Germinal Vesicle Breakdown In Response Research Articles

Nitric oxide (NO) inhibition of meiotic G2-M1 transition in Anabas testudineus oocytes: Participation of cAMP-dependent protein kinase (PKA) in regulation of intra-oocyte signaling events

Nitric oxide (NO) regulation of ovarian function in mammals has been studied extensively. However, relatively less information is available on NO action on meiotic G2-M1 transition in teleost oocytes. In the present study using follicle-enclosed oocytes of Anabas testudineus, NO regulation of intra-oocyte signaling events during meiotic G2-M1 transition were examined. Priming with NO donor, sodium nitroprusside (SNP) prevented 17α,20β-dihydroxy-4-pregenen-3-one (17,20β-P)-induced germinal vesicle break down (GVBD) in dose- and duration-dependent manner. Impaired GVBD response in SNP-treated groups corroborated well with reduced p34Cdc2 (Thr161) phosphorylation. Immunoblot analysis revealed that congruent with elevated cAMP-dependent protein kinase (PKA) phosphorylation (activation), NO inhibition of meiotic maturation involves down regulation of Cdc25 activation, Mos synthesis and MAPK3/1 (ERK1/2) phosphorylation. However, priming with PKA inhibitor (H89) could reverse SNP attenuation of oocyte GVBD significantly. Collectively our results indicate that negative influence of NO on meiotic G2-M1 transition in perch oocytes might involve PKA activation.

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1.

Binding of 17β-estradiol (E2) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17α,20β-dihydroxy-4-pregnen-3-one (DHP)- and Igf-mediated signalling cascades in alleviating E2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E2, on OM in vitro. While priming of denuded oocytes with E2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E2 inhibition of meiosis resumption in zebrafish oocytes.

Regulation of recombinant human insulin-induced maturational events in Clarias batrachus (L.) oocytes in vitro.

Regulation of insulin-mediated resumption of meiotic maturation in catfish oocytes was investigated. Insulin stimulation of post-vitellogenic oocytes promotes the synthesis of cyclin B, histone H1 kinase activation and a germinal vesicle breakdown (GVBD) response in a dose-dependent and duration-dependent manner. The PI3K inhibitor wortmannin abrogates recombinant human (rh)-insulin action on histone H1 kinase activation and meiotic G2-M1 transition in denuded and follicle-enclosed oocytes in vitro. While the translational inhibitor cycloheximide attenuates rh-insulin action, priming with transcriptional blocker actinomycin D prevents insulin-stimulated maturational response appreciably, albeit in low amounts. Compared with rh-insulin, human chorionic gonadotrophin (hCG) stimulation of follicle-enclosed oocytes in vitro triggers a sharp increase in 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DHP) secreted in the incubation medium at 12 h. Interestingly, the insulin, but not the hCG-induced, maturational response shows less susceptibility to steroidogenesis inhibitors, trilostane or dl-aminoglutethimide. In addition, priming with phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) or cell-permeable dbcAMP or adenylyl cyclase activator forskolin reverses the action of insulin on meiotic G2-M1 transition. Conversely, the adenylyl cyclase inhibitor, SQ 22536, or PKA inhibitor H89 promotes the resumption of meiosis alone and further potentiates the GVBD response in the presence of rh-insulin. Furthermore, insulin-mediated meiotic maturation involves the down-regulation of endogenous protein kinase A (PKA) activity in a manner sensitive to PI3K activation, suggesting potential involvement of a cross-talk between cAMP/PKA and insulin-mediated signalling cascade in catfish oocytes in vitro. Taken together, these results suggest that rh-insulin regulation of the maturational response in C. batrachus oocytes involves down-regulation of PKA, synthesis of cyclin B, and histone H1 kinase activation and demonstrates reduced sensitivity to steroidogenesis and transcriptional inhibition.

