Plasma membrane destination of the classical Xenopus laevis progesterone receptor accelerates progesterone-induced oocyte maturation

Xenopus laevis oocyte maturation is induced by the steroid hormone progesterone through a non-genomic mechanism initiated at the cell membrane. Recently, two Xenopus oocyte progesterone receptors have been cloned; one is the classical progesterone receptor (xPR-1) involved in genomic actions and the other a putative seven-transmembrane-G-protein-couple receptor. Both receptors are postulated to be mediating the steroid-induced maturation process in the frog oocyte. In this study, we tested the hypothesis that the classical progesterone receptor, associated to the oocyte plasma membrane, is participating in the reinitiation of the cell cycle. Addition of a myristoilation and palmytoilation signal at the amino terminus of xPR-1 (mp xPR-1), increased the amount of receptor associated to the oocyte plasma membrane and most importantly, significantly potentiated progesterone-induced oocyte maturation sensitivity. These findings suggest that the classical xPR-1, located at the plasma membrane, is mediating through a non-genomic mechanism, the reinitiation of the meiotic cell cycle in the X. laevis oocyte.     

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.     

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.     

Steroids involved with final oocyte maturation in the winter flounder.

A number of androgens and progestogens including 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17,20-P) were examined in female winter flounder as possible maturation inducing steroids (MIS). During final oocyte maturation serum levels of testosterone (T) and 17 beta-hydroxy-5 beta-androsten-3-one (5 beta-T) peaking at over 200 ng/ml and pregnenolone (PE) at 40 ng/ml were the predominant steroids found from each major group. High levels of T and 5 beta-T were correlated with oocyte stages characterized by germinal vesicle migration. Of the PEs measured, maximum serum levels of PE, 3 beta,17 alpha-hydroxy-5-pregnen-20-one (17-PE) and 3 beta,17 alpha, 20 beta-dihydroxy-5-pregnene (17,20-PE) were found during later oocytes stages associated with germinal vesicle breakdown. Levels of 17,20-P, an established MIS in most fish, were almost non-detectable (less than 0.1 ng/ml serum) in females throughout all stages of final oocyte maturation. Incubations of ovarian follicles in vitro with physiological concentrations of T and 5 beta-T indicated that these steroids could induce all stages of final oocyte maturation. Similar in vitro incubations showed that 17-PE and 17,20-PE were only effective on germinal vesicle breakdown. The principal conclusions are that T, 5 beta-T and the PEs can be considered as MISs in winter flounder and the PE pathway predominates during the final stages of oocyte maturation in winter flounder in contrast to progesterones which predominate in other fish species, mostly salmonids, studies to date.     

Induction and inhibition of oocyte maturation by EDCs in zebrafish

Background  

Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH), which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), a nonsteroidal estrogen.     

Characterization of Luteinizing Hormone and Luteinizing Hormone Receptor and Their Indispensable Role in the Ovulatory Process of the Medaka

The molecular properties and roles of luteinizing hormone (Lh) and its receptor (Lhcgrbb) have not been studied for the medaka (Oryzias latipes), which is an excellent animal model for ovulation studies. Here, we characterized the medaka Lh/Lhcgrbb system, with attention to its involvement in the ovulatory process of this teleost fish. In the medaka ovary, follicle-stimulating hormone receptor mRNA was expressed in small and medium-sized follicles, while lhcgrbb mRNA was expressed in the follicle layers of all growing follicles. Experiments using HEK 293T cells expressing medaka Lhcgrbb in vitro revealed that gonadotropin from pregnant mare’s serum and medaka recombinant Lh (rLh) bound to the fish Lhcgrbb. The fish gonadotropin subunits Gtha, Fshb, and Lhb were essentially expressed at fairly constant levels in the pituitary of the fish during a 24-h spawning cycle. Using medaka rLh, we developed a follicle culture system that allowed us to follow the whole process of oocyte maturation and ovulation in vitro. This follicle culture method enabled us to determine that the Lh surge for the preovulatory follicle occurred in vivo between 19 and 15 h before ovulation. The present study also showed that oocyte maturation and ovulation were delayed several hours in vitro compared with in vivo. Treatment of large follicles with medaka rLh in vitro significantly increased the expression of Mmp15, which was previously demonstrated to be crucial for ovulation in the fish. These findings demonstrate that Lh/Lhcgrbb is critically involved in the induction of oocyte maturation and ovulation.     

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.     

Multiple rapid progestin actions and progestin membrane receptor subtypes in fish

Progestin hormones exert rapid, nongenomic actions on a variety of target tissues in fish. The induction of oocyte maturation and the progestin membrane receptor (mPR) that mediates this action of progestins have been well characterized in fishes. Progestins also act on Atlantic croaker spermatozoa via an mPR to rapidly increase sperm motility. Preliminary results indicate that progestins can also exert rapid actions in the preoptic anterior hypothalamus (POAH) in this species to down-regulate gonadotropin-releasing hormone (GnRH) secretion. Recently, we reported the cloning, sequencing and characterization of a novel cDNA in a closely related species, spotted seatrout, that has the characteristics of the mPR involved in the progestin induction of oocyte maturation. Three distinct mPR subtypes, named alpha, beta, and gamma, have been identified in both fishes and mammals. The tissue distribution of the mPRalpha protein in seatrout suggests the alpha-subtype mediates progestin actions on GnRH secretion, sperm motility and oocyte maturation. However, mPRbeta antisense experiments in zebrafish oocytes suggest the beta-subtype also participates in the control of oocyte maturation in zebrafish.     

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.     

Luteinizing hormone-dependent activation of the epidermal growth factor network is essential for ovulation

In the preovulatory ovarian follicle, mammalian oocytes are maintained in prophase meiotic arrest until the luteinizing hormone (LH) surge induces reentry into the first meiotic division. Dramatic changes in the somatic cells surrounding the oocytes and in the follicular wall are also induced by LH and are necessary for ovulation. Here, we provide genetic evidence that LH-dependent transactivation of the epidermal growth factor receptor (EGFR) is indispensable for oocyte reentry into the meiotic cell cycle, for the synthesis of the extracellular matrix surrounding the oocyte that causes cumulus expansion, and for follicle rupture in vivo. Mice deficient in either amphiregulin or epiregulin, two EGFR ligands, display delayed or reduced oocyte maturation and cumulus expansion. In compound-mutant mice in which loss of one EGFR ligand is associated with decreased signaling from a hypomorphic allele of the EGFR, LH no longer signals oocyte meiotic resumption. Moreover, induction of genes involved in cumulus expansion and follicle rupture is compromised in these mice, resulting in impaired ovulation. Thus, these studies demonstrate that LH induction of epidermal growth factor-like growth factors and EGFR transactivation are essential for the regulation of a critical physiological process such as ovulation and provide new strategies for manipulation of fertility.     

GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.     

Gonadotropin releasing hormone: regulation of phospholipid turnover and prostaglandin production in ovarian granulosa cells

The direct effect of gonadotropin releasing hormone (GnRH) upon ovarian function, is initiated by a rapid receptor-mediated increase in phosphatidylinositol (PI) turnover (approximately 5 min) followed by prostaglandin E (PGE, 120 min) and progesterone (120 min) formation, oocyte maturation and induction of ovulation. In contrast, luteinizing hormone (LH) stimulation of oocyte maturation and induction of ovulation is mediated by increased adenosine 3',5'-monophosphate (cAMP, 15 min), progesterone (30 min) and PGE (180 min) production. Both LH and GnRH stimulation of oocyte maturation are inhibited by dibutyryl cAMP and 3-isobutyl-1-methylxanthine, whereas induction of ovulation by the two hormones is blocked by indomethacin. GnRH and LH differ, therefore, in the mechanism leading to PGE formation, but thereafter share a common mechanism responsible for oocyte maturation and independently for induction of ovulation.     

Reproductive biology and endocrine regulation of final oocyte maturation of captive white bass

The ovarian development, and plasma levels of gonadotropin II (GtH II) and sex-steroid hormones at the end of vitellogenesis were examined in captive white bass Morone chrysops. The changes in plasma hormone levels and oocyte morphology associated with gonadotropinreleasing hormone agonist (GnRHa)-induced final oocyte maturation (FOM) were studied. Although plasma 17β-oestradiol (E₂) and oocyte diameter increased, there were no changes in GtH II, testosterone (T), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) or 17,20β,21-dihydroxy-4-pregnen-3-one (17,20β,21-P) in non-hormone-treated females, and no FOM was observed. Treatment with a sustained-release GnRHa delivery system (GnRHa implant) induced two FOM cycles separated by about 24 h, with the release of approximately equal numbers of eggs in each spawn. Plasma GtH II levels were elevated significantly throughout FOM, reaching a maximum of 9·07 ± 1·55 ng ml^?1 in ovulated fish. Both plasma E₂ and T increased soon after the GnRHa treatment, but E₂ declined in fish undergoing germinal vesicle (GV) migration. Plasma T increased further during FOM (7·55 ± 2·87 ng ml^?1), but declined precipitously at ovulation. A surge in plasma 17,20β-P and 17,20β,21-P (4·11 ± 0·97 ng ml^?1 and 3·10 ± 0·77 ng ml^?1, respectively) was observed in females undergoing GV breakdown (GVBD). Based on the involvement of different sex-steroid hormones, FOM was separated into two stages. Early FOM included lipid-droplet coalescence and GV migration, and was associated with elevations in plasma GtH II and T. Late FOM included GVBD and yolk-globule coalescence, and was associated with elevations in plasma GtH II, 17,20β-P and 17,20β,21-P. The results of this study point to the absence of a surge in plasma GtH II as the missing link in the reproductive axis responsible for the failure of captive white bass to undergo FOM at the end of vitellogenesis. Sustained elevation of plasma GtH II via treatment with a GnRHa implant induced two consecutive spawns with an overall egg production two- to eightfold higher than previously obtained from captive broodstocks, and similar to annual egg production Values reported for wild fish.     

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.     

Daily spawning and development of sensitivity to gonadotropin and maturation-inducing steroid in the oocytes of the bambooleaf wrasse, Pseudolabrus japonicus

The cycle of oocyte development of the bambooleaf wrasse, Pseudolabrus japonicus, was studied to elucidate the endocrinological mechanism of oocyte maturation in a marine teleost. A single female reared with two males spawned every day for 17 days in captivity, indicating that this species is a daily spawner. Ovarian histology revealed that germinal vesicle migration of the largest oocytes progressed from 12:00 to 3:00 h, and germinal vesicle breakdown (GVBD) was completed at 6:00 h. Ovulation and spawning occurred between 6:00 and 9:00 h. The effectiveness of human chorionic gonadotropin (HCG) and 17,20-dihydroxy-4-pregnen-3-one (17,20-P), which is one of the most potent steroidal inducers of GVBD in bambooleaf wrasse oocytes, in inducing final oocyte maturation was examined at eight different times of the day. The responsiveness of the oocyte to HCG and steroid differed at different times of the day. The GVBD could be induced by HCG but not 17,20-P at 9:00 h. Between 12:00 and 18:00 h, not only HCG but also 17,20-P induced GVBD. Both GVBD and ovulation spontaneously occurred between 0:00 and 6:00 h without any hormonal treatment. These results clearly showed that the oocyte of the bambooleaf wrasse possessed a diurnal maturation cycle. Responsiveness of oocytes to HCG appeared earlier than responsiveness to 17,20-P. This suggests that sensitivity to 17,20 -P is induced by gonadotropic hormone (GTH).     

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.     

Serotonin inhibition of steroid-induced meiotic maturation in the teleost Fundulus heteroclitus: Role of cyclic AMP and protein kinases

The transduction of the serotonin (5-HT) signal in Fundulus heteroclitusovarian follicles leading to the inhibition of oocyte meiosis reinitiation (oocyte maturation) in vitro induced by the naturally occurring maturation-inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) was investigated. Steroid-induced oocyte maturation was inhibited by 5-HT in a dose-dependent manner; maximum inhibition (90%) was observed with 10⁻⁴ M 5-HT. Groups of follicle-enclosed oocytes were cultured in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and treated with increasing doses of 5-HT. Serotonin was found to slightly increase the levels of follicular 3′,5′-cyclic adenosine monophosphate (cAMP) in a dose-dependent manner; 10⁻⁴ M 5-HT induced approximately a 3-fold increase in cAMP with respect to the controls. The changes in cAMP were then evaluated in follicles treated with 17,20βP in IBMX-free culture media in the presence or absence of 10⁻⁴ M 5-HT. The exposure of follicles to 17,20βP alone produced a small and transient reduction in cAMP (40%) within 1–3 hr of steroid stimulation, and these early changes in cAMP appeared associated with a high incidence of germinal vesicle breakdown (80% GVBD) by 24 hr of incubation. Under these conditions, treatment of follicles with 5-HT also increased significantly the production of cAMP, and when 5-HT was combined with 17,20βP, the steroid-mediated reduction in cAMP was prevented and the levels of GVBD inhibited by 95%. Meiosis also was reinitiated with either the protein kinase A (PKA) inhibitor H8 or the protein kinase C (PKC) activator PMA, and the 5-HT inhibitory action on GVBD was found to be 100-fold reduced or completely ineffective, respectively. Preincubation of follicles with the PKC inhibitor GF109203x abolished PMA-induced GVBD in a dose-dependent manner, whereas this inhibitor had no effect on 17,20βP-triggered meiotic maturation, indicating that activation of PKC is apparently sufficient but not necessary to reinitiate meiosis. Taken together, these findings suggest that 5-HT may inhibit 17,20βP-induced meiotic reinitiation through the activation of a cAMP-PKA transduction pathway and that PKC possibly induces oocyte maturation by a different pathway than the steroid and thus is not affected by 5-HT. Mol. Reprod. Dev. 49:333–341, 1998. © 1998 Wiley-Liss, Inc.