The metaphase II arrest in mouse oocytes is controlled through microtubule-dependent destruction of cyclin B in the presence of CSF.

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long-term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF-like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II-arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three-dimensional organization of the spindle.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

c-mos proto-oncogene product is partly degraded after release from meiotic arrest and persists during interphase in mouse zygotes.

Recently, it has been shown that the product of the c-mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c-mos product in metaphase II mouse oocytes and soon after activation. The amount of c-mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c-mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c-mos is detectable during the first zygotic interphase. Our data show that the degradation of c-mos is not involved in the release from the metaphase arrest.     

A characterization of cytostatic factor activity from Xenopus eggs and c-mos-transformed cells

In Xenopus oocytes, the mos proto-oncogene product is required during meiosis I for the activation of maturation promoting factor (MPF) and the subsequent breakdown of the germinal vesicle (GVBD). In addition, the mos product has been shown to be a candidate "initiator" of meiotic maturation and is an active component of cytostatic factor (CSF), an activity responsible for metaphase II arrest. Here we demonstrate that pp39mos is required throughout oocyte maturation. We found that in progesterone stimulated oocytes, depletion of mos RNA immediately before GVBD terminally decreased MPF. Likewise, oocytes depleted of mos RNA and induced to mature with crude MPF proceeded through GVBD but lacked the MPF activity required to arrest mature oocytes at metaphase II. Thus, during maturation the mos product is required, directly or indirectly, to sustain MPF activity. On the other hand, mouse NIH/3T3 cells transformed by the constitutive expression of pp39mosxc possessed CSF activity but lacked constitutive levels of MPF or its associated histone H1 kinase activity. Moreover, cytosols prepared from transformed NIH/3T3 cells or Xenopus eggs had similar levels of CSF activity, but pp39mos levels were greater than 40-fold higher in the transformed cell extract. These analyses show that maintenance of CSF during interphase does not result in the maintenance of MPF.     

Interplay between Cdc2 kinase and the c-Mos/MAPK pathway between metaphase I and metaphase II in Xenopus oocytes

Xenopus oocytes arrested in prophase I resume meiotic division in response to progesterone and arrest at metaphase II. Entry into meiosis I depends on the activation of Cdc2 kinase [M-phase promoting factor (MPF)]. To better understand the role of Cdc2, MPF activity was specifically inhibited by injection of the CDK inhibitor, Cip1. When Cip1 is injected at germinal vesicle breakdown (GVBD) time, Cdc25 and Plx1 are both dephosphorylated and Cdc2 is rephosphorylated on tyrosine. The autoamplification loop characterizing MPF is therefore not only required for MPF generation before GVBD, but also for its stability during the GVBD period. The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), responsible for cyclin degradation, is also under the control of Cdc2; therefore, Cdc2 activity itself induces its own inactivation through cyclin degradation, allowing the exit from the first meiotic division. In contrast, cyclin accumulation, responsible for Cdc2 activity increase allowing entry into metaphase II, is independent of Cdc2. The c-Mos/mitogen-activated protein kinase (MAPK) pathway remains active when Cdc2 activity is inhibited at GVBD time. This pathway could be responsible for the sustained cyclin neosynthesis. In contrast, during the metaphase II block, the c-Mos/MAPK pathway depends on Cdc2. Therefore, the metaphase II block depends on a dynamic interplay between MPF and CSF, the c-Mos/MAPK pathway stabilizing cyclin B, whereas in turn, MPF prevents c-Mos degradation.     

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.     

The Mos/MAPK pathway is involved in metaphase II arrest as a cytostatic factor but is neither necessary nor sufficient for initiating oocyte maturation in goldfish

Mos plays a crucial role in meiotic cell division in vertebrates. In Xenopus, Mos is involved in the initiation of oocyte maturation as an initiator and in the arrest at the metaphase II stage (MII) as a component of the cytostatic factor (CSF). The function of Mos is mediated by MAP kinase (MAPK). We investigated the function of the Mos/MAPK pathway during goldfish oocyte maturation induced by 17α,20β-dihydroxy-4-pregnen-3-one (17α,20β-DP), a natural maturation-inducing hormone in fishes. Mos was absent in immature goldfish oocytes. It appeared before the onset of germinal vesicle breakdown (GVBD), increased to a maximum in mature oocytes arrested at MII and disappeared after fertilization. MAPK was activated after Mos synthesis but before maturation-promoting factor (MPF) activation, and its activity reached maximum at MII. Injection of either Xenopus or goldfish c-mos mRNA into one blastomere of 2-cell-stage Xenopus and goldfish embryos induced metaphase arrest, suggesting that goldfish Mos has a CSF activity. Injection of constitutively active Xenopus c-mos mRNA into immature goldfish oocytes induced MAPK activation, but neither MPF activation nor GVBD occurred. Conversely, the injection of goldfish c-mos antisense RNA inhibited both Mos synthesis and MAPK activation in the 17α,20β-DP-treated oocytes, but these oocytes underwent GVBD. These results indicate that the Mos/MAPK pathway is not essential for initiating goldfish oocyte maturation despite its general function as a CSF. We discuss the general role of Mos/MAPK during oocyte maturation, with reference to the difference in contents of inactive MPF (pre-MPF) stored in immature oocytes. Received: 10 February 2000 / Accepted: 25 April 2000     

Maintenance of sister chromatid attachment in mouse eggs through maturation-promoting factor activity

Mammalian eggs naturally arrest at metaphase of the second meiotic division, until sperm triggers a series of Ca(2+) spikes that result in activation of the anaphase-promoting complex/cyclosome (APC/C). APC/C activation at metaphase targets destruction-box containing substrates, such as cyclin B1 and securin, for degradation, and as such eggs complete the second meiotic division. Cyclin B1 degradation reduces maturation (M-phase)-promoting factor (MPF) activity and securin degradation allows sister chromatid separation. Here we examined the second meiotic division in mouse eggs following expression of a cyclin B1 construct with an N-terminal 90 amino acid deletion (Delta 90 cyclin B1) that was visualized by coupling to EGFP. This cyclin construct was not an APC/C substrate, and so following fertilization, sperm were incapable of stimulating Delta 90 cyclin B1 degradation. In these eggs, chromatin remained condensed and no pronuclei formed. As a consequence of the lack of pronucleus formation, sperm-triggered Ca(2+) spiking continued indefinitely, consistent with a current model in which the sperm-activating factor is localized to the nucleus. Because Ca(2+) spiking was not inhibited by Delta 90 cyclin B1, the degradation timing of securin, visualized by coupling it to EGFP, was unaffected. However, despite rapid securin degradation, sister chromatids remained attached. This was a direct consequence of MPF activity because separation was induced following application of the MPF inhibitor roscovitine. Similar observations regarding the ability of MPF to prevent sister chromatid separation have recently been made in Xenopus egg extracts and in HeLa cells. The results presented here show this mechanism can also occur in intact mammalian eggs and further that this mechanism appears conserved among vertebrates. We present a model in which metaphase II arrest is maintained primarily by MPF levels only.     

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. We here present evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which we term endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals.     

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.     

The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes

In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.     

Maturation promoting factor destabilization mediates human chorionic gonadotropin induced meiotic resumption in rat oocytes

Human chorionic gonadotropin (hCG) mimics the action of luteinizing hormone (LH) and triggers meiotic maturation and ovulation in mammals. The mechanism by which hCG triggers meiotic resumption in mammalian oocytes remains poorly understood. We aimed to find out the impact of hCG surge on morphological changes, adenosine 3′,5′‐cyclic monophosphate (cAMP), guanosine 3′,5′‐cyclic monophosphate (cGMP), cell division cycle 25B (Cdc25B), Wee1, early mitotic inhibitor 2 (Emi2), anaphase‐promoting complex/cyclosome (APC/C), meiotic arrest deficient protein 2 (MAD2), phosphorylation status of cyclin‐dependent kinase 1 (Cdk1), its activity and cyclin B1 expression levels during meiotic resumption from diplotene as well as metaphase‐II (M‐II) arrest in cumulus oocyte complexes (COCs). Our data suggest that hCG surge increased cyclic nucleotides level in encircling granulosa cells but decreased their level in oocyte. The reduced intraoocyte cyclic nucleotides level is associated with the decrease of Cdc25B, Thr161 phosphorylated Cdk1 and Emi2 expression levels. On the other hand, hCG surge increased Wee1, Thr14/Tyr15 phosphorylated Cdk1, APC/C as well as MAD2 expression levels. The elevated APC/C activity reduced cyclin B1 level. The changes in phosphorylation status of Cdk1 and reduced cyclin B1 level might have resulted in maturation promoting factor (MPF) destabilization. The destabilized MPF finally triggered resumption of meiosis from diplotene as well as M‐II arrest in rat oocytes.     

Vesicular traffic at the cell membrane regulates oocyte meiotic arrest

Vertebrate oocytes are maintained in meiotic arrest for prolonged periods of time before undergoing oocyte maturation in preparation for fertilization. Cyclic AMP (cAMP) signaling plays a crucial role in maintaining meiotic arrest, which is released by a species-specific hormonal signal. Evidence in both frog and mouse argues that meiotic arrest is maintained by a constitutively active G-protein coupled receptor (GPCR) leading to high cAMP levels. Because activated GPCRs are typically targeted for endocytosis as part of the signal desensitization pathway, we were interested in determining the role of trafficking at the cell membrane in maintaining meiotic arrest. Here we show that blocking exocytosis, using a dominant-negative SNAP25 mutant in Xenopus oocytes, releases meiotic arrest independently of progesterone. Oocyte maturation in response to the exocytic block induces the MAPK and Cdc25C signaling cascades, leading to MPF activation, germinal vesicle breakdown and arrest at metaphase of meiosis II with a normal bipolar spindle. It thus replicates all tested aspects of physiological maturation. Furthermore, inhibiting clathrin-mediated endocytosis hinders the effectiveness of progesterone in releasing meiotic arrest. These data show that vesicular traffic at the cell membrane is crucial in maintaining meiotic arrest in vertebrates, and support the argument for active recycling of a constitutively active GPCR at the cell membrane.     

