Changes in the activity of the maturation-promoting factor during meiotic maturation and following activation of amphibian and starfish oocytes: their correlations with protein phosphorylation

Changes in the extent of protein phosphorylation and their possible correlation with changes in the activity of maturation-promoting (MPF) factor were investigated throughout meiotic maturation and following activation of amphibian and starfish oocytes. Despite several exceptions in the pattern of phosphorylation of individual proteins, high and low levels of protein phosphorylation were found to be correlated with high and low levels of MPF activity. Both the extent of protein phosphorylation and MPF activity were found to drop upon parthenogenetic activation and to cycle synchronously thereafter in the amphibian. In contrast no drop in MPF activity or in the extent of protein phosphorylation was observed following activation of starfish oocytes with ionophore A23187. This suggests that changes of protein phosphorylation and of MPF activity are rather related to the progression of the cell cycle than directly to Ca2+-dependent activation reaction. In amphibians global protein kinase activity in homogenates was found to drop with MPF activity following activation. Changes in the ratio of threonine vs serine phosphorylation were also investigated during the course of meiotic maturation and activation in both amphibian and starfish oocytes: changes in the activity of MPF were found to be better correlated with changes in threonine than serine phosphorylation.     

Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs

We have examined the regulation of maturation-promoting factor (MPF) activity in the mitotic and meiotic cell cycles of Xenopus laevis eggs and oocytes. To this end, we developed a method for the small scale extraction of eggs and oocytes and measured MPF activity in extracts by a dilution end point assay. We find that in oocytes, MPF activity appears before germinal vesicle breakdown and then disappears rapidly at the end of the first meiotic cycle. In the second meiotic cycle, MPF reappears before second metaphase, when maturation arrests. Thus, MPF cycling coincides with the abbreviated cycles of meiosis. When oocytes are induced to mature by low levels of injected MPF, cycloheximide does not prevent the appearance of MPF at high levels in the first cycle. This amplification indicates that an MPF precursor is present in the oocyte and activated by posttranslational means, triggered by the low level of injected MPF. Furthermore, MPF disappears approximately on time in such oocytes, indicating that the agent for MPF inactivation is also activated by posttranslational means. However, in the absence of protein synthesis, MPF never reappears in the second meiotic cycle. Upon fertilization or artificial activation of normal eggs, MPF disappears from the cytoplasm within 8 min. For a period thereafter, the inactivating agent remains able to destroy large amounts of MPF injected into the egg. It loses activity just as endogenous MPF appears at prophase of the first mitotic cycle. The repeated reciprocal cycling of MPF and the inactivating agent during cleavage stages is unaffected by colchicine and nocodazole and therefore does not require the effective completion of spindle formation, mitosis, or cytokinesis. However, MPF appearance is blocked by cycloheximide applied before mitosis; and MPF disappearance is blocked by cytostatic factor. In all these respects, MPF and the inactivating agent seem to be tightly linked to, and perhaps participate in, the cell cycle oscillator previously described for cleaving eggs of Xenopus laevis (Hara, K., P. Tydeman, and M. Kirschner, 1980, Proc. Natl. Acad. Sci. USA, 77:462- 466).     

Critical effect of pigWee1B on the regulation of meiotic resumption in porcine immature oocytes

