A role for the MEK-MAPK pathway in okadaic acid-induced meiotic resumption of incompetent growing mouse oocytes

Fully grown competent mouse oocytes spontaneously resume meiosis in vitro when released from their follicular environment, in contrast to growing incompetent oocytes, which remain blocked in prophase I. The cell cycle regulators, maturation promoting factor (MPF; [p34(cdc2)/cyclin B kinase]) and mitogen-activated protein (MAP) kinases (p42(MAPK) and p44(MAPK)), are implicated in meiotic competence acquisition. Incompetent oocytes contain levels of p42(MAPK), p44(MAPK), and cyclin B proteins that are comparable to those in competent oocytes, but their level of p34(cdc2) is markedly lower. Okadaic acid (OA), an inhibitor of phosphatases 1 and 2A, induces meiotic resumption of incompetent oocytes. The kinetics and the percentage of germinal vesicle breakdown depends on whether or not oocytes have been cultured before OA treatment. We show that the fast kinetics and the high percentage of germinal vesicle breakdown induced by OA following 2 days in culture is neither the result of an accumulation of p34(cdc2) protein, nor to the activation of MPF in incompetent oocytes, but rather by the premature activation of MAP kinases. Indeed, a specific inhibitor of MAPK kinase (MEK) activity, PD98059, inhibits activation of MAP kinases and meiotic resumption. Altogether, these results indicate that the MEK-MAPK pathway is implicated in OA-induced meiotic resumption of incompetent mouse oocytes, and that the MEK-MAPK pathway can induce meiotic resumption in the absence of MPF activation.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Characterization of ribosomal S6 protein kinase p90rsk during meiotic maturation and fertilization in pig oocytes: mitogen-activated protein kinase-associated activation and localization

Mitogen-activated protein kinase (MAPK) becomes activated during the meiotic maturation of pig oocytes, but its physiological substrate is unknown. The 90-kDa ribosome S6 protein kinase (p90rsk) is the best known MAPK substrate in Xenopus and mouse oocytes. The present study was designed to investigate the expression, phosphorylation, subcellular localization, and possible roles of p90rsk in porcine oocytes during meiotic maturation, fertilization, and parthenogenetic activation. This kinase was partially phosphorylated in oocytes at germinal vesicle (GV) stage through a MAPK-independent mechanism, but its full phosphorylation is dependent on MAPK activity. After fertilization or electrical activation, p90rsk was dephosphorylated shortly before pronucleus formation, which coincided with the inactivation of MAPK. A protein phosphatase inhibitor, okadaic acid, accelerated the phosphorylation of p90rsk during meiotic maturation and induced its rephosphorylation in activated eggs. MAPK kinase (MAPKK or MEK) inhibitor U0126 inhibited the activation of MAPK and p90rsk in both cumulus-enclosed and denuded pig oocytes, but prevented GV breakdown (GVBD) only in cumulus-enclosed oocytes. Active MAPK and p90rsk were detected in pig cumulus cells, and U0126 induced their dephosphorylation. In meiosis II arrested eggs, U0126 led to the inactivation of MAPK and p90rsk, as well as the interphase transition of the eggs. P90rsk was distributed evenly in GV oocytes, but it accumulated in the nucleus before GVBD. It was localized to the meiotic spindle after GVBD and concentrated in the spindle mid zone during emission of the polar bodies. All these results suggest that p90rsk is downstream of MAPK and plays functional roles in the regulation of nuclear status and microtubule organization. Although MAPK and p90rsk activity are not essential for the spontaneous meiotic resumption in denuded oocytes, activation of this cascade in cumulus cells is indispensable for the gonadotropin-induced meiotic resumption of pig oocytes.     

