Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes cultured in?vitro

The present study was aimed to investigate whether a decrease of nitric oxide (NO) level is beneficial for sponateous resumptiom of meiosis in diplotene-arrested oocytes cultured in vitro. For this purpose, diplotene-arrested oocytes were collected from ovary of immature female rats after a single subcutaneous injection of 20 IU pregnant mare’s serum gonadotropins (PMSG) for 48 h. In vitro effects of S-nitroso-l-acetyl penicillamine (SNAP; an NO donor) and aminoguanidine (AG; an inducible NOS [iNOS] inhibitor), intracellular NO, cyclic guanosine monophosphate (cGMP), Cdc25B, Thr-14/Tyr-15 and Thr-161 phosphorylated cyclin-dependent kinase-1 (CDK1), and cyclin B1 levels were analyzed. The SNAP inhibited spontaneous meiotic resumption form diplotene arrest in a concentration-dependent manner, while AG-induced meiotic resumption form diplotene in 0.1 mmol/L 3-isobutyl-1-methylxanthine (IBMX)-treated oocytes in a concentration-dependent manner. The intracellular NO as well as cGMP levels were decreased significantly during spontaneous meiotic resumption from diplotene arrest. The reduction of Cdc25B expression level was associated with the accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. However, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels were reduced significantly during meiotic resumption from diplotene arrest. Taken together, these data suggest that the inhibition of iNOS expression leads to a decrease of NO and cGMP levels thereby decreasing Cdc25B level. The reduced CDC25 B level leads to accumulation of Thr-14/Tyr-15 phosphorylated CDK1 level. As a result, Thr-161 phosphorylated CDK1 as well as cyclin B1 levels are decreased leading to maturation-promoting factor (MPF) inactivation. The inactive MPF finally induced meiotic resumption from diplotene stage in rat oocytes cultured in vitro.     

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.     

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro-metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD-inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ-tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.     

Aurora kinase A controls meiosis I progression in mouse oocytes

Aurora kinase A (AURKA), which is a centrosome-localized serine/threonine kinase crucial for cell cycle control, is critically involved in centrosome maturation and spindle assembly in somatic cells. Active T288 phosphorylated AURKA localizes to the centrosome in the late G2 and also spreads to the minus ends of mitotic spindle microtubules. AURKA activates centrosomal CDC25B and recruits cyclin B1 to centrosomes. We report here functions for AURKA in meiotic maturation of mouse oocytes, which is a model system to study the G2 to M transition. Whereas AURKA is present throughout the entire GV-stage oocyte with a clear accumulation on microtubule organizing centers (MTOC), active AURKA becomes entirely localized to MTOCs shortly before germinal vesicle breakdown. In contrast to somatic cells in which active AURKA is present at the centrosomes and minus ends of microtubules, active AURKA is mainly located on MTOCs at metaphase I (MI) in oocytes. Inhibitor studies using Roscovitine (CDK1 inhibitor), LY-294002 (PI3K inhibitor) and SH-6 (PKB inhibitor) reveal that activation of AURKA localized on MTOCs is independent on PI3K-PKB and CDK1 signaling pathways and MOTC amplification is observed in roscovitine- and SH-6- treated oocytes that fail to undergo nuclear envelope breakdown. Moreover, microinjection of Aurka mRNA into GV-stage oocytes cultured in 3-isobutyl-1-methyl xanthine (IBMX)-containing medium to prevent maturation also results in MOTC amplification in the absence of CDK1 activation. Over-expression of AURKA also leads to formation of an abnormal MI spindle, whereas RNAi-mediated reduction of AURKA interferes with resumption of meiosis and spindle assembly. Results of these experiments indicate that AURKA is a critical MTOC-associated component involved in resumption of meiosis, MTOC multiplication, proper spindle formation and the metaphase I-metaphase II transition.     

CDC6 requirement for spindle formation during maturation of mouse oocytes

A master regulator of DNA replication, CDC6 also functions in the DNA-replication checkpoint by preventing DNA rereplication. Cyclin-dependent kinases (CDKs) regulate the amount and localization of CDC6 throughout the cell cycle; CDC6 phosphorylation after DNA replication initiation leads to its proteolysis in yeast or translocation to the cytoplasm in mammals. Overexpression of CDC6 during the late S phase prevents entry into the M phase by activating CHEK1 kinase that then inactivates CDK1/cyclin B, which is essential for the G2/M-phase transition. We analyzed the role of CDC6 during resumption of meiosis in mouse oocytes, which are arrested in the first meiotic prophase with low CDK1/cyclin B activity; this is similar to somatic cells at the G2/M-phase border. Overexpression of CDC6 in mouse oocytes does not prevent resumption of meiosis. The RNA interference-mediated knockdown of CDC6, however, reveals a new and unexpected function for CDC6; namely, it is essential for spindle formation in mouse oocytes.     