High cAMP attenuation of insulin-stimulated meiotic G2-M1 transition in zebrafish oocytes: Interaction between the cAMP-dependent protein kinase (PKA) and the MAPK3/1 pathways

High intra-cellular cyclic nucleotide (cAMP) ensures prophase-I arrest and prevent steroid-induced meiotic G2-M1 transition in full-grown oocytes; however, relatively less information is available for cAMP regulation of growth factor-stimulated signalling events in the oocyte model. Here using zebrafish oocytes, we show that priming with dibutyryl cAMP (dbcAMP) or cAMP modulators, e.g. adenylate cyclase activator, forskolin or phosphodiesterase inhibitors (IBMX/cilostamide) block insulin action on germinal vesicle breakdown (GVBD) and histone H1 kinase activation. Though high cAMP priming attenuates insulin-induced MAPK3/1 (ERK1/2) phosphorylation (activation), following 2h of insulin stimulation it fails to block MAPK activation and GVBD. Further, insulin stimulation promotes down regulation of phospho-PKAc (inactivation) and PKA inhibition by H89/PKI-(6-22)-amide overcomes negative regulation by cAMP and induces GVBD and MAPK activation. Moreover, MEK1/2 inhibitor U0126 has no influence on H89-induced GVBD; however, it delays GVBD response in insulin-stimulated oocytes. MAPK activation by okadaic acid (OA) promotes GVBD; however, high dbcAMP abrogates OA action suggesting cross-talk between cAMP/PKA and MAPK-mediated signalling pathways may contribute significantly in maturing zebrafish oocyte.

In vitro effects of 2-hydroxyestradiol-17β on ovarian follicular steroid secretion in the catfish Heteropneustes fossilis and identification of the receptor and signaling mechanisms

Ovarian pieces containing postvitellogenic follicles were incubated in vitro with different concentrations of the catecholestrogen 2-hydroxyestradiol-17β (2-OHE 2) to evaluate its effects on steroid production and germinal vesicle breakdown (GVBD) in the catfish Heteropneustes fossilis. The incubation with 2-OHE 2 induced a shift in steroidogenic pattern: the C 19 and C 18 steroids testosterone (T) and estradiol-17β (E 2), respectively were significantly decreased with a concomitant significant increase in the C 21 steroids progesterone (P 4), 17-hydroxyprogesterone (17-OHP), 17,20β-dihydroxy-4-pregnen-3-one (17,20β-DP), 17,20α-dihydroxy-4-pregnen-3-one (17,20α-DP) and cortisol (F). Concomitantly, the catecholestrogen induced dose-dependently GVBD response, the first sign of meiosis resumption. The co- and pre-incubations of the ovarian pieces with 2-OHE 2, and adrenergic (phentolamine, α-blocker and propranolol, β-blocker) or estrogen (tamoxifen) receptor blockers resulted in inhibition of the stimulatory effect of the catecholestrogen on C 21 steroids and reversed the inhibition of testosterone and E 2. The α-blocker was more effective than the β-blocker. Our results suggest that 2-OHE 2 appears to employ both adrenergic (α-type) and estrogen receptor mechanisms in mediating the effects. The co- or pre-incubation of ovarian pieces with IBMX (a cAMP elevating drug), H89 (a protein kinase A inhibitor), and PD098059 (a MAP kinase kinase inhibitor) significantly inhibited the stimulatory effect of 2-OHE 2 on the C 21 steroids. The effect of chelerythrine (a protein kinase C inhibitor), on the other hand, varied with the incubation condition. In the co-incubation, the steroids showed varied effects: 17,20β-DP, testosterone and E 2 were elevated, and P 4 and 17-OHP were decreased. In the pre-incubation set up, all the steroids were inhibited except E 2. The inhibition by the blockers was higher in the pre-incubation groups. Taken together, the data suggest the involvement cAMP–protein kinase A, protein kinase C and MAP kinase pathways in the modulation of the steroidogenic activity.

