Ovarian steroidogenesis in white sturgeon (Acipenser transmontanus) during oocyte maturation and induced ovulation

Ovarian follicles and plasma were collected from two female white sturgeon, Acipenser transmontanus, injected with sturgeon pituitary homogenate followed 12h later with GnRHa to induce ovulation. The oocytes of one female underwent germinal vesicle breakdown (GVBD) but ovulation did not occur in response to hormonal stimulation (Female 1), while the oocytes of the other female underwent GVBD and ovulation (Female 2). Follicles collected 12h after the first injection to induce ovulation were incubated with radioinert pregnenolone (P5) or tritiated-P5 ([3H]P5) plus radioinert P5. Steroids were extracted from media and intact follicles, and the extracts were fractionated by high performance liquid chromatography (HPLC). Fractions from the incubation with radioinert precursor were used in a bioassay to determine the potency of the steroid products to induce GVBD. Plasma levels of testosterone (T), estradiol, and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were measured by radioimmunoassay during induced ovulation, and plasma collected at the time of ovulation (actual or expected) was analyzed by HPLC. A peak in plasma 17,20beta-P was detected at the time of the second injection to induce ovulation in Female 2 (the time at which follicles were collected for incubation with [3H]P5). The HPLC analysis revealed several progestins in the plasma of Female 2 at ovulation that were not present in Female 1 at the time of expected ovulation. A variety of C19 and C21 steroids were produced in vitro by ovarian follicles from both females. The "suggestive" identities of the major metabolites were 11-deoxycortisol, androstenedione, 17-hydroxyprogesterone (17OHP), and 17,20beta-P in Female 1 and cortisol, 17,20beta, 21-trihydroxyprogesterone (20beta-S), 11-deoxycortisol, T, 17OHP, and 17,20beta-P in Female 2. Several of the steroids were active in a GVBD bioassay, but the fractions that contained the steroid coeluting the authentic 11-deoxycortisol on the HPLC and 17,20beta-P (positively identified by gas chromatography-mass spectrometry) were found to be the most potent. The results from this study combined with the results of Webb et al. (2001b) suggest the potential roles of 11-deoxycortisol, 17,20beta-P, 20beta-S, and P4 as maturation-inducing steroids in sturgeon.     

Relative effects of estradiol-17beta (E2), catecholestrogens and clomiphene citrate on in vitro oocyte maturation in the catfish Heteropneustes fossilis (Bloch) and E2 inhibition of 2-hydroxyestradiol-induced maturation

In vitro effects of estradiol-17beta (E(2)), the catecholestrogens 2-hydroxyE(2) (2-OHE(2)) and 2-methoxyE(2), and the nonsteroidal antiestrogen clomiphene citrate (clomid) on oocyte maturation were investigated in the catfish Heteropneustes fossilis. Incubation of postvitellogenic follicles with 2-OHE(2) induced germinal vesicle breakdown (GVBD; 86% at 5 microM for 30 h) and progression of meiosis up to metaphase II, as evident from the presence of Hoechst stained metaphase chromosomes and anti-alpha-tubulin-positive bipolar spindles. The response was both concentration (1, 2.5, 5, 10, and 20 microM)- and duration (0, 3, 6, 12, 24, and 30 h)-dependent. The diameter of the follicles increased and about 20% follicles elicited ovulation. Incubation of the follicles with clomid (20 microM) induced only about 29-35% GVBD at 30 h. This might be due to the dual properties of clomid with estrogenic (cis-isomer) and antiestrogenic (trans-isomer) actions or due to estrogen receptor binding dynamics. Incubations of the follicles with E(2) or 2-methoxyE(2) did not induce oocyte maturation. The higher concentrations of 2-methoxyE(2) caused degenerative changes in the follicles. In competition studies, E(2) inhibited the GVBD response of 2-OHE(2) (5 microM) significantly in a concentration (1, 5, 10, and 20 microM) or duration (2, 4, and 6 h)-dependent manner after pre-incubation with 20 microM E(2) (P<0.001, one-way ANOVA, P<0.05, Newman-Keuls' test). The results show that 2-OHE(2) induces maturational activity while the parent estrogen is a strong inhibitor, alone or in combination with 2-OHE(2).     

