Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis-specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP-E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP-E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP-E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP-E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP-E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP-E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP-E survived through the transition even in cycloheximide-treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP-E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side-by-side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI.     

Biphasic activation of Aurora-A kinase during the meiosis I- meiosis II transition in Xenopus oocytes

Xenopus Aurora-A (also known as Eg2) is a member of the Aurora family of mitotic serine/threonine kinases. In Xenopus oocytes, Aurora-A phosphorylates and activates a cytoplasmic mRNA polyadenylation factor (CPEB) and therefore plays a pivotal role in MOS translation. However, hyperphosphorylation and activation of Aurora-A appear to be dependent on maturation-promoting factor (MPF) activation. To resolve this apparent paradox, we generated a constitutively activated Aurora-A by engineering a myristylation signal at its N terminus. Injection of Myr-Aurora-A mRNA induced germinal vesicle breakdown (GVBD) with the concomitant activation of MOS, mitogen-activated protein kinase, and MPF. Myr-Aurora-A-injected oocytes, however, appeared to arrest in meiosis I with high MPF activity and highly condensed, metaphase-like chromosomes but no organized microtubule spindles. No degradation of CPEB or cyclin B2 was observed following GVBD in Myr-Aurora-A-injected oocytes. In the presence of progesterone, the endogenous Aurora-A became hyperphosphorylated and activated at the time of MPF activation. Following GVBD, Aurora-A was gradually dephosphorylated and inactivated before it was hyperphosphorylated and activated again. This biphasic pattern of Aurora-A activation mirrored that of MPF activation and hence may explain meiosis I arrest by the constitutively activated Myr-Aurora-A.     

Evidence that protein kinase C (PKC) participates in the meiosis I to meiosis II transition in mouse oocytes.

Oocytes from LTXBO mice exhibit a delayed entry into anaphase I and frequently enter interphase after the first meiotic division. This unique oocyte model was used to test the hypothesis that protein kinase C (PKC) may regulate the meiosis I-to-meiosis II transition. PKC activity was detected in LTXBO oocytes at prophase I and increased with meiotic maturation, with the highest (P < 0.05) activity observed at late metaphase I (MI). Treatment of late MI-stage oocytes with the PKC inhibitor, bisindolylmaleimide I (BIM), transiently reduced (P < 0.05) M-phase-promoting factor (MPF) activity and promoted (P < 0.05) progression to metaphase II (MII), while mitogen-activated protein kinase (MAPK) activity remained elevated during the MI-to-MII transition. Confocal microscopy analysis of LTXBO oocytes during this transition showed PKC-delta associated with the meiotic spindle and then with the chromosomes at MII. Inhibition of PKC activity also prevented untimely entry into interphase, but only when PKC activity was reduced in oocytes before the progression to MII and thus indicates that the transition into interphase is directly associated with the delayed triggering of anaphase I. Moreover, the defect(s) that initiate activation occur upstream of MAPK, as suppression of PKC activity failed to prevent activation by Mos(tm1Ev)/ Mos(tm1Ev) LTXBO oocytes expressing no detectable MAPK activity. In summary, PKC participates in the regulatory mechanisms that delay entry into anaphase I in LTXBO oocytes, and the disruption promotes untimely entry into interphase. Thus, loss of regulatory control over PKC activity during oocyte maturation disrupts the critical MI-to-MII transition, leading to a precocious exit from meiosis.     

Xkid chromokinesin is required for the meiosis I to meiosis II transition in Xenopus laevis oocytes

Xkid chromokinesin is required for chromosome alignment on the metaphase plate of spindles formed in Xenopus laevis egg extracts. We have investigated the role of Xkid in Xenopus oocyte meiotic maturation, a progesterone-triggered process that reinitiates the meiotic cell cycle in oocytes arrested at the G2/M border of meiosis I. Here we show that Xkid starts to accumulate at the time of germinal vesicle breakdown and reaches its largest quantities at metaphase II in oocytes treated with progesterone. Both germinal vesicle breakdown and spindle assembly at meiosis I can occur normally in the absence of Xkid. But Xkid-depleted oocytes cannot reactivate Cdc2/cyclin B after meiosis I and, instead of proceeding to meiosis II, they enter an interphase-like state and undergo DNA replication. Expression of a Xkid mutant that lacks the DNA-binding domain allows Xkid-depleted oocytes to complete meiotic maturation. Our results show that Xkid has a role in the meiotic cell cycle that is independent from its role in metaphase chromosome alignment.     