Signaling pathways in ascidian oocyte maturation: The roles of cAMP/Epac, intracellular calcium levels, and calmodulin kinase in regulating GVBD

Most mature ascidian oocytes undergo germinal vesicle breakdown (GVBD) when released by the ovary into sea water (SW). Acidic SW blocks this but they can be stimulated by raising the pH, increasing intracellular cAMP levels by cell permeant forms, inhibiting its breakdown or causing synthesis. Boltenia villosa oocytes undergo GVBD in response to these drugs. However, the cAMP receptor protein kinase A (PKA) does not appear to be involved, as oocytes are not affected by the kinase inhibitor H-89. Also, the PKA independent Epac agonist 8CPT-2Me-cAMP stimulates GVBD in acidic SW. GVBD is inhibited in calcium free sea water (CaFSW). The intracellular calcium chelator BAPTA-AM blocks GVBD at 10 µM. GVBD is also inhibited when the ryanodine receptors (RYR) are blocked by tetracaine or ruthenium red but not by the IP(3) inhibitor D-609. However, dimethylbenzanthracene (DMBA), a protein kinase activator, stimulates GVBD in BAPTA, tetracaine or ruthenium red blocked oocytes. The calmodulin kinase inhibitor KN-93 blocks GVBD at 10 µM. This and preceding papers support the hypothesis that the maturation inducing substance (MIS) produced by the follicle cells in response to increased pH causes activation of a G protein which triggers cAMP synthesis. The cAMP then activates an Epac molecule, which causes an increase in intracellular calcium from the endoplasmic reticulum ryanodine receptor. The increased intracellular calcium subsequently activates calmodulin kinase, which causes an increase in cdc25 phosphatase activity, activating MPF and the progression of the oocyte into meiosis.

Signaling pathways in ascidian oocyte maturation: The role of cyclic AMP and follicle cells in germinal vesicle breakdown

Many ascidian oocytes undergo 'spontaneous' germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 nm in a reversible manner.

2-hydroxyestradiol-17β-induced oocyte maturation: involvement of cAMP–protein kinase A and okadaic acid-sensitive protein phosphatases,and their interplay in oocyte maturation in the catfishHeteropneustes fossilis

In Heteropneustes fossilis, in vitro incubation of postvitellogenic follicles with 2-hydroxyestradiol-17beta (2-OHE2, 5 micromol l(-1)) decreased significantly the total cAMP level, concomitant with germinal vesicle breakdown (GVBD). The incubation of the follicles with cAMP or cAMP-elevating drugs [phosphodiesterase (PDE) inhibitors], such as IBMX (3-isobutyl-1-methyl-xanthine), theophylline and caffeine, inhibited the 2-OHE2-induced GVBD in a concentration-dependent manner. The magnitude of the response varied: both cAMP and IBMX were effective at all concentrations (0.1-2.0 mmol l(-1)), followed by theophylline (0.5-2.0 mmol l(-1)) and caffeine (1-2.0 mmol l(-1)). The protein kinase A (PKA) inhibitor H89 stimulated oocyte maturation in a concentration-dependent manner. However, when co-incubated with 2-OHE2 for 24 h it produced a biphasic effect: low concentrations (0.1 and 1.0 micromol l(-1)) did not alter the 2-OHE2-induced GVBD, but high concentrations (5 and 10 micromol l(-1)) inhibited it. The incubation of the follicles with H89 lowered the inhibitory effect of IBMX on the 2-OHE2-induced GVBD. The incubation of the follicles with okadaic acid (OA), a protein phosphatase 1 and 2A inhibitor did not affect GVBD but when co-incubated with 2-OHE2, it enhanced the GVBD response. OA reversed the inhibitory effect of IBMX. The results suggest that OA may overcome the inhibition of 2-OHE2-induced GVBD by IBMX at a step distal to the cAMP-PKA pathway.

