Chromosome condensation in pig oocytes: lack of a requirement for either cdc2 kinase or MAP kinase activity

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases (cdk), is shown to inhibit germinal vesicle breakdown (GVBD) in pig oocytes. Oocytes treated with 100 microM BL I were arrested in the germinal vesicle (GV)-stage and displayed low activity of cdc2 kinase and MAP kinase. Nevertheless, chromosome condensation occurred and highly condensed bivalents were seen within an intact GV after a 24-hr culture in the presence of BL I. The inhibitory effect of BL I on MAP kinase activation during culture was likely mediated through a cdk-dependent pathway, since MAP kinase activity present in extracts derived from metaphase II eggs was not inhibited by BL I. The block of GVBD could be released by treating oocytes with okadaic acid (OA), an inhibitor of type 1 and 2A phosphatases; 82% of the oocytes treated with the combination of OA/BL I underwent GVBD, and MAP kinase became activated, while cdc2 kinase remained inhibited. These results suggest that both chromosome condensation and GVBD could occur without activation of cdc2 kinase, whereas an increase in MAP kinase activity may be a requisite for GVBD in pig oocytes in conditions when cdc2 kinase activation is blocked by BL I.     

Protein kinase C activity, protein phosphorylation and germinal vesicle breakdown in Spisula oocytes

To test the possible role of protein kinase C (C-kinase) in regulating germinal vesicle breakdown (GVBD) in Spisula oocytes, we studied the effects of phorbol esters and antagonists of C-kinase on GVBD and protein phosphorylation. Responses to these agents were compared to those elicited by fertilization or increased extracellular K+. The tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent agonist of C-kinase, elicited GVBD with half-maximal stimulation at 20 nM. By contrast, 4 alpha-phorbol-12,13-didecanoate, a phorbol ester which does not stimulate C-kinase, did not trigger GVBD. TPA accelerated GVBD when induced by excess K+, but it did not affect the time course of the process when initiated by fertilization. Three structurally different antagonists of C-kinase (W-7, H-7, and retinol) all blocked GVBD when induced by fertilization or TPA. When oocytes were preincubated with [32P]orthophosphate and then stimulated to undergo GVBD by fertilization, TPA, or 45 mM K+, protein phosphorylation was greatly increased, especially for a polypeptide(s) of about 45 kDa. Phosphorylation increased prior to GVBD. Retinol inhibited phosphorylation in activated eggs. C-kinase activity was demonstrated in oocyte extracts. These results strongly suggest that protein phosphorylation by C-kinase is involved in the pathway that regulates GVBD in Spisula oocytes.     

Control of chromosome behavior in amphibian oocytes. I. The activity of maturing oocytes inducing chromosome condensation in transplanted brain nuclei

The capability of oocyte cytoplasm to induce chromosome condensation was studied by transplantation of isolated brain nuclei into Rana pipiens oocytes induced to undergo maturation in vitro by progesterone treatment. It was found that the chromosome condensation activity (CCA) first appeared in the cytoplasm of maturing oocytes shortly after germinal vesicle breakdown (GVBD), persisted in fully mature oocytes, but rapidly disappeared when the oocytes were artificially activated. A comparison of the time course of the oocyte chromosome condensation cycle and of brain chromosome condensation in maturing and activated oocytes revealed a close temporal correlation between the two, suggesting that both are under the control of the same cytoplasmic factor(s). Oocytes enucleated before GVBD always failed to develop CCA. The CCA could be restored in enucleated oocytes by injecting nucleoplasm obtained from oocytes that had not yet undergone GVBD although this same nucleoplasm was incapable of producing CCA when mixed with the cytoplasm of oocytes that had not reached the stage of GVBD. It was therefore suggested that the CCA had a dual origin involving both cytoplasmic maturation and GV materials.     

Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocytes

The activity of maturation promoting factor (MPF) which causes chromosome condensation and subsequent oocyte maturation was investigated in mouse oocytes using polyethylene-glycol-mediated cell fusion technique. Fully grown oocytes were bisected at germinal vesicle (GV) stage or shortly after germinal vesicle breakdown (GVBD) into anucleate and nucleate fragments. After 2-3 or 15-17 hr of culture these fragments were fused with interphase blastomeres from two-cell embryos. It was found that almost all the anucleate oocyte fragments cultured for a short term (2-3 hr), regardless of whether they were produced at GV stage or after GVBD, induced premature chromosome condensation in the blastomere nuclei, whereas only about 20% of those cultured for a long term (15-17 hr) could do so. On the other hand, the nucleate fragments always retain the cytoplasmic activity to induce chromosome condensation. Thus we suggested that the MPF initially could appear in mouse oocytes independently of the GV, that the mixing of GV material with the oocyte cytoplasm following GVBD had no effect on the activity of MPF in anucleate fragments, and that oocyte chromosomes or some components associated with them could play a significant role in maintaining the MPF activity.     