Erp1/Emi2 is essential for the meiosis I to meiosis II transition in Xenopus oocytes

Erp1 (also called Emi2), an inhibitor of the APC/C ubiquitin ligase, is a key component of cytostatic factor (CSF) responsible for Meta-II arrest in vertebrate eggs. Reportedly, however, Erp1 is expressed even during meiosis I in Xenopus oocytes. If so, it is a puzzle why normally maturing oocytes cannot arrest at Meta-I. Here, we show that actually Erp1 synthesis begins only around the end of meiosis I in Xenopus oocytes, and that specific inhibition of Erp1 synthesis by morpholino oligos prevents entry into meiosis II. Furthermore, we demonstrate that premature, ectopic expression of Erp1 at physiological Meta-II levels can arrest maturing oocytes at Meta-I. Thus, our results show the essential role for Erp1 in the meiosis I/meiosis II transition in Xenopus oocytes and can explain why normally maturing oocytes cannot arrest at Meta-I.     

Involvement of Aurora A kinase during meiosis I-II transition in Xenopus oocytes

The Aurora kinase family has been involved both in vivo and in vitro in the stability of the metaphase plate and chromosome segregation. However, to date only one member of this family, the protein kinase Aurora B, has been implicated in the regulation of meiotic division in Caenorhabditis elegans. In this species, disruption of Aurora B results in the failure of polar body extrusion. To investigate whether Aurora A is also required in meiosis, we microinjected highly specific alpha-Aurora A antibodies in Xenopus oocytes. We demonstrated that microinjected oocytes fail to extrude the first polar body and are arrested with condensed chromosomes on a typical metaphase I plate, which has not performed its normal 90 degrees rotation. We additionally found that, although the failure of first polar body extrusion observed in alpha-Aurora A-microinjected oocytes is likely mediated by Eg5, the impairment of the metaphase plate rotation does not involve this kinesin-like protein. Surprisingly, although chromosomes remain condensed at a metaphase I stage in alpha-Aurora A-microinjected oocytes, the cytoplasmic cell cycle events progress normally through meiosis until metaphase II arrest. Moreover, these oocytes are able to undergo parthenogenetic activation. We conclude that Aurora A and Eg5 are involved in meiosis I to meiosis II transition in Xenopus oocytes.     

Fertilization initiates the transition from anaphase I to metaphase II during female meiosis in C. elegans

Oocytes from most animals arrest twice during the meiotic cell cycle. The universally conserved prophase I arrest is released by a maturation hormone that allows progression to a second arrest point, typically metaphase I or II. This second arrest allows for short-term storage of fertilization-competent eggs and is released by signaling that occurs during fertilization. Nematodes are unique in that the maturation hormone is secreted by sperm rather than by the mother's somatic tissues. We have investigated the nature of the second arrest in matured but unfertilized Caenorhabditis elegans embryos using time-lapse imaging of GFP-tubulin or GFP-histone. Unfertilized embryos completed anaphase I but did not form polar bodies or assemble meiosis II spindles. Nevertheless, unfertilized embryos assembled female pronuclei at the same time as fertilized embryos. Analysis of embryos fertilized by sperm lacking the SPE-11 protein indicated that fertilization promotes meiotic cytokinesis through the SPE-11 protein but assembly of the meiosis II spindle is initiated through an SPE-11-independent pathway.     

Aurora kinase A controls meiosis I progression in mouse oocytes

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6- treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Over-expression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.     