Effects of gonadotrophin in vivo and 2-hydroxyoestradiol-17β in vitro on follicular steroid hormone profile associated with oocyte maturation in the catfish Heteropneustes fossilis

An HPLC method was used to tentatively identify progesterone (P4) and its metabolites (17-hydroxyprogesterone (17-P4) and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P)), corticosteroids (cortisol and corticosterone) and testosterone in ovary/follicular preparations of the catfish Heteropneustes fossilis associated with in vivo or in vitro oocyte maturation/ovulation. A single i.p. injection of human chorionic gonadotrophin (100 IU/fish, sampled at 0, 8 and 16 h) induced oocyte maturation and ovulation, which coincided with significant and progressive increases in 17,20beta-P, and P4 and 17-P4, the precursors of the former. Both cortisol and corticosterone also increased significantly. Conversely, testosterone decreased significantly and progressively over time. Under in vitro conditions, incubation of post-vitellogenic (intact) follicles or follicular envelope (layer) with 2-hydroxyoestradiol (2-OHE2, 5 microM for 0, 6 and 24 h) elicited a sharp significant increase in 17,20beta-P, the increase being higher in the follicular envelope incubate. P4 and 17-P4 also registered significant increases over the time with the peak values at 24 h. Cortisol and corticosterone increased significantly in the intact follicle, but not in the follicular envelope incubate. Testosterone decreased significantly in the intact follicle, but increased significantly (24 h) in the follicular envelope incubate. Coincident with these changes, the percentage of germinal vesicle breakdown (GVBD) increased over the time in the intact follicle incubate (48.9% at 6 h and 79.8% at 24 h). Denuded oocytes on incubation with 2-OHE2 (5 microM) did not produce any significant change in the percentage of GVBD or in the steroid profile. While corticosterone and 17,20beta-P were undetected, P4, 17-P4, cortisol and testosterone were detected in low amounts. The results show that the 2-OHE2-induced GVBD response seems to be mediated through the production of 17,20beta-P and corticosteroids. It is suggested that hydroxyoestrogens seem to be a component in the gonadotrophin cascade of regulation of oocyte maturation/ovulation in the catfish.

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-I Induces Oocyte Maturational Competence but Not Meiotic Resumption in White Bass (Morone chrysops) Follicles In Vitro: Evidence for Rapid Evolution of Insulin-Like Growth Factor Action1

A combination of recombinant human (rh) insulin-like growth factor-I (IGF-I) (25 nM) and the maturation-inducing hormone (MIH), 17,20beta,21-trihydroxy-4-pregnen-3-one (20beta-S; 72.5 nM), induced germinal vesicle breakdown (GVBD) in ovarian follicles of white bass incubated in vitro, whereas a four times greater concentration of each hormone was ineffective alone. These results indicate that IGF-I induces oocyte maturational competence (OMC) but not meiotic resumption in white bass. Culture medium concentrations of 20beta-S remained below detection limits for ovarian fragments incubated with rhIGF-I. Actinomycin D blocked GVBD in response to hCG but not to rhIGF-I plus 20beta-S, suggesting that IGF-I requires de novo translation but not transcription to induce OMC. Gap junction uncouplers, 1-octanol and 1-heptanol, and the phosphatidylinositiol 3-kinase (PI 3-K) inhibitors, wortmannin and LY 294002, attenuated hCG-, 20beta-S-, and rhIGF-I plus 20beta-S-induced GVBD. Although these inhibitors reduced hCG-induced progestin release, PI 3-K inhibitors did not alter MIH synthesis in some incubations and addition of 20beta-S to the incubations did not fully overcome the effects of either class of inhibitors, suggesting that decreasing MIH production is not their only inhibitory effect on gonadotropin (GtH) action. Our data suggest that gap junctions and PI 3-K activity are necessary for GtH and IGF-I to induce and maintain OMC in white bass. The induction of OMC but not meiotic resumption by IGF-I in white bass, compared with the induction of meiotic resumption but not OMC by IGF-I discovered in the congeneric striped bass suggests rapid evolution of the reproductive actions of IGF-I among temperate basses (genus Morone).

Periovulatory changes in catfish ovarian oestradiol-17beta, oestrogen-2-hydroxylase and catechol-O-methyltransferase during GnRH analogue-induced ovulation and in vitro induction of oocyte maturation by catecholoestrogens.