Pleiotropic effect of okadaic acid on maturing mouse oocytes.

Okadaic acid (OA), a potent inhibitor of types 1 and 2A protein phosphatases, was shown recently to induce chromatin condensation and germinal vesicle breakdown (GVBD) in mouse oocytes arrested at the dictyate stage by dibutyryl cAMP (dbcAMP), isobutyl methylxanthine (IBMX) and 12,13-phorbol dibutyrate (PDBu). We confirm these results using IBMX and another phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) and show that OA also bypasses the inhibitory effect of 6-dimethylaminopurine (6-DMAP). It has been concluded that protein phosphatases 1 and/or 2A (PP1, 2A), involved in the negative control of MPF activation, are thus operating downstream from both the protein kinase A and protein kinase C catalysed phosphorylation steps that prevent the breakdown of GV. Similar enzymatic activities are also able to counteract the general inhibition of protein phosphorylation. However, PP1 and/or PP2A are positively involved in the activation of pericentriolar material (PCM) into microtubule organizing centres (MTOCs). This explains the inhibitory effect of OA on spindle assembly. Finally, OA interferes with the integrity and/or function of actomyosin filaments. This results in a dramatic ruffling of the plasma membrane leading to the internalization of large vacuoles, the inhibition of chromosome centrifugal displacement and, consequently, the prevention of polar body extrusion.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

Two phases of calcium requirement during starfish meiotic maturation

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.     

Effect of cycloheximide upon maturation of bovine oocytes

Germinal vesicle breakdown (GVBD) of bovine oocytes was completely blocked by cycloheximide added to culture medium at concentrations of 1-20 micrograms/ml. Nevertheless, under such conditions a certain degree of chromatin condensation inside the germinal vesicle was observed. The inhibitory effect was not influenced by the presence or absence of cumulus cells and was fully reversible; but the process of GVBD was then significantly accelerated. The critical period in which the proteins necessary for GVBD are synthesized lasts approximately the first 5 h of culture. When germinal vesicle-arrested oocytes are fused to maturing bovine oocytes containing condensed chromosomes, GVBD of immature oocytes occurs within 3 h, even in the presence of cycloheximide. In the mouse, GVBD cannot be inhibited by protein synthesis inhibitors. When immature mouse oocytes are fused with immature bovine oocytes and the giant cells are then cultured in cycloheximide-supplemented medium, both GVs are observed, or only mouse GVBD occurs in common cytoplasm after 8 h of culture. We conclude that protein synthesis is necessary for GVBD of bovine oocytes. Our results also suggest that maturation-promoting factor (MPF) is not autocatalytically amplified in mammalian oocytes.     

Involvement of cumulus cells stimulated by FSH in chromatin condensation and the activation of maturation-promoting factor in bovine oocytes

The effects of FSH-stimulated cumulus cells on the regulatory mechanisms of chromatin condensation and maturation-promoting factor (MPF) activation around the time of germinal vesicle breakdown (GVBD) in bovine oocytes were examined. Chromatin condensation occurred in oocytes arrested at the germinal vesicle (GV) stage by protein synthesis inhibitor, cycloheximide, but this condensation was blocked by FSH-stimulated cumulus cells. However, treatment with cyclic AMP (cAMP)-dependent protein kinase inhibitor, H-8, dramatically increased the proportion of oocytes possessing GVs with condensed bivalents. Under the condition of inhibited protein synthesis, the phosphorylation form of p34cdc2 kinase was not changed due to chromatin condensation, although the activity of histone H1 kinase was significantly increased compared with that of oocytes possessing GVs with filamentous bivalents. The cycloheximide-dependent GVBD block was overcome by okadaic acid (OA) in 48 and 13% of the oocytes in the absence and presence of FSH, respectively. An initial 6-h culture period critical for protein synthesis was necessary for OA to counteract the inhibitory effect exerted by cycloheximide on the induction of GVBD and activation of histone H1 kinase in the absence of FSH, whereas this first culture period was prolonged for 2 h in the presence of FSH. Furthermore, even in FSH-stimulated oocytes, H-8 facilitated an OA-counteracted overcome of the cycloheximide-dependent GVBD block after 2 h of initial culture for protein synthesis. From these results, it is concluded that cAMP-dependent protein kinase activity regulated by cumulus cells following FSH-stimulation requests plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes.     