Zinc requirement during meiosis I-meiosis II transition in mouse oocytes is independent of the MOS-MAPK pathway

Zinc is essential for many biological processes, including proper functioning of gametes. We recently reported that zinc levels rise by over 50% during oocyte maturation and that attenuation of zinc availability during this period could be achieved using the membrane-permeable heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). This zinc insufficiency resulted in formation of large polar bodies, failure to establish metaphase II arrest, and impaired establishment of cortical polarity. As these phenotypes resemble those of MOS null oocytes, we examined the impact of zinc insufficiency on the MOS-MAPK pathway. Reduced levels of both MOS protein and phosphorylation of MAP2K1/2 are observed in zinc-insufficient oocytes; however, these differences appear only after completion of the first meiotic division. In addition, activation of the downstream effector of the MOS pathway, MAPK3/1, is not affected by zinc insufficiency, and reduced MOS levels are observed only with the presence of TPEN after the first polar body extrusion. These data are inconsistent with the hypothesis that reduced MOS mediates the observed phenotype. Finally, MOS overexpression does not rescue the phenotype of zinc-insufficient oocytes, confirming that the observed disruption of asymmetric division and spindle abnormalities cannot be attributed to impaired MOS signaling. Zinc-insufficient oocytes do not increase maturation promoting factor (MPF) activity following the first meiotic division, and increasing MPF activity through expression of nondegradable cyclin B1 partially rescues the ability of zinc-insufficient oocytes to enter metaphase II. Although we have shown that zinc has a novel role in the meiotic cell cycle, it is not mediated through the MOS-MAPK pathway.     

Newly assembled cyclin B-cdc2 kinase is required to suppress DNA replication between meiosis I and meiosis II in starfish oocytes.

Micro-injection of catalytically inactive GST-cdc2-K33R or GST-cdk2-K33R fusion proteins, each of which efficiently titrates cyclin B in oocytes and prevents assembly of cyclin B-cdc2 complexes, readily induces premature DNA replication in starfish oocytes after emission of the first polar body. Moreover, partial ablation of cyclin B mRNA by micro-injection of antisense oligonucleotides facilitates premature DNA replication induced by the dominant-negative cdc2 and cdk2 mutant proteins. We thus propose that enhanced translation of cyclin B after GVBD, a universal feature of oocyte maturation in the animal kingdom, and subsequent assembly of cyclin B-cdc2 complexes, are part of the checkpoint that prevents DNA replication in the oocyte after emission of the first polar body. MAPK inactivation is neither required for premature DNA replication after the first meiotic cell cycle nor for DNA replication after completion of meiotic maturation. However, micro-injection of a N-terminally truncated form of the budding yeast STE11 protein, that constitutively maintains MAPK active after the second meiotic cleavage, prevents fertilized eggs from proceeding into embryogenesis, and arrests them at G2, as is the case in unfertilized eggs that cannot inactivate MAPK after the second meiotic cleavage. We thus propose that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development.     

Degradation of pig cyclin B1 molecules precedes MAP kinase dephosphorylation during fertilisation of the oocytes

Pig oocytes at metaphase II were activated by penetration of spermatozoa in cycloheximide-free and cycloheximide-containing fertilisation media. The precise nuclear stage, and the kinetics of degradation of cyclin B1 and dephosphorylation of MAP kinase were assessed after insemination. After maturation culture, 96% of oocytes reached metaphase II. At 6 h after insemination in cycloheximide-free medium, 68% of the oocytes were activated and had progressed to anaphase II or beyond. After 8 h, 89% of the oocytes were activated: a female pronucleus had formed and the heads of penetrating spermatozoa had enlarged and changed to male pronuclei. In the cycloheximide-containing medium, activation of oocytes started earlier than in cycloheximide-free medium. After 4 h, 43% of the oocytes were activated, and the percentage increased to 97% after 6 h. Pig cyclin B1 disappeared in the oocytes at 6 h after insemination in both cycloheximide-containing and cycloheximide-free media. Pig oocytes at metaphase II contained two types of MAP kinase--ERK 1 and ERK 2--in their active phosphorylated forms. At 8 h after insemination ERK 2 changed to the fast-migrating inactive form in the oocytes cultured in both cycloheximide-containing and cycloheximide-free media, although the shift-down was not complete. The change was delayed by 2 h after the degradation of cyclin B1 molecules. These results demonstrate that degradation of pig cyclin B1 molecules corresponds to the transition of the oocytes from metaphase II arrest to anaphase II/telophase II and was followed by MAP kinase dephosphorylation.     

