Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

Pharmacology of the serotonergic inhibition of steroid-induced reinitiation of oocyte meiosis in the teleost Fundulus heteroclitus

Serotonin (5-HT) was found to inhibit steroid (17α,20β-dihydroxy-4-pregnen-3-one; 17,20βP)-induced resumption of oocyte meiosis (oocyte maturation) in vitro in the teleost Fundulus heteroclitus. Serotonin inhibited both follicle-enclosed and denuded oocytes, which indicates the presence of oocyte-associated 5-HT sensitive sites. The response of oocytes to 5-HT was characterized pharmacologically, i.e., the capacity of serotonergic agonists and antagonists to mimic or block the 5-HT inhibition of the steroid-induced oocyte maturation was assessed by the changes in the percentage of oocyte germinal vesicle breakdown (GVBD). Dose-response curves for each compound were drawn and compared. The rank order of potency among the agonists was: 5-HT > 5-methoxytryptamine > tryptamine = 5,6-diHT = 5-carboxidotryptamine > 5,7-diHT = 5-methoxy-dimethyltryptamine > α-methyl-5-HT > 2-methyl-5-HT. Incubation of ovarian follicles with high doses of some antagonists (mianserin and metergoline) induced oocyte GVBD, although this effect was associated with high levels of oocyte atresia during GVBD or shortly after maturation. Consequently, doses of the antagonist too low to induce GVBD were tested for their ability to block the 5-HT inhibitory action; the rank order of potency was: MDL-72222 = metoclopramide > metergoline > propanolol > ketanserin. Dopamine, acetylcholine, epinephrine, and norepinephrine could also inhibit 17,20βP-induced GVBD, although at doses much higher than those of 5-HT; melatonin and histamine had no effect on oocyte maturation. These results suggest that specific receptors mediate the inhibitory action of 5-HT on the steroid-triggered meiosis resumption. The pharmacological profile of these 5-HT receptors is different from those of any known mammalian 5-HT receptor, although they showed some similarities to the 5-HT_1A, 5-HT₂, and 5-HT₃ receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs. Mol. Reprod. Dev. 48:282–291, 1997. © 1997 Wiley-Liss, Inc.     

The effect of four C21-steroids on oocyte maturation in Siberian sturgeon (<Emphasis Type="Italic">Acipenser baeri</Emphasis> Brandt)

The effect of four C21-steroids, progesterone (P₄), 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), 17,20β,21-trihydroxy-4-pregnen-3-one (20βS) and 11-desoxycortisol (S), on in vitro oocyte maturation in Siberian sturgeon (Acipenser baeri Brandt) females was demonstrated using short-term (5 and 30 min) exposures of ovarian follicles to steroid solutions followed by incubation in steroid-free medium. The study aimed to find out which of the four candidates for a maturationinducing steroid (P₄, 17,20βP, 20βS or S) induces a fastest germinal vesicle breakdown (GVBD) in oocytes of Siberian sturgeon. Dissolution of the oocyte nucleus or GVBD was taken as a criterion of oocyte maturation. Dose-response profiles of hormone activities as well as effects of the hormones under short-term exposures of follicles to their equal doses were compared. P₄ was found to be a most active GVBD inducer compared to other C21-steroids, S was the second in its activity, whereas 17,20βP and 20βS were less efficient. A comparison of the present and previously obtained data on the dynamics of C21-steroids in vivo and their effect on ovarian follicles in vitro indicates an important role of the above hormones, particularly P₄ and 20βS, in the regulation of the final stage of oocyte maturation in sturgeons.     

