Mitosis-specific monoclonal antibodies block cleavage in amphibian embryos

By microinjecting monoclonal antibodies that bind specifically to mitotic and meiotic cells of a variety of species, we studied the biological activity of antigens recognized by these antibodies. The antibodies recognize a family of phosphoprotein antigens that are found throughout the cytoplasm of mitotic cells and particularly at microtubule organizing centers, including centrosomes and kinetochores. Their binding is dependent on phosphorylation of the polypeptides. Immunoglobulins were introduced into Xenopus laevis and Rana pipiens oocytes or cleaving embryos using glass micropipettes. The ability of the antibody-injected oocytes to undergo mitosis or meiosis was compared with those injected with control mouse immunoglobulins. The antibodies failed to block chromosome condensation and germinal vesicle breakdown in progesterone-treated oocytes. However, functional mitotic spindles were not assembled in cleavage stage frog embryos injected with antibodies. In vitro, the binding of the antibodies to the antigens inhibited the dephosphorylation of the antigens by alkaline phosphatase. The antibody binding to the activated microtubule organizing centers (MTOC) seems to block not only the nucleation of microtubules and the organization of the mitotic spindle, but also the dephosphorylation of proteins associated with the MTOC that normally occurs at the mitosis-G1 transition.     

Acentriolar microtubule organization centers and Ran‐mediated microtubule formation pathways are both required in porcine oocytes

Mammalian oocytes lack centrioles but can generate bipolar spindles using several different mechanisms. For example, mouse oocytes have acentriolar microtubule organization centers (MTOCs) that contain many components of the centrosome, and which initiate microtubule polymerization. On the contrary, human oocytes lack MTOCs and the Ran‐mediated mechanisms may be responsible for spindle assembly. Complete knowledge of the different mechanisms of spindle assembly is lacking in various mammalian oocytes. In this study, we demonstrate that both MTOC‐ and Ran‐mediated microtubule nucleation are required for functional meiotic metaphase I spindle generation in porcine oocytes. Acentriolar MTOC components, including Cep192 and pericentrin, were absent in the germinal vesicle and germinal vesicle breakdown stages. However, they start to colocalize to the spindle microtubules, but are absent in the meiotic spindle poles. Knockdown of Cep192 or inhibition of Polo‐like kinase 1 activity impaired the recruitment of Cep192 and pericentrin to the spindles, impaired microtubule assembly, and decreased the polar body extrusion rate. When the Ran^GTP gradient was perturbed by the expression of dominant negative or constitutively active Ran mutants, severe defects in microtubule nucleation and cytokinesis were observed, and the localization of MTOC materials in the spindles was abolished. These results demonstrate that the stepwise involvement of MTOC‐ and Ran‐mediated microtubule assembly is crucial for the formation of meiotic spindles in porcine oocytes, indicating the diversity of spindle formation mechanisms among mammalian oocytes.     

The dual-specificity phosphatase CDC14B bundles and stabilizes microtubules

The Cdc14 dual-specificity phosphatases regulate key events in the eukaryotic cell cycle. However, little is known about the function of mammalian CDC14B family members. Here, we demonstrate that subcellular localization of CDC14B protein is cell cycle regulated. CDC14B can bind, bundle, and stabilize microtubules in vitro independently of its catalytic activity. Basic amino acid residues within the nucleolar targeting domain are important for both retaining CDC14B in the nucleolus and preventing microtubule bundling. Overexpression of CDC14B resulted in the formation of cytoplasmic CDC14B and microtubule bundles in interphase cells. These microtubule bundles were resistant to microtubule depolymerization reagents and enriched in acetylated alpha-tubulin. Expression of cytoplasmic forms of CDC14B impaired microtubule nucleation from the microtubule organization center. CDC14B is thus a novel microtubule-bundling and -stabilizing protein, whose regulated subcellular localization may help modulate spindle and microtubule dynamics in mitosis.     