The meiosis I-to-meiosis II transition in mouse oocytes requires separase activity

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.     

Mitogen-activated protein kinase phosphorylation patterns in pig oocytes and cumulus cells during gonadotrophin-induced resumption of meiosis in vitro

The present study investigated the phosphorylation pattern of mitogen-activated protein kinase (MAPK) in cumulus-oocyte complexes (COCs) during spontaneous and FSH/LH-induced in vitro maturation (IVM). Both isoforms of MAPK were unphosphorylated in oocytes recovered immediately after liberation from follicles and became phosphorylated following 25 h incubation, corresponding to the time of germinal vesicle breakdown (GVBD). In contrast, MAPK was already phosphorylated in minimal amounts in cumulus cells at the time of liberation from follicles and phosphorylation of MAPK increased after 0.5 h incubation. Supplementation of medium with gonadotrophins intensified phosphorylation at 0.5 h incubation, demonstrating the early and rapid action of FSH/LH on MAPK phosphorylation. Phosphorylation of MAPK in cumulus cells peaked after 21 h of incubation, whereas MAPK was almost completely dephosphorylated at the end of incubation (45 h). During subsequent incubation in the absence of added gonadotrophins, between 5 and 10 h exposure to FSH/LH-supplemented medium was required to induce resumption of meiosis in COCs. Phosphorylation of MAPK in oocytes was prevented by the MEK inhibitor U0126, but the inhibitor reduced phosphorylation of MAPK in cumulus cells only during the first 2 h of IVM. The data support the hypothesis that two different MAPK phosphorylation events occurred following gonadotrophin stimulation, one in cumulus cells and the other in oocytes. In cumulus cells, FSH/LH induced early and rapid U0126-insensitive phosphorylation of MAPK, whereas U0126-susceptible MAPK phosphorylation took place in the oocyte itself around the time of GVBD.     

The critical role of the MAP kinase pathway in meiosis II in Xenopus oocytes is mediated by p90(Rsk)

BACKGROUND: During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, we used U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90(Rsk), a MAP kinase target. RESULTS: Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes failed to form metaphase I spindles, failed to reaccumulate cyclin B to a high level and failed to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes entered S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90(Rsk) were able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. CONCLUSIONS: The MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90(Rsk), is sufficient to mediate these effects during oocyte maturation.     

Acquisition of meiotic competence in growing pig oocytes correlates with their ability to activate Cdc2 kinase and MAP kinase

Meiotic maturation of mammalian oocytes is under the control of cell cycle molecules Cdc2 kinase and MAP kinase (mitogen-activated protein kinase). In the present study, we investigated the relationship between the ability to activate Cdc2 kinase and MAP kinase and the acquisition of meiotic competence during pig oocyte growth. Growing and fully grown pig oocytes were collected from four groups of antral follicles of various diameters (A, 0.5-0.7 mm; B, 1.0-1.5 mm; C, 2.0-2.5 mm; D, 4.0-6.0 mm) and cultured in vitro. Fully grown oocytes from class D follicles, which have full competence to mature to metaphase II, had the ability to activate both Cdc2 kinase and MAP kinase. In contrast, growing oocytes from class A follicles, which have limited competence to resume meiosis, had no such ability. Cyclin B1 molecules did accumulate, however, with phosphorylated 35 and 36 kDa bands of p34cdc2 appearing in the cultured oocytes. Of the growing oocytes from class B follicles, 60% resumed meiosis but arrested at metaphase I. Some of the oocytes in this class were capable of activating Cdc2 kinase, although they did not appear to have established a MAP kinase-activating pathway or the ability to activate MEK. These results suggest that limited meiotic competence in growing oocytes from class A follicles is due to their inability to activate Cdc2 kinase and their incomplete MEK-MAP-kinase pathway, although the oocytes are capable of accumulating cyclin B1 molecules. During the final growth phase, pig oocytes acquire the ability to activate Cdc2 kinase and then establish the MEK-MAP-kinase pathway for full meiotic competence.     