In the catfish Heteropneustes fossilis and Clarias batrachus, ovarian oestrogen-2-hydroxylase (OE-2-H) activity increased significantly at 8 h after the injection of an ovulatory dose (0.15 microg/g body weight) of a mammalian GnRH analogue ([d -Ala(6)-Pro(9)]-LHRH ethylamide) and was restored to the 0 h (control) level after egg-stripping at 16 h. On the other hand, ovarian oestradiol-17beta (OE2) level and catechol-O-methyltransferase (COMT) activity decreased significantly at 8 h. While the OE2 level was restored to the 0 h level, COMT activity increased significantly at 16 h. Changes in ovarian OE2 level and enzymes indicate higher synthesis of 2-hydroxylated catecholoestrogens and their degradation during the periovulatory period. Under in vitro conditions, the synthetic catecholoestrogens (CEs, 2- and 4-hydroxylated oestradiol17beta and oestrone (OE1)) induced germinal vesicle break down (GVBD) in a dose- (0.01-10 microg/ml) and duration- (1-36 h) dependent manner, the mean values of the responses being in the order 2-OH OE2>4-OH OE2> 2-OH OE1>4-OH OE1. The CE-induced GVBD response (8 h induction) was not blocked by prior and subsequent incubations with steroid synthesis inhibitors (cyanoketone, epostane and aminoglutethimide) up to 36 h, suggesting that de novo steroidogenesis is not essential for the response. The percentage of GVBD response to 2-h induction by CEs was significantly inhibited by actinomycin D (a transcriptional inhibitor) and cycloheximide (a translational inhibitor), indicating the involvement of both RNA and protein synthesis. The CE-induced 8-h stimulation of GVBD was mildly blocked by propranolol, the beta-adrenergic inhibitor, suggesting the response was partly mediated through a beta-adrenergic receptor mechanism. Incubations with phentolamine, an alpha-adrenergic inhibitor, did not interfere with the CE-induced GVBD response. The results demonstrate CE-related enzymatic changes in teleost (catfish) ovaries and maturation-inducing substance activity of CEs.

Time-related effect of GnRH on histone H1 kinase activity in the goldfish follicle-enclosed oocyte

The level of cyclin B-associated cdc2 kinase, a component of maturation promoting factor (MPF), is known to be high during metaphase of the meiotic maturation of oocytes. The time-related action of gonadotropin-realising hormones (GnRH) on histone H1 kinase activity (known to reflect cdc2 kinase activity) was investigated in vitro in follicle-enclosed goldfish oocytes. Germinal vesicle breakdown (GVBD) and testosterone production were also investigated in the same follicle-enclosed goldfish oocytes to determine the temporal relationship between GnRH-induced histone H1 kinase activity and the reinitiation of meiosis and steroidogenesis. Treatments with gonadotropin (GTH) or GnRH stimulated the histone H1 kinase activity to the same maximum level. However, sGnRH- and cGnRH-II-induced histone H1 kinase activity were significantly higher compared with controls after 2 hours of treatment, whereas the GTH-induced increase became significantly higher after 6-8 hours of incubation. Overall, the results demonstrate a close temporal relationship between GVBD response and histone H1 kinase activity induced by GTH and sGnRH-cGnRH-II.Key words: GnRH, oocyte meiosis, testosterone, H1 kinase, goldfish.

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin.

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.

Time-related effect of GnRH on histone H1 kinase activity in the goldfish follicle-enclosed oocyte

The level of cyclin B-associated cdc2 kinase, a component of maturation promoting factor (MPF), is known to be high during metaphase of the meiotic maturation of oocytes. The time-related action of gonadotropin-releasing hormones (GnRH) on histone H1 kinase activity (known to reflect cdc2 kinase activity) was investigated in vitro in follicle-enclosed goldfish oocytes. Germinal vesicle breakdown (GVBD) and testosterone production were also investigated in the same follicle-enclosed goldfish oocytes to determine the temporal relationship between GnRH-induced histone H1 kinase activity and the reinitiation of meiosis and steroidogenesis. Treatments with gonadotropin (GTH) or GnRH stimulated the histone H1 kinase activity to the same maximum level. However, sGnRH- and cGnRH-II-induced histone H1 kinase activity were significantly higher compared with controls after 2 hours of treatment, whereas the GTH-induced increase became significantly higher after 6-8 hours of incubation. Overall, the results demonstrate a close temporal relationship between GVBD response and histone H1 kinase activity induced by GTH and sGnRH-cGnRH-II.

Change in Intracellular Ca 2+Is Not Involved in Serotonin-Induced Meiosis Reinitiation from the First Prophase in Oocytes of the Marine Bivalve Crassostrea gigas

In response to the neurohormone serotonin (5-hydroxytryptamine, 5-HT), prophase-arrested oocytes of the marine bivalve Crassostrea gigas(oyster) reinitiate meiosis, undergo germinal vesicle breakdown (GVBD), and are arrested again at metaphase I. We examined the pharmacological characteristics of 5-HT receptors and the signal transduction pathway following 5-HT stimulation in oyster oocytes. Among 5-HT agonists tested, only α-methyl 5-HT, a 5-HT 2agonist, induced GVBD, although it was 1000 times less sensitive than 5-HT. The rank order of the potency of 5-HT antagonists to inhibit GVBD was propranolol, cyproheptadine > metoclopramide > mianserin. These results are quite different from those reported for other mollusks, suggesting the presence of unique 5-HT receptors on oyster oocytes. Using the fluorescent Ca 2+dyes fura-2 and calcium green and the pH indicator 1-hydroxypyrene-3,6,8-trisulfonic acid, we examined changes in intracellular Ca 2+([Ca 2+] i) and intracellular pH (pH i) during 5-HT-induced meiosis reinitiation. 5-HT did not trigger any changes in [Ca 2+] i. However, an increase in pH iwas observed during the 5-HT-induced meiosis reinitiation. The increased pH ilevel was rather small before GVBD and not necessary for GVBD, because lowering pH iby sodium acetate seawater (pH 7.0) did not prevent 5-HT-induced GVBD. Measurement of the kinase activity toward a peptide substrate specific to cdc2 demonstrated that maturation-promoting factor (MPF) was activated in accordance with the occurrence of GVBD in response to 5-HT. Therefore, it is likely that in oyster oocytes the signal transduction pathways and intracellular effectors participating in 5-HT-induced meiosis reinitiation via the activation of MPF are insensitive to [Ca 2+] iand pH i.

Regulation of maturation-promoting factor by protein kinase C inChaetopterusoocytes

Summary We present the results of a variety of studies showing that activation of protein kinase C (PKC) in oocytes of Chaetopterus pergamentaceus results in germinal vesicle breakdown (GVBD). Phorbol esters and diacylglycerol can initiate a morphologically normal GVBD accompanied by a spectrum of associated biochemical processes, including increased protein phosphorylation, a shift in protein synthesis and activation of a protein kinase, maturation promoting factor (MPF). MPF activation is essential for GVBD in response to phorbol esters. In addition, inhibitors of PKC can block naturally-induced GVBD. We also present evidence that PKC can phosphorylate p34cde2, the catalytic subunit of MPF and that phosphorylation by PKC increases the histone H1 kinase activity of immunoprecipitated MPF. Immunoblot studies show that Chaetopterus oocyte p34cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34cdc2. Since PKC is the intracellular receptor for and is directly activated by tumor-promoters, tumor promotion might involve acceleration of the cell cycle through modification of the enzymatic activity of MPF by PKC.

