CDC25A phosphatase controls meiosis I progression in mouse oocytes

CDK1 is a pivotal regulator of resumption of meiosis and meiotic maturation of oocytes. CDC25A/B/C are dual-specificity phosphatases and activate cyclin-dependent kinases (CDKs). Although CDC25C is not essential for either mitotic or meiotic cell cycle regulation, CDC25B is essential for CDK1 activation during resumption of meiosis. Cdc25a −/− mice are embryonic lethal and therefore a role for CDC25A in meiosis is unknown. We report that activation of CDK1 results in a maturation-associated decrease in the amount of CDC25A protein, but not Cdc25a mRNA, such that little CDC25A is present by metaphase I. In addition, expression of exogenous CDC25A overcomes cAMP-mediated maintenance of meiotic arrest. Microinjection of Gfp-Cdc25a and Gpf-Cdc25b mRNAs constructs reveals that CDC25A is exclusively localized to the nucleus prior to nuclear envelope breakdown (NEBD). In contrast, CDC25B localizes to cytoplasm in GV-intact oocytes and translocates to the nucleus shortly before NEBD. Over-expressing GFP-CDC25A, which compensates for the normal maturation-associated decrease in CDC25A, blocks meiotic maturation at MI. This MI block is characterized by defects in chromosome congression and spindle formation and a transient reduction in both CDK1 and MAPK activities. Lastly, RNAi-mediated reduction of CDC25A results in fewer oocytes resuming meiosis and reaching MII. These data demonstrate that CDC25A behaves differently during female meiosis than during mitosis, and moreover, that CDC25A has a function in resumption of meiosis, MI spindle formation and the MI-MII transition. Thus, both CDC25A and CDC25B are critical for meiotic maturation of 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.     

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.     

The CDC14A phosphatase regulates oocyte maturation in mouse

The final steps of oogenesis occur during oocyte maturation that generates fertilization-competent haploid eggs capable of supporting embryonic development. Cyclin-dependent kinase 1 (CDK1) drives oocyte maturation and its activity and actions on substrates are tightly regulated. CDC14 is a dual-specificity phosphatase that reduces CDK1 activity and reverses the actions of CDK1 during mitosis. In budding yeast, Cdc14 is essential for meiosis, but it is not known whether its mammalian homolog CDC14A is required for meiosis in females. Here, we report that CDC14A is concentrated in the nucleus of meiotically incompetent mouse oocytes but is dispersed throughout meiotically competent oocytes. During meiotic progression CDC14A has no specific sub-cellular localization except between metaphase of meiosis I (Met I) and metaphase of meiosis II (Met II) when it co-localizes with the central portion of the meiotic spindle. Over-expression of CDC14A generally delays meiotic progression after resumption of meiosis whereas microinjection of oocytes with an antibody against CDC14A specifically delays exit from Met I. Each of these perturbations generates eggs with chromosome alignment abnormalities and eggs that were injected with the CDC14A antibody had an elevated incidence of aneuploidy. Collectively, these data suggest that CDC14A regulates oocyte maturation and functions to promote the meiosis I-to-meiosis II transition as its homolog does in budding yeast.     

A role for cyclin A1 in the activation of MPF and G2-M transition during meiosis of male germ cells in mice

Cell-cycle transition at G2-M is controlled by MPF (M-phase-promoting factor), a complex consisting of the Cdc2 kinase and a B-type cyclin. We have shown that in mice, targeted disruption of an A-type cyclin gene, cyclin A1, results in a block of spermatogenesis prior to the entry into metaphase I. The meiotic arrest is accompanied by a defect in Cdc2 kinase activation at the G2--M transition, raising the possibility that a cyclin A1-dependent process dictates the activation of MPF. Here we show that like Cdc2, the expression of B-type cyclins is retained in cyclin A1-deficient spermatocytes, while their associated kinases are kept at inactive states. Treatment of arrested germ cells with the protein phosphatase type-1 and -2A inhibitor okadaic acid restores the MPF activity and induces entry into M phase and the formation of normally condensed chromosome bivalents, concomitant with hyperphosphorylation of Cdc25 proteins. Conversely, inhibition of tyrosine phosphatases, including Cdc25s, by vanadate suppresses the okadaic acid-induced metaphase induction. The highest levels of Cdc25A and Cdc25C expression and their subcellular localization during meiotic prophase coincide with that of cyclin A1, and when overexpressed in HeLa cells, cyclin A1 coimmunoprecipitates with Cdc25A. Furthermore, the protein kinase complexes consisting of cyclin A1 and either Cdc2 or Cdk2 phosphorylate both Cdc25A and Cdc25C in vitro. These results suggest that in normal meiotic male germ cells, cyclin A1 participates in the regulation of other protein kinases or phosphatases critical for the G2-M transition. In particular, it may be directly involved in the initial amplification of MPF through the activating phosphorylation on Cdc25 phosphatases.     