CDC20 downregulation impairs spindle morphology and causes reduced first polar body emission during bovine oocyte maturation

The cell division cycle protein 20 (CDC20) is an essential regulator of cell division, encoded by the CDC20 gene. However, the role of CDC20 in bovine oocyte maturation is unknown. In this study, CDC20 morpholino antisense oligonucleotides (MOs) were microinjected into the cytoplasm of bovine oocytes to block the translation of CDC20 mRNA. CDC20 downregulation significantly reduced the rate of first polar body emission (PB1). Further analysis indicated that oocytes treated with CDC20 MO arrested before or at meiotic stage I with abnormal spindles. To further confirm the functions of CDC20 during oocyte meiotic division, CDC20 MOs were microinjected into oocytes together with a supplementary PB1. The results showed that newly synthesized CDC20 was not necessary at the meiosis II-to-anaphase II transition. Our data suggest that CDC20 is required for spindle assembly, chromosomal segregation, and PB1 extrusion during bovine oocyte maturation.     

Cdc25C expression in meiotically competent and incompetent goat oocytes

Change in Cdc25C expression and localization during maturation and meiotic competence acquisition was investigated in goat oocytes. Western blot analysis revealed that Cdc25C is constitutively expressed throughout meiosis in competent goat oocytes, with changes in its phosphorylation level. Cdc25C was detected at 55 and 70 kDa, representing the nonphosphorylated form and the hyperphosphorylated active form, respectively. During the G2-M transition at meiosis resumption, Cdc25C was hyperphosphorylated as evidenced by a clear shift from 55 to 70 kDa. Okadaic acid which induced premature meiosis resumption associated with MPF activation also involved a premature shift from 55 to 70 kDa in goat competent oocytes. After artificial activation of goat oocytes, Cdc25C returned to its 55 kDa form. By indirect immunofluorescence, Cdc25C was found essentially localized in the nucleus at the germinal vesicle stage, suggesting that Cdc25C functions within the nucleus to regulate MPF activation. Concomitantly with germinal vesicle breakdown, Cdc25C was redistributed throughout the cytoplasm. The amount of Cdc25C, very low in incompetent oocytes, increased with meiosis competence acquisition. On the other hand, during oocyte growth while the expression of Cdc25C increased, its phosphorylation level increased concomitantly as well as its nuclear translocation. These results suggest that meiosis resumption needs a sufficient amount of Cdc25C which must be completely phosphorylated and nuclear and that the amount of Cdc25C may be a limiting factor for meiotic competence acquisition. We could consider that Cdc25C nuclear translocation and phosphorylation, during oocyte growth, prepare the oocytes in advance for the G2-M phase transition occurring during meiosis resumption.     

Specialization of CDK1 and cyclin B paralog functions in a coenocystic mode of oogenic meiosis

Oogenesis in the urochordate, Oikopleura dioica, occurs in a large coenocyst in which vitellogenesis precedes oocyte selection in order to adapt oocyte production to nutrient conditions. The animal has expanded Cyclin-Dependant Kinase 1 (CDK1) and Cyclin B paralog complements, with several expressed during oogenesis. Here, we addressed functional redundancy and specialization of CDK1 and cyclin B paralogs during oogenesis and early embryogenesis through spatiotemporal analyses and knockdown assays. CDK1a translocated from organizing centres (OCs) to selected meiotic nuclei at the beginning of the P4 phase of oogenesis, and its knockdown impaired vitellogenesis, nurse nuclear dumping, and entry of nurse nuclei into apoptosis. CDK1d-Cyclin Ba translocated from OCs to selected meiotic nuclei in P4, drove meiosis resumption and promoted nuclear envelope breakdown (NEBD). CDK1d-Cyclin Ba was also involved in histone H3S28 phosphorylation on centromeres and meiotic spindle assembly through regulating Aurora B localization to centromeres during prometaphase I. In other studied species, Cyclin B3 commonly promotes anaphase entry, but we found O. dioica Cyclin B3a to be non-essential for anaphase entry during oogenic meiosis. Instead, Cyclin B3a contributed to meiotic spindle assembly though its loss could be compensated by Cyclin Ba.     

CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation

Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We report that CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdc2a mRNA, which activates CDC2A, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDC2A following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible.     