A novel oocyte maturation arresting factor in the central nervous system of scallops inhibits serotonin-induced oocyte maturation and spawning of bivalve mollusks

Serotonin (5-hydroxytriptamine; 5-HT) is a major neurotransmitter that triggers oocyte maturation and sequential spawning in bivalve mollusks. A proteinous and heat-labile substance that proved to be a novel inhibitor of 5-HT-induced egg release from ovarian tissue was found in the cerebral and pedal ganglia (CPG) of the scallop Patinopecten yessoensis. The same inhibitory activity was also observed in the proteinous fraction from the supernatant of hemolymph. Histological observation demonstrated that the novel inhibitor prevented 5-HT from inducing oocyte maturation in the scallop ovary and that no prostaglandin F2alpha (PGF2alpha) inhibited 5-HT-induced oocyte maturation, although PGF2alpha strongly prohibited 5-HT-induced egg release through the gonoduct from ovarian tissue. The novel inhibitor from the scallop CPG also prohibited 5-HT-induced oocyte maturation of other bivalve species as well as scallops. The novel inhibitor, mediated through a receptor mechanism on oocyte membranes, blocked extracellular Ca2+ uptake into oocytes, which was observed in 5-HT-induced oocyte maturation. It is suggested that the novel inhibitor with a molecular mass of 60 kDa, named oocyte maturation arresting factor, which appears to be a universal substance for bivalve species, may be transported from the CPG to the ovary via hemolymph and may prohibit 5-HT-induced oocyte maturation due to the interference of extracellular Ca2+ influx into oocytes, eventually resulting in the inhibition of spawning. On the other hand, it seems that PGF2alpha inhibits 5-HT-induced transport of mature eggs through the gonoduct.     

Involvement of estradiol-17β and its membrane receptor, G protein coupled receptor 30 (GPR30) in regulation of oocyte maturation in zebrafish, Danio rario

The orphan G protein coupled receptor, GPR30, has the characteristics of a high affinity, specific estrogen membrane receptor on Atlantic croaker oocytes and mediates estrogen inhibition of oocyte maturation in this perciform fish. In order to determine the broad applicability of these findings to other teleosts, similar experiments were conducted in a cyprinid fish, zebrafish, in the present study. GPR30 mRNA expression was detected in zebrafish oocytes but not in the ovarian follicular cells. Both spontaneous and 17, 20β-dihyroxy-4-pregnen-3-one (DHP)-induced maturation of follicle-enclosed zebrafish oocytes was significantly decreased when they were incubated with either estradiol-17β, or the GPR30 agonists, ICI 182 780 and tamoxifen, or with the GPR30 specific agonist G-1. On the other hand spontaneous oocyte maturation increased two-fold when zebrafish ovarian follicles were incubated with an aromatase inhibitor, ATD. Moreover, the stimulatory effects of ATD on germinal vesicle breakdown (GVBD) were partially reversed by co-treatment with 100 nM of E2 or G-1. These results suggest that endogenous estrogens acting through GPR30 are involved in maintaining meiotic arrest of zebrafish oocytes.     

Melatonin protects oocyte quality from Bisphenol A‐induced deterioration in the mouse

Bisphenol A (BPA) has been reported to adversely affect the mammalian reproductive system in both sexes. However, the underlying mechanisms regarding how BPA disrupts the mammalian oocyte quality and how to prevent it have not been fully defined. Here, we document that BPA weakens oocyte quality by impairing both oocyte meiotic maturation and fertilization ability. We find that oral administration of BPA (100 μg/kg body weight per day for 7 days) compromises the first polar body extrusion (78.0% vs 57.0%, P<.05) by disrupting normal spindle assembly, chromosome alignment, and kinetochore‐microtubule attachment. This defect could be remarkably ameliorated (76.7%, P<.05) by concurrent oral administration of melatonin (30 mg/kg body weight per day for 7 days). In addition, BPA administration significantly decreases the fertilization rate of oocytes (87.2% vs 41.1%, P<.05) by reducing the number of sperm binding to the zona pellucida, which is consistent with the premature cleavage of ZP2 as well as the mis‐localization and decreased protein level of ovastacin. Also, the localization and protein level of Juno, the sperm receptor on the egg membrane, are strikingly impaired in BPA‐administered oocytes. Finally, we show that melatonin administration substantially elevates the in vitro fertilization rate (63.0%, P<.05) by restoring above defects of fertilization proteins and events, which might be mediated by the improvement of oocyte quality via reduction of ROS levels and inhibition of apoptosis. Collectively, our data reveal that melatonin has a protective action against BPA‐induced deterioration of oocyte quality in mice.     