CDC25B蛋白亚细胞定位调控小鼠1-细胞期受精卵发育

为探讨小鼠细胞分裂周期25B（CDC25B）蛋白149位丝氨酸磷酸化状态对小鼠1 细胞期受精卵中CDC25B的亚细胞定位和发育的影响,应用定制的CDC25B-pS149位的 磷酸化和非磷酸化抗体检测小鼠1-细胞期受精卵各细胞时期的磷酸化和非磷酸化状 态;应用免疫荧光观察各期受精卵中CDC25B蛋白的定位情况;将质粒pEGFP-CDC25B -WT、pEGFP-CDC25B-S149A和pEGFP-CDC25B-S149D融合质粒及空载体质粒显微注射入 G1期受精卵中,观察不同显微注射组小鼠1-细胞期受精卵中外源性CDC25B蛋白亚细 胞定位．结果显示,CDC25B-S149位丝氨酸在G1和S期被磷酸化,在G2和M期去磷酸化 ．1-细胞期受精卵从G2向M期的转换过程中,发生了CDC25B向细胞核区的移位,到2- 细胞初期,部分CDC25B蛋白又从细胞核回到细胞浆．实验结果提示,小鼠1-细胞期受精卵G2/M期转换过程中,CDC25B 的S149位点磷酸化修饰可能是对CDC25B细胞内定 位及其活性的精确调节方式．     

Behavior of delta-tubulin during spindle formation in Xenopus oocytes: requirement of cytoplasmic dynein-dependent translocation

Vertebrate oocytes do not contain centrosomes and therefore form an acentrosomal spindle during oocyte maturation. gamma-Tubulin is known to be essential for nucleation of microtubules at centrosomes, but little is known about the behaviour and role of gamma-tubulin during spindle formation in oocytes. We first observed sequential localization of gamma-tubulin during spindle formation in Xenopus oocytes. gamma-Tubulin assembled in the basal regions of the germinal vesicle (GV) at the onset of germinal vesicle breakdown (GVBD) and remained on the microtubule-organizing centre (MTOC) until a complex of the MTOC and transient-microtubule array (TMA) reached the oocyte surface. Prior to bipolar spindle formation, oocytes formed an aggregation of microtubules and gamma-tubulin was concentrated at the centre of the aggregation. At the late stage of bipolar spindle formation, gamma-tubulin accumulated at each pole. Anti-dynein antibody disrupted the localization of gamma-tubulin, indicating that the translocation described above is dependent on dynein activity. We finally revealed that XMAP215, a microtubule-associated protein cooperating with gamma-tubulin for the assembly of microtubules, but not gamma-tubulin, was phosphorylated during oocyte maturation. These results suggest that gamma-tubulin is translocated by dynein to regulate microtubule organization leading to spindle formation and that modification of the molecules that cooperate with gamma-tubulin, but not gamma-tubulin itself, is important for microtubule reorganization.     

Stable kinetochore–microtubule attachments restrict MTOC position and spindle elongation in oocytes

In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball‐like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore–microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore–microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore–microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles. These findings suggest a role for stable kinetochore–microtubule attachments in fine‐tuning acentrosomal spindle bipolarity.     

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.     

Organization, nucleation, and acetylation of microtubules in Xenopus laevis oocytes: a study by confocal immunofluorescence microscopy

Anti-tubulin immunofluorescence and laser-scanning confocal microscopy were used to examine microtubule organization during Xenopus oogenesis (Dumont stages I-VI). Stage I oocytes contained a poorly ordered microtubule array, characterized by concentrations of microtubule in the cortex, surrounding the germinal vesicle, and associated with the mitochondrial mass. No focus of microtubule organization was detectable by optical sectioning or in microtubule regrowth experiments, suggesting that stage I oocytes lack a functional MTOC. The microtubule array becomes progressively more complex and polarized during oogenesis; an extensive array of microtubules and microtubule bundles was apparent in the animal hemisphere of stage VI oocytes, and a less ordered array was observed in the vegetal hemisphere. A dense network of microtubules surrounded the germinal vesicle, apparently extending from a tubulin- and microtubule-rich region of cytoplasm adjacent to the vegetal surface of the GV. The organization of microtubules in normal oocytes, in oocytes recovering from cold-induced microtubule depolymerization, and in enucleated oocytes, suggested that the germinal vesicle serves as an MTOC in stage VI oocytes. Antibodies to acetylated alpha-tubulin revealed numerous acetylated, presumably stable, microtubules in stage I and stage VI oocytes. The array of oocyte microtubules thus might function as a stable framework for the localization of developmentally important molecules and organelles during oogenesis.     

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.     

