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.     

Involvement of histone H3 (Ser10) phosphorylation in chromosome condensation without Cdc2 kinase and mitogen-activated protein kinase activation in pig oocytes

When oocytes resume meiosis, chromosomes start to condense and Cdc2 kinase becomes activated. However, recent findings show that the chromosome condensation does not always correlate with the Cdc2 kinase activity in pig oocytes. The objectives of this study were to examine 1) the correlation between chromosome condensation and histone H3 phosphorylation at serine 10 (Ser10) during the meiotic maturation of pig oocytes and 2) the effects of protein phosphatase 1/2A (PP1/ PP2A) inhibitors on the chromosome condensation and the involvement of Cdc2 kinase, MAP kinase, and histone H3 kinase in this process. The phosphorylation of histone H3 (Ser10) was first detected in the clump of condensed chromosomes at the diakinesis stage and was maintained until metaphase II. The kinase assay showed that histone H3 kinase activity was low in oocytes at the germinal vesicle stage (GV) and increased at the diakinesis stage and that high activity was maintained until metaphase II. Treatment of GV-oocytes with okadaic acid (OA) or calyculin-A (CL-A), the PP1/PP2A inhibitors, induced rapid chromosome condensation with histone H3 (Ser10) phosphorylation after 2 h. Both histone H3 kinase and MAP kinase were activated in the treated oocytes, although Cdc2 kinase was not activated. In the oocytes treated with CL-A and the MEK inhibitor U0126, neither Cdc2 kinase nor MAP kinase were activated and no oocytes underwent germinal vesicle breakdown (GVBD), although histone H3 kinase was still activated and the chromosomes condensed with histone H3 (Ser10) phosphorylation. These results suggest that the phosphorylation of histone H3 (Ser10) occurs in condensed chromosomes during maturation in pig oocytes. Furthermore, the chromosome condensation is correlated with histone H3 kinase activity but not with Cdc2 kinase and MAP kinase activities.     

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

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

Raf-1 protein kinase is important for progesterone-induced Xenopus oocyte maturation and acts downstream of mos.

In somatic cells, the Raf-1 serine/threonine protein kinase is activated by several polypeptide growth factors. We investigated the role of Raf-1 in progesterone-induced meiotic maturation of Xenopus laevis oocytes. Raf-1 enzymatic activity and phosphorylation (reflected by a mobility shift on sodium dodecyl sulfate gels) were increased in oocytes following progesterone stimulation. The increase in Raf-1 activity was concurrent with an elevation in the activity of mitogen-activated protein (MAP) kinase. When RNA encoding an oncogenic form of Raf-1 (v-Raf) was injected into immature oocytes, MAP kinase mobility shift, germinal vesicle breakdown, and histone H1 phosphorylation increased markedly. When RNA encoding a dominant-negative version of Raf-1 was injected, progesterone-induced oocyte maturation was blocked. When RNA encoding Xenopus mos (mosxe) was injected into oocytes, Raf-1 and MAP kinase mobility shifts were observed after several hours. Also, when antisense mosxe oligonucleotides were injected into oocytes, progesterone-induced Raf-1 and MAP kinase mobility shifts were blocked. Finally, when antisense mosxe oligonucleotides were coinjected with v-Raf RNA into oocytes, histone H1 kinase activation, germinal vesicle breakdown, and MAP kinase mobility shift occurred. These findings suggest that Raf-1 activity is required for progesterone-induced oocyte maturation and that Raf-1 is downstream of mosxe activity.     

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.     

Oocyte nucleus controls progression through meiotic maturation

We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei.     