Porcine immature oocytes require protein synthesis for meiotic resumption, thus the importance of Cdc2 inhibitory phosphorylation in their meiotic arrest remains controversial. We examined the involvement of Cdc2 phosphorylation in the meiotic arrest of porcine oocytes with a special focus on Wee1B, an oocyte-specific Wee1 family member recently reported in mouse oocytes. We cloned a Wee1B homologue of pig by RT-PCR followed by 5’- and 3’-RACE. Overexpression of pigWee1B in porcine immature oocytes by the injection of pigWee1B mRNA almost completely blocked the germinal vesicle breakdown (GVBD) under the low cAMP concentration, which could not block their spontaneous meiotic resumption by itself. The MPF activation and cyclin B synthesis were inhibited in these oocytes. Conversely, downregulation of pigWee1B expression by the injection of specific antisense mRNA induced GVBD in the oocytes, the spontaneous meiotic resumption of which was blocked by the high concentration of cAMP (dbcAMP). In these oocytes, the MPF activity was elevated and cyclin B was accumulated. Downregulation of pigMyt1, another Wee1 family member, could not induce the GVBD under the same condition. The inhibition of tyrosine phosphatase by vanadate blocked the GVBD even in the pigWee1B-downregulated oocytes. These results suggest that the inhibitory phosphorylation of CDC2, which is catalyzed by pigWee1B, but not pigMyt1, is involved in the meiotic arrest of porcine oocytes, and that the inactivation of Wee1B in combination with the phosphatase activation induces the conversion of pre-MPF to the active MPF and starts the cyclin B synthesis, follwed by a further increase of MPF and meiotic resumption.     

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.     

Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation

During mouse oocyte maturation the regulation of the activity of a cytoplasmic maturation-promoting factor (MPF) was examined. The mouse MPF activity was determined based on its ability to induce maturation in immature starfish oocytes after microinjection with the cytoplasm from mouse oocytes. MPF appeared initially at germinal vesicle breakdown (GVBD), and its activity fluctuated in exact correspondence with meiotic cycles, reaching a peak at each metaphase and almost disappearing at the time of emission of the first polar body. Cycloheximide affected neither the initial MPF appearance nor GVBD. Thereafter, however, in the presence of cycloheximide the meiotic spindle was not formed and MPF disappeared, although the chromosomes remained condensed. After removing cycloheximide, MPF reappeared and was followed by the first metaphase and subsequently by polar body emission. Finally the meiotic cycle progressed to the second metaphase. Thus, for the appearance of MPF, there is a critical period shortly before the first metaphase, after which protein synthesis is required. In the presence of either cytochalasin D or colcemid, MPF activity remained at elevated levels. Addition of cycloheximide to such cytochalasin-treated oocytes, in which the meiotic cycle was arrested at the first metaphase, caused the MPF levels to decrease and was followed by movement of chromosomes to both poles where they decondensed and two nucleus-like structures were formed. Thus, the disappearance of MPF may initiate the metaphase-anaphase transition. Furthermore, detailed cytological examination revealed that chromosomes in cytochalasin-treated oocytes were monovalent while those treated only with cycloheximide were divalent, suggesting that dissociation of the synapsis is a prerequisite for chromosome decondensation after the disappearance of MPF. In all these respects, MPF seems to be a metaphase-promoting factor rather than just a maturation-promoting factor.     

Changes in protein phosphorylation accompanying maturation of Xenopus laevis oocytes

Protein phosphorylation has been measured after injection of [³²P]phosphate into oocytes of Xenopus laevis undergoing progesterone-induced meiotic maturation. As oocytes mature, there is a burst of nonyolk protein phosphorylation several hours after progesterone exposure and shortly before germinal vesicle breakdown (GVBD). This burst is not due to changes in the specific activity of the phosphate or ATP pool. Enucleated oocytes exposed to progesterone also experience the burst, indicating the cytoplasmic location of phosphoprotein formation. When an oocyte receives an injection of cytoplasm containing the maturation-promoting factor (MPF), a burst of protein phosphorylation occurs immediately, and GVBD occurs shortly thereafter, even in the presence of cycloheximide. Under a variety of conditions promoting or blocking maturation, oocytes which undergo GVBD are the only ones to have experienced the phosphorylation burst. The results suggest that the protein phosphorylation burst is a necessary step in the mechanism by which MPF promotes GVBD.     

Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes

In starfish oocytes, activity of the maturation-promoting factor (MPF) and that of a major cAMP-independent protein kinase dropped at the time of meiotic cleavage, and rose again after the first but not the second meiotic cleavage. Protein synthesis was required before the first meiotic cleavage for both MPF and protein kinase activity to rise again after the first meiotic cleavage. Microinjection of either leupeptin or soybean trypsin inhibitor early enough prior to first polar body emission suppressed both the meiotic cleavage and the associated drop of MPF activity. Microinjection of leupeptin or soybean trypsin inhibitor during the 10-min period before the first meiotic cleavage also suppressed cytokinesis but did not prevent a decrease in MPF activity at the normal time of cytokinesis. The lysosomotropic inhibitor ammonia neither suppressed cytokinesis nor the drop of MPF activity at the time of first meiotic cleavage. Activity of neutral proteases sensitive to leupeptin and soybean trypsin inhibitor was demonstrated in oocyte homogenates prepared at the time of first meiotic cleavage. It is proposed that such proteases might be involved in degradation of protein kinase(s) and in the drop of MPF activity at the time of first meiotic cleavage.     

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.     

Interaction of cell cycle kinases, microtubules, and chromatin in ascidian oocytes during meiosis

We used kinase assays and confocal microscopy to study the interaction of cell cycle proteins with microtubule organising centres (MTOC) and chromatin in ascidian oocytes during meiosis. The activity of maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK) appear not to be correlated in control oocytes. MPF activity peaks during metaphase I and II of the meiotic cell cycle whereas the activity of MAPK peaks at telophase I and is subsequently degraded to remain at low levels for the remainder of meiosis. The protein synthesis inhibitor emetine induces the degradation in MPF activity in unfertilized metaphase-I (M-I) oocytes, while MAPK is unaffected. Emetine does not alter the activities of these cell cycle kinases in fertilized oocytes during meiosis I but MPF activity remains low while MAPK activity is high for an elongated time period and oocytes do not complete meiosis I. Emetine induces maternal MTOC duplication in unfertilized M-I oocytes and prevents sperm aster growth in fertilized oocytes, but it does not alter the M-I meiotic apparatus in unfertilized oocytes. These experiments suggest that neither MPF alone nor emetine-sensitive proteins are responsible for M-I arrest in ascidian oocytes, MAPK may ensure this stability. In addition, we showed that the maternal MTOC is present at M-I but suppressed from duplicating in an emetine-sensitive manner.     

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.     

Urea-Induced Meiosis Reinitiation in Oocytes of the Starfish, Marthasterias glacialis

In the absence of hormone stimulation, prophase-blocked oocytes of Marthasterias glacialis have been induced to undergo meiosis reinitiation up to female pronucleus formation by pulse incubation in isoosmotic urea-sea water solutions. Even when this procedure was not effective all along the breeding season, it could trigger full maturation when applied to so-called "incompetent oocytes" that did not complete maturation following microinjection-induced mixing of their nucleoplasm and cytoplasm.
³²P phosphate incorporation into proteins and cell fusion experiments demonstrate that this treatment produces an increased protein phosphorylation which appears tightly associated with the production of M-phase promoting factor (MPF). Instead, when oocytes are maintained in the inducing medium, dephosphorylation soon occurs and MPF is no longer present to support meiosis. Under these conditions, the GV-disrupted oocytes present a permanent nucleolus and do not form a meiotic spindle. The same cytological aspect was also obtained when the oocytes were treated in the presence of 90 μM emetine or 150 μM of the intracellular chelator Quin 2-AM.
These data suggest that urea-induced maturation may involve an intracellular Ca²⁺ shift which would be required to activate both MPF precursor molecules and the resting female centers which stand in the animal cortex outside the nucleus and give rise to the poles of the first maturation spindle. They also show that nuclear disruption alone, without protein phosphorylation, cannot trigger meiosis reinitiation of incompetent oocytes.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer

Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are key regulators of both meiotic and mitotic cycles. Oocytes arrested at metaphase of the second meiotic division (MII) contain high levels of both kinases; however, these activities decline with age. Caffeine (an inhibitor of Myt1/Wee1 activity) can increase MPF and MAPK activities in ovine oocytes; however, the effects of caffeine treatment on the activation, nuclear configuration and developmental potential of ovine SC nuclear transfer (SCNT) embryos were unknown. We examined the effects of aging and caffeine treatment on MPF and MAPK activities, activation, development, and nuclear remodeling of SCNT embryos. Both kinases reached maximum activities at 24-h postonset of maturation (hpm) and then decreased with time. The decline in MPF activity occurred rapidly, whereas MAPK activity declined more slowly. Caffeine treatment (10.0 mM) of aging oocytes prevented the decline in activities associated with both kinases and prevented the acquisition of activation competence by a single activation stimulus. However, treatment of aged oocytes with caffeine could not increase kinase activities or reverse the acquisition of activation competence. Enucleation did not affect kinase activities, but caffeine treatment significantly increased both. Caffeine treatment did not affect the decline in MPF or MAPK activities following activation or significantly affect development of parthenogenetically activated oocytes. When SCNT reconstructed embryos were treated with caffeine following fusion, no increase in the frequency of development to blastocyst was observed; however, a significant increase in the occurrence of nuclear envelope break-down (NEBD) and an increase in total cell numbers occurred.     

Partial purification and characterization of the maturation-promoting factor from eggs of Xenopus laevis

Maturation-promoting factor (MPF) was purified 20- to 30-fold from unfertilized eggs of Xenopus laevis, by ammonium sulfate precipitation and chromatography on pentyl-agarose and arginine-agarose. The final material induces maturation in 50% of the recipient oocytes when 5 ng of protein is injected in a volume of 20 ml. The maturation response includes precocious germinal vesical breakdown, elevated protein phosphorylation, amplification of cytoplasmic MPF, and formation of an activatable egg blocked at second meiotic metaphase. These eggs are capable of cleavage and, in some cases, of gastrulation. A quantitative in vivo assay of MPF is described and a unit of MPF activity is defined as that amount causing a 50% maturation frequency when oocytes are injected each with a 20-nl test volume. Maturation frequency has a very high-order dependence on MPF concentration. The purification procedure selects simultaneously for endogenous protein phosphorylation systems containing kinases, protein substrates, and phosphatases. This fact, as well as the finding that ATP enhances MPF activity at least twofold when included in the dilution medium for assay, is discussed in terms of the possible involvement of protein phosphorylation in MPF activation and inactivation.     

Inhibition of protein synthesis affects histone H1 kinase,but not chromosome condensation activity,during the first meiotic division of pig oocytes

The influence of protein synthesis on the regulation of the first meiotic division was studied in pig oocytes. We show that histone H1 kinase activity gradually increases during in vitro culture of pig oocytes, reaching maximum in metaphase I stage after 24 hr of culture. However, in the presence of the protein synthesis inhibitor cycloheximide, histone H1 kinase is not activated during the whole culture period, and after 24 hr it is approximately at the same level as in prophase-stage oocytes. The gradual increase in phosphorylation of six proteins of molecular weights 39, 48, 53, 66, 96, and 120 kDa, observed during the first 24 hr of culture, was not detected when cycloheximide was added to the culture medium. Similarly, the decrease in phosphorylation of a 90-kDa protein was not seen in cycloheximide-treated oocytes. On the other hand, the levels of both MPF components, p34^cdc2 and cyclin B, which were found to be nearly constant during the first meiotic division, were not influenced by cycloheximide treatment as revealed by Western blotting. The process of germinal vesicle breakdown (GVBD) was totally blocked by cycloheximide. The condensation of chromatin, however, was not influenced, suggesting that GVBD and chromosome condensation could be regulated independently. The different degrees of MPF activation involved in these processes, as well as the nature of the protein(s) which must be synthesized for triggering GVBD, are discussed. © 1995 Wiley-Liss, Inc.     