Butyrolactone I reversibly inhibits meiotic maturation of bovine oocytes,Without influencing chromosome condensation activity

In this study, butyrolactone I (BL I), a potent and specific inhibitor of cyclin-dependent kinases, was shown to block germinal vesicle (GV) breakdown (GVBD) in bovine oocytes in a concentration-dependent manner; GVBD was almost totally inhibited over the course of 24-48 h of culture when 100 microM BL I was included in tissue culture medium 199 containing either polyvinyl alcohol or BSA. Correlated with this inhibition was the failure of either p34(cdc2) kinase or mitogen-activated protein (MAP) kinase to become activated, and it was unlikely that BL I directly inhibited MAP kinase, since 100 microM BL I did not inhibit MAP kinase activity present in extracts obtained from metaphase II-arrested bovine eggs that possess high levels of MAP kinase activity. Nevertheless, the formation of highly condensed bivalents was observed in 78% of the BL I-treated GV-intact oocytes. This result suggests that chromosome condensation during first meiosis in bovine oocytes does not require the activity of either p34(cdc2) kinase or MAP kinase. Treatment of BL I-arrested oocytes with okadaic acid (OA) did not result in either the activation of p34(cdc2) kinase or MAP kinase, or inducement of GVBD. The BL I-induced block of GVBD for 24 h was reversible, and a subsequent 24-h culture resulted in 90% of oocytes reaching metaphase II with emission of the first polar body. Correlated with the progression to and arrest at metaphase II was the full activation of both p34(cdc2) and MAP kinases. The reversibility after 48 h of culture in BL I was partially decreased when compared to that achieved after an initial 24-h culture. Fertilization in vitro of these eggs resulted in a high incidence of both sperm penetration and pronucleus formation (88% and 70%, respectively).     

Evidence for Involvement of Protein Kinase C in Germinal Vesicle Breakdown in Chaetopterus

We have examined the possible involvement of protein kinase C (C-kinase) in the initiation of germinal vesicle breakdown (GVBD) in Chaetopterus oocytes. Two tumor-promoting phorbol esters (phorbol-12, 13-dibezoate and 12-0-tetradecanoylphorbol-13-acetate [TPA]) and a permeant diacylglycerol (1-oleoyl-2-acetylglycerol), potent activators of C-Kinase, triggered GVBD. Two other phorbol esters (phorbol-13-monoacetate and 4α-phorbol-12, 13-didecanoate), which do not activate C-kinase, were inactive. Three C-kinase antagonists (W-7, H-7 and retinol) inhibited both naturally-and TPA-induced GVBD, whereas W–5, a much less inhibitory W–7 analog, had no effect on GVBD. Triggering of GVBD by TPA was independent of extracellular Ca²⁺. Although naturally-induced GVBD was blocked by micromolar concentrations of the calmodulin antagonist, calmidazolium (R24571), and by millimolar concentrations of the permeant cAMP analog, dibutryryl cAMP, TPA-induced GVBD was not affected by these agents. These results support the hypothesis that both C-kinase and calmodulin are involved in the sequence of events leading to GVBD in this species.     

Combined effects of protein synthesis and phosphorylation inhibitors on maturation of mouse oocytes in vitro

In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 micrograms/ml), followed by further 3 hours in cycloheximide only, lowered the rate of GVBD (93% and 92%, respectively). It means that 3 and 5 hours preincubation in cycloheximide did not impair the ability of mouse oocytes to resume meiosis in medium with the protein synthesis inhibitor. To test the combined effects of inhibition of protein phosphorylation and protein synthesis, oocytes were cultured for 3, 4, or 5 hours in 2 mM of 6-DMAP and subsequently for 3 hours in 10 micrograms/ml cycloheximide. The incubation in 6-DAMP for 4 or 5 hours diminished (63% or 35% of GVBD, respectively) the ability of mouse oocytes to resume meiosis when subsequent protein synthesis was blocked by cycloheximide. However, the highly condensed bivalents were always visible in GVs. Thus the above treatment did not prevent chromatin condensation although GVBD was blocked.     