Melatonin protects against paraquat‐induced damage during in vitro maturation of bovine oocytes

Paraquat (PQ), a broad‐spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ‐induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro‐produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic‐related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ‐exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ‐induced p38 mitogen‐activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl‐histone H3 lysine 4 (H3K4me3) and trimethyl‐histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ‐induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.     

Involvement of PI3 kinase and MAP kinase in IGF-I- and insulin-induced oocyte maturation in Cyprinus carpio

Previously, we observed that in vitro germinal vesicle breakdown (GVBD) in Cyprinus carpio oocytes was induced by recombinant human insulin-like growth factor-I (IGF-I) and bovine insulin (b-insulin) and this induction was steroid-independent. To investigate further the early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during oocyte maturation was examined. IGF-I- and b-insulin-induced oocyte maturation was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase after 90 min of their treatment. Both IGF-I and b-insulin were found to activate cdc2 kinase at 21 h of treatment. We examined the relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in IGF-I- and b-insulin-induced oocyte maturation in C. carpio. MAP kinase was rapidly phosphorylated and activated (30–150 min) in response to exposure of the oocytes with IGF-I and b-insulin. This response preceded the phosphorylation and activation of cdc2 by several hours (almost 19 h). A potent and selective inhibitor of MEK, PD98059, the protein kinase that phosphorylates and activate MAP kinase, blocked the phosphorylation and activation of MAP kinase and cdc2 kinase and GVBD induction. Likewise, PI3 kinase inhibitors strongly inhibited phosphorylation and activation of MAP kinase, which was increased during oocyte maturation. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes MAP kinase, and MPF activation during IGF-I- and b-insulin-induced oocyte maturation in C. carpio.     

Melatonin reduces mortality from Aleutian disease in mink (Mustela vison)

Abstract: Aleutian disease (AD) results from a persistent parvoviral infection that results in marked hypergammaglobulinemia and immune complex mediated lesions of the kidney, liver, lungs and, arteries. Melatonin protected both a wild type or demi strain and a demi/dark crossed strain of mink from AD. The biogenic amine also afforded protection against other non-diagnosed diseases naturally found on mink farms when it was available from a subcutaneously-placed reservoir. Some genetic strains of mink apparently differed in the resistance of mink to the virus and in the protective ability of melatonin. The demi strain was the most resistant followed by pastels, mahogany, darks, and those strains with the double recessive Aleutian gene. The protective action of melatonin appeared to result from melatonin's ability to scavenge free radicals, but it could also be due to the induction of antioxidant enzymes or to the modulation of immunity. Melatonin also protected mink against distemper.     

Melatonin improves inflammation processes in liver of senescence-accelerated prone male mice (SAMP8)

Aging is associated with an increase in oxidative stress and inflammation. The aim of this study was to investigate the effect of aging on various physiological parameters related to inflammation in livers obtained from two types of male mice models: Senescence-accelerated prone (SAMP8) and senescence-accelerated-resistant (SAMR1) mice, and to study the influence of the administration of melatonin (1 mg/kg/day) for one month on old SAMP8 mice on these parameters.     

Melatonin (an antioxidant) does not ameliorate alcohol-induced Purkinje cell loss in the developing cerebellum