An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation

The activity of a Ca2+- and cyclic nucleotide-independent protein kinase(s) which catalyzes hyperphosphorylation of a set of endogenous proteins, including a 95-kDa soluble phosphoprotein, is found to fluctuate in both the meiotic and mitotic cell cycles of Xenopus oocytes and activated eggs. The activity is high in M-phase and hardly detectable in interphase. The activity copurifies with a major histone kinase(s) throughout four purification steps: ammonium sulfate precipitation, DEAE-cellulose chromatography, high-performance liquid chromatography on TSK G3000, and CM-Sepharose chromatography. This suggests that a single enzyme shares activity against endogenous proteins and added histones. Changes in the activity of the M-phase-specific protein kinase(s) as assayed in vitro correlate with changes in the extent of protein phosphorylation in oocytes pulse-labeled with 32P-phosphate by microinjection during meiotic maturation and the early embryonic cell cycle. This suggests that the kinase(s) has a broad specificity and plays a key role in the increased protein phosphorylation which occurs at the transition to M-phase. Microinjection of the maturation-promoting factor (MPF) into immature oocytes triggers, after a 10-min lag period, the activation of the M-phase specific kinase(s), even in the absence of protein synthesis. In contrast MPF microinjection does not induce kinase activation in cycloheximide-treated oocytes arrested after completion of the first meiotic cell cycle or in activated eggs arrested in S-phase by incubation in cycloheximide. This suggests that immature oocytes contain an inactive kinase precursor (prokinase) which is synthesized at each of the following cell cycles. In the absence of MPF addition, the prokinase to kinase transition occurs "spontaneously" after a 2-hr lag period in high-speed supernatants prepared from prophase-arrested oocytes if low-molecular-weight metabolites are eliminated by gel filtration. Addition of ATP, but not of AMP-PNP (adenylyl-imidodiphosphate), prevents spontaneous kinase activation in gel-filtered extracts. We propose that MPF activates the M-phase-specific protein kinase in the intact cell by inactivating a factor which requires phosphorylation conditions to inhibit the prokinase to kinase transition.     

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.     

Activation of ribosomal S6 kinase (RSK) during porcine oocyte maturation

The normal kinetics of ribosomal S6 kinase (RSK) during the meiotic maturation of porcine oocytes were examined. The phosphorylation states of RSK and extracellular signal-regulated kinase (ERK), major mitogen-activated protein (MAP) kinases in maturating porcine oocytes, were detected by Western blotting analysis. The S6 protein kinase activity was assayed using a specific substrate peptide which contained the major phosphorylation sites of S6 kinase. Full phosphorylation of RSK was correlated with ERK phosphorylation and was observed before germinal vesicle breakdown. S6 kinase activity was low in both freshly isolated and 20 h cultured oocytes. S6 kinase activity was significantly elevated in matured oocytes to a level about 6 times higher than that in freshly isolated oocytes. Furthermore, full phosphorylation of RSK was inhibited when oocytes were treated with U0126, a specific MAP kinase kinase inhibitor, in dose-dependent manner, indicating that RSK is one of the substrates of MAP kinase. These results suggest that the activation of RSK is involved in the regulation of meiotic maturation of porcine oocytes.     

Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes.

Porcine oocytes cultured in follicular fluid for various periods of up to 48 h were stained with Hoechst-33342 and classified according to maturation before assaying. Histone H1 kinase activity at metaphase I was approximately 10 times that at the germinal vesicle stage. An abrupt reduction in activity was observed in oocytes emitting the first polar body; then the activity increased again to the same level as at metaphase I. This pattern is similar to those reported in non-mammalian species and supports the concepts that histone H1 kinase is ubiquitous in eukaryotes and controls the meiotic cell cycle in mammals.     

Inhibition of phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase leads to suppression of p34(cdc2) kinase activity and meiotic progression beyond the meiosis I stage in porcine oocytes surrounded with cumulus cells

