Research On The Roles Of Nek9and ERK8in Meiotic Maturation Of Mouse Oocytes And Their Locations In Early Embryo Mitosis

Mouse oocyte maturation is a multi-stage, precisely orchestrated and orderly process.Germinal vesicle breakdown (GVBD) marks the beginning of oocyte maturation. After GVBD, microtubules