Regulation of asymmetrical cytokinesis by cAMP during meiosis I in mouse oocytes

Mammalian oocytes undergo an asymmetrical first meiotic division, extruding half of their chromosomes in a small polar body to preserve maternal resources for embryonic development. To divide asymmetrically, mammalian oocytes relocate chromosomes from the center of the cell to the cortex, but little is known about the underlying mechanisms. Here, we show that upon the elevation of intracellular cAMP level, mouse oocytes produced two daughter cells with similar sizes. This symmetrical cell division could be rescued by the inhibition of PKA, a cAMP-dependent protein kinase. Live cell imaging revealed that a symmetrically localized cleavage furrow resulted in symmetrical cell division. Detailed analyses demonstrated that symmetrically localized cleavage furrows were caused by the inappropriate central positioning of chromosome clusters at anaphase onset, indicating that chromosome cluster migration was impaired. Notably, high intracellular cAMP reduced myosin II activity, and the microinjection of phospho-myosin II antibody into the oocytes impeded chromosome migration and promoted symmetrical cell division. Our results support the hypothesis that cAMP plays a role in regulating asymmetrical cell division by modulating myosin II activity during mouse oocyte meiosis I, providing a novel insight into the regulation of female gamete formation in mammals.     

Reduced expression of MAD2, BCL2, and MAP kinase activity in pig oocytes after in vitro aging are associated with defects in sister chromatid segregation during meiosis II and embryo fragmentation after activation

This study was conducted to examine expression of centromere protein B (CENPB), spindle checkpoint protein MAD2 (mitotic arrest deficient protein), and antiapoptotic protein BCL2; activities of MAPK (mitogen-activated protein kinase) and mitochondria distribution in pig oocytes during aging, and their relationship with sister chromatid separation during meiosis II and embryo fragmentation and apoptosis after activation. After immature oocytes were cultured for 40-72 h, CENPB, MAD2, tubulin, BCL2, and MAPK in the oocytes were examined by immunoblotting. Spindles, chromosomes, kinetochores, and mitochondria were examined by immunofluorescence staining and apoptosis was examined by TUNEL assay. It was found that tubulin and CENPB was not changed during 40-72 h of culture. However, the expression of MAD2 and BCL2 and the activity of MAPK were gradually reduced during oocyte aging. The percentages of oocytes with normal spindle, chromosomes, and kinetochores were also reduced as oocyte aged from 9.5% at 40 h to 17.3%, 34.6%, and 42.9% at 48, 60, and 72 h, respectively. Aggregated mitochondria were found in the aged oocytes as compared with the uniform distribution in young oocytes. After activation, the proportions of oocytes with abnormal anaphase II were significantly increased in aged oocytes. More (P<0.001) oocytes cultured for 60-72 h fragmented and showed apoptosis after activation as compared with the oocytes cultured for 40-48 h. This study indicates that aging reduces expression in spindle checkpoint protein and antiapoptosis protein and MAPK activity in pig oocytes. These events in turn cause abnormal sister chromatid segregation during meiosis II, embryo fragmentation, and apoptosis.     

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.     

Interplay between CDC2 kinase and MAP kinase pathway during maturation of mammalian oocytes

Two principal kinases, p34cdc2 kinase and MAP kinase play a pivotal role in maturation of mammalian oocytes. In the porcine and bovine oocytes both kinases are activated around the time of germinal vesicle breakdown (GVBD). Butyrolactone I (BL I), a specific inhibitor of cdk kinases, prevents effectively and reversibly resumption of meiosis in the porcine and bovine oocytes. Neither p34cdc2 kinase nor MAP kinase are activated in oocytes inhibited in the GV stage. The bovine oocytes maintained for 48 h in the medium supplemented with BL I, progress subsequently to metaphase II in 91%, their cumuli expand optimally and after in vitro fertilization they possess two pronuclei. When the cdc2 kinase is blocked in the porcine oocytes by BL I, MAP kinase, activated by okadaic acid treatment, is able to substitute cdc2 kinase and induce GVBD. The histone H1 kinase activity sharply decreases in the metaphase II oocytes treated by BL I and one or two female pronuclei are formed. These data indicate that BL I is a useful tool either for the two step in vitro culture of mammalian oocytes or for their activation in nuclear transfer experiments.