BRCA1 is required for meiotic spindle assembly and spindle assembly checkpoint activation in mouse oocytes

BRCA1 as a tumor suppressor has been widely investigated in mitosis, but its functions in meiosis are unclear. In the present study, we examined the expression, localization, and function of BRCA1 during mouse oocyte meiotic maturation. We found that expression level of BRCA1 was increased progressively from germinal vesicle to metaphase I stage, and then remained stable until metaphase II stage. Immunofluorescent analysis showed that BRCA1 was localized to the spindle poles at metaphase I and metaphase II stages, colocalizing with centrosomal protein gamma-tubulin. Taxol treatment resulted in the presence of BRCA1 onto the spindle microtubule fibers, whereas nocodazole treatment induced the localization of BRCA1 onto the chromosomes. Depletion of BRCA1 by both antibody injection and siRNA injection caused severely impaired spindles and misaligned chromosomes. Furthermore, BRCA1-depleted oocytes could not arrest at the metaphase I in the presence of low-dose nocodazole, suggesting that the spindle checkpoint is defective. Also, in BRCA1-depleted oocytes, gamma-tubulin dissociated from spindle poles and MAD2L1 failed to rebind to the kinetochores when exposed to nocodazole at metaphase I stage. Collectively, these data indicate that BRCA1 regulates not only meiotic spindle assembly, but also spindle assembly checkpoint, implying a link between BRCA1 deficiency and aneuploid embryos.     

CDC25B Overexpression Stabilises Centrin 2 and Promotes the Formation of Excess Centriolar Foci

CDK-cyclin complexes regulate centriole duplication and microtubule nucleation at specific cell cycle stages, although their exact roles in these processes remain unclear. As the activities of CDK-cyclins are themselves positively regulated by CDC25 phosphatases, we investigated the role of centrosomal CDC25B during interphase. We report that overexpression of CDC25B, as is commonly found in human cancer, results in a significant increase in centrin 2 at the centrosomes of interphase cells. Conversely, CDC25B depletion causes a loss of centrin 2 from the centrosome, which can be rescued by treatment with the proteasome inhibitor MG132. CDC25B overexpression also promotes the formation of excess centrin 2 “foci”. These foci can accumulate other centrosome proteins, including γ-tubulin and PCM-1, and can function as microtubule organising centres, indicating that these represent functional centrosomes. Formation of centrin 2 foci can be blocked by specific inhibition of CDK2 but not CDK1. CDK2-mediated phosphorylation of Monopolar spindle 1 (Mps1) at the G1/S transition is essential for the initiation of centrosome duplication, and Mps1 is reported to phosphorylate centrin 2. Overexpression of wild-type or non-degradable Mps1 exacerbated the formation of excess centrin 2 foci induced by CDC25B overexpression, while kinase-dead Mps1 has a protective effect. Together, our data suggest that CDC25B, through activation of a centrosomal pool of CDK2, stabilises the local pool of Mps1 which in turn regulates the level of centrin 2 at the centrosome. Overexpression of CDC25B may therefore contribute to tumourigenesis by perturbing the natural turnover of centrosome proteins such as Mps1 and centrin 2, thus resulting in the de novo assembly of extra-numerary centrosomes and potentiating chromosome instability.     

A monoclonal antibody to animal centrosomes recognizes components of the basal-body root complex inChlamydomonas

Summary The mammalian centrosome monoclonal antibody MPM-13 recognized component(s) of the well defined MTOC basal-body root complex in the green plantChlamydomonas. The antibody reaction coincided in location with the basal-body root complex and the cruciate nature of the staining pattern corresponded to the configuration of the root microtubules. During mitosis the behaviour of MPM-13 stained material mirrored the duplication, separation and migration to the spindle poles of the basal body-root complex. It is suggested that conserved MTOC components were recognized and that these may have retained a similar, perhaps universal, function in microtubule organization.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidine-2-phenylindole dihydrochloride - mt mating type - MT microtubule - MTOC microtubule organizing centre - PFA paraformaldehyde - PBS phosphate buffered saline     

The "starter" and "gas pedal" of mitosis reside at the centrosome: Commentary on "Characterization of centrosomal localization and dynamics of CDC25C phosphatase in mitosis" by Bonnet et al.