Meiosis requires a translational positive loop where CPEB1 ensues its replacement by CPEB4

Meiotic progression is driven by the sequential translational activation of maternal messenger RNAs stored in the cytoplasm. This activation is mainly induced by the cytoplasmic elongation of their poly(A) tails, which is mediated by the cytoplasmic polyadenylation element (CPE) present in their 3′ untranslated regions. Although polyadenylation in prophase I and metaphase I is mediated by the CPE‐binding protein 1 (CPEB1), this protein is degraded during the first meiotic division. Thus, raising the question of how the cytoplasmic polyadenylation required for the second meiotic division is achieved. In this work, we show that CPEB1 generates a positive loop by activating the translation of CPEB4 mRNA, which, in turn, replaces CPEB1 and drives the transition from metaphase I to metaphase II. We further show that CPEB1 and CPEB4 are differentially regulated by phase‐specific kinases, generating the need of two sequential CPEB activities to sustain cytoplasmic polyadenylation during all the meiotic phases. Altogether, this work defines a new element in the translational circuit that support an autonomous transition between the two meiotic divisions in the absence of DNA replication.     

The Cdc14 phosphatase and the FEAR network control meiotic spindle disassembly and chromosome segregation

During meiosis, DNA replication is followed by two consecutive rounds of chromosome segregation. Cells lacking the protein phosphatase CDC14 or its regulators, SPO12 and SLK19, undergo only a single meiotic division, with some chromosomes segregating reductionally and others equationally. We find that this abnormal chromosome behavior is due to an uncoupling of meiotic events. Anaphase I spindle disassembly is delayed in cdc14-1, slk19Delta, or spo12Delta mutants, but the chromosome segregation cycle continues, so that both meiotic chromosome segregation phases take place on the persisting meiosis I spindle. Our results show that Cdc14, Slk19, and Spo12 are not only required for meiosis I spindle disassembly but also play a pivotal role in establishing two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.     

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome–cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development.     

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.     

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

What are the spermatocyte's requirements for successful meiotic division?

The consequences of error during meiotic division in spermatogenesis can be serious: aneuploid spermatozoa, embryonic lethality, and developmental abnormalities. Recombination between homologs is essential to ensure normal segregation; thus the spermatocyte must time division precisely so that it occurs after recombination between chromosomes and accumulation of the cell-cycle machinery necessary to ensure an accurate segregation of chromosomes. We use two systems to investigate meiotic division during spermatogenesis in the mouse: pharmacological induction of meiotic metaphase in cultured spermatocytes and transillumination-mediated dissection of stage XII seminiferous tubule segments to monitor progress through the division phase. By these approaches we can assess timing of acquisition of competence for the meiotic division phase and the temporal order of events as division proceeds. Competence for the meiotic division arises in the mid-pachytene stage of meiotic prophase, after chromosomes have synapsed and coincident with the accumulation of the cell-cycle regulatory protein CDC25C. The activity of both MPF and topoisomerase II are required. The earliest hallmarks of the division phase are nuclear envelope breakdown, followed by phosphorylation of histone H3 and chromosome condensation. These events are likely to be monitored by checkpoint mechanisms since checkpoint proteins can be localized in nuclei and DNA-damaging agents delay entry into the meiotic division phase. Understanding how the spermatocyte regulates its entry into the meiotic division phase can help clarify the natural mechanisms ensuring accurate chromosome segregation and preventing aneuploidy. J. Exp. Zool. (Mol. Dev. Evol.) 285:243-250, 1999.     

The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation

In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1+ as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.     

Cdc25B phosphatase participates in maintaining metaphase II arrest in mouse oocytes

Cdc25B is an essential regulator for meiotic resumption in mouse oocytes. However, the role of this phosphatase during the later stage of the meiotic cell cycle is not known. In this study, we investigated the role of Cdc25B during metaphase II (MII) arrest in mouse oocytes. Cdc25B was extensively phosphorylated during MII arrest with an increase in the phosphatase activity toward Cdk1. Downregulation of Cdc25B by antibody injection induced the formation of a pronucleus-like structure. Conversely, overexpression of Cdc25B inhibited Ca²⁺-mediated release from MII arrest. Moreover, Cdc25B was immediately dephosphorylated and hence inactivated during MII exit, suggesting that Cdk1 phosphorylation is required to exit from MII arrest. Interestingly, this inactivation occurred prior to cyclin B degradation. Taken together, our data demonstrate that MII arrest in mouse oocytes is tightly regulated not only by the proteolytic degradation of cyclin B but also by dynamic phosphorylation of Cdk1.     