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.     

The anaphase-promoting complex/cyclosome inhibitor Emi2 is essential for meiotic but not mitotic cell cycles

Vertebrate oocytes awaiting fertilization are arrested at metaphase of meiosis II by cytostatic factor (CSF). This arrest is due to inhibition of the anaphase-promoting complex/cyclosome, in part by a newly identified protein, Emi2 (xErp1). Emi2 is required for maintenance of CSF arrest in egg extracts, but its function in CSF establishment in oocytes and the normal embryonic cell cycle is unknown. Here we show that during oocyte maturation, Emi2 appears only after metaphase I, and its level peaks at CSF arrest (metaphase II). In M phase, Emi2 undergoes a phosphorylation-dependent electrophoretic shift. Microinjection of antisense oligonucleotides against Emi2 into stage VI oocytes blocks progression through meiosis II and the establishment of CSF arrest. Recombinant Emi2 rescues CSF arrest in these oocytes and also causes CSF arrest in egg extracts and in blastomeres of two-cell embryos. Fertilization triggers rapid, complete degradation of Emi2, but it is resynthesized in the first embryonic cell cycle to reach levels 5-fold lower than during CSF arrest. However, depletion of the protein from cycling egg extracts does not prevent mitotic cell cycle progression. Thus, Emi2 plays an essential role in meiotic but not mitotic cell cycles.     

Transient reactivation of CSF in parthenogenetic one-cell mouse embryos

During meiosis, the cytostatic factor (CSF) activity stabilizes the activity of the M-phase promoting factor (MPF) in metaphase II arrested vertebrate oocytes. Upon oocyte activation, the inactivation of both MPF and CSF enables the entry into the first embryonic mitotic cell cycle. Using a biological assay based on cell-fusion (hybrid between a parthenogenetically activated egg entering the first mitotic division and an activated oocyte), we observed that in activated mouse oocytes a first drop in CSF activity is detectable as early as 20 min post-activation. This suggests that CSF is inactivated upon MPF inactivation. However, CSF activity increases again to reach a maximum 60 min post-activation and gradually disappears during the following 40 min. Thus, in activated mouse oocytes (undergoing the transition to interphase) CSF activity fluctuates before definitive inactivation. We found that hybrids arrested in M-phase, thus containing CSF activity after oocyte activation, have activated forms of MAP kinases while hybrids in interphase have inactive forms of these enzymes. We postulate that CSF inactivation in mouse oocytes proceeds in two steps. The initial inactivation of CSF, required for MPF inactivation, is transient and does not require MAP kinase inactivation. The final inactivation of CSF, required for normal embryonic cell cycle progression, is dependent upon the inactivation of MAP kinases.     

Oocyte Maturation,Mos and Cyclins—A Matter of Synthesis: Two Functionally Redundant Ways to Induce Meiotic Maturation

ABSTRACT The development of an immature oocyte into a fertilizable gamete is a process known as meiotic maturation. In vertebrates, it corresponds to the transition from the prophase arrest of the first meiotic division (usually considered as a late G2 phase) to the metaphase arrest of the second meiotic division. This transition is controlled by modulating the activity of the cyclin B-Cdc2 complex, MPF (M-phase promoting factor), the universal regulator of the G2/M transition. Meiotic maturation of frog oocytes is triggered by steroid hormones through a rapid, necessary and sufficient suppression of PKA and requires ongoing protein synthesis. A long-standing question has been to identify key protein(s) required to trigger the activation of MPF in response to the hormonal signal. Here we will discuss data supporting the view that steroids bring about meiotic maturation through functionally redundant pathways involving synthesis of Mos or of cyclin proteins, reinforcing the robustness of the system.     

Xenopus oocyte maturation does not require new cyclin synthesis

Progesterone induces fully grown, stage VI, Xenopus oocytes to pass through meiosis I and arrest in metaphase of meiosis II. Protein synthesis is required twice in this process: in order to activate maturation promoting factor (MPF) which induces meiosis I, and then again after the completion of meiosis I to reactivate MPF in order to induce meiosis II. We have used antisense oligonucleotides to destroy maternal stores of cyclin mRNAs, and demonstrate that new cyclin synthesis is not required for entry into either meiosis I or II. This finding is consistent with the demonstration that stage VI oocytes contain a store of B-type cyclin polypeptides (Kobayashi, H., J. Minshull, C. Ford, R. Golsteyn, R. Poon, and T. Hunt. 1991. J. Cell Biol. 114:755-765). Although approximately 70% of cyclin B2 is destroyed at first meiosis, the surviving fraction, together with a larger pool of surviving cyclin B1, must be sufficient to allow the reactivation of MPF and induce entry into second meiotic metaphase. Since stage VI oocytes do not contain any cyclin A, our results show that cyclin A is not required for meiosis in Xenopus. We discuss the possible nature of the proteins whose synthesis is required to induce meiosis I and II.     

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.