Mos limits the number of meiotic divisions in urochordate eggs

Mos kinase is a universal mediator of oocyte meiotic maturation and is produced during oogenesis and destroyed after fertilization. The hallmark of maternal meiosis is that two successive M phases (meiosis I and II) drive two rounds of asymmetric cell division (ACD). However, how the egg limits the number of meioses to just two, thereby preventing gross aneuploidy, is poorly characterized. Here, in urochordate eggs, we show that loss of Mos/MAPK activity is necessary to prevent entry into meiosis III. Remarkably, maintaining the Mos/MAPK pathway active after fertilization at near physiological levels induces additional rounds of meiotic M phase (meiosis III, IV and V). During these additional rounds of meiosis, the spindle is positioned asymmetrically resulting in further rounds of ACD. In addition, inhibiting meiotic exit with Mos prevents pronuclear formation, cyclin A accumulation and maintains sperm-triggered Ca(2+) oscillations, all of which are hallmarks of the meiotic cell cycle in ascidians. It will be interesting to determine whether Mos availability in mammals can also control the number of meioses as it does in the urochordates. Our results demonstrate the power of urochordate eggs as a model to dissect the egg-to-embryo transition.     

Mechanistic studies of the mitotic activation of Mos

The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2beta. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix alphaC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix alphaC.     

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.     

The casein kinase II beta subunit binds to Mos and inhibits Mos activity.

Mos is a germ cell-specific serine/threonine kinase and is required for Xenopus oocyte maturation. Active Mos stimulates a mitogen-activated protein kinase (MAPK) by directly phosphorylating and activating MAPK kinase (MKK). We report here that the Xenopus homolog of the beta subunit of casein kinase II (CKII beta) binds to and regulates Mos. The Mos-interacting region of CKII beta was mapped to the C terminus. Mos bound to CKII beta in somatic cells ectopically expressing Mos and CKII beta as well as in unfertilized Xenopus eggs. CKII beta inhibited Mos-mediated MAPK activation in rabbit reticulocyte lysates and repressed MKK activation by v-Mos in a coupled kinase assay. In addition, microinjection of CKII beta mRNA into Xenopus oocytes inhibited progesterone-induced meiotic maturation and MAPK activation, presumably by binding of CKII beta to Mos and thereby inhibiting MAPK activation. Moreover, this inhibitory phenotype could be rescued by another protein that binds to CKII beta, CKII alpha. The ability of ectopic CKII beta to inhibit meiotic maturation and the detection of a complex between endogenous Mos and CKII beta suggest that CKII beta may act as an inhibitor of Mos during oocyte maturation, perhaps setting a threshold beyond which Mos protein must accumulate before it can activate the MAPK pathway.     

Mos is not required for the initiation of meiotic maturation in Xenopus oocytes

In Xenopus oocytes, the c-mos proto-oncogene product has been proposed to act downstream of progesterone to control the entry into meiosis I, the transition from meiosis I to meiosis II, which is characterized by the absence of S phase, and the metaphase II arrest seen prior to fertilization. Here, we report that inhibition of Mos synthesis by morpholino antisense oligonucleotides does not prevent the progesterone-induced initiation of Xenopus oocyte meiotic maturation, as previously thought. Mos-depleted oocytes complete meiosis I but fail to arrest at metaphase II, entering a series of embryonic-like cell cycles accompanied by oscillations of Cdc2 activity and DNA replication. We propose that the unique and conserved role of Mos is to prevent mitotic cell cycles of the female gamete until the fertilization in Xenopus, starfish and mouse oocytes.     

Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca(2+) store depletion from store-operated Ca(2+) entry.

During oocyte maturation, eggs acquire the ability to generate specialized Ca(2+) signals in response to sperm entry. Such Ca(2+) signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca(2+) entry (SOCE), an important Ca(2+) influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos-mitogen-activated protein kinase (MAPK)-maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca(2+) influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.     