Gonadotropin-induced steroidogenic shift towards maturation-inducing hormone in Japanese yellowtail during final oocyte maturation

Intact ovarian follicles, obtained from untreated and human chorionic gonadotropin (HCG) treated Japanese yellowtail Seriola quinqueradiata during different maturational stages, were incubated with radioactive [3H]pregnenolone, [³H]17‐hydroxyprogesterone or [¹⁴C] androstenedione and steroid metabolites identified by thin layer chromatography (TLC) followed by recrystallization to constant specific activity. In untreated late vitellogenic (0 h) follicles, androstenedione was the major product with smaller amounts of testosterone and oestradiol‐17α. In post‐vitellogenic (12 h post‐injection) intact follicles, androstenedione predominated, and although testosterone and oestradiol‐17α were not produced, there were small amounts of 17, 20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) and 17,21‐dihydroxy‐4‐pregnene‐3, 20‐dione (11‐deoxycortisol). In HCG‐treated fish, a steroidogenic shift resulted in the disappearance of testosterone and oestradiol‐17 coinciding with the appearance of 17, 20β‐P. During early and late final oocyte maturation FOM (24 and 36 h post‐injection), there was a five‐ to seven‐fold increase in the production of 17, 20β‐P, whereas production of 11‐deoxycortisol remained almost the same. During FOM, in addition to 17,20β‐P, its 5β‐reduced metabolite, 17,20β‐dihydroxy‐5β‐pregnan‐3‐one (5β‐17,20β‐P) was synthesized, suggesting a decrease in maturation‐inducing 17,20β‐P activity. 17, 20β,21‐Trihydroxy‐4‐pregnen‐3‐one (20β‐S) was not synthesized by ovarian fragments in Japanese yellowtail at any maturational stage. The metabolites identified on TLC during FOM were tested to evaluate their maturation‐inducing activity in an in vitro bioassay. Of the steroids tested, 17,20β‐P was the most effective inducer of germinal vesicle breakdown (GVBD), followed by 5β‐17,20β‐P. Timely synthesis of 17,20β‐P immediately prior to and during FOM as well as its great potency in inducing GVBD in vitro supports the evidence for a physiological role of 17,20β‐P as a maturation‐inducing hormone in Japanese yellowtail.     

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.     

Induction and inhibition of meiotic maturation of amphibian (Rana dybowskii) follicular oocytes by forskolin and cAMP in vitro

Previous studies have demonstrated that direct or indirect elevation of cAMP levels in cultured amphibian ovarian follicles simultaneously stimulated production of oocyte maturation-inducing steroid (progesterone) by the follicles and inhibited oocyte maturation induced by endogenous or exogenous hormone. The duration of cAMP stimulation influenced arrest and reinitiation of oocyte meiotic maturation in ovarian follicles of Rana dybowskii. Addition of forskolin (adenylate cyclase stimulator) to cultured follicles inhibited both progesterone- and frog pituitary homogenate (FPH)-induced oocyte maturation. Similar inhibitory results were obtained when hormone-treated follicles were cultured in the continual presence of cAMP. Oocyte maturation increasingly occurred in follicular oocytes when cAMP or forskolin addition was delayed following treatment with FPH or progesterone. Transient exposure (6-8 hr) of ovarian follicles to forskolin or cAMP markedly stimulated oocyte maturation as well as accumulation of progesterone as measured by radioimmunoassay within the ovarian follicles. Forskolin was more effective than cAMP, at the dose tested, in stimulating progesterone production and accumulation by the follicles. The data demonstrate that transient manipulation (elevation) of cAMP levels in cultured follicles, without added FPH or steroid, was sufficient to initiate oocyte maturation. Results suggest that, with transient exposure to forskolin or exogenous cAMP, there is a sequential increase and decrease in endogenous cAMP levels in the somatic cells and germ cell components of the ovarian follicle. These changes appear to mediate production of maturation-inducing steroid and secondarily allow its effects on the oocyte to be expressed.     