Xtr, a plural tudor domain-containing protein, is involved in the translational regulation of maternal mRNA during oocyte maturation in Xenopus laevis

Xtr in the fertilized eggs of Xenopus has been demonstrated to be a member of a messenger ribonucleoprotein (mRNP) complex that plays a crucial role in karyokinesis during cleavage. Since the Xtr is also present both in oocytes and spermatocytes and its amount increases immediately after spematogenic cells enter into the meiotic phase, this protein was also predicted to act during meiotic progression. Taking advantage of Xenopus oocytes' large size to microinject anti-Xtr antibody into them for inhibition of Xtr function, we examined the role of Xtr in meiotic progression of oocytes. Microinjection of anti-Xtr antibody into immature oocytes followed by reinitiation of oocyte maturation did not affect germinal vesicle break down and the oscillation of Cdc2/cyclin B activity during meiotic progression but caused abnormal spindle formation and chromosomal alignment at meiotic metaphase I and II. Immunoprecipitation of Xtr showed the association of Xtr with FRGY2 and mRNAs such as RCC1 and XL-INCENP mRNAs, which are involved in the progression of karyokinesis. When anti-Xtr antibody was injected into oocytes, translation of XL-INCENP mRNA, which is known to be repressed in immature oocytes and induced after reinitiation of oocyte maturation, was inhibited even if the oocytes were treated with progesterone. A similar translational regulation was observed in oocytes injected with a reporter mRNA, which was composed of an enhanced green fluorescent protein open reading frame followed by the 3' untranslational region (3'UTR) of XL-INCENP mRNA. These results indicate that Xtr regulates the translation of XL-INCENP mRNA through its 3'UTR during meiotic progression of oocyte.     

Regulation of the Aurora B chromosome passenger protein complex during oocyte maturation in Xenopus laevis

The dynamics of the Aurora B protein kinase during Xenopus oocyte meiotic maturation were examined. Resting G2 oocytes express inactive Aurora B that is not associated with other subunits of the chromosome passenger complex (CPC). Activity increases near the time of germinal vesicle breakdown in progesterone-treated oocytes, and this increase is correlated with the synthesis of inner centromere protein (INCENP) and survivin, components of the CPC. Ablation of INCENP synthesis led to the failure of progesterone treatment to activate Aurora B, but biochemical progression through the meiosis I-to-II transition and arrest at metaphase II were not affected. At fertilization, Aurora B was deactivated in concert with the degradation of INCENP, and the levels of Aurora B kinase activity and INCENP oscillated in subsequent embryonic cell cycles. Prevention of the decrease in Aurora B activity at fertilization by expression of ectopic wild-type INCENP, but not kinase-dead Aurora B INCENP, blocked calcium-induced exit from metaphase arrest in egg extracts.     

CDC25B acts as a potential target of PRKACA in fertilized mouse eggs

Protein kinase A (PRKACA) has been documented as a pivotal regulator in meiosis and mitosis arrest. Although our previous work has established that PRKACA regulates cell cycle progression of mouse fertilized eggs by inhibiting M-phase promoting factor (MPF), little is known about the intermediate factor between PRKACA and MPF in the mitotic cell cycle. In this study, we investigated the role of the PRKACA/CDC25B pathway on the early development of mouse fertilized eggs. Overexpression of unphosphorylatable CDC25B mutant (Cdc25b-S321A or Cdc25b-S229A/S321A) rapidly caused G2-phase eggs to enter mitosis. Microinjection of either Cdc25b-WT or Cdc25b-S229A mRNA also promoted G2/M transition, but much less efficiently than Cdc25b-S321A and Cdc25b-S229A/S321A. Moreover, mouse fertilized eggs overrode the G2 arrest by microinjection of either Cdc25b-S321A or Cdc25b-S229A/S321A mRNA, which efficiently resulted in MPF activation by directly dephosphorylating CDC2A-Tyr15, despite culture under conditions that maintained exogenous dibutyryl cAMP. Using a highly specific antibody against phospho-Ser321 of CDC25B in Western blotting, we showed that CDC25B-Ser321 was phosphorylated at the G1 and S phases, whereas Ser321 was dephosphorylated at the G2 and M phases in vivo. Our findings identify CDC25B as a potential target of PRKACA and show that PRKACA regulates G2/M transition by phosphorylating CDC25B-Ser321 but not CDC25B-Ser229 on the first mitotic division of mouse fertilized eggs.