BACKGROUND: One popular mechanism proposed to account for alcohol-induced brain damage is the generation of free radicals after alcohol exposure. Therefore, it is reasonable to hypothesize that administration of an antioxidant should reduce the severity of alcohol-induced brain damage. Recently, melatonin has been shown to be an effective free-radical scavenger. In this study, the ability of melatonin to attenuate alcohol-induced cerebellar Purkinje cell loss in the cerebellar vermis and lobule I was assessed. METHODS: Sprague-Dawley rat pups were used in this study. These neonatal pups were exposed to alcohol (4.5 g/kg), melatonin (10 mg/kg), both alcohol and melatonin, or control vehicle via artificial-rearing methods from postnatal day (PD) 4 to PD 9. Alcohol, melatonin, or control vehicle was mixed with milk formula in 2 of the daily 12 feedings. Pups were killed 90 min after the beginning of the second alcohol feeding on PD 9. RESULTS: Alcohol significantly reduced the Purkinje cell numbers in the vermis and lobule I, with a higher percentage of cell loss in lobule I compared with the vermis. However, melatonin, per se, neither affected the Purkinje cell number nor diminished alcohol-induced Purkinje cell loss. CONCLUSIONS: Melatonin was not effective in attenuating alcohol-induced loss of Purkinje cells in our neonatal rat model system, even though such a dosage of melatonin is capable of reversing free radical-induced damage in other tissues.     

The role of the insulin-like growth factor (IGF) system in zebrafish (Danio rerio) ovarian development

The presence of an ovarian IGF system in teleosts suggests a distinct role in reproductive physiology. This study investigates the role of the ovarian IGF system in oocyte maturation, the acquisition of maturational competence and steroidogenesis in the zebrafish (Danio rerio). Recombinant human IGF-I and IGF-II stimulated germinal vesicle breakdown (GVBD) in early vitellogenic (EV; 0.35-0.44 mm), midvitellogenic (MV; 0.45-0.56 mm) and full grown (FG; 0.57-0.65 mm) follicles incubated in vitro. By comparison, the maturation inducing steroid 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P) only induced GVBD in MV and FG follicles. Collectively these studies suggest that IGF is involved in oocyte maturation and that follicles become responsive to IGFs at an earlier stage compared to 17,20β-P. IGF-I also increased the responsiveness of the follicle to 17,20β-P, suggesting a role in promoting maturational competence. IGF-I alone and in combination with human chorionic gonadotropin (hCG) stimulated the production of 17,20β-P by ovarian follicles incubated in vitro. However, IGF-I had no effect on the production of 17β-estradiol (E₂) or the expression of genes involved in steroidogenesis (20β-hydroxysteroid dehydrogenase; 20β-HSD and P450c17-II). These results provide evidence that the IGF system plays an important role in the promotion of oocyte maturation and ovarian development in the zebrafish.     

Involvement of corticotropin-releasing factor and adrenocorticotropic hormone in the ovarian maturation, seawater acclimation, and induced spawning of Liza ramada

In the present study, we investigated the distribution and activities of corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH) immunoreactive (ir) cells in the brain and pituitary of Liza ramada during ovarian maturation, seawater acclimation, and induction of spawning. Using immunohistochemistry, we detected that CRF-ir cell bodies exist in different brain regions: medulla oblongata (MO), midbrain tegmentum, habenula, nucleus preopticus (NPO), and in a ventral hypothalamic region corresponding to the nucleus lateralis tuberis (NLTP). In the pituitary gland, we detected some ACTH-producing cells in the rostral pars distalis (RPD) containing CRF immunoreactivity. The synthetic and secretory activity of CRF-ir cells in the NPO and MO as well as ACTH-ir cells in the pituitary were enhanced during ovarian maturation. During seawater acclimation, CRF-ir cells in the NPO and MO and ACTH-ir cells in the pituitary showed dramatic increases in their synthetic activity. These cells showed dramatic increase in their secretory activity during spawning induced by human chorionic gonadotropin (HCG) injection in L. ramada. Finally, hormonally induced ovulation was accompanied with elevation of plasma cortisol and depletion of CRF and ACTH immunoreactivity within the brain and the pituitary gland, respectively. Taken together, our findings suggest that mature breeders of L. ramada may respond to stress resulting from ovarian maturation, and seawater acclimation as well as induced spawning. Mechanisms include enhancement of the synthetic and/or secretory activity of CRF-ir cells in the NPO and MO as well as ACTH-ir cells in the pituitary gland along with a rise in plasma cortisol during ovulation, supporting the possible role of these hormones during stress and reproduction in L. ramada.     

An involvement of vasotocin in oocyte hydration in the catfish Heteropneustes fossilis: A comparison with effects of isotocin and hCG

In the present investigation, in vitro effects of vasotocin (VT) on oocyte (follicular) hydration during germinal vesicle breakdown (GVBD) and ovulation were demonstrated in hCG-primed and non-primed catfish. The data were compared with that of groups incubated with isotocin, and hCG alone or in combination with VT. The priming with hCG resulted in significant increases on percentage GVBD and ovulation, and stimulated follicular hydration, as judged by the increase in diameter, volume, water content, osmolality and Ca²⁺ concentration. However, Na⁺, K⁺ ATPase activity, and concentrations of Na⁺, K⁺ and Mg²⁺ did not alter significantly. The incubations with hCG or VT stimulated all the above parameters. In the non-primed fish, the response of hCG was significantly higher on follicular diameter, volume and osmolality, and that of VT on ovulation. In the primed fish, the VT response was significantly higher on GVBD, ovulation, Na⁺, K⁺ ATPase activity and divalent cation concentrations. The co-incubation with both hCG and VT produced maximal increases in all the parameters with significantly higher effects in the primed fish. The effects of IT on various parameters were relatively low compared to hCG or VT effects. The results indicate that VT may play an important role in oocyte (follicular) hydration, which is consistent with its role in osmoregulation of fish.     

Expression and gonadotropin regulation of membrane progestin receptor alpha in Atlantic croaker (Micropogonias undulatus) gonads: Role in gamete maturation

Recent results suggest that membrane progestin receptor alpha (mPRα) mediates nongenomic actions of progestin hormones to induce oocyte maturation and sperm hypermotility in several teleost species. The role of mPRα in gamete and gonadal physiology was further evaluated in the present study by examining gonadal expression of mPRα during gamete maturation in Atlantic croaker (Micropogonias undulatus), a well-characterized teleost model of oocyte maturation and sperm motility. Sequencing of the croaker mPRα gene isolated from croaker ovaries showed it is 98% homologous at the nucleotide level to spotted seatrout mPRα. The mPRα mRNA and protein were detected in both somatic and gonadal tissues. In croaker ovaries, the mPRα protein was present throughout the gonadal cycle and was upregulated by gonadotropin in vitro, coincident with the acquisition of oocyte maturational competence (i.e., ability to respond to progestin hormones and complete oocyte maturation). Both mPRα mRNA and protein were also expressed in croaker testes throughout the gonadal cycle. Expression of mPRα protein was weakly upregulated in testes after 18 h of in vitro gonadotropin treatment. Immunocytochemical staining showed mPRα was localized to both germ and interstitial cells. Finally, elevated levels of mPRα protein in croaker sperm were associated with high sperm motility. Taken together, these data strongly support the hypothesis that mPRα mediates progestin induction of oocyte maturation and upregulation of sperm motility in teleosts.     

Melatonin and 5-methoxytryptophol (5-ML) in nervous and/or neurosensory structures of a gastropod mollusc (Helix aspersa maxima): synthesis and diurnal rhythms

Daily patterns of melatonin and 5-methoxytryptophol (5-ML) concentrations and of aryl alkylamine N-acetyltransferase (AA-NAT) and hydroxyindole-O-methyltransferase (HIOMT) activities have been measured in the cerebroid ganglions, visceral ganglions, and ocular tentacles of the gastropod mollusc Helix aspersa maxima. Melatonin concentrations are very low in all the studied structures, except a small peak at the end of the night in the cerebroid ganglions. 5-ML, which is quite undetectable in the cerebroid and visceral ganglions, shows clear daily variations in the ocular tentacles with low values in the middle of the light period and high values during the night. These results are opposite to what is known on daily variations of 5-ML in vertebrates. AA-NAT activity was not detected, while the presence of an HIOMT-like activity supports the hypothesis that 5-ML is synthesized in the ocular tentacles. The temporal relationships existing between the 5-ML rhythm in the ocular tentacles and the hemolymph suggest that 5-ML could be released in the general circulation. These preliminary results suggest that 5-ML could be an informative molecule involved in adaptative processes in the snail and they reinforce the hypothesis that the different 5-methoxyindoles could be implicated in the integration of environmental information.     