MEK inhibitors block AICAR-induced maturation in mouse oocytes by a MAPK-independent mechanism

The present study was carried out to assess the possible role of mitogen-activated protein kinase (MAPK) in the meiosis-inducing action of the AMP-activated protein kinase (AMPK) activator, 5-aminoimidazole-4-carboxamide 1-beta-ribofuranoside (AICAR). Cumulus cell-enclosed oocytes (CEO) or denuded oocytes (DO) from immature, eCG-primed mice were cultured 4 hr in Eagle's minimum essential medium containing dbcAMP plus increasing concentrations of AICAR or okadaic acid (OA). OA is a phosphatase inhibitor known to stimulate both meiotic maturation and MAPK activation and served as a positive control. Both OA and AICAR were potent inducers of meiotic resumption in mouse oocytes and brought about the phosphorylation (and thus, activation) of MAPK, but by different kinetics: MAPK phosphorylation preceded GVB in OA-treated oocytes, while that resulting from AICAR treatment appeared only after GVB. The MEK inhibitors, PD98059 and U0126, blocked the meiotic resumption induced by AICAR but not that induced by OA. Although the MEK inhibitors suppressed MAPK phosphorylation in both OA- and AICAR-treated oocytes, meiotic resumption was not causally linked to MAPK phosphorylation in either group. Furthermore, AICAR-induced meiotic resumption in Mos-null oocytes (which are unable to stimulate MAPK) was also abrogated by PD98059 treatment. A non-specific effect of the MEK inhibitors on AICAR accessibility to the oocyte was discounted by showing that they failed to suppress either nucleoside uptake or AICAR-stimulated phosphorylation of acetyl CoA carboxylase (ACC), a substrate of AMPK. The suppression of AICAR-induced maturation by MEK inhibitors must, therefore, be occurring by actions unrelated to MEK stimulation of MAPK; consequently, it would be prudent to consider this possible non-specific action of the inhibitors when they are used to block MAPK activation in mouse oocytes.     

Activation of p34(cdc2) kinase around the meiotic resumption in bovine oocytes cultured in vitro

The p34(cdc2) kinase has been identified as a protein factor that is a regulator of meiotic maturation in mammalian oocytes. To investigate the regulatory function of the meiotic resumption in bovine oocytes cultured in vitro, the changes in the phosphorylation states of p34(cdc2) kinase and the histone H1 kinase activity were examined around germinal vesicle breakdown (GVBD). All bovine oocytes just after isolation from their follicles were arrested at the germinal vesicle (GV) stage, and these extracts exhibited two (upper and lower) bands of p34(cdc2) kinase on SDS-PAGE followed by immunoblotting with an antibody against C-terminal peptide of p34(cdc2). When these oocytes were cultured for 24 h in a medium supplemented with 100 microg/ml genistein, tyrosine phosphorylation inhibitor, GVBD was induced in 85% of oocytes, indicating that the upper band of p34(cdc2) kinase in bovine oocytes at the GV stage was already fully phosphorylated tyrosine residue prior to culture. Another (middle) band of p34(cdc2) kinase between the upper and lower bands appeared in the extracts of the oocytes cultured for 4 h, and significant activation of the histone H1 kinase was found in these oocytes (67 +/- 18 fmol/h/oocyte) as compared to that in oocytes cultured for 0 h (46 +/- 11 fmol/h/oocyte). The staining intensity of the middle band and the activity of the histone H1 kinase were further increased after the initiation of GVBD at 6 h of culture, but the quantitative changes of upper and lower bands were not detected throughout the 12 h of culture. Thus, it is concluded that the dephosphorylation of p34(cdc2) kinase followed by activation of the histone H1 kinase after the onset of culture plays a key role in the resumption of meiosis in bovine oocytes.     

Phosphorylation of mitogen-activated protein kinase is regulated by protein kinase C, cyclic 3',5'-adenosine monophosphate, and protein phosphatase modulators during meiosis resumption in rat oocytes

Mitogen-activated protein (MAP) kinase, protein kinase C (PKC), cAMP, and okadaic acid (OA)-sensitive protein phosphatases (PPs) have been suggested to be involved in oocyte meiotic resumption. However, whether these protein kinases and phosphatases act by independent pathways or interact with each other in regulating meiosis resumption is unknown. In the present study, we aimed to determine the regulation of meiosis resumption and MAP kinase phosphorylation by PKC, cAMP, and OA-sensitive PPs in rat oocytes using an in vitro oocyte maturation system and Western blot analysis. We found that ERK1 and ERK2 isoforms of MAP kinases existed in a dephosphorylated (inactive) form in germinal vesicle breakdown (GVBD)-incompetent and GVBD-competent germinal vesicle intact (GVI) oocytes as well as GVBD oocytes at equivalent levels. These results indicate that MAP kinases are not responsible for the initiation of normal meiotic resumption in rat oocytes. However, when GVBD-incompetent and GVBD-competent oocytes were incubated in vitro for 5 h, MAP kinases were phosphorylated (activated) in GVBD-competent oocytes, but not in meiotic-incompetent oocytes, suggesting that oocytes acquire the ability to phosphorylate MAP kinase during acquisition of meiotic competence. We also found that both meiosis resumption and MAP kinase phosphorylation were inhibited by PKC activation or cAMP elevation. Moreover, these inhibitory effects were overcome by OA, which inhibited PP1/PP2A activities. These results suggest that both cAMP elevation and PKC activation inhibit meiosis resumption and MAP kinase phosphorylation at a step prior to OA-sensitive protein phosphatases. In addition, inhibitory effects of cAMP elevation on meiotic resumption and MAP kinase phosphorylation were not reversed by calphostin C-induced PKC inactivation, indicating that cAMP inhibits both meiotic resumption and MAP kinase activation in a PKC-independent manner.     

Zinc Regulates Meiotic Resumption in Porcine Oocytes via a Protein Kinase C-Related Pathway

Zinc is an extremely important trace element that plays important roles in several biological processes. However, the function of zinc in meiotic division of porcine oocytes is unknown. In this study, we investigated the role of zinc during meiotic resumption in in vitro matured porcine oocytes. During meiotic division, a massive release of zinc was observed. The level of free zinc in the cytoplasm significantly increased during maturation. Depletion of zinc using N, N, N′, N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a Zn²⁺ chelator, blocked meiotic resumption in a dose dependent manner. The level of phosphorylated mitogen activated protein kinase (MAPK) and p34^cdc2 kinase activity were reduced when zinc was depleted. Moreover, zinc depletion reduced the levels of phosphorylated protein kinase C (PKC) substrates in a dose dependent manner. Real-time PCR analysis showed that expression of the MAPK- and maturation promoting factor related genes C-mos, CyclinB1, and Cdc2 was downregulated following zinc depletion. Treatment with the PKC agonist phorbol 12-myristate 13-acetate (PMA) increased phosphorylation of PKC substrates and MAPK and increased p34^cdc2 kinase activity. This rescued the meiotic arrest, even in the presence of TPEN. Activation of PKC by PMA increased the level of zinc in the cytoplasm. These data demonstrate that zinc is required for meiotic resumption in porcine oocytes, and this appears to be regulated via a PKC related pathway.     

Effect of dexamethasone on the expression of p34(cdc2) and cyclin B1 in pig oocytes in vitro

The meiotic division in oocytes is arrested in the G2 phase of the cell cycle. Resumption of meiosis, also known as oocyte maturation, entails a G2 to M transition. At the G2-M boundary, maturation promoting factor (MPF) activation is usually induced via several ways, including tyrosine dephosphorylation of p34(cdc2) and synthesis of cyclin B according to cell type and species. Previous studies in our laboratory demonstrated that glucocorticoids directly inhibit the meiotic maturation of pig oocytes in vitro. The aim of this study was therefore to investigate the influence of glucocorticoids on the expression of p34(cdc2) and cyclin B1 in resumption of meiosis of pig oocytes. We detected the relative levels and association of p34(cdc2) and cyclin B1. Isolated cumulus-enclosed oocytes were cultured in Waymouth MB752/1 medium supplemented with sodium pyruvate (50 microgram/ml), LH (0.5 microgram/ml), FSH (0.5 microgram/ml), and estradiol-17beta (1 microgram/ml) in the presence or absence of dexamethasone (DEX) for 24 hr; they then were cultured without hormonal supplements in the presence or absence of DEX for an additional 24 hr. We found that cyclin B1, as well as p34(cdc2), was already present in fully grown G2-arrested pig oocytes when removed from the follicle. In these oocytes, cyclin B1 and p34(cdc2) were already associated in complex. Treatment with DEX at concentrations of 1 microgram/ml or above decreased the level of cyclin B1, but had no effect on the level of p34(cdc2). The exposure of oocytes to DEX also decreased the amount of complexed p34(cdc2)-cyclin B1. These findings suggest that the inhibitory action of DEX on meiotic maturation could be due, at least in part, to the reduced amount of p34(cdc2)-cyclin B1 complex.     

Several signaling pathways are involved in the control of cattle oocyte maturation

The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment.     

Okadaic acid-sensitive phosphatase is related to MII/G1 transition in mouse oocytes

It is reported that okadaic acid (OA)-sensitive phosphatase is related to mitogen-activated protein kinase (MAPK)/p90rsk activation in mammalian oocytes. OA is also involved in the positive feedback loop between M phase-promoting factor (MPF) and cdc25c in Xenopus oocytes during meiotic maturation. However, the effect of phosphatase inhibition by OA on MPF and MAPK activities at the MII/G1 in oocytes remains unknown. The aim of this study is to clarify the relationship between OA-sensitive phosphatase and mitosis MII/G1 transition in mouse oocytes. MII-arrested oocytes were, isolated from mice, inseminated and cultured in TYH medium (control group) or TYH medium supplemented with 2.5 μM of OA (OA group). Histone H1 kinase and myelin basic protein (MBP) kinase activities were measured as indicators of MPF and p42 MAPK activities after insemination. Phosphorylation of cdc25c after insemination was analized in OA and control group by western blotting. Seven hours after insemination a pronucleus (PN) was formed in 84.1% (69/85) of oocytes in the control group. However, no PN was formed in oocytes of the OA group (p < 0.001). Although MPF and MAPK activities in the control group significantly decreased at 3, 4, 5, and 7 h after insemination, these decreases were significantly inhibited by OA addition (p < 0.05). Furthermore, OA addition prevented cdc25c dephosphorylation 7 h after insemination. In conclusion, OA-sensitive phosphatase correlates with inactivation of MPF and MAPK, and with the dephosphorylation of cdc25c at the MII/G1 transition in mouse oocytes.     

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.     

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.     

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.     

Antibodies to phosphotyrosine injected in Xenopus laevis oocytes modulate maturation induced by insulin/IGF-I.

Xenopus oocytes carry IGF-I receptors, and undergo meiotic maturation in response to binding of IGF-I or insulin to the IGF-I receptor. Maturation is initiated upon activation of the IGF-I receptor tyrosine kinase and requires tyrosine dephosphorylation of p34cdc2, the kinase component of maturation promoting factor (MPF). To further evaluate the role of tyrosine phosphorylation in the signalling pathway triggered by insulin/IGF-I, we have injected antibodies to phosphotyrosine into oocytes and examined their effects on oocyte maturation. Antibodies at a low concentration (40 ng/oocyte, corresponding to a concentration of 40 micrograms/ml), enhanced specifically insulin-, but not progesterone-induced maturation. In contrast, at 150 ng/oocyte, the same antibodies decreased maturation induced by insulin, progesterone, or microinjected MPF. In cell-free systems, antibodies to phosphotyrosine recognized the oocyte IGF-I receptor and modulated its ligand-induced tyrosine kinase activity in a biphasic manner, with a stimulation at 40 micrograms/ml and an inhibition at higher concentrations. Moreover, antibodies at 150 ng/oocyte neutralized the kinase activity of a crude MPF extract. This neutralization was not accompanied by a rephosphorylation of p34cdc2, but by a decrease in tyrosine phosphorylation of a 60-kDa protein, which was present in M phase extracts and undetectable in G2-arrested oocytes. Taken together, these results point to at least two levels of anti-phosphotyrosine antibody action: (i) the IGF-I receptor signalling system, and (ii) a regulatory step of MPF activation, which might be distinct of the well-documented inactivating phosphorylation of p34cdc2.     

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.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.