The CDC25 phosphatases play an essential role in the spatial and temporal regulation of the control of entry into mitosis. These enzymes dephosphorylate and activate the CDK-cyclin complexes, in particular CDK1-cyclin B1, the master regulator of mitosis. Three CDC25 genes in exist in humans (CDC25A, CDC25B and CDC25C), and the original model of their function proposed that they acted sequentially at discrete cell cycle transitions, i.e., that CDC25A was dedicated to the activation of the G1/S progression-associated CDKs, CDC25B controlled early prophase events, while CDC25C was thought to achieve the full activation of CDK1-cyclin B1 at entry into mitosis. Indeed, the situation appears much more complicated than this, and current evidence shows that all three CDC25 phosphatases act at a variety of mitotic stages, with and considerable experimental evidence to indicate that all three are involved in orchestrating cell cycle progression in mitosis.1 Previous work has led to the proposal that CDC25B acts as the starter of mitosis. Additionally, a number of recent studies have shown that CDC25B also localizes to the centrosome where its activating role on CDK-cyclin complexes appears to be regulated by multiple activatory and inhibitory kinases.2-5 As such, it has been proposed that CDC25B might act as a central centrosomal integrator and a trigger for the initial events that set up the sequence of events leading to mitosis.6 As a target of the first small pool of activated CDK1-cyclin B1 that translocates to the nucleus, CDC25C was thought to subsequently be responsible for the massive activation of the nuclear pool of CDK1-cyclin B1 that occurs at entry into mitosis. A report from the group headed by May Morris presented in this issue of Cell Cycle (Bonnet et al., pp. 1990–7) provides new insight into the dynamics of these events and in the understanding of the involvement of both CDC25B and CDC25C in the earliest stages of the G2/M transition. Bonnet and collaborators show for the first time, as has long been suspected but until now never observed, the localization of a fraction of CDC25C at the centrosome during interphase. This centrosomal localization occurs from S-phase onward and is also present during mitosis. Using FRAP analysis, their study elegantly shows that this centrosomal population of CDC25C is highly dynamic. Furthermore, the authors show that mutations of CDC25C that impair its catalytic activity or its binding to its CDK-cyclin substrates promote its centrosomal accumulation, thus suggesting an active role in the dephosphorylation and activation of CDK-cyclins at this location. Together with previous reports showing that the activity of CDC25C is amplified following its mitotic phosphorylation by CDK1-cyclin B1 while the activity of CDC25B is not,7 these new findings lead to the proposition of an alternative regulatory model for the control of the G2/M transition. In this model, the CDK1-cyclin B1 complex is activated at the centrosomal level both by the initial action of CDC25B (as has already been suggested8) as well as by the centrosomal pool of activated CDC25C that subsequently amplifies the process through its own phosphorylation and activation (Fig. 1). While CDC25B can be considered as a “starter”, CDC25C plays the role of the “gas pedal” that speeds up entry into mitosis by amplifying the signaling cascade from the centrosome and finally increasing nuclear levels. This model is certainly too simplistic and does not integrate many major issues that remain to be investigated. Among these unsolved questions is the role that the multiple splice variants of the CDC25 phosphatases might play. There are at least five variants for both CDC25B and CDC25C whose specific regulation and roles in the dephosphorylation of individual CDK-cyclins substrates is still unknown.5 Likely related to this question is the issue of the presence of both CDC25B and CDC25C until late stages of mitosis. Why is CDC25C associated with the centrosome when, according to the dogma, the entire pool of CDK1-cyclin B1 has been fully activated? An attractive hypothesis is to speculate that the CDC25 phosphatases might continue to play discrete roles in the dephosphorylation and the activation of sub-populations of CDK-cyclins throughout the entire process of mitosis to ensure a fine tuning of the kinase activities that are involved in the many architectural and functional aspects of the mitotic figure. Centrosomes are made up of numerous proteins whose amino acid sequence suggests a coiled-coil tertiary structure. Increasing evidence indicates that this molecular structure may be well-designed for the organization of multiprotein scaffolds that can anchor a diversity of activities ranging from protein complexes involved in microtubule nucleation to multicomponent pathways for cellular regulation.9 By physically linking components of a common pathway, molecular scaffolds can increase the local concentration of components, limit nonspecific interactions, and provide spatial control for regulatory pathways by positioning by positioning them at specific sites in proximity to downstream targets or upstream modulators. On the basis of the increasing number of regulatory molecules anchored at the centrosome, it is likely that this organelle serves as a centralized control center for regulating a diversity of cellular activities. Recent studies have provided some of the first functional links between centrosomes and regulatory networks in cell cycle transitions from G1 to S-phase, G2 to M-phase and metaphase to anaphase. The findings by Bonnet et al. support this line of evidence.