Inhibitory effects of cAMP and protein kinase C on meiotic maturation and MAP kinase phosphorylation in porcine oocytes

The regulation of MAP kinase phosphorylation by cAMP and protein kinase C (PKC) modulators during pig oocyte maturation was studied by Western immunoblotting. We showed that both forskolin and IBMX inhibited MAP kinase phosphorylation and meiosis resumption in a dose-dependent manner, and this inhibitory effect was overcome by the protein phosphatase inhibitor, okadaic acid. Pharmacological PKC activator phorbol myristate acetate or physiological PKC activator diC8 also delayed MAP kinase phosphorylation and meiosis resumption, and their effect was abrogated by PKC inhibitors, staurosporine, and calphostin C. The results suggest that meiotic resumption is inhibited by elevation of cAMP or delayed by activation of PKC probably via down-regulation of MAP kinase activation, which is mediated by protein phosphatase, during pig oocyte maturation.     

Steroidogenesis in Fundulus heteroclitus. IV. Dichotomous effects of a phorbol ester on ovarian steroid production and oocyte maturation.

The possible role of protein kinase C (PKC) activation in mediating the stimulatory actions of a Fundulus pituitary extract (FPE) on ovarian steroidogenesis and oocyte maturation was investigated. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), alone slightly increased basal 17 alpha-hydroxy,20 beta-dihydroprogesterone (DHP) and 17 beta-estradiol (E2) synthesis and significantly stimulated germinal vesicle breakdown (GVBD). Addition of FPE promoted synthesis of DHP, testosterone (T), and E2, and initiated GVBD. Phorbol ester inhibited FPE-induced steroidogenesis but increased the number of oocytes that underwent GVBD. Phorbol ester also markedly impeded induction of steroidogenesis by dibutyryl cAMP and differentially affected the conversion of 25-hydroxycholesterol, pregnenolone, or progesterone to DHP, T, and E2: DHP production was not affected; T production diminished; and E2 synthesis increased (T aromatization also increased). These results suggest an inhibitory role for the PKC pathway on FPE-induced ovarian steroid production, with PMA appearing to affect various steroidogenic steps. The stimulatory action of PMA on oocyte maturation seems to be independent of follicular steroid production since aminoglutethimide, an inhibitor of steroidogenesis, did not block PMA-induced GVBD. Moreover, PMA had a marked stimulatory effect on GVBD in denuded oocytes. Thus, in contrast to the inhibitory role found for the PKC pathway on ovarian follicular steroidogenesis, activation of PKC in the oocyte may serve as a signal-transducing mechanism leading to GVBD.     

Effect of defolliculation and 17α-hydroxy, 20β-dihydroprogesterone on cyclic AMP level in full-grown oocytes of the rainbow trout,Salmo gairdneri

The changes in cAMP were followed in trout oocytes incubated in vitro after defolliculation performed by either enzymatic or manual dissection. Both defolliculation methods induced a highly significant rise in oocyte cAMP level (4.5 times the basal level of control [follicle-enclosed oocytes], after 6 h). Treatment of defolliculated oocytes with 17α-hydroxy,20β-dihydroprogesterone (17α,20β-OH-P) (10^?6 M), which induced oocyte maturation (germinal vesicle breakdown [GVBD]) was able, first, to interrupt the increase of oocyte cAMP level promoted by defolliculation and then to lower this level significantly down to values that still remained higher than folliculated controls. Very low concentrations of 17α,20β-OH-P (1.38–55.6 10^?9 M), or physiological doses of testosterone (0.35 10^?6 M, in the range found in vivo before ovulation) were able to induce a similar decrease of oocyte cAMP level without inducing GVBD. Under the same experimental conditions estradiol (0.35 10^?6 M) exhibited no action. These results suggest that some factor(s) originating in the follicle (FIF), inhibit the oocytes' tendency to accumulate cAMP before the final surge of 17α,20β-OH-P. This factor might be a follicular steroid such as testosterone or nonmaturing concentrations of 17α,20β-OH-P. Moreover our data favour the hypothesis that the final surge of 17α,20β-OH-P could induce distinct intraoocyte mechanisms: the first induces an irreversible blockage of cAMP level before the inhibitory action of the FIF is suppressed by ovulation, and the second mechanism leads to GVBD.     

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms

In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.     

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.     