SGK1 is essential for meiotic resumption in mammalian oocytes

In mammalian females, oocytes are stored in the ovary and meiosis is arrested at the diplotene stage of prophase I. When females reach puberty oocytes are selectively recruited in cycles to grow, overcome the meiotic arrest, complete the first meiotic division and become mature (ready for fertilization). At a molecular level, the master regulator of prophase I arrest and meiotic resumption is the maturation-promoting factor (MPF) complex, formed by the active form of cyclin dependent kinase 1 (CDK1) and Cyclin B1. However, we still do not have complete information regarding the factors implicated in MPF activation.In this study we document that out of three mammalian serum-glucocorticoid kinase proteins (SGK1, SGK2, SGK3), mouse oocytes express only SGK1 with a phosphorylated (active) form dominantly localized in the nucleoplasm. Further, suppression of SGK1 activity in oocytes results in decreased CDK1 activation via the phosphatase cell division cycle 25B (CDC25B), consequently delaying or inhibiting nuclear envelope breakdown. Expression of exogenous constitutively active CDK1 can rescue the phenotype induced by SGK1 inhibition. These findings bring new insights into the molecular pathways acting upstream of MPF and a better understanding of meiotic resumption control by presenting a new key player SGK1 in mammalian oocytes.     

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.     

Specific deletion of Cdc42 does not affect meiotic spindle organization/migration and homologous chromosome segregation but disrupts polarity establishment and cytokinesis in mouse oocytes

Mammalian oocyte maturation is distinguished by highly asymmetric meiotic divisions during which a haploid female gamete is produced and almost all the cytoplasm is maintained in the egg for embryo development. Actin-dependent meiosis I spindle positioning to the cortex induces the formation of a polarized actin cap and oocyte polarity, and it determines asymmetric divisions resulting in two polar bodies. Here we investigate the functions of Cdc42 in oocyte meiotic maturation by oocyte-specific deletion of Cdc42 through Cre-loxP conditional knockout technology. We find that Cdc42 deletion causes female infertility in mice. Cdc42 deletion has little effect on meiotic spindle organization and migration to the cortex but inhibits polar body emission, although homologous chromosome segregation occurs. The failure of cytokinesis is due to the loss of polarized Arp2/3 accumulation and actin cap formation; thus the defective contract ring. In addition, we correlate active Cdc42 dynamics with its function during polar body emission and find a relationship between Cdc42 and polarity, as well as polar body emission, in mouse oocytes.     

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.     

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.     

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.     

Regulation of Cdc25C Activity During the Meiotic G2/M Transition

The Cdc25C phosphatase is a key activator of Cdc2/cyclin B that controls M-phase entry in eukaryotic cells. Here we discuss the regulation of Cdc25C by phosphorylation during the meiotic maturation of Xenopus oocytes. In G2 arrested oocytes, Cdc25C is phosphorylated on Ser287 and associated with 14-3-3 proteins. Entry of the oocytes into M-phase of meiosis is triggered by progesterone, which activates a signaling pathway leading to the dephosphorylation of Ser287, probably mediated by the PP1 phosphatase. The activation of Cdc25C during oocyte maturation correlates also with its phosphorylation on multiple sites. These phosphorylations involve several signaling pathways, including Polo kinases and MAP kinases, and might require also the inhibition of the PP2A phosphatase. Finally, Cdc25C is further phosphorylated by its substrate Cdc2/cyclin B, as part of an auto-amplification loop that ensures the high Cdc2/cyclin B activity level required to drive the oocyte through the meiotic cell cycle.     

Role of Greatwall kinase in release of mouse oocytes from diplotene arrest

In eukaryotes, mitosis entry is induced by activation of maturation‐promoting factor (MPF), which is regulated by a network of kinases and phosphatases. It has been suggested that Greatwall (GWL) kinase was crucial for the M‐phase entry and could maintain cyclin B–Cdc2 activity through regulation of protein phosphatase 2A (PP2A), a counteracting phosphatase of MPF. Here, the role of GWL was assessed during release of mouse oocytes from prophase I arrest. GWL was crucial for meiotic maturation in mouse oocytes. As a positive regulator for meiosis resumption, GWL was continually expressed in germinal vesicle (GV) and MII stage oocytes and two‐cell stage embryos. Additionally, GWL localized to the nucleus and dispersed into cytoplasm during GV breakdown (GVBD). Furthermore, downregulation of GWL or overexpression of catalytically‐inactive GWL inhibited partial meiotic maturation. This prophase I arrest induced by GWL depletion could be rescued by the PP2A inhibition. However, both GWL‐depleted and rescued oocytes had severe spindle defects that hardly reached MII. In contrast, oocytes overexpressing wild‐type GWL resumed meiosis and progressed to MII stage. Thus, our data demonstrate that GWL acts in a pathway with PP2A which is essential for prophase I exit and metaphase I microtubule assembly in mouse oocytes.