Germinal Vesicle Breakdown In Oocytes Research Articles

Antioxidants stimulate meiosis resumption, but inhibit mitogen-activated protein kinase phosphorylation and further cell cycle progression in porcine oocytes

In the present study the effects of two cell-permeant antioxidants, 2(3)-tert-butyl-4-hydroxyanisole (BHA) and nordihydroguaiaretic acid (NDGA), on porcine oocyte meiosis resumption, chromatin behaviour and spindle assembly were investigated. The antioxidants BHA and NDGA stimulated meiosis resumption in a dose-dependent manner in both cumulus-enclosed and denuded porcine oocytes. After in vitro culture for 8 h, few oocytes underwent germinal vesicle breakdown (GVBD) in control groups, whereas GVBD occurred in high percentages of oocytes treated with BHA or NDGA at concentrations that inhibit GVBD in rodent oocytes, although mitogen-activated protein (MAP) kinase was not phosphorylated as revealed by Western immunoblots. Orcein staining and fluorescein isothiocyanate-anti-alpha-tubulin labelling showed that chromosome and spindle formation, respectively, and further meiosis progression were inhibited 20 and 25 h after culture. Instead, chromatin was highly condensed or existed in scattered condensed clusters. Correspondingly, MAP kinase phosphorylation was inhibited by both BHA and NDGA in a dose-dependent manner. The inhibitory effects of BHA on meiosis completion and MAP kinase phosphorylation was reversible. These results suggest that, unlike in rodent oocytes, antioxidants stimulate GVBD in the absence of MAP kinase activation, but inhibit MAP kinase phosphorylation, meiotic apparatus formation and thus the further progression of the meiosis of porcine oocytes.

Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes

The present study was conducted to examine effects of hormones and osmolarity on germinal vesicle breakdown (GVBD) and histone H1 kinase (HIK) activity in porcine oocytes cultured in vitro. The basic medium used for culture of oocytes was modified Tyrode's solution in which the osmolarity was adjusted to 134 to 495 mOsm by changing the concentration of sodium chloride (NaCl). When the hormones were present, osmolarity of medium that allows GVBD of oocytes was less than 400 mOsm. However, the range of osmolarity of medium that allows meiotic maturation of oocytes was 210 to 362 mOsm. On the other hand, without hormonal supplement, the incidence of GVBD in oocytes decreased as the osmolarity of the medium increased in the rage of 210 to 362 mOsm. By increasing the osmolarity of the medium from 210 to 362 mOsm by addition with sorbitol instead of NaCl, the incidence decreased from 89.1 % to 13.3 %. In oocytes cultured in medium of 210 mOsm without hormones, the percentage of oocytes that underwent GVBD and had increased H1K activity 20 h after culture was significantly higher (P<0.05) than those of oocytes cultured in the same medium supplemented with hormones or medium of 362 mOsm. These results indicate that in vitro induction of GVBD in porcine oocytes is strongly affected by osmolarity of the medium in the absence of hormones. The results also suggest that, under low osmolarity (210 mOsm), GVBD is accelerated with rapid increase of H1K activity.

MAP kinase activity is downregulated by phorbol ester during mouse oocyte maturation and egg activation in vitro.

The effects of protein kinase C (PKC) stimulator, phorbol 12-myriatate 13-acetate (PMA), on meiotic cell cycle regulation and mitogen-activated protein (MAP) kinase changes have been studied in mouse oocytes and eggs. The results showed that MAP kinase activation itself was not necessary for germinal vesicle breakdown (GVBD), but the ability of the ooplasm to phosphorylate MAP kinase was a prerequisite for this event. At concentrations of 1.6 nM, PMA effectively inhibited GVBD and MAP kinase activation, suggesting that PMA inhibits GVBD by inhibiting molecule(s) upstream to MAP kinase. At concentrations of 16.2 nM, PMA induced metaphase-interphase transition more effectively in eggs collected 19 hr after human chorionic gonadotropin (hCG) administration than in those collected 15 hr after hCG administration. The degree of MAP kinase activity decrease was well correlated with the time course and proportion of pronuclear formation. On the other hand, when the effect of PMA on cell cycle progression was abolished by protein phosphatase inhibitor, okadaic acid, MAP kinase was superactivated. The biologically inactive 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) had no evident effects on either GVBD and interphase transition or on MAP kinase activity. Furthermore, the effects of PMA on oocyte GVBD, egg activation, and MAP kinase activity could be overcome by the specific PKC inhibitor, calphostin C, suggesting the possible involvement of this enzyme in the regulation of MAP kinase activity. The results suggest that activation of PKC by PMA entrains a cascade of events that ultimately inhibits MAP kinase activation and GVBD in mouse oocytes and induces MAP kinase inactivation and metaphase-interphase transition in mouse eggs.