In this study, the effects of U0126 that inhibits the activity of mitogen-activated protein (MAP) kinase kinase (MEK), and LY294002, which is a phosphatidylinositol (PI) 3-kinase inhibitor, on meiotic progression beyond the metaphase I (MI) stage in porcine oocytes were examined. Cumulus-oocyte complexes (COCs) were cultured for 22 h with 50 microM LY294002 or 10 microM U0126 following cultivation for the initial 22 h. MAP kinase activity in oocytes cultured with LY294002 or U0126 was significantly lower than that in control oocytes cultured for up to 44 h. U0126 and LY294002 significantly decreased p34(cdc2) kinase activity and the proportion of oocytes reaching the MII stage compared to those in control oocytes. Oocytes denuded after COCs had been cultured for 22 h were cultured further for 22 h with U0126 or LY294002. In the denuded oocytes, U0126 suppressed MAP kinase activity, p34(cdc2) kinase activity, and meiotic progression to the MII stage; however, LY294002 did not significantly affect the activity of these kinases and meiotic progression. These results suggest that increasing MAP kinase activity in oocytes via the PI 3-kinase signaling pathway in cumulus cells is involved in the stimulation of maturation promoting factor, leading to meiotic progression beyond the MI to MII stage in porcine oocytes.     

Activation of p34cdc2 protein kinase activity in meiotic and mitotic cell cycles in mouse oocytes and embryos.

p34cdc2 protein kinase is a universal regulator of M-phase in eukaryotic cell cycle. To investigate the regulation of meiotic and mitotic cell cycle in mammals, we examined the changes in phosphorylation states of p34cdc2 and its histone H1 kinase activity in mouse oocytes and embryos. We showed that p34cdc2 has three different migrating bands (referred to as upper, middle and lower bands) on SDS-PAGE followed by immunoblotting with anti-PSTAIR antibody, and that the upper and middle bands are phosphorylated forms since these two bands shifted to the lower one by alkaline phosphatase treatment. In meiotic cell cycle, only germinal vesicle (GV) stage oocytes had the three forms. The phosphorylated forms decreased gradually in oocytes up to 2 h after isolation from follicles, and thereafter the phosphorylation states did not change significantly until metaphase II. However, the histone H1 kinase activity oscillated, being activated at the first and second metaphase in meiosis and inactivated at the time of the first polar body extrusion. These results suggest that changes in phosphorylation states of p34cdc2 triggered its activation at the first metaphase, but not inactivation and reactivation at the first and second metaphase, respectively. In mitotic cell cycle, phosphorylated forms appeared at 4 h after insemination, increased greatly just before metaphase, and were dephosphorylated in metaphase. Histone H1 kinase activity was high only at metaphase. This kinase activation is probably triggered by dephosphorylation of p34cdc2.     

Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC

Here we show that during the meiotic maturation of Xenopus oocytes, histone H3 becomes phosphorylated on serine-10 at about the time of maturation promoting factor activation and meiosis I entry. However, overexpression of cAMP-dependent protein kinase that blocks entry into M phase, also leads to massive serine-10 phosphorylation of histone H3 in intact Xenopus oocytes but does not cause chromosome condensation. We also show that the phosphorylation of histone H3 during oocyte maturation requires the activation of the mitogen-activated protein kinase/p90Rsk pathway. Our results indicate that in G2-arrested oocytes, which are about to enter M phase, histone H3 phosphorylation is not sufficient for chromosome condensation.     

Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation

MPF extracted from starfish oocytes copurifies with an M phase-specific H1 histone kinase encoded by a homolog of the fission yeast cell cycle control gene cdc2+. The most purified preparations contain p34cdc2 as the only major protein. Activation of the p34cdc2 kinase is correlated with appearance of the MPF activity both in vivo and in vitro. The increase in protein kinase activity is associated with p34cdc2 dephosphorylation and the decrease in protein kinase activity on leaving M phase with rephosphorylation. Microinjection of a peptide perfectly conserved in p34cdc2 from yeast to humans induces meiotic maturation, suggesting that an inhibitory component in G2 arrested oocytes interacts with this region of the p34cdc2 kinase. We propose that initiation of M phase is brought about by the dephosphorylation of p34cdc2, leading to increase in its protein kinase activity.     