A new role for Mos in Xenopus oocyte maturation: targeting Myt1 independently of MAPK

The resumption of meiosis in Xenopus arrested oocytes is triggered by progesterone, which leads to polyadenylation and translation of Mos mRNA, then activation of MAPK pathway. While Mos protein kinase has been reported to be essential for re-entry into meiosis in Xenopus, arrested oocytes can undergo germinal vesicle breakdown (GVBD) independently of MAPK activation, leading us to question what the Mos target might be if Mos is still required. We now demonstrate that Mos is indeed necessary, although is independent of the MAPK cascade, for conversion of inactive pre-MPF into active MPF. We have found that Myt1 is likely to be the Mos target in this process, as Mos interacts with Myt1 in oocyte extracts and Mos triggers Myt1 phosphorylation on some sites in vivo, even in the absence of MAPK activation. We propose that Mos is involved, not only in the MAPK cascade pathway, but also in a mechanism that directly activates MPF in Xenopus oocytes.     

Translation of incenp during oocyte maturation is required for embryonic development in Xenopus laevis

The chromosome passenger complex (CPC) consists of Aurora-B kinase and several other subunits. One of these, incenp, binds Aurora-B and regulates its kinase activity. During Xenopus oocyte maturation, incenp accumulates through translation, contributing to aurora-b activation. A previous study has demonstrated that inhibition of incenp translation during oocyte maturation diminishes aurora-b activation but does not interfere with oocyte maturation, characterized by normal maturation-specific cyclin-b phosphorylation, degradation, and resynthesis. Here we have extended these findings, showing that inhibition of incenp translation during oocyte maturation did not interfere with meiosis I or II, as indicated by the normal emission of the first polar body and metaphase II arrest, followed by the successful emission of the second polar body upon parthenogenetic egg activation. Most importantly, however, when transferred to host frogs and subsequently ovulated, the incenp-deficient eggs were fertilized but failed to undergo mitotic cleavage. Thus, translation of incenp during oocyte maturation appears to be part of oocyte cytoplasmic maturation, preparing the egg for the rapid mitosis following fertilization.     

Metaphase arrest by cyclin E-Cdk2 requires the spindle-checkpoint kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.     

Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation

The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Dissection of c-MOS degron

c-MOS, a MAP kinase kinase kinase, is a regulator of oocyte maturation. The concentration of c-MOS is controlled in part through its conditional degradation. Previous studies proposed the "second-codon rule", according to which the N-terminal proline (Pro) of c-MOS is a destabilizing residue that targets c-MOS for degradation. We analyzed the degradation signal (degron) of c-MOS in Xenopus oocytes, found it to be a portable degron, and demonstrated that, contrary to the model above, the N-terminal Pro residue of c-MOS is entirely dispensable for its degradation if Ser-2 (encoded Ser-3) of c-MOS is replaced by a small non-phosphorylatable residue such as Gly. The dependence of c-MOS degradation on N-terminal Pro is shown to be caused by a Pro-mediated downregulation of the net phosphorylation of Ser-2, a modification that halts c-MOS degradation in oocytes. Thus, the N-terminal Pro residue of c-MOS is not a recognition determinant for a ubiquitin ligase, in agreement with earlier evidence that Pro is a stabilizing residue in the N-end rule.     

Regulation of the G2/M transition in oocytes of xenopus tropicalis

The molecular events regulating hormone-induced oocyte activation and meiotic maturation are probably best understood in Xenopus laevis. In X. laevis, progesterone activates the G2-arrested oocyte, induces entry into M phase of meiosis I (MI) and resumption of the meiotic cell cycles, and leads to the formation of a mature, fertilizable egg. Oocytes of Xenopus tropicalis offer several practical advantages over those of X. laevis, including faster and more synchronous meiotic cell cycle progression, less seasonal variability, and the availability of transgenic approaches. Previous work found several similarities in the pathways regulating oocyte maturation in the two species. Here, we report several additional ones that are conserved in X. tropicalis. (1). Injection of Mos mRNA into G2-arrested oocytes activates the MAP kinase cascade and induces the G2/MI transition. (2). Injection of the beta subunit of the kinase CK2 (a negative regulator of Mos and oocyte activation) delays the G2/MI transition. (3). Elevating PKA activity blocks progesterone-induced maturation; repressing PKA activity induces entry into MI in the absence of progesterone. (4). LF (anthrax lethal factor), which cleaves certain MAP kinase kinases, strongly reduces both the rate and extent of entry into MI. In contrast to the one previously reported major difference between oocytes of the two species, we find that injection of egg cytoplasm ("MPF activity") into G2-arrested X. tropicalis oocytes induces entry into meiosis I even when protein synthesis is blocked, just as it does in oocytes of X. laevis. These results indicate that much of what we have learned from studies of X. laevis oocytes holds for those of X. tropicalis, and suggest that X. tropicalis oocytes offer a good experimental system for investigating certain questions that require a rapid, synchronous progression through the G2/meiosis I transition.     