Inhibition of S6 Kinase by Rapamycin Blocks Maturation ofRana dybowskiiOocytes

Studies were carried out to define the hormone-induced signal transduction pathway during maturation ofRana dybowskiioocytes. Rapamycin, a specific inhibitor of S6 kinase, blocked progesterone-induced oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner indicating that S6 kinase is required for meiotic maturation ofRanaoocytes. Addition of rapamycin within 3 h, but not 6 h, of progesterone treatment inhibited GVBD. In contrast, cycloheximide, a general protein synthesis inhibitor, blocked GVBD even when added 9 h after progesterone addition. A twofold increase in S6 kinase activity occurred within 1 h of progesterone stimulation and rapamycin inhibited this activity. Rapamycin also suppressed, in a dose-dependent manner, progesterone-induced protein synthesis during the first 12 h of culture but less effectively later. Histone H1 kinase activity (maturation-promoting factor, MPF) was observed in oocyte extracts at two different times (between 6 and 9 h and at 24 h) following progesterone stimulation. Rapamycin blocked H1 kinase activity between 6 and 9 h of culture but not that observed at 24 h. In contrast, cycloheximide suppressed progesterone-induced H1 kinase activity as well as protein synthesis throughout the course of incubation. Such results indicate that rapamycin and cycloheximide have common and unique effects on oocyte maturation and suggest that progesterone-induced S6 kinase activity is closely associated with induction of protein synthesis and activation of MPF during oocyte maturation. Results inRanacontrast with those obtained inXenopuswhere rapamycin inhibited S6 kinase but failed to inhibit GVBD or protein synthesis. Differences in the response ofRanaandXenopusoocytes to rapamycin are discussed in relation to seasonal, biochemical, and species variations.

Identification of Residues in the Cysteine-rich Domain of Raf-1 That Control Ras Binding and Raf-1 Activity

We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.

Involvement of meiotic resumption in the disruption of gap junctions between cumulus cells attached to pig oocytes.

The present study was undertaken to examine the mechanisms by which cumulus cells regulate meiotic resumption in pig oocytes using microinjections of lucifer yellow into cumulus-oocyte complexes combined with a fluorescent assay. Some cumulus-oocyte complexes cultured for 0, 8, 16, 24, 32, 40 and 48 h were denuded to assess the nuclear status of oocytes; the remaining complexes were injected with lucifer yellow and monitored for the transfer from the oocyte to the surrounding cumulus cells using confocal laser scanning microscopy. The proportion of oocytes undergoing germinal vesicle breakdown at 16, 24 and 32 h of cultivation was much higher than that of cumulus-oocyte complexes in which all gap junctions within cumulus cells and between cumulus cells and oocyte were disrupted (16 h: 22.7% versus 8.8%, 24 h: 66.7% versus 40.3%, 32 h: 84.0% versus 69.4%), showing that the disruption does not trigger meiotic resumption in the pig oocyte. A significant positive correlation (r = 0.99, P < 0.01) was established between the proportion of germinal vesicle breakdown oocytes and that of cumulus-oocyte complexes exhibiting loss of gap junctions within all cumulus cell layers excluding the innermost layer. From these results, it is concluded that meiotic resumption in pig oocytes is induced by the disruption of gap junctions within cumulus cells, rather than that between the oocyte and cumulus cells, which blocks the conduction of meiosis inhibitory signals from the outer cumulus cells to the oocyte.

