Cell cycle control by daf-21/Hsp90 at the first meiotic prophase/metaphase boundary during oogenesis in Caenorhabditis elegans

DAF-21, a Caenorhabditis elegans homologue of Hsp90, is expressed primarily in germline cells. Although mutations in the daf-21 gene affect animal fertility, its cellular roles have remained elusive. To phenocopy daf-21 mutations, we impaired the daf-21 function by RNA interference (RNAi), and found that oocytes skipped the diakinesis arrest and displayed a defective diakinesis arrest, which led to the production of endomitotic oocytes with polyploid chromosomes (Emo phenotype). The same Emo phenotype was also observed with RNAi against wee-1.3. To identify a cause for Emo, we examined the CDK-1 (Cdc2) phosphorylation status in Emo animals, since CDK-1 is a key regulator of the prophase/metaphase transition and is kept inactivated by WEE-1.3 kinase during prophase. We immunostained both daf-21(RNAi) and wee-1.3(RNAi) animals with anti-phosphorylated-CDK-1 antibody and observed no detectable phosphates on CDK-1 in either of the animals. We also examined WEE-1.3 expression in daf-21(RNAi) and found a significant reduction of WEE-1.3. These results indicate that CDK-1 was not phosphorylated in either daf-21(RNAi) or wee-1.3(RNAi) animals, and suggest that daf-21 was necessary for producing functional WEE-1.3. Thus, all together, we propose that DAF-21 indirectly regulates the meiotic prophase/metaphase transition during oocyte development by ensuring the normal function of WEE-1.3.     

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.     

Activation of both Mos and Cdc25 is required for G2-M transition in perch oocyte

Resumption of meiosis from diplotene arrest during the first meiotic prophase in vertebrate oocytes is universally controlled by MPF, a heterodimer of Cdk1 and cyclin B. Activation of MPF depends on the withdrawal of Cdk1 inhibition by Wee1/Myt1 kinase on the one hand and the activation of Cdk1 by Cdc25 phosphatase on the other. It is relevant to know whether both these pathways are necessary to rescue diplotene arrest or if either one of them is sufficient. In MIH (17alpha, 20beta dihydroxy-4-pregnen-3-one) incubated perch (Anabas testudineus) oocytes we have examined these possibilities. Perch oocyte extract following MIH incubation showed a significant increase in Myt1 phosphorylation from 12 to 16 hr indicating its progressive deactivation. MIH induced Mos expression markedly increased at 16 hr effecting 95% GVBD. Cycloheximide inhibited MIH induced Mos expression and its phosphorylation, which in turn reduced Myt1 phosphorylation and GVBD. Myt1 phosphorylation was blocked in Mos immunodepleted oocytes. All these suggest the involvement of Mos in Myt1 phosphorylation. Oocytes incubated in MIH for 16 hr activated Cdc25, but such activation could not rescue the inhibition of GVBD due to Myt1 in Mos immunodepleted oocytes. Blocking Cdc25 with an antisense oligo significantly inhibited GVBD even though Myt1 remained deactivated during this period. Taken together, our findings indicate that MIH requires both pathways for perch oocyte maturation: the expression and activation of Mos, which is linked to Myt1 deactivation on the one hand, and the activation of Cdc25 on the other, as blocking either pathway compromised G2-M transition in perch oocytes.     

Dominant mutations in the Caenorhabditis elegans Myt1 ortholog wee-1.3 reveal a novel domain that controls M-phase entry during spermatogenesis

Regulatory phosphorylation of the Cdc2p kinase by Wee1p-type kinases prevents eukaryotic cells from entering mitosis or meiosis at an inappropriate time. The canonical Wee1p kinase is a soluble protein that functions in the eukaryotic nucleus. All metazoa also have a membrane-associated Wee1p-like kinase named Myt1, and we describe the first genetic characterization of this less well-studied kinase. The Caenorhabditis elegans Myt1 ortholog is encoded by the wee-1.3 gene, and six dominant missense mutants prevent primary spermatocytes from entering M phase but do not affect either oocyte meiosis or any mitotic division. These six dominant wee-1.3(gf) mutations are located in a four amino acid region near the C terminus and they cause self-sterility of hermaphrodites. Second-site intragenic suppressor mutations in wee-1.3(gf) restore self-fertility to these dominant sterile hermaphrodites, permitting genetic dissection of this kinase. Ten intragenic wee-1.3 suppressor mutations were recovered and they form an allelic series that includes semi-dominant, hypomorphic and null mutations. These mutants reveal that WEE-1.3 protein is required for embryonic development, germline proliferation and initiation of meiosis during spermatogenesis. This suggests that a novel, sperm-specific pathway negatively regulates WEE-1.3 to allow the G2/M transition of male meiosis I, and that dominant wee-1.3 mutants prevent this negative regulation.     

Expression of cell-cycle regulators during Xenopus oogenesis

In full-grown Xenopus oocytes, cell-cycle regulators and an inactive form of maturation/M phase promoting factor (pre-MPF) are stored ready to bring about a specific cell cycle for oocyte maturation. We examined the expression pattern of these cell-cycle regulators as well as pre-MPF formation during oogenesis. Cdc2 and Cyclin B2 were already present in stage I oocytes and pre-MPF formation was also detected in stage I oocytes. Some negative regulators of MPF, Myt1 and Chk1, were synthesized early in oogenesis. In contrast, positive regulators of MPF, MEK, MAPK and Cdc25C, were mainly synthesized late in oogenesis. Northern blotting analysis suggested that the synthesis of these cell-cycle regulators was controlled by translation.     

Wee1B is an oocyte-specific kinase involved in the control of meiotic arrest in the mouse

In most species, the meiotic cell cycle is arrested at the transition between prophase and metaphase through unclear somatic signals. Activation of the Cdc2-kinase component of maturation promoting factor (MPF) triggers germinal vesicle breakdown after the luteinizing hormone (LH) surge and reentry into the meiotic cell cycle. Although high levels of cAMP and activation of protein kinase A (PKA) play a critical role in maintaining an inactive Cdc2, the steps downstream of PKA in the oocyte remain unknown. Using a small-pool expression-screening strategy, we have isolated several putative PKA substrates from a mouse oocyte cDNA library. One of these clones encodes a Wee1-like kinase that prevents progesterone-induced oocyte maturation when expressed in Xenopus oocytes. Unlike the widely expressed Wee1 and Myt1, mWee1B mRNA and its protein are expressed only in oocytes, and mRNA downregulation by RNAi injection in vitro or transgenic overexpression of RNAi in vivo causes a leaky meiotic arrest. Ser15 residue of mWee1B is the major PKA phosphorylation site in vitro, and the inhibitory effects of the kinase are enhanced when this residue is phosphorylated. Thus, mWee1B is a key MPF inhibitory kinase in mouse oocytes, functions downstream of PKA, and is required for maintaining meiotic arrest.     

A critical balance between Cyclin B synthesis and Myt1 activity controls meiosis entry in Xenopus oocytes

In fully grown oocytes, meiosis is arrested at first prophase until species-specific initiation signals trigger maturation. Meiotic resumption universally involves early activation of M phase-promoting factor (Cdc2 kinase-Cyclin B complex, MPF) by dephosphorylation of the inhibitory Thr14/Tyr15 sites of Cdc2. However, underlying mechanisms vary. In Xenopus oocytes, deciphering the intervening chain of events has been hampered by a sensitive amplification loop involving Cdc2-Cyclin B, the inhibitory kinase Myt1 and the activating phosphatase Cdc25. In this study we provide evidence that the critical event in meiotic resumption is a change in the balance between inhibitory Myt1 activity and Cyclin B neosynthesis. First, we show that in fully grown oocytes Myt1 is essential for maintaining prophase I arrest. Second, we demonstrate that, upon upregulation of Cyclin B synthesis in response to progesterone, rapid inactivating phosphorylation of Myt1 occurs, mediated by Cdc2 and without any significant contribution of Mos/MAPK or Plx1. We propose a model in which the appearance of active MPF complexes following increased Cyclin B synthesis causes Myt1 inhibition, upstream of the MPF/Cdc25 amplification loop.     

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.     

Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

A gain-of-function mutation in oma-1, a C. elegans gene required for oocyte maturation,results in delayed degradation of maternal proteins and embryonic lethality