2-Hydroxyestradiol-17beta-induced oocyte maturation in catfish (Heteropneustes fossilis) involves protein kinase C and its interaction with protein phosphatases

In vitro effects of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, calphostin C (PKC inhibitor) and okadaic acid [OA, a protein phosphatase (PP; PP1 and PP2A) inhibitor] on 2-hydroxyestradiol-17β (2-OHE₂)-induced oocyte maturation were investigated in the catfish Heteropneustes fossilis. Incubations of postvitellogenic follicles with PMA or OA alone did not induce oocyte maturation. However, co-incubations with 2-OHE₂ and PMA (0.05, 0.5 and 5 μM) or 2-OHE₂ and OA (0.5, 1.0 or 2.0 μM) increased germinal vesicle breakdown (GVBD) significantly over that of 2-OHE₂. Incubation of follicles with calphostin C elicited varied effects on GVBD, low (0.005 and 0.01 μM) and high (5.0 and 10.0 μM) concentrations did not affect GVBD, but medium concentrations (0.05, 0.1, 0.5, 1.0 and 2.5 μM) stimulated it. The medium concentrations elicited a biphasic stimulatory response with peak GVBD at 0.1 μM (54%). Calphostin C (≥ 2.5 μM) inhibited the 2-OHE₂-induced GVBD in a concentration-dependent manner during the 24 h incubation. Pre- or post-treatment with calphostin C inhibited the steroid-induced GVBD only at 6 h. In co-incubation studies, both PMA and OA reversed the inhibitory effect of calphostin C: the former partially and the latter fully. The results of the present study show that PKC appears to modulate the 2-OHE₂-induced oocyte maturation. The OA-sensitive PP may be involved in the PKC modulation of steroid-induced oocyte maturation.     

Involvement of cAMP in initiating maturation of the brittle-star Amphipholis kochii oocytes: induction of oocyte maturation by inhibitors of cyclic nucleotide phosphodiesterase and activators of adenylate cyclase

The maturation of brittle-star (Amphipholis kochii) oocytes, i.e., the reinitiation of meiosis accompanied by germinal vesicle breakdown (GVBD) and the acquisition of fertilizability, was induced by acid (pH 3.0) seawater containing 10 mM cAMP. Oocyte maturation was also induced by seawater of normal pH (pH 8.0) that contained either an inhibitor of cyclic nucleotide phosphodiesterase (25 mM theophylline, 25 mM caffeine) or an activator of adenylate cyclase (100 microM forskolin, 0.6 microM cholera toxin). Experiments in which the oocytes were treated with forskolin or theophylline for various periods of time demonstrated that there was a positive correlation between the oocyte cAMP level measured by radioimmunoassay and the extent of GVBD induced in each treatment: both increased as the treatment period became longer and about a threefold increase in cAMP level induced 50% GVBD. These results indicate that an increase in cAMP level initiates maturation of the brittle-star oocytes.     

Regulation of maturation-promoting factor by protein kinase C in Chaetopterus oocytes

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 p34^cde2, 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 p34^cdc2 is not tyrosine phosphorylated prior to the initiation of GVBD, indicating that activation of MPF at GVBD in this species does not require p80^cdc25, the activator of MPF at mitosis. These results suggest that PKC is an essential regulator of GVBD which can directly phosphorylate and regulate p34^cdc2. 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.     

Effects of cAMP forskolin and cyanoketone on invitro oocyte maturation in the catfish,Clarias batrachus