Pharmacological characterization of serotonin receptor in the oocyte membrane of bivalve molluscs and its formation during oogenesis

Serotonin (5-hydroxytryptamine, 5-HT) is a major neurotransmitter that triggers the spawning of bivalve molluscs and oocyte germinal vesicle breakdown (GVBD) of oocytes. The present study employed [3H] 5-HT binding assay to characterize a putative receptor on oocyte membranes prepared from scallops and oysters. Results demonstrated a profile of hormone control of 5-HT receptor formation during oocyte growth. The dissociation constants for 5-HT on the surfaces of scallop and oyster oocytes were estimated to be 2.64 μM and 2.01 μM, respectively, and the maximal binding capacity of the oocyte surface was 152 pmol/mg protein in scallops and 529 pmol/mg protein in oysters. The inhibition of [3H] 5-HT bound by various agonists and antagonists provided pharmacological evidence for a 5-HT receptor in each oocyte, a mixed profile of 5-HT1/5-HT2 in scallops and a single profile of 5-HT1 in oyster. Formation of 5-HT receptor on the surface of the scallop oocyte was induced by estradiol-17β(E2), which was suggested as a primary promoter of the effect of E2 on sensitivity to 5-HT, resulting in the induction of spawning. This formation occurred during oocyte growth controlled by E2. J. Exp. Zool. 281:124–131, 1998. © 1998 Wiley-Liss, Inc.

Phospholipase C β1, the PLC isoform present at the nuclear level, regulates the IP3-dependent Ca 2+ oscillations responsible for mouse oocyte germinal vesicle breakdown

Phospholipase C β1, the PLC isoform present at the nuclear level, regulates the IP3-dependent Ca 2+ oscillations responsible for mouse oocyte germinal vesicle breakdown

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 (17 alpha,20 beta-dihydroxy-4-pregnen-3-one; 17,20 beta P)-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 > alpha-methyl-5HT > 2-methyl-5HT. 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 17,20 beta P-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-HT1A, 5-HT2, and 5-HT3 receptors, as well as to 5-HT receptors on oocytes of some bivalve molluscs.

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.

Effects of the Isoquinolinesulfonamide H-8 on Fundulus heteroclitus Ovarian Follicles: Role of Cyclic Nucleotide-Dependent Protein Kinases on Steroidogenesis and Oocyte Maturation In Vitro

The possible role of cyclic nucleotide-dependent protein kinases in mediating the stimulatory actions of Fundulus heteroclitus pituitary extract (FPE) during ovarian steroidogenesis and oocyte maturation in vitro was investigated. Follicle-enclosed oocytes were cultured in the presence of FPE and/or N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), a compound that inhibits protein kinase A (PKA) and cGMP-dependent protein kinase. H-8 alone (0.1–1 mM) promoted oocyte germinal vesicle breakdown (GVBD) in a dose-dependent manner. However, the process of GVBD initiated by H-8 was much slower than that triggered by 17 α,20 β-dihydroxy-4-pregnen-3-one (17,20 βP), the natural inducer of oocyte maturation in F. heteroclitus. Treatment with H-8 also increased 17,20 βP production by the follicles and the accumulation of this steroid in the media was much slower than that initiated by FPE. However, in contrast to the FPE action on the oocyte, which is mediated by 17,20 βP, the stimulatory action of H-8 on GVBD appears to be independent of follicular steroid production, since aminoglutethimide (AGI), an inhibitor of steroidogenesis, did not-block H-8-induced GVBD while inhibiting H-8-induced 17,20 βP production. Moreover, addition of H-8 to FPE-treated follicles significantly reduced 17,20 βP secretion and the percentage of GVBD. These results provide further support for the involvement of PKA in the mechanism by which FPE stimulates ovarian steroidogenesis in F. heteroclitus. Furthermore, the fact that H-8 alone increased 17,20 βP levels may imply that basal follicular production of this steroid could be induced by inactivation of cyclic nucleotide-dependent protein kinases. Data also indicate that inhibition of PKA and/or c-GMP-dependent protein kinase in the oocyte may be involved in the mechanism leading to resumption of meiosis in this species.

Maturation, fertilization, and development of marmoset monkey oocytes in vitro.

