Functional interaction between p90Rsk2 and Emi1 contributes to the metaphase arrest of mouse oocytes

Vertebrate eggs arrest at metaphase of the second meiotic division before fertilization under the effect of a cytostatic factor (CSF). This arrest is established during oocyte maturation by the MAPK kinase module, comprised of Mos, MEK, MAPKs and p90Rsk. Maintenance of CSF arrest at metaphase requires inhibitors of the anaphase-promoting complex (APC) like Emi1, which sequesters the APC activator Cdc20. Although it was proposed that the Mos pathway and Emi1 act independently, neither one alone is sufficient to entirely reproduce CSF arrest. Herein we demonstrate that p90Rsk2 associates with and phosphorylates Emi1 upstream of the binding region for Cdc20, thus stabilizing their interaction. Experiments in transfected cells and two-cell embryos indicate that Emi1 and p90Rsk2 cooperate to induce the metaphase arrest. Moreover, oocyte maturation was impaired by interfering with the interaction between p90Rsk2 and Emi1 or by RNA interference of Emi1. Our results indicate that p90Rsk2 and Emi1 functionally interact during oocyte maturation and that the Mos pathway establishes CSF activity through stabilization of an APC-inhibitory complex composed by Emi1 and Cdc20 before fertilization.     

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.     

Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

Emi2 at the Crossroads: Where CSF Meets MPF

Vertebrate eggs arrest at metaphase of meiosis II due to an activity known as cytostatic factor (CSF). CSF antagonizes the ubiquitin ligase activity of the anaphase-promoting complex/cyclosome (APC/C), preventing cyclin B destruction and meiotic exit until fertilization occurs. A puzzling feature of CSF arrest is that APC/C inhibition is leaky. Ongoing cyclin B synthesis is counterbalanced by a limited amount of APC/C-mediated cyclin B destruction; thus, cyclin B/Cdc2 activity remains at steady state. How the APC/C can be slightly active toward cyclin B, and yet restrained from ubiquitinating cyclin B altogether, is unknown. Emi2/XErp1 is the critical CSF component directly responsible for APC/C inhibition during CSF arrest. Fertilization triggers the Ca2+-dependent destruction of Emi2, releasing the APC/C to ubiquitinate the full pool of cyclin B and initiate completion of meiosis. Previously, we showed that a phosphatase maintains Emi2’s APC/C-inhibitory activity in CSF-arrested Xenopus egg extracts. Here, we demonstrate that phosphatase inhibition permits Emi2 phosphorylation at thr-545 and -551, which inactivates Emi2. Furthermore, we provide evidence that adding excess cyclin B to CSF extracts stimulates Cdc2 phosphorylation of these same residues, antagonizing Emi2-APC/C association. Our findings suggest a model wherein the pool of Emi2 acts analogously to a rheostat by integrating Cdc2 and phosphatase activities to prevent cyclin B overaccumulation and Cdc2 hyperactivity during the indefinite period of time between arrival at metaphase II and eventual fertilization. Finally, we propose that inactivation of Emi2 by Cdc2 permits mitotic progression during early embryonic cleavage cycles.     

A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest

BACKGROUND: Vertebrate oocytes are arrested in metaphase II of meiosis prior to fertilization by cytostatic factor (CSF). CSF enforces a cell-cycle arrest by inhibiting the anaphase-promoting complex (APC), an E3 ubiquitin ligase that targets Cyclin B for degradation. Although Cyclin B synthesis is ongoing during CSF arrest, constant Cyclin B levels are maintained. To achieve this, oocytes allow continuous slow Cyclin B degradation, without eliminating the bulk of Cyclin B, which would induce release from CSF arrest. However, the mechanism that controls this continuous degradation is not understood. RESULTS: We report here the molecular details of a negative feedback loop wherein Cyclin B promotes its own destruction through Cdc2/Cyclin B-mediated phosphorylation and inhibition of the APC inhibitor Emi2. Emi2 bound to the core APC, and this binding was disrupted by Cdc2/Cyclin B, without affecting Emi2 protein stability. Cdc2-mediated phosphorylation of Emi2 was antagonized by PP2A, which could bind to Emi2 and promote Emi2-APC interactions. CONCLUSIONS: Constant Cyclin B levels are maintained during a CSF arrest through the regulation of Emi2 activity. A balance between Cdc2 and PP2A controls Emi2 phosphorylation, which in turn controls the ability of Emi2 to bind to and inhibit the APC. This balance allows proper maintenance of Cyclin B levels and Cdc2 kinase activity during CSF arrest.     

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.     

Regulation of meiotic maturation in the mammalian oocyte: interplay between exogenous cues and the microtubule cytoskeleton.

Mammalian oocytes exhibit a series of cell cycle transitions that coordinate the penultimate events of meiosis with the onset of embryogenesis at fertilization. The execution of these cell cycle transitions, at G2/M of meiosis-I and metaphase/anaphase of meiosis I and II, involve both biosynthetic and post-translational modifications that directly modulate centrosome and microtubule behavior. Specifically, somatic cells alter the signal transduction pathways in the oocyte and influence the expression of maturation promoting factor (MPF) and cytostatic factor (CSF) activity through a microtubule-dependent mechanism. The regulation of the oocytes' cell cycle machinery by hormone-mediated somatic cell signals, involving both positive and negative stimuli, ensures that meiotic cell cycle progression is synchronized with the earliest pivotal events of mammalian reproduction.     