The cell cycle can occur in starfish oocytes and embryos without the production of transferable MPF (maturation-promoting factor)

All cells undergoing the transition from interphase to metaphase have been postulated to contain a "maturation-promoting factor" (MPF) capable of causing meiotic maturation when injected into immature oocytes. We have shown in an accompanying paper (A. Picard, M. C. Harricane, J. C. Labbe, and M. Doreé, 1988, Dev. Biol. 128, 121-128) that the basic oscillator driving the cell cycle still operates in maturing starfish oocytes and fertilized eggs in the absence of germinal vesicle (GV) material. Under such conditions of enucleation, we now show, however, that MPF activity cannot be detected after hormonal stimulation of prophase-arrested oocytes in Astropecten or after the normal time of second meiotic cleavage in Marthasterias. In contrast, cell cycles occur with the production of transferable MPF activity in embryos from which both pronuclei have been removed after fertilization. Reinjection of the entire contents of a GV after the normal time of second meiotic cleavage restores the ability of cytoplasm to induce meiotic maturation in immature recipient oocytes after transfer. Transduction of the hormonal stimulus at the level of the plasma membrane, stimulation of the phosphorylation of cytoplasmic proteins, and activation of a cycling Ca2+- and cyclic nucleotide-independent histone kinase still occur in the absence of GV material. Since previous studies have demonstrated that the presence of GV material in the recipient oocytes is absolutely required in starfish for the amplification of microinjected MPF (Kishimoto et al., 1981; Picard and Doree, 1984), we propose that some unidentified component of the GV is required, at least after the normal time of second meiotic cleavage in donor oocytes and at any time in recipient oocytes, for the successful transfer of MPF activity in starfish.     

Female infertility in PDE3A-/- mice: Polo-like kinase 1 (Plk1) may be a target of protein kinase A (PKA) and involved in meiotic arrest of oocytes from PDE3A-/- mice

Mechanisms of cAMP/PKA-induced meiotic arrest in oocytes are not completely identified. In cultured, G₂/M-arrested PDE3A^-/- murine oocytes, elevated PKA activity was associated with inactivation of Cdc2 and Plk1, and inhibition of phosphorylation of histone H3 (S10) and of dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15). In cultured WT oocytes, PKA activity was transiently reduced and then increased to that observed in PDE3A^-/- oocytes; Cdc2 and Plk1 were activated, phosphorylation of histone H3 (S10) and dephosphorylation of Cdc25B (S323) and Cdc2 (Thr14/Tyr15) were observed. In WT oocytes, PKAc were rapidly translocated into nucleus, and then to the spindle apparatus, but in PDE3A^-/- oocytes, PKAc remained in the cytosol. Plk1 was reactivated by incubation of PDE3A^-/- oocytes with PKA inhibitor, Rp-cAMPS. PDE3A was co-localized with Plk1 in WT oocytes, and co-immunoprecipitated with Plk1 in WT ovary and Hela cells. PKAc phosphorylated rPlk1 and Hela cell Plk1 and inhibited Plk1 activity in vitro. Our results suggest that PKA-induced inhibition of Plk1 may be critical in oocyte meiotic arrest and female infertility in PDE3A^-/- mice.Key words: mice oocytes, PDE3A, cAMP, PKA, polo-like kinase 1, centrosome, prophase arrested     

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.     

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.     

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.     

Involvement in Meiotic Prophase of H1 Histone Kinase and p34cdc2 Homologues in Lily (Lilium longiflorum) Microsporocytes

We have taken advantage of the synchrony of meiotic prophase I in Lilium microsporocytes to investigate the presence and involvement in four stages of meiotic prophase I (leptotene, zygotene, pachytene, and diplotene) of the p34^cdc2 H1 histone kinase, a component of MPF and a key participant in division control in other eukaryotes. H1 kinase activity showed a peak pattern during meiotic prophase I with the highest kinase activity at pachytene. A monoclonal antibody directed against a highly conserved region of p34^cdc2 (termed the 'PSTAIR') recognized three major protein forms by immunoblotting. The highest level of the fastest-migrating form was observed at pachytene, coinciding with the highest activity of H1 kinase. Both the proteins recognized by the anti-PSTAIR antibody and H1 histone kinase activity were retained on beads conjugated with p13^suc1, a protein known to physically associate with p34^cdc2. These observations suggest that p34^cdc2 or protein(s) highly homologous to p34^cdc2 is a component of Lilium H1 histone kinase and plays a role in regulating meiotic prophase I.