Antagonistic effects of xenobiotics on steroid-induced final maturation of Atlantic croaker oocytes in vitro

The antagonistic effects of Kepone (chlordecone) and o,p'-DDD on maturation-inducing steroid (17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S) induced final maturation of Atlantic croaker ( Micropogonias undulatus). Oocytes were investigated in an in vitro germinal vesicle breakdown (GVBD) bioassay. Exposure to Kepone or o,p'-DDD (100 nM-100 μM) prevented the majority of the oocytes from completing GVBD in response to 20β-S; many of them were arrested at the lipid coalescence or germinal vesicle migration stages when exposed to high xenobiotic concentrations (100 μM). In addition, clearing of the ooplasm, oil droplet formation and hydration were incomplete in xenobiotic-exposed oocytes that underwent GVBD. The xenobiotics inhibited GVBD in a concentration-dependent manner. One minute exposure to either of the xenobiotics was sufficient to block GVBD. Washing the follicle-enclosed oocytes after xenobiotic exposure restored their ability to undergo GVBD in response to 20β-S. These results suggest that o,p'-DDD and Kepone exert antagonistic effects on 20β-S action, possibly by competing for the ovarian 20β-S membrane receptor.

Insulin and Insulin-like Growth Factors I and II Induce Final Maturation of Oocytes of Red Seabream, Pagrus major, in Vitro

The effects of various growth factors and peptides on in vitro germinal vesicle breakdown (GVBD) were examined in oocytes of the red seabream, Pagrus major , a daily spawning marine teleost. The oocytes used underwent GVBD in response to HCG but not to 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Insulin (1 μ M) and insulin-like growth factor-1 (IGF-I, 10 n M) and IGF-II (13 n M) induced GVBD. Salmon gonadotropin-releasing hormone (GnRH), chicken GnRH (100 n M), angiotensin II and III (10 μg/ml), inhibin A, activin A (200 ng/ml), and epidermal growth factor (1 n M) were ineffective. IGF-I was the most potent inducer of GVBD. In the presence of DHP, oocytes underwent GVBD in response to low doses of IGF-I or -II or insulin which alone were ineffective. GVBD occurred in response to DHP after oocytes were preincubated with IGF-I or with HCG. Actinomycin D inhibited GVBD induced by HCG alone or in combination with DHP, but actinomycin D did not block GVBD induced by IGF-I alone or in combination with DHP. Cycloheximide totally inhibited induction of GVBD by HCG or IGF-I, alone or in combination with DHP. These results suggest, for the first time in teleost, that growth factors, such as IGF-I, are involved in the induction of GVBD of oocytes. IGF-I appears to act directly on oocytes, and not via maturation-inducing hormone production, to induce GVBD.

Development of Maturational Competence of Oocytes of Red Seabream, Pagrus major, after Human Chorionic Gonadotropin Treatment in Vitro Requires RNA and Protein Synthesis

The effects of human chorionic gonadotropin (HCG) and 17α,20β-dihydroxy-4-pregnen-3-one (DHP) on in vitro germinal vesicle breakdown (GVBD) in oocytes obtained at different times of day from a daily spawning marine teleost, the red seabream Pagrus major, were investigated. Oocytes isolated at 0800 hr underwent GVBD in response to HCG (10 IU/ml) but not to DHP (10 ng/ml). GVBD could be induced in oocytes isolated at 1600 hr by either HCG or DHP. Oocytes underwent GVBD in response to DHP after DHP-insensitive oocytes were incubated with HCG for 30, 60, or 120 min. The effects of actinomycin D (a transcriptional inhibitor) and cycloheximide (a translational inhibitor) on HCG- and DHP-induced GVBD were also investigated. Actinomycin D (1 μg/ml) totally inhibited HCG-induction of GVBD in oocytes isolated at 0600 and 0800 hr. Actinomycin D also significantly inhibited GVBD induced by HCG in combination with DHP in oocytes taken at 0600 and 0800 hr. Cycloheximide (1 μg/ml) completely inhibited the induction of GVBD by HCG or DHP, alone or in combination, in oocytes obtained at 0600, 1000, and 1400 hr. These results indicate that gonadotropin induces maturational competence (responsiveness to maturation-inducing steroid) in oocytes of red seabream. These results also suggest that gonadotropin-induced synthesis of new protein through a mechanism dependent on RNA is essential for the development of maturational competence in oocytes of red seabream.