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.     

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.     

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.     

Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.     

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.     

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.     

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.     

Degradation of Ccnb3 is essential for maintenance of MII arrest in oocyte

Before fertilization, ovulated mammalian oocytes are arrested at the metaphase of second meiosis (MII), which is maintained by the so-called cytostatic factor (CSF). It is well known that the continuous synthesis and accumulation of cyclin B is critical for maintaining the CSF-mediated MII arrest. Recent studies by us and others have shown that Ccnb3 is required for the metaphase-to-anaphase transition during the first meiosis of mouse oocytes, but whether Ccnb3 plays a role in MII arrest and exit remains unknown. Here, we showed that the protein level of Ccnb3 gradually decreased during oocyte meiotic maturation, and exogenous expression of Ccnb3 led to release of MII arrest, degradation of securin, separation of sister chromatids, extrusion of the second polar body (PB2), and finally entry into interphase. These phenotypes could be rescued by inhibition of Wee1B or CDK2. Our results indicate that Ccnb3 plays a critical regulatory role in MII arrest and exit in mouse oocytes.     

Effect of physiological levels of phytoestrogens on mouse oocyte maturation in vitro

Phytoestrogens are a group of naturally occurring compounds that have weak estrogenic activity. Genistein and daidzein are major phytoestrogens produced by soybeans. It has been reported previously that at high concentration, some phytoestrogens inhibit cell cycle progression of mouse germinal vesicle (GV) oocytes, but the environmentally relevant level is much lower. Here we show the effects of low concentrations of the isoflavones genistein, daidzein and the daidzein metabolite, equol, on mouse oocyte maturation. GV oocytes denuded of cumulus cells were cultured in TaM medium containing low levels (5 μM) of genistein, daidzein. or equol. In all cases, the oocytes underwent normal GV break down, first polar body extrusion and became arrested at metaphase II (mII). As judged by fluorescence microscopy, the treated mII oocytes exhibited normal distributions of actin microfilaments, cortical granules and metaphase spindle formation with condensed metaphase chromatin. Moreover, mRNA expression levels of the cytostatic factors Emi2 and Mos were similar to those of their respective controls. These data suggest that exposure of maturing GV oocytes to environmental levels of genistein, daidzein or equol in vitro do not cause negative effects on maturation to produce mII oocytes.     

Calcium elevation at fertilization coordinates phosphorylation of XErp1/Emi2 by Plx1 and CaMK II to release metaphase arrest by cytostatic factor

BACKGROUND: Vertebrate oocytes are arrested at second meiotic metaphase by cytostatic factor (CSF) while awaiting fertilization. Accumulating evidence has suggested that inhibition of the anaphase-promoting complex/cyclosome (APC/C) is responsible for this arrest. Xenopus polo-like kinase 1 (Plx1) is required for activation of the APC/C at the metaphase-anaphase transition, and calcium elevation, upon fertilization/activation of eggs, acting through calmodulin-dependent kinase II (CaMKII) is sufficient to activate the APC/C and terminate CSF arrest. However, connections between the Plx1 pathway and the CaMKII pathway have not been identified. RESULTS: Overexpression of Plx1 causes CSF release in the absence of calcium, and depletion of Plx1 from egg extracts blocks induction of CSF release by calcium and CaMKII. Prior phosphorylation of the APC/C inhibitor XErp1/Emi2 by CaMK II renders it a good substrate for Plx1, and phosphorylation by both kinases together promotes its degradation in egg extracts. The pathway is enhanced by the ability of Plx1 to cause calcium-independent activation of CaMKII. The results identify the targets of CaMKII and Plx1 that promote egg activation and define the first known pathway of CSF release in which an APC/C inhibitor is targeted for degradation only when both CaMKII and Plx1 are active after calcium elevation at fertilization. CONCLUSIONS: Plx1 with an intact polo-box domain is necessary for release of CSF arrest and sufficient when overexpressed. It acts at the same level as CaMKII in the pathway of calcium-induced CSF release by cooperating with CaMKII to regulate APC/C regulator(s), such as XErp1/Emi2, rather than by directly activating the APC/C itself.     

Wee1B,Myt1, and Cdc25 function in distinct compartments of the mouse oocyte to control meiotic resumption

After a long period of quiescence at dictyate prophase I, termed the germinal vesicle (GV) stage, mammalian oocytes reenter meiosis by activating the Cdc2–cyclin B complex (maturation-promoting factor [MPF]). The activity of MPF is regulated by Wee1/Myt1 kinases and Cdc25 phosphatases. In this study, we demonstrate that the sequestration of components that regulate MPF activity in distinct subcellular compartments is essential for their function during meiosis. Down-regulation of either Wee1B or Myt1 causes partial meiotic resumption, and oocytes reenter the cell cycle only when both proteins are down-regulated. Shortly before GV breakdown (GVBD), Cdc25B is translocated from the cytoplasm to the nucleus, whereas Wee1B is exported from the nucleus to the cytoplasm. These movements are regulated by PKA inactivation and MPF activation, respectively. Mislocalized Wee1B or Myt1 is not able to maintain meiotic arrest. Thus, cooperation of Wee1B, Myt1, and Cdc25 is required to maintain meiotic arrest and relocation of these components before GVBD is necessary for meiotic reentry.