The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signaling pathway.

Quiescent Xenopus oocytes are activated by progesterone, which binds to an unidentified surface-associated receptor. Progesterone activates a poorly understood signaling pathway that results in the translational activation of mRNA encoding Mos, a MAP kinase kinase kinase necessary for the activation of MAP kinase and MPF, the resumption of meiosis, and maturation of the oocyte into the sperm-responsive egg. We have designed a screen to identify early signaling proteins based on the premise that some of these proteins would be phosphorylated or otherwise modified within minutes of progesterone addition. This screen has revealed Eg2, a Ser/Thr kinase. We find that Eg2 is phosphorylated soon after progesterone stimulation and provide evidence that it functions in the signaling pathway. Overexpression of Eg2 via mRNA microinjection shortens the time between progesterone stimulation and the appearance of new Mos protein, accelerates activation of MAP kinase and advances entry into the meiotic cell cycle. Finally, overexpression of Eg2 dramatically reduces the concentration of progesterone needed to trigger oocyte activation. These results argue that the kinase Eg2 is a component of the progesterone-activated signaling pathway that releases frog oocytes from cell cycle 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.     

蛋白激酶在卵母细胞减数分裂和受精中的作用

脊椎动物卵母细胞的减数分裂和受精过程受到多种蛋白激酶的调节。近年来对于卵母细胞成熟、活化和受精的分子机制研究取得了长足进步 ,发现促成熟因子 (MPF)和促分裂原活化蛋白激酶 (MAPK)是调节卵母细胞细胞周期的关键分子 ,二者的激活和失活导致了减数分裂的恢复、阻滞和完成。许多蛋白激酶通过调节MPF和MAPK活性来影响减数分裂。Polo like激酶活化MPF ,Mos激活MAPK而启动成熟分裂并维持中期阻滞。CaMKII通过泛素途径灭活MPF使卵突破MII期阻滞。另外 ,p90 rsk作为MAPK的下游分子参与减数分裂调节 ,蛋白激酶C(PKC)诱导皮质颗粒排放并抑制MAPK激活 ,酪氨酸蛋白激酶家族成员介导受精诱发的Ca2 释放。这些蛋白激酶的协同作用推动了卵母细胞正常的成熟与受精     

Function of the Mos/MAPK pathway during oocyte maturation in the Japanese brown frog Rana japonica

Fully grown immature oocytes acquire the ability to be fertilized with sperm after meiotic maturation, which is finally accomplished by the formation and activation of the maturation-promoting factor (MPF). MPF is the complex of Cdc2 and cyclin B, and its function in promoting metaphase is common among species. The Mos/mitogen-activated protein kinase (MAPK) pathway is also commonly activated during vertebrate oocyte maturation, but its function seems to be different among species. We investigated the function of the Mos/MAPK pathway during oocyte maturation of the frog Rana japonica. Although MAPK was activated in accordance with MPF activation during oocyte maturation, MPF activation and germinal vesicle breakdown (GVBD) was not initiated when the Mos/MAPK pathway was activated in immature oocytes by the injection of c-mos mRNA. Inhibition of Mos synthesis by c-mos antisense RNA and inactivation of MAPK by CL100 phosphatase did not prevent progesterone-induced MPF activation and GVBD. However, continuous MAPK activation and MAPK inhibition through oocyte maturation accelerated and delayed MPF activation, respectively. Furthermore, Mos induced a low level of cyclin B protein synthesis in immature oocytes without the aid of MAPK. These results suggest that the general function of the Mos/MAPK pathway, which is not essential for MPF activation and GVBD in Rana oocytes, is to enhance cyclin B translation by Mos itself and to stabilize cyclin B protein by MAPK during oocyte maturation.     

The ''second-codon rule'' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes.

The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.     

Mos mediates the mitotic activation of p42 MAPK in Xenopus egg extracts

The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. Here we have partially purified a Cdc2-cyclin B-induced MEK-activating protein kinase from mitotic Xenopus egg extracts and identified it as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts.