced GVBD. These data suggest that PI-3 kinase activity is required for IGF-I, b-insulin and HCG induction of GVBD in C. carpio.

Insulin-Like Growth Factor-Binding Protein (IGFBP)-1, -2, -3, -4, -5, and -6 and IGFBP-Related Protein 1 during Rainbow Trout Postvitellogenesis and Oocyte Maturation: Molecular Characterization, Expression Profiles, and Hormonal Regulation

In the present study we report the full coding sequence of rainbow trout IGF-binding protein-1 (IGFBP1), -2, -3, -5, and -6 and IGFBP-related protein-1 (IGFBP-rP1) mRNAs as well as the partial coding sequence of IGFBP-4 mRNA. To our knowledge, this is the first report of IGFBP4, IGFBP6, and IGFBP-rP1 in a nonmammalian species. The tissue distribution of all mRNAs was studied, and the ovarian expression profiles of IGFBP2 to -6 and IGFBP-rP1 between late vitellogenesis and oocyte maturation were characterized. In addition, in vitro hormonal regulation by the maturation-inducing steroid 17,20beta-dihydroxy-4-pregnen-3-one (17,20betaP), gonadotropin, and estradiol were studied. We observed that besides IGFBP1, which was only found in liver, IGFBP2 to -6 and IGFPB-rP1 were expressed in the preovulatory ovary. IGFBP3 was also detected in liver, trunk, kidney, skin, and gills, whereas IGFBP2 to -6 and IGFBP-rP1 exhibited a wider tissue distribution. In the preovulatory ovary, IGFBP3 was strongly down-regulated during the postvitellogenesis period, whereas IGFBP5 exhibited a limited up-regulation. In addition, IGFBP6 and IGFBP-rP1 were up-regulated during oocyte maturation. Hormonal regulation data indicated that all ovarian IGFBPs and IGFBP-rP1 transcripts are regulated under gonadotropic stimulation at a concentration that induced 100% oocyte maturation. In addition, IGFBP2 to -5 transcripts are regulated by 17,20betaP and estradiol. Together, our observations strongly suggest that during final oocyte maturation, a down-regulation of IGFBP3, -4, and -5 occurs in the oocyte in response to gonadotropic and 17,20betaP (IGFBP3 and -5) stimulation, whereas an up-regulation of IGFBP2 and -6 occurs in follicular layers or extrafollicular tissue in response to gonadotropic stimulation.

Non-invasive assessment of reproductive status and cycle of sex steroid levels in a captive wild broodstock of Senegalese sole Solea senegalensis (Kaup)

Senegalese sole, Solea senegalensis, intensive culture is currently limited mainly due to the low control on reproduction in captivity. Comprehensive knowledge of reproductive biology and physiology for this species is needed in order to improve tank spawning success. This work describes for the first time the seasonal profiles of plasma levels of sex steroids (17β-estradiol, testosterone, 11-ketotestosterone, and 17,20β-dihydroxy-4-pregnen-3-one [17,20β-P]) in a S. senegalensis captive wild broodstock held under natural conditions, during two consecutive reproductive cycles. Changes in apparent maturation in females, dynamics of sperm release in males, and the condition factor ( K) were monitored. Six maturation stages were established for females according to apparent size of the ovary and external abdominal swelling: early, intermediate and final ovarian development (F2+, F3+ and F4+, respectively), and partially spawned, mid spawned and spawned out or regressed (F3−, F2−, and F1−, respectively). During summer, F1− and non-running males (NRM) were predominant in association with low K and plasma steroid levels. At the end of summer, a new cycle of gonadal development started, denoted by the increase in reproductive parameters ( K and steroid levels) and the appearance of F2+. By middle autumn, some females reached advanced maturation stages (F3+ and F4+) while the proportion of running males (RM) showed a maximum. An occasional spawning could be registered during this season (November 2002). Towards the end of winter and beginning of spring, ovarian development reached its maximum. At this point, the proportion of F3+, F4+ and RM, K (specially in females), and steroid concentrations were the highest in concordance with the starting of the main spawning period (lasting from January to June 2003 and from March to June 2004). Throughout this period, concomitantly with oocyte and sperm release, the proportion of F3−, F2−, F1− and NRM progressively increased, while steroid levels and K progressively declined (concentration of steroids could fluctuate under a decreasing trend). The relatively elevated levels of 17,20β-P correlating with some parts of the spawning periods makes it a candidate for the role of the maturation-inducing steroid in S. senegalensis. Seasonal variations of measured parameters were consistent with the reproductive cycle of this species in the wild, and comparable to those found in other asynchronous multi-spawning fish.

Biochemical characterization of the Arctic char (Salvelinus alpinus) 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 (Kd, 13.8 ± 1.1 nM), low capacity (Bmax, 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 t1/2 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.

Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor: effects on the hydration mechanism

In teleost oocytes, yolk proteins (YPs) derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation before ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration, which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish (Fundulus heteroclitus), in which oocyte hydration is driven by K+. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. In this study, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B, and F (CatL, CatB, and CatF, respectively), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, whereas CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, underwent maturation in vitro, but acidification of yolk globules, activation of CatB, and proteolysis of YPs were prevented. In addition, MIS plus bafilomycin A1-treated oocytes accumulated less K+ than those stimulated with MIS alone; hence, oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a vacuolar-type H+-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.

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.

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 ( K d ∼ 20 nM), limited capacity ( B max 0.08 nM), specific and displaceable binding for progestins that is characteristic of mPRs. The MIS (17,20β,21-trihydroxy-4-pregnen-3-one, 20β-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 (mPRα). Immunocytochemistry of whole croaker spermatozoa with the mPRα antibody showed that staining was primarily localized on the midpiece, consistent with a role of the mPRα 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.

Membrane-bound progestin receptors in channel catfish and zebrafish ovary: Changes in gene expression associated with the reproductive cycles and hormonal reagents

Membrane-bound progestin receptors (mPRs) are potential intermediaries in meiotic maturation of fish oocytes and other physiological processes. In this study, gene expression of the mPRs in the ovary of catfish and zebrafish during the reproductive cycle and the hormonal regulation of the expression were investigated. The transcript abundance of catfish mPRα gradually increased in conjunction with ovarian growth and then decreased prior to spawning period whereas the ovarian gene expression of mPRβ varied little throughout the reproductive cycle. In contrast, mPRγ gene expression peaked early in the mid-vitellogenic stage. The transcript abundance of zebrafish mPRα and β was low in ovarian follicles at early stages of oogenesis and gradually increased after the onset of vitellogenic growth and, thereafter, the gene expression did not vary. Gonadotropic treatment did not modulate the ovarian expression of mPRα and β genes in either catfish or zebrafish. On the other hand, exposure to 17,20β-dihydroxy-4-pregenen-3-one (the maturation-inducing steroid in this species) resulted in the down-regulation of mPRα in catfish ovary whereas gene expression was significantly induced by estradiol-17β. Taken together, these findings suggest that gonadotropin-induced final oocyte maturation may not require an induction of mPR(s) expression or that the gonadotropin stimulates mPR protein production at the post-transcriptional level, presuming these receptors are indispensable for oocyte maturation.

Particularities of reproduction and oogenesis in teleost fish compared to mammals

Compared to mammals, teleost reproduction presents many original features. Reproductive strategies of species are diversified into numerous adaptations to a large variety of aquatic environments. This diversity may concern sexuality, spawning and parental behaviour, sensitivity to environmental factors, and specific features of gametogenesis such as the duration of vitellogenesis, and egg morphology. Sexuality presents a variety of natural modalities, from gonochorism to hermaphrodism. The absence of definitive arrest of body growth in the adult of most species gives a particular interest to the practical control of growth-reproduction interactions. Vitellogenesis, which represents an important metabolic effort for the maternal organism, involves the synthesis of vitellogenin, a specific glycolipo-phosphoprotein produced in the liver under estradiol stimulation, and its incorporation into oocytes by a receptor mediated process. Both estradiol synthesis in follicle cells and vtg uptake by vitellogenic follicles appear to be mainly controlled by FSH. Oocyte maturation is directly triggered by a progestin, or MIS (maturation inducing steroid) synthesised in follicle cells mainly under LH control, and acting through the non-genomic activation of a membrane receptor. Practical applications of some of these particularities result mainly from the external character of the fertilisation process and of embryonic development, which allows manipulating respectively egg chromosome stocks and sex differentiation. Moreover, the sensitivity of sex differentiation to exogenous factors favours the development of practical methods to control the sex of farmed populations. Finally, the sensitivity of reproductive mechanisms to xenobiotics has led to various kinds of bioassays for putative pollutants.

Identification of 17,20β,21-trihydroxy-4-pregnen-3-one as an oocyte maturation-inducing steroid in black porgy, Acanthopagrus schlegeli

The identity of the maturation-inducing steroid (MIS) in black porgy, Acanthopagrus schlegeli, a marine protandrous teleost, is unknown. Previous studies demonstrated that two teleost MISs, the progestins 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S) and 17,20β-dihydroxy-4-pregnen-3-one (DHP) can induce maturation of black porgy oocytes in vitro. The purpose of the present study was to identify the major progestin produced during oocyte maturation (OM) in black porgy and investigate whether its secretion increases during this process. Females were injected twice with a LHRH analog to induce OM. Ovarian follicles undergoing OM were incubated in vitro with tritiated [ 3H]pregnenolone precursor and the tritiated products were extracted, purified, and identified by HPLC, TLC, acetylation, and recrystallization. Significant amounts of tritiated products were biosynthesized from [ 3H]pregnenolone that co-migrated with 20β-S but not with DHP on HPLC and TLC. Similar TLC profiles were obtained with the tritiated products isolated from the HPLC/TLC 20β-S fraction and standard 20β-S after the acetylation reaction. The identity of the tritiated products as 20β-S was confirmed by recrystallization. 20β-S had a slightly higher potency than DHP in the inducing in vitro final oocyte maturation. Plasma 20β-S concentrations increased significantly during the oocyte maturation after injection with a LHRH analog. The present data suggest that 20β-S is the MIS in black porgy.

274 Insulin-like growth factor-binding protein 3: single-agent and synergistic effects with paclitaxel in breast tumour models

Increasing evidence suggests a regulatory role for the IGF system in teleost oocyte maturation (OM). Our objectives were to determine if IGF-I and IGF-II regulate different stages of OM in southern flounder (Paralichthys lethostigma) and to identify the likely maturation-inducing steroid (MIS) in this species. The most abundant final product of ovarian steroidogenesis assays eluted at the position of 17,20β,21-trihydroxy-4-pregnen-3-one (20β-S). 20β-S was also more potent in inducing germinal vesicle breakdown (GVBD) of maturationally-competent oocytes than other teleost MISs. IGF-II (100 nM) induced maturational competence (OMC), as greater GVBD was induced after incubation with IGF-II + 20β-S compared to that of the 20β-S + 20β-S or IGF-II + no treatment group. Incubation with IGF-II (100 nM) for 4–8 h significantly increased ovarian membrane progestin receptor alpha (mPRα or Paqr7b) mRNA levels 12–15% and mPRα protein levels 75–101%. Further, the IGF-II-induced increase in mPRα protein concentrations was partially blocked by pretreatment with Wortmannin, a Pik3 inhibitor, and PD 098,059, a Mapk inhibitor. Both IGF-I and -II (100 nM) induced GVBD of maturationally-competent oocytes was blocked by incubation with cycloheximide. Incubation with d,l-Aminoglutethimide decreased IGF-II-induced GVBD but had no effect on IGF-I-induced GVBD. IGF-I and -II were also able to induce GVBD of maturationally-incompetent oocytes, and elicited 75% and 135% greater GVBD, respectively, than hCG + 20β-S at 100 nM. In conclusion, we show that 20β-S is the likely MIS in this species and that IGF-I and -II are also able to induce GVBD. Further, IGF-II not only induces OMC but also up-regulates ovarian mPRα mRNA and protein through Pik3- and Mapk-dependent pathways. This is the first demonstration of mPRα regulation by an IGF in any vertebrate species.

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.

Temperature effects along the reproductive axis during spawning induction of grass carp ( Ctenopharyngodon idella)

For grass carp ( Ctenopharyngodon idella) raised in the Ivory Coast (with water temperatures of 26–31 °C), induced spawning is obligatory for fry production. However, ovulation rates following hormonal treatment are often low. We hypothesized that high temperatures are an inhibiting factor for the reproductive axis (brain–pituitary–gonad) in these conditions. By in vivo and in vitro experiments, we tried to determine the thermosensitive steps during spawning induction. We compared gonadotropin and maturation-inducing steroid (MIS) profiles during a spawning induction at controlled temperatures of 24 and 28 °C in relation to ovulation success. We performed pituitary cell cultures and ovarian fragment incubations at controlled temperatures. The ovulation rate was lower at 28 °C (10%) than at 24 °C (36%). At the pituitary level, we found only minor thermal impacts on GnRH-stimulated LH release, but our data suggest an increase of the dopaminergic inhibition by high temperatures. The main effects were found at the ovary level, where ovary responsiveness to gonadotropin by MIS synthesis was disturbed, as well as oocyte responsiveness to MIS triggering final maturation, and probably ovulation. These results show the importance of regulating temperature during spawning induction to ensure a high rate of ovulation.

Annual reproductive cycle of black rockfish Sebastes schlegeli in captivity

: The gonadal development and serum profiles of vitellogenin and sex steroids in rearing Sebastes schlegeli were monitored for one full year. Female fish began vitellogenesis from November and completed it in March. Gestation occurred from April, and parturition occurred in June. A thin chorion and scanty cortical alveoli are oogenetic peculiarities of this fish. Male fish began spermatogenesis from June, and matured in November and December. It appears that copulation occurs in November and December, and that the sperm are stored freely in the ovary during the early vitellogenic period and under the ovigerous lamellae epithelium during the late vitellogenic period. Serum vitellogenin levels in female fish had a good correlation with oocyte growth. Serum estradiol-17β (E2) levels in female fish were elevated from November to February, suggesting that E2 controls vitellogenesis. Serum 17,20β-dihydroxy-4-pregnen-3-one (DHP) in female fish increased in the late vitellogenic period, suggesting that DHP was a maturation-inducing steroid. High levels of serum DHP during gestation suggest that it may be one of the endocrine factors for maintaining gestation. Serum 11-ketotestosterone (11-KT) levels in male fish were elevated from June to October, suggesting that 11-KT controls spermatogenesis. Serum DHP in male fish had a single peak in October, suggesting that DHP plays some role in the late stages of spermatogenesis.