This study investigated the interactive effects of cyanoketone (CK), an inhibitor of 3β-hydroxysteroid dehydrogenase on the effects of cAMP and forskolin (FK) on oocyte maturation inClarias batrachus using an in vitro incubation technique. When the oocytes were incubated in the presence of 1 Μg/ml 17α, 20β-dihydroxy-4-pregnen-3-one[l7α, 20Β-DP, the maturation-inducing steroid (MIS) of this species] for 6h, they matured [85.3 + 1.36% germinal vesicle breakdown (GVBD)] normally after additional incubation for 20–30 h in plain medium. On the other hand, exposure to 1.0 and 8 0 mM of cAMP after MIS stimulation caused significant inhibition of GVBD but lower concentrations (0.1 and 0.5 mM) of cAMP were noninhibitory. However, when the oocytes were preincubated for 1 h with 1 μg/mI CK, a significant inhibition in the percentage of GVBD was recorded including the lower concentrations of cAMP. FK, an activator of adenylate cyclase, could significantly induce GVBD at all of its concentrations (0.1, 0.5, 1.0 and 10.0 μM) in a dose- and time-dependent manner. However, when the oocytes were exposed to 1 μg/ml CK for 1 h, prior to FK stimulation, a complete inhibition of GVBD occurred but when CK treatment was given after the FK stimulation, only a partial inhibition of maturation was observed. Taken together, these data indirectly suggest that FK induces catfish oocyte maturation probably by stimulating follicular production of Δ⁴ steroid ( 17α,20 β-DP)through an adenylate cyclase-c AMP-mediated pathway, a mechanism identical to the gonadotropin-induced oocyte maturation.     

Cyclic AMP level and phosphodiesterase activity during 17alpha,20beta-dihydroxy-4-pregnen-3-one induction and theophylline inhibition of oocyte maturation in the catfish,Clarias batrachus

This study directly tested the hypothesis that the induction of oocyte maturation in the catfish Clarias batrachus is followed by a transient decrease in oocyte cyclic AMP (cAMP) level that is due to an increase in phosphodiesterase (PDE) activity. Further, the PDE inhibitor theophylline was used to investigate the possible role of PDE in the maturation-inducing action of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), the physiological maturation-inducing steroid of this catfish species. The results obtained from batches of oocytes taken from the same donor at the same time clearly show a close relationship between dose-dependent induction of germinal vesicle breakdown (GVBD) and PDE activity with a concomitant decrease in cAMP in the oocytes treated with different concentrations of 17alpha,20beta-DP. In contrast, theophylline prevents GVBD and inhibits PDE activity by promoting cAMP accumulation in oocytes. A time-dependent decrease in PDE activity and an increase in cAMP content with a marked inhibition of GVBD were recorded even in oocytes pre-stimulated with 1 microgram/ml 17alpha,20beta-DP for 6 h and then treated with 1 mM theophylline for various times. These results suggest that cAMP plays a key role in the regulation of oocyte maturation in C. batrachus which may be mediated by PDE activity.     

Regulation of ovarian steroidogenesis in vitro by follicle-stimulating hormone and luteinizing hormone during sexual maturation in salmonid fish

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20beta-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17alpha-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17beta production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20beta-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.     

Vasoactive intestinal peptide stimulates oocyte maturation, steroidogenesis, and cyclic adenosine 3',5'-monophosphate production in isolated preovulatory rat follicles

Vasoactive intestinal peptide (VIP) is present in the rat ovary and has been shown to stimulate cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone production in cultured rat granulosa cells. In the present study, VIP-stimulated cAMP production has been studied in relation to steroid accumulation and oocyte maturation in isolated preovulatory rat follicles. VIP stimulated resumption of meiosis (oocyte maturation) in up to 60% of the follicle-enclosed oocytes after 6 h at 1 microM (control, 1.8%; luteinizing hormone 99%). The effect was time- and dose-dependent up to 6 h and was seen with both natural and synthetic VIP. VIP also stimulated the accumulation of steroids (estrogen, 2.3-fold; testosterone, 2.0-fold; and progesterone, 1.6-fold increase after 6 h of incubation) and lactate (2.6-fold) by the follicles. VIP-increased tissue levels of cAMP in the follicle were dose- and time-dependent. This effect was potentiated by a phosphodiesterase inhibitor. When isolated oocyte-cumulus complexes were studied, VIP caused a transient inhibition of spontaneous oocyte maturation, and demonstrated no effect on denuded oocytes. These results extend earlier preliminary observations on the ability of VIP to induce meiotic maturation of follicle-enclosed oocytes. Our results also show that VIP can stimulate steroid and lactate accumulation in the isolated follicles. The pattern of steroids produced suggests an effect both on the theca- and granulosa cells. We also show that VIP can delay spontaneous oocyte maturation. These effects appeared, at least partially, to be mediated by cAMP.     