Hormonal action of relaxin-like gonad-stimulating substance (GSS) on starfish ovaries in growing and fully grown states

Gonad-stimulating substance (GSS) of starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, we purified GSS from the radial nerves of the starfish Asterina pectinifera and identified the chemical structure as a relaxin-like peptide. This study examined the hormonal action of GSS on ovaries in the growing (stage IV) and fully grown states (stage V) of the starfish. The sensitivity of oocytes to 1-methyladenine (1-MeAde) as starfish maturation-inducing hormone was enhanced as oocytes enlarged in stage V. GSS-stimulated 1-MeAde production by ovarian follicle cells was also correlated with the size of oocytes. Although 1-MeAde production was observed in whole ovaries in stage V, GSS failed to induce 1-MeAde production in young ovaries (stage IV). This suggests that follicle cells in ovaries in a growing state (stage IV) are still unresponsive to the hormonal action of GSS. According to competitive experiments using radioiodinated and radioinert GSS, however, dissociation constant (K_d) values and the number of binding sites for GSS were mostly constant in the ovaries from stages IV to V. These results strongly suggest that GSS receptors are expressed in follicle cells of ovaries in the growing state. The failure of GSS to induce 1-MeAde production in young ovaries may be due to the uncoupling of signal transduction from the receptor to 1-MeAde biosynthesis in follicle cells.     

Role of gap junctions and protein kinase A during the development of oocyte maturational competence in Ayu (Plecoglossus altivelis)

Meiotic resumption in teleost oocytes is induced by a maturation-inducing hormone (MIH). The sensitivity of oocytes to MIH, also known as oocyte maturational competence (OMC), is induced by LH via mechanisms that are not fully understood. A previous study of Ayu (Plecoglossus altivelis) showed the presence of functional heterologous gap junctions (GJs) between oocytes and their surrounding granulosa cells. The objectives of this study were to determine the role of ovarian GJs and of protein kinase A (PKA) during the acquisition of OMC. We examined the effects of the specific GJ inhibitor carbenoxolone (CBX) and 18alpha-glycyrrhetinic acid (alpha-GA) on the LH-(hCG)-dependent acquisition of OMC and on MIH-(17,20beta-dihydroxy-4-pregnen-3-one)-dependent meiotic resumption; measured the cAMP content of ovarian follicles during the hCG-dependent acquisition of OMC; and determined the effects of PK activators and inhibitors on hCG-dependent OMC. Production of follicular cAMP increased during the hCG-dependent acquisition of OMC. Both GJ inhibitors and the PKA inhibitor H8-dihydrochloride, but not the PKC inhibitor GF109203X, suppressed the hCG-dependent acquisition of OMC in a dose-dependent manner. The PKA activator forskolin induced OMC with a similar potency to hCG. Unlike previous observations with teleosts where disruption of heterologous GJ either blocks or stimulates meiotic resumption, treatment with GJ inhibitors did not affect MIH-dependent meiotic resumption in maturationally competent follicles of Ayu. These observations suggest that ovarian GJs are essential for LH-dependent acquisition of OMC but not for MIH-dependent meiotic resumption, and that the stimulation of OMC by LH is mediated by cAMP-dependent PKA. They are also consistent with the view that a precise balance between GJ-mediated signals (positive or negative) and oocyte maturational readiness is required for hormonally regulated meiotic resumption.     

Effects of pinealectomy and melatonin on gonadotropin-releasing hormone (GnRH) gene expression in the male rat brain

Melatonin, a pineal hormone, is known to be an important neurohormonal factor involved in the timing of reproductive events which occur seasonally in various mammalian species. In order to evaluate the influence of melatonin on neurons which are producing gonadotropin-releasing hormone (GnRH), we studied the effect of light-dark cycle as well as pinealectomy and melatonin administration on GnRH gene expression in the adult male rat medial preoptic area (MPOA) using quantitativein situ hybridization. The animals were kept under artificial light (light on 6:00 h–20:00 h). In animals which were sacrificed at 24:00 h (when endogenous melatonin levels are high), the hybridization signal was higher than that detected in animals sacrificed at 20:00 h (before the onset of darkness). Administration of melatonin during the light period (16:00 h) induced a 15% increase in the amount of GnRH mRNA after 4 h. Three weeks after pinealectomy mRNA levels were decreased by 35%. Injection of melatonin to pinealectomized rats 4 h before sacrifice increase the amount of GnRH mRNA, completely reversing the decrease in mRNA induced by pinealectomy. These results strongly suggest that melatonin produced by the pineal gland exerts a positive influence on GnRH neuronal activity in the male rat.