This study was conducted to investigate 1) the capacity of in vitro-matured (IVM) marmoset oocytes to be fertilized and to support embryonic development in vitro and 2) oocyte meiotic maturation in relation to in vivo FSH administration, follicle size, and oocyte-cumulus cell status. Pairs of ovaries were collected on Day 4 of the follicular phase from adult females receiving either 1) human FSH (3 IU; n = 5) or 2) control (saline; n = 5) daily for 4 days. Antral follicles were excised from ovaries and separated into classes according to size: class 1 (660-840 microm), class 2 (> 840-1000 microm), class 3 (> 1000-1400 microm), and class 4 (> 1400 microm). A total of 823 partially naked and cumulus-enclosed oocytes (CEOs) were released from follicles and cultured in vitro. Cumulus cells remaining after 22 h were removed, metaphase II (MII) oocytes were inseminated with epididymal sperm, and resulting embryos were cultured until developmental arrest. Fluorescence microscopy was used to assess oocyte meiotic and embryo developmental progression. Oocyte germinal vesicle breakdown (GVB)- and MII-competencies increased significantly with follicular size (p < 0.01 and p < 0.0001, respectively), although they were independent of oocyte-cumulus cell associations. After 24 and 32 h in vitro, 69% and 93%, respectively, of CEOs with MII competence had completed meiotic maturation, and the rate of nuclear maturation increased progressively with follicle size (p < 0.01) and with the association of cumulus cells (p < 0.01). In vivo FSH priming slightly improved oocyte GVB- and MII-competencies (p < 0.01 and p < 0.05, respectively) and decreased the time required to achieve MII (p < 0.01). IVM oocytes from all follicle sizes fertilized (78-92%) in vitro, with 27% developing to morula- and 4% to blastocyst-stage embryos. This study demonstrates for the first time that IVM New World primate oocytes are able to support advanced preimplantation embryonic development in vitro. Oocyte meiotic competence and the time course of nuclear maturation are profoundly influenced by their follicular origin, and marginally by FSH treatment.

A receptor for the oocyte maturation-inducing hormone 17alpha,20beta,21-trihydroxy-4-pregnen-3-one on ovarian membranes of striped bass.

Previous studies have shown that blood plasma levels of 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) and 17alpha, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S) increase in striped bass (Morone saxatilis) undergoing final oocyte maturation (FOM). Both hormones are produced by ovarian fragments undergoing hCG-induced germinal vesicle breakdown (GVBD) in vitro. In the present study, we investigated binding of DHP and 20beta-S to ovarian membranes from striped bass undergoing FOM. Saturable binding sites for DHP were not detected. Saturation of 20beta-S binding sites with 5 nM [3H]20beta-S occurred within 40 min at 0 degrees C (at 3 min, half of the maximum specific binding of steroid was calculated to have occurred), and the binding was pH-dependent. Scatchard analyses revealed the presence of a single class of high-affinity (dissociation constant [Kd] = 1.4 +/- 0.2 nM), limited-capacity (estimated concentration [Bmax] = 2.7 +/- 0.3 pmol/g ovary) 20beta-S binding sites on membranes from striped bass ovaries undergoing FOM. In contrast, only low levels of specific binding (Bmax < 0.04 pmol/g tissue) were detected on membranes from testes, liver, brain, and muscle. Ovarian membranes prepared from vitellogenic females also had low levels (Bmax < 0.1 pmol/g ovary) of specific 20beta-S binding, less than 5% of that found during FOM. Results of competition assays showed that DHP was approximately 250 times less effective than 20beta-S for displacing 20beta-S from ovarian membranes. In contrast, 20beta, 21-dihydroxy-4-pregnen-3-one was a very effective competitor, although it is only a weak inducer of oocyte GVBD in vitro. Of several other steroids tested, only progesterone showed affinity for the 20beta-S binding site within a physiological range of concentrations. Taken together with previous studies of striped bass FOM, these findings indicate that 20beta-S is the oocyte maturation-inducing steroid hormone in striped bass.

Meiotic abnormalities of c-mos knockout mouse oocytes: activation after first meiosis or entrance into third meiotic metaphase.

In Xenopus oocytes, Mos activates the mitogen-activated protein kinase (MAPK) signal transduction cascade and regulates meiosis. In mammalian oocytes, however, the functions of Mos are still unclear. In the present study, we used c-mos knockout mouse oocytes and examined the roles of Mos in mouse oocyte maturation and fertilization, including whether Mos controls MAPK and maturation promoting factor (MPF) activity. The kinetics of germinal vesicle breakdown (GVBD) and the first polar body emission were similar in wild-type, heterozygous mutant, and homozygous mutant mice. Activities of MPF were also not significantly different among the three genotypes until the first polar body emission. In contrast, MAPK activity in c-mos knockout oocytes did not significantly fluctuate throughout maturation, and the oocytes had abnormal diffused spindles and loosely condensed chromosomes, although a clear increase in MAPK activities was observed after GVBD in wild-type and heterozygous mutant oocytes that had normal spindles and chromosomes. After the first polar body emission, 38% of c-mos knockout oocytes formed a pronucleus instead of undergoing second meiosis, indicating the crucial role of Mos in MPF reactivation after first meiosis. When oocytes that reached second metaphase were fertilized or stimulated by ethanol, many c-mos knockout oocytes emitted a second polar body and progressed into third meiotic metaphase instead of interphase, although all fertilized or activated oocytes in the heterozygote progressed to interphase, indicating that Mos deletion leads to compensatory factors that might not be degraded after fertilization or parthenogenetic activation. These results suggest that Mos is located upstream of MAPK in mouse oocytes as in Xenopus oocytes but is independent of MPF activity, and that Mos/MAPK is not necessary go GVBD and first polar body emission. Our results also suggest that Mos plays a crucial role in normal spindle and chromosome morphology and the reactivation of MPF after first meiosis.

The Dominant Negative Effects of H-Ras Harboring a Gly to Ala Mutation at Position 60

v-H-Ras harboring the Gly-60 to Ala mutation (G60A) lacks the ability to induce germinal vesicle breakdown in Xenopus oocytes. Moreover, this mutant is capable of inhibiting the activity of v-H-Ras to induce oocyte germinal vesicle breakdown when co-injected. The duration and the extent of inhibition depends on the molar ratio of v-H-Ras(G60A) to v-H-Ras. The inhibition is not due to a general toxicity of v-H-Ras(G60A) to oocytes because oocytes injected with v-H-Ras(G60A) can be readily induced to mature by other mitogenic agents, such as insulin, insulin-like growth factor 1, insulin-like growth factor 2, and phosphatidylcholine-specific phospholipase C. The dominant negative effect of v-H-Ras(G60A) requires proper membrane attachment of v-H-Ras(G60A). By using a competition assay, it was concluded that the dominant negative phenotype of v-H-Ras(G60A) resulted from sequestering H-Ras downstream effector(s). Raf-1 was identified as one of the sequestered targets.

Effect of gonadotropins on the ability of 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) to inhibit germinal vesicle breakdown in bovine oocytes

The transcriptional inhibitor, 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB), prevents germinal vesicle breakdown (GVBD) in bovine oocytes only in the presence of gonadotropins. The objectives of this study were to examine the ability of gonadotropins to facilitate transcriptional inhibition of GVBD in bovine oocytes and to examine the effect of gonadotropins on transcriptional inhibition of cumulus expansion. Cumulus-oocyte complexes (COC) from 2 to 7 mm follicles were cultured in TCM-199 with 1 μg/ml estradiol, 50 μg/ml gentamicin and hormonal treatments for 20 to 24 h at 39 °C in 5% CO 2 in air. After culture, COC were assessed for degree of cumulus expansion and oocytes were then denuded, fixed and stained to determine stage of meiosis. In the presence of LH and FSH the proportion of oocytes arrested at germinal vesicle (GV) stage was significantly increased with DRB treatment (58 vs 3% GV for LH/FSH + DRB vs LH/FSH-DRB; P < 0.001). However, maximal inhibition of GVBD by treatment with DRB could also be achieved in the presence of FSH alone (60% GV for FSH + DRB). The ability of DRB to block GVBD was significantly reduced in the presence of LH alone (20 to 28% GV for LH + DRB; P < 0.05 vs FSH + DRB), and treatment with DRB did not block GVBD in the presence of hCG (6.8 to 13.3% GV for hCG + DRB; P < 0.001 vs FSH + DRB). Inhibition of cumulus expansion by treatment with DRB occurred in the presence of either FSH or LH. Based on these results, it is suggested that DRB prevents GVBD in cultured bovine COC by interfering with a transcriptional event mediated primarily by FSH.

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17β (E2), testosterone (T), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and 17α,20β,21-tri-hydroxy-4-pregnen-3-one (20β-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20β-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E2 and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20β-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E2 and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20β-S and oocyte GVBD by white perch follices. DHP and 20β-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20β-S in the control of FOM in white perch and white bass.

Human Dual Specificity Phosphatase VHR Activates Maturation Promotion Factor and Triggers Meiotic Maturation in Xenopus Oocytes

Bacterially expressed, dual specificity phosphatase VHR protein induced germinal vesicle breakdown (GVBD) when microinjected into Xenopus oocytes, albeit with slower kinetics than that observed in progesterone- or insulin-induced maturation. A mutant VHR protein missing an essential cysteine residue for its in vitro phosphatase activity completely lacked activity in injected oocytes. VHR injection done in conjunction with progesterone or insulin treatment resulted in highly synergized GVBD responses showing much faster kinetics than that produced by VHR or either hormone alone. The delayed kinetics of VHR-induced GVBD and the synergistic responses obtained in the presence of hormones suggested that this protein may be promoting G2/M transition by weakly mimicking the action of cdc25, the dual specificity phosphatase that physiologically activates the maturation promotion factor. Various experimental observations are consistent with such a role for the injected VHR in oocytes: 1) as opposed to hormone-treated oocytes, histone H1 kinase activation is not preceded by MAPK activation in the process of GVBD in VHR-injected oocytes; 2) incubation of purified VHR with highly concentrated cell-free extracts of untreated oocytes resulted in activation of histone H1 kinase activity in the lysates; 3) coinjection of VHR with activated Ras proteins resulted in synergized responses, faster than those produced by either protein alone; 4) coinjection of VHR with the purified amino-terminal SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase (which blocks insulin-induced GVBD) does not affect VHR-induced maturation. The biological actions of VHR in oocytes clearly distinguish it from other dual specificity phosphatases, which have shown inhibitory effects when tested in oocytes. We speculate that VHR may represent a dual specificity phosphatase responsible for activation of cdk-cyclin complex(es) at a still undetermined stage of the cell cycle.

Requirement for protein synthesis during the onset of meiosis in bovine oocytes and its involvement in the autocatalytic amplification of maturation-promoting factor.

The present study was carried out using the method of electrofusion, or treatment with okadaic acid (OA), to determine whether protein synthesis at the onset of culture was required for the meiotic resumption of bovine follicular oocytes. Germinal vesicle breakdown (GVBD) occurred in bovine oocytes at 6 hr after separation from their follicles in vitro. Following this, immature germinal vesicle (GV) oocytes, preincubated for 0, 2, 4, and 6 hr, were fused to mature oocytes. When immature oocytes, preincubated for 0 hr, were fused to mature oocytes and then cultured for 3 hr in basic medium, GVBD was observed in all fused cells, whereas in the case of cultivation in medium supplemented with the protein synthesis inhibitor (25 micrograms/ml cycloheximide; CX), 39% of the fused cells possessed an intact GV within their cytoplasm. In immature oocytes preincubated for 4 or 6 hr, however, this proportion was significantly reduced to 7% and 4%, respectively, without protein synthesis after fusion. In addition, the CX-dependent block of GVBD could be overcome in only 13% of bovine follicular oocytes by the addition of 2 microM OA, although 51% of oocytes which synthesized the protein during the first 6 hr of culture induced GVBD in subsequent culture with CX plus OA. Thus, we conclude that the initiation of GVBD in bovine oocytes requires protein synthesized at the onset of meiosis, which is related to the autocatalytic amplification of the maturation-promoting factor.

Ras-dependent maturation of Xenopus oocytes is blocked by modified peptides of GTPase activating protein (GAP).

Guanosine triphosphatase activating protein (GAP) is an important modulator of p21ras (Ras)-dependent signal transduction in mammalian cells and in insulin-induced maturation of Xenopus oocytes. A synthetic octapeptide from the catalytic domain of GAP, residues 899-906 (F899VFLRLIC906), inhibited GAP-stimulated hydrolysis of GTP to GDP by Ras in an in vitro biochemical assay (IC50 = 12 microM). The peptide was assayed for its ability to block insulin- (Ras-dependent) and progesterone- (Ras-independent) induced maturation of stage VI Xenopus laevis oocytes, marked by germinal vesicle breakdown (GVBD). Microinjection of 50 pmol of the peptide inhibited insulin- but not progesterone-induced GVBD by 50%. A 7-residue peptide lacking F899, GAP(900-906)-NH2, failed to inhibit GAP-stimulated GTPase activity and did not block GVBD. Replacement of the cysteine residue at position 906 with methionine resulted in a peptide with prolonged inhibitory activity in the oocyte. Moreover, sequential replacement of specific L-amino acid residues with the corresponding D-amino acids produced a peptide with a two-fold increased half-life after injection into oocytes. None of the peptides tested affected progesterone induced GVBD, suggesting that the modifications did not result in loss of specificity. These studies show that (a) peptides that were able to inhibit GAP-stimulated Ras GTPase activity in vitro were also able to block Ras-dependent GVBD in oocytes, and (b) specific substitutions in these peptides can result in improved stability in oocytes.