Does the M-Phase Promoting Factor (MPF) Activate a Major Ca2+-and Cyclic Nucleotide-Independent Protein Kinase in Starfish Ocytes?

In starfish, the activity of a major Ca²⁺-and cyclic nuleotide-independent protein kinase has been shown to fluctuate in phase with that of MPF along meiotic and mitotic cell cycle (23, 25). Microinjection of α-naphthylphosphate (α-NP), a potent phosphatase inhibitor, increased considerably (from 15 to 546 picomoles/min/mg protein) the activity of this major cycling kinase in homogenates. Although this result supported the view that kinase phosphorylation might induce its own activation, this hypothesis was eliminated because injection of cytoplasm from hormone-stimulated enucleated oocytes, which contained the fully activated kinase but no MPF, failed to trigger kinase activation in recipient oocytes. In contrast, kinase activation was induced in recipient oocytes injected with either cytoplasm taken from nucleated maturing oocytes, which contained high MPF and kinase activities, or cytoplasm taken later from hormone-stimulated and ATP-γ-S-injected oocytes which contained high MPF but low kinase activites. These results indicate that inhibiting dephosphorylation of some regulatory protein activates the M-phase-specific protein kinase. The possibility that the M-phase or maturation-promoting factor (MPF) might be this regulatory protein is discussed.     

Activity of maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) after parthenogenetic activation of ovine oocytes

The maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are the key regulators of both meiotic and mitotic cell cycles. Knowledge of the dynamics of these two kinases during the transition from meiosis to mitosis would be of great importance for cloning by nuclear transfer. In this study, experiments were designed to assay the changes of MPF and MAP kinase activity of in vitro matured ovine oocytes after chemical activation and culture in 0 mM or 2 mM 6-dimethylaminopurine (6-DMAP) for 12 h. Moreover, to determine the biological significance of the fluctuations of MPF, activated oocytes were fused with GV-staged partners. The biochemical results showed that the high MPF activity of MII oocytes fell to basal level precipitously within the first hour after activation, started to increase at 6-8 h, rising to 80 +/- 4% of MII after 12 h. MAPK activity decreased to a low level 4 h after activation, increased between 6-12 h, but remained below 30 +/- 3.6% of MII values. The incubation with 6-DMAP had no effect on the kinetics of MPF and MAP kinase activity. Fusion of MII oocytes to GV partners induced rapid breakdown of the GV, whereas no breakdown occurred when GV were fused with eggs in the first hours post activation. Interestingly, the high biochemical levels of MPF activity at 8-12 h after activation were not able to induce GVBD in fusion partners.     

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.     

Bovine embryo cloning: Characterization of the recipient cytoplasts by phosphorylation patterns and kinase activities

When in vitro -matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro -matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.     

Maturation-promoting factor governs mitogen-activated protein kinase activation and interphase suppression during meiosis of rat oocytes

Meiosis is a particular example of a cell cycle, characterized by two successive divisions without an intervening interphase. Resumption of meiosis in oocytes is associated with activation of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK). The activity of MPF declines during the transition between the two meiotic divisions, whereas the activity of MAPK is sustained. Attempts to disclose the interplay between these key regulators of meiosis in both amphibian and mammalian oocytes generated contradictory results. Furthermore, the enzyme that governs the suppression of interphase in mammals is still unidentified. To our knowledge, we provide herein the first demonstration in a mammalian system that inhibition of MPF at reinitiation of meiosis abrogated Mos expression and MAPK activation. We also show that oocytes, in which reactivation of MPF at completion of the first telophase was prevented, exhibited an interphase nucleus with decondensed chromosomes. Inhibition of MAPK did not interfere with the progression to the second meiotic metaphase but, rather, resulted in parthenogenic activation. We conclude that in rat oocytes, MPF regulates MAPK activation and its timely reactivation prevents the oocytes from entering interphase.