References

Boutros R, Dozier C, Ducommun B. The when and wheres of CDC25 phosphatases. Curr Opin Cell Biol 2006; 18:185-91. Dutertre S, Cazales M, Quaranta M, Froment C, Trabut V, Dozier C, Mirey G, Bouche J, Theis-Febvre N, Schmitt E, Monsarrat B, Prigent C, Ducommun B. Phosphorylation of CDC25B by Aurora-A at the centrosome contributes to the G2/M transition. J Cell Science 2004; 117:2523-31. Schmitt E, Boutros R, Froment C, Monsarrat B, Ducommun B, Dozier C. CHK1 phosphorylates CDC25B during the cell cycle in the absence of DNA damage. J Cell Sci 2006; 119:4269-75. Boutros R, Ducommun B. Asymmetric localization of the CDC25B phosphatase to the mother centrosome during interphase. Cell Cycle 2008; 7:401-6. Boutros R, Lobjois V, Ducommun B. CDC25 phosphatases in cancer cells: key players? Good targets? Nat Rev Cancer 2007; 7:495-507. Lindqvist A, Kallstrom H, Lundgren A, Barsoum E, Rosenthal CK. Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome. J Cell Biol 2005; 171:35-45. Baldin V, Pelpel K, Cazales M, Cans C, Ducommun B. Nuclear Localization of CDC25B1 and Serine 146 Integrity Are Required for Induction of Mitosis. J Biol Chem 2002; 277:35176-82. Jackman M, Lindon C, Nigg EA, Pines J. Active cyclin B1-Cdk1 first appears on centrosomes in prophase. Nat Cell Biol 2003; 5:143-8. Kramer A, Lukas J, Bartek J. Checking out the centrosome. Cell Cycle 2004; 3:1390-3.     

CDC25B associates with a centrin 2-containing complex and is involved in maintaining centrosome integrity

Background information. CDC25 (cell division cycle 25) phosphatases function as activators of CDK (cyclin‐dependent kinase)–cyclin complexes to regulate progression through the CDC. We have recently identified a pool of CDC25B at the centrosome of interphase cells that plays a role in regulating centrosome numbers. Results. In the present study, we demonstrate that CDC25B forms a close association with Ctn (centrin) proteins at the centrosome. This interaction involves both N‐ and C‐terminal regions of CDC25B and requires CDC25B binding to its CDK—cyclin substrates. However, the interaction is not dependent on the enzyme activity of CDC25B. Although CDC25B appears to bind indirectly to Ctn2, this association is pertinent to CDC25B localization at the centrosome. We further demonstrate that CDC25B plays a role in maintaining the overall integrity of the centrosome, by regulating the centrosome levels of multiple centrosome proteins, including that of Ctn2. Conclusions. Our results therefore suggest that CDC25B associates with a Ctn2‐containing multiprotein complex in the cytoplasm, which targets it to the centrosome, where it plays a role in maintaining the centrosome levels of Ctn2 and a number of other centrosome components.     

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.     

NEK5 regulates cell cycle progression during mouse oocyte maturation and preimplantation embryonic development

NEK5, a member of never in mitosis‐gene A‐related protein kinase, is involved in the regulation of centrosome integrity and centrosome cohesion at mitosis in somatic cells. In this study, we investigated the expression and function of NEK5 during mouse oocyte maturation and preimplantation embryonic development. The results showed that NEK5 was expressed from germinal vesicle (GV) to metaphase II (MII) stages during oocyte maturation with the highest level of expression at the GV stage. It was shown that NEK5 localized in the cytoplasm of oocytes at GV stage, concentrated around chromosomes at germinal vesicle breakdown (GVBD) stage, and localized to the entire spindle at prometaphase I, MI and MII stages. The small interfering RNA‐mediated depletion of Nek5 significantly increased the phosphorylation level of cyclin‐dependent kinase 1 in oocytes, resulting in a decrease of maturation‐promoting factor activity, and severely impaired GVBD. The failure of meiotic resumption caused by Nek5 depletion could be rescued by the depletion of Wee1B. We found that Nek5 depletion did not affect CDC25B translocation into the GV. We also found that NEK5 was expressed from 1‐cell to blastocyst stages with the highest expression at the blastocyst stage, and Nek5 depletion severely impaired preimplantation embryonic development. This study demonstrated for the first time that NEK5 plays important roles during meiotic G2/M transition and preimplantation embryonic development.     

Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis

Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.