PKB/AKT is involved in resumption of meiosis in mouse oocytes

BACKGROUND INFORMATION: In fully grown mouse oocytes, a decrease in cAMP concentration precedes and is linked to CDK1 (cyclin-dependent kinase 1) activation. The molecular mechanism for this coupling, however, is not defined. PKB (protein kinase B, also called AKT) is implicated in CDK1 activation in lower species. During resumption of meiosis in starfish oocytes, MYT1, a negative regulator of CDK1, is phosphorylated by PKB in an inhibitory manner. It can imply that PKB is also involved in CDK1 activation in mammalian oocytes. RESULTS: We monitored activation of PKB and CDK1 during maturation of mouse oocytes. PKB phosphorylation and activation preceded GVBD (germinal vesicle breakdown) in oocytes maturing either in vitro or in vivo. Activation was transient and PKB activity was markedly reduced when virtually all of the oocytes had undergone GVBD. PKB activation was independent of CDK1 activity, because although butyrolactone I prevented CDK1 activation and GVBD, PKB was nevertheless transiently phosphorylated and activated. LY-294002, an inhibitor of phosphoinositide 3-kinase-PKB signalling, suppressed activation of PKB and CDK1 as well as resumption of meiosis. OA (okadaic acid)-sensitive phosphatases are involved in PKB-activity regulation, because OA induced PKB hyperphosphorylation. During resumption of meiosis, PKB phosphorylated on Ser(473) is associated with nuclear membrane and centrosome, whereas PKB phosphorylated on Thr(308) is localized on centrosome only. CONCLUSIONS: The results of the present paper indicate that PKB is involved in CDK1 activation and resumption of meiosis in mouse oocytes. The presence of phosphorylated PKB on centrosome at the time of GVBD suggests its important role for an initial CDK1 activation.     

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C.

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.     

Cytoplasmic control of nuclear maturation in mouse oocytes

Oocytes with germinal vesicles were cut into anucleate and nucleate fragments. At the time of germinal vesicle breakdown (GVBD) in nucleate fragments (after 2–3 h of culture) sister anucleate fragments were fused with the help of inactivated Sendai virus with interphase blastomeres from 2-cell embryos. The hybrid cells were examined after h and 20 h. The anucleate fragments induced chromosome condensation in the nuclei of interphase blastomeres immediately after fusion. On this basis it may be concluded that GVBD and nuclear maturation in mouse oocytes is induced by a cytoplasmic factor which is produced or unmasked independently of the nucleus.     

MAP and cdc2 Kinase Activities at Germinal Vesicle Breakdown inChaetopterus

In frog oocytes, activation of mitogen-activated protein kinase (MAPK, ERK) leads to activation of cdc2 and germinal vesicle breakdown (GVBD). By contrast, in starfish, MAPK is activated after GVBD. Here we have examined the relative involvements of MAPK and cdc2 in GVBD ofChaetopterusoocytes. MAPK was rapidly tyrosine-phosphorylated and activated (within 1–2 min) in response to exposure of the oocytes either to natural seawater (the normal trigger of GVBD in this organism) or to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which can also elicit GVBD. This response preceded the tyrosine dephosphorylation and activation of cdc2 by several minutes. MAPK phosphorylation and activation were transient, lasting only until GVBD occurred and the spindle migrated to the cortex. The enzyme was not phosphorylated again as a result of egg activation. These results are consistent with the hypothesis that the activation of MAPK has a role in GVBD. However, PD 98059, a potent and selective inhibitor of MEK, the protein kinase that phosphorylates and activates MAPK, blocked the phosphorylation of MAPK but did not block GVBD, the dephosphorylation and activation of cdc2, or spindle formation and migration. Oocytes that underwent GVBD in PD 98059 could be fertilized and cleaved normally. Ionophore A23187, although it caused germinal vesicles to disappear and caused transient phosphorylation of MAPK, did not cause dephosphorylation of cdc2, and therefore this disappearance is artifactual. These results suggest that MAPK activation is neither obligatory nor sufficient for either GVBD or meiotic metaphase arrest inChaetopterusand that activation of MAPK and cdc2 occur on independent, parallel pathways.     

Proper chromatin condensation and maintenance of histone H3 phosphorylation during mouse oocyte meiosis requires protein phosphatase activity

We have shown okadaic acid (OA) and calyculin-A (CLA) inhibition of mouse oocyte phosphoprotein phosphatase 1 (PPP1C) and/or phosphoprotein phosphatase 2A (PPP2CA) results in aberrant chromatin condensation, as evidenced by the inability to resolve bivalents. Phosphorylation of histone H3 at specific residues is thought to regulate chromatin condensation. Therefore, we examined changes in histone H3 phosphorylation during oocyte meiosis and the potential regulation by protein PPPs. Western blot and immunocytochemical analysis revealed histone H3 phosphorylation changed during mouse oocyte meiosis, with changes in chromatin condensation. Germinal vesicle-intact (GV-intact; 0 h) oocytes had no phospho-Ser10 but did have phospho-Ser28 histone H3. Oocytes that had undergone germinal vesicle breakdown (GVBD; 2 h) and progressed to metaphase I (MI; 7 h) and MII (16 h) had phosphorylated Ser10 and Ser28 histone H3 associated with condensed chromatin. To determine whether OA-induced aberrations in chromatin condensation were due to alterations in levels of histone H3 phosphorylation, we assessed phosphorylation of Ser10 and Ser28 residues following PPP inhibition. Oocytes treated with OA (1 microM) displayed increased phosphorylation of histone H3 at both Ser10 and Ser28 compared with controls. To begin to elucidate which OA-sensitive PPP is responsible for regulating chromatin condensation and histone H3 phosphorylation, we examined spatial and temporal localization of OA-sensitive PPPs, PPP1C, and PPP2CA. PPPC2A did not localize to condensed chromatin, whereas PPP1beta (PPP1CB) associated with condensing chromatin in GVBD, MI, and MII oocytes. Additionally, Western blot and immunocytochemistry confirmed presence of the PPP1C regulatory inhibitor subunit 2 (PPP1R2) in oocytes at condensed chromatin during meiosis and indicated a change in PPP1R2 phosphorylation. Inhibition of oocyte glycogen synthase kinase 3 (GSK3) appeared to regulate phosphorylation of PPP1R2. Furthermore, inhibition of GSK3 resulted in aberrant oocyte bivalent formation similar to that observed following PPP inhibition. These data suggest that PPP1CB is the OA/CLA-sensitive PPP that regulates oocyte chromatin condensation through regulation of histone H3 phosphorylation. Furthermore, GSK3 inhibition results in aberrant chromatin condensation and appears to regulate phosphorylation of PPP1R2.     

Protein phosphatases are involved in the in vivo activation of histone H1 kinase in mouse oocyte

Histone H1 kinase and protein phosphorylation have been studied in mouse oocyte. Histone H1 kinase activity increases when the oocyte enters M-phase at the time of GVBD and is paralleled with a burst of protein phosphorylation. This activity dramatically drops after parthenogenetic activation induced by puromycin. Okadic acid (OA), a potent inhibitor of protein phosphatases, induces GVBD when oocytes are arrested in the first meiotic prophase by dbc-AMP; the continuous presence of the phosphatase inhibitor, however, inhibits the polymerization of metaphase microtubules. Following activation of metaphase II-arrested mouse eggs by puromycin, OA can induce the breakdown of the nuclear envelope and the activation of histone H1 kinase. This indicates that in the absence of protein synthesis, and therefore of cyclin synthesis, inhibition of protein phosphatases may be sufficient to induce the entry into M-phase during the first cell cycle of the mouse parthenogenetic activated oocyte.     

Inhibition of nuclear maturation in fully grown porcine and mouse oocytes after their fusion with growing porcine oocytes

Porcine ovarian oocytes, isolated from follicles of 5 mm in diameter (large oocytes), were fused either together or with oocytes isolated from follicles of 0.5 mm in diameter (small oocytes). In giant cells composed of two large oocytes (control) germinal vesicle breakdown (GVBD) occurred and two metaphase I chromosome sets (M I) were observed 24 to 30 h after fusion. By contrast, in giant cells composed of one large and one small porcine oocyte, both germinal vesicles (GVs) remained well conserved after 24-30 h of culture. An identical situation was observed after fusion and cultivation of small porcine and large mouse oocytes isolated from preovulatory follicles. The results demonstrate the presence of inhibiting activity in the ooplasm of small porcine oocytes that prevents nuclear maturation of large porcine and mouse oocytes fused to them. This maturation inhibiting activity can be overcome by preincubating large porcine oocytes for more than 14 h before fusion with small oocytes. During preincubation the ooplasm produces sufficient amount of maturation promoting factor (MPF) to overcome the inhibiting activity present in small porcine oocytes thus inducing GVBD and chromatin condensation both in small and large oocytes.     

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.