Protein kinase C and mitogen-activated protein kinase cascade in mouse cumulus cells: cross talk and effect on meiotic resumption of oocyte

Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in cumulus cells are involved in FSH-induced meiotic resumption of cumulus-enclosed oocytes (CEOs), but their regulation and cross talk are unknown. The present experiments were designed to investigate 1) the possible involvement of MAPK cascade in PKC-induced meiotic resumption; 2) the regulation of PKC on MAPK activity in FSH-induced oocyte maturation; and 3) the pattern of PKC and MAPK function in induced meiotic resumption of mouse oocytes. PKC activators, phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), induced the meiotic resumption of CEOs and activation of MAPK in cumulus cells, whereas this effect could be abolished by PKC inhibitors, calphostin C and chelerythrine, or MEK inhibitor U0126. These results suggest that PKC might induce the meiotic reinitiation of CEOs by activating MAPK in cumulus cells. Both PKC inhibitors and U0126 inhibited the FSH-induced germinal vesicle breakdown (GVBD) of oocytes and MAPK activation in cumulus cells, suggesting that PKC and MAPK are involved in FSH-induced GVBD of mouse CEOs. Protein synthesis inhibitor cycloheximide (CHX) inhibited FSH- or PMA-induced oocyte meiotic resumption, but not the MAPK activation in cumulus cells. FSH and PKC activators induced the GVBD in denuded oocytes cocultured with cumulus cells in hypoxanthine (HX)-supplemented medium, and this effect could be reversed by U0126. Thus, when activated by FSH and PKC, MAPK may stimulate the synthesis of specific proteins in cumulus cells followed by secretion of an unknown positive factor that is capable of inducing GVBD in oocytes.     

Regulation of mouse oocyte maturation: involvement of cyclic AMP phosphodiesterase and calmodulin

A decrease in mouse oocyte cAMP occurs during commitment to resume meiosis (R. M. Schultz, R. R. Montgomery, and J. R. Belanoff, 1983, Dev. Biol. 97, 264-273). Experiments described in this report were performed to ascertain if oocyte cyclic nucleotide phosphodiesterase (PDE) is involved in this decrease. PDE activity was found in extracts of mouse oocytes. The activity appeared soluble and not membrane bound. For each of three different PDE inhibitors, a positive correlation was found between the ability of increasing concentrations of each compound to inhibit PDE in oocyte extracts and to inhibit germinal vesicle breakdown (GVBD). Moreover, the more potent the PDE inhibitor, the more effectively it inhibited GVBD. The possibility that calmodulin (CaM) plays a role in maturation was examined since CaM modulates PDE activity in other systems. About 0.3% of total oocyte protein is CaM as determined by radioimmunoassay and activation of exogenous PDE. A CaM-dependent step in maturation was suggested since the CaM inhibitors trifluoperazine and calmidizolium inhibited GVBD in a dose-dependent manner. In addition, the CaM inhibitors W7 and W13 inhibited GVBD at lower concentrations than the less-active corresponding congeners W5 and W12. Oocyte extracts contained a CaM-modulated PDE. Activity was inhibited about 50% by addition of EGTA, and fully restored by addition of exogenous CaM and excess calcium. cAMP hydrolysis was inhibited in a dose-dependent manner by either trifluoperazine, calmidizolium, or W7; maximal inhibition was also about 50%. CaM-modulated PDE, however, did not appear to be the target for the effects of CaM inhibitors on GVBD, since concentrations of W7 that inhibited maturation did not inhibit cAMP hydrolysis in the oocyte. Results from these studies suggest that oocyte PDE is involved in the decrease in cAMP associated with resumption of meiosis, but that the CaM-dependent step occurs subsequent to or concurrently with the drop in cAMP.