In vertebrates, oocytes undergo maturation, arrest in metaphase II, and can then be fertilized by sperm. Fertilization initiates molecular events that lead to the activation of early embryonic development. In Caenorhabditis elegans, where no delay between oocyte maturation and fertilization is apparent, oocyte maturation and fertilization must be tightly coordinated. It is not clear what coordinates the transition from an oocyte to an embryo in C. elegans, but regulated turnover of oocyte-specific proteins contributes to the process. We describe here a gain-of-function mutation (zu405) in a gene that is essential for oocyte maturation, oma-1. In wild type animals, OMA-1 protein is expressed at a high level exclusively in oocytes and newly fertilized embryos and is degraded rapidly after the first mitotic division. The zu405 mutation results in improper degradation of the OMA-1 protein in embryos. In oma-1(zu405) embryos, the C blastomere is transformed to the EMS blastomere fate, resulting in embryonic lethality. We show that degradation of several maternally supplied cell fate determinants, including SKN-1, PIE-1, MEX-3, and MEX-5, is delayed in oma-1(zu405) mutant embryos. In wild type embryos, SKN-1 functions in EMS for EMS blastomere fate specification. A decreased level of maternal SKN-1 protein in the C blastomere relative to EMS is believed to be responsible for this cell expressing the C, instead of the EMS, fate. Delayed degradation of maternal SKN-1 protein in oma-1(zu405) embryos and resultant elevated levels in C blastomere is likely responsible for the observed C-to-EMS blastomere fate transformation. These observations suggest that oma-1, in addition to its role in oocyte maturation, contributes to early embryonic development by regulating the temporal degradation of maternal proteins in early C. elegans embryos.     

Several signaling pathways are involved in the control of cattle oocyte maturation

The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment.     

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.     

Developmental control of oocyte maturation and egg activation in metazoan models

Production of functional eggs requires meiosis to be coordinated with developmental signals. Oocytes arrest in prophase I to permit oocyte differentiation, and in most animals, a second meiotic arrest links completion of meiosis to fertilization. Comparison of oocyte maturation and egg activation between mammals, Caenorhabditis elegans, and Drosophila reveal conserved signaling pathways and regulatory mechanisms as well as unique adaptations for reproductive strategies. Recent studies in mammals and C. elegans show the role of signaling between surrounding somatic cells and the oocyte in maintaining the prophase I arrest and controlling maturation. Proteins that regulate levels of active Cdk1/cyclin B during prophase I arrest have been identified in Drosophila. Protein kinases play crucial roles in the transition from meiosis in the oocyte to mitotic embryonic divisions in C. elegans and Drosophila. Here we will contrast the regulation of key meiotic events in oocytes.     

Gas6 downregulation impaired cytoplasmic maturation and pronuclear formation independent to the MPF activity

Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr(2+), Gas6-silenced MII oocytes had markedly reduced Ca(2+) oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation.     

Participation of MAPK, PKA and PP2A in the regulation of MPF activity in Bufo arenarum oocytes

The objectives of the present paper were to study the involvement and possible interactions of both cAMP-PKA and protein phosphatases in Bufo arenarum oocyte maturation and to determine if these pathways are independent or not of the MAP kinase (MAPK) cascade. Our results indicated that the inhibition of PKA by treatment with H-89, an inhibitor of the catalytic subunit of PKA, was capable of inducing GVBD in a dose-dependent manner by a pathway in which Cdc25 phosphatase but not the MAPK cascade is involved. The injection of 50 nl of H-89 10 μM produced GVBD percentages similar to those obtained with treatment with progesterone. In addition, the assays with okadaic acid (OA), a PP2A inhibitor, significantly enhanced the percentage of oocytes that resumed meiosis by a signal transducing pathway in which the activation of the MEK-MAPK pathway is necessary, but in which Cdc25 phosphatase was not involved. Treatment with H-89, was able to overcome the inhibitory effect of PKA on GVBD; however, the inhibition of Cdc25 activity with NaVO3 was able to overcome the induction of GVBD by H-89. Although the connections between PKA and other signalling molecules that regulate oocytes maturation are still unclear, our results suggest that phosphatase Cdc25 may be the direct substrate of PKA. In Xenopus oocytes it was proposed that PP2A, a major Ser/Thr phosphatase present, is a negative regulator of Cdc2 activation. However, in Bufo arenarum oocytes, inhibition of Cdc25 with NaVO? did not inhibit OA-induced maturation, suggesting that the target of PP2A was not the Cdc25 phosphatase. MAPK activation has been reported to be essential in Xenopus oocytes GVBD. In B. arenarum oocytes we demonstrated that the inhibition of MAPK by PD 98059 prevented the activation of MPF induced by OA, suggesting that the activation of the MAPK cascade produced an inhibition of Myt1 and, in consequence, the activation of MPF without participation of the Cdc25 phosphatase. Our results suggest that in incompetent oocytes of B. arenarum two signal transduction pathways may be involved in the control of MPF activation: (1) the inhibition of phosphatase 2A that through the MEK-MAPK pathway regulates the activity of the Myt1; and (2) the inhibition of AMPc-PKA, which affects the activity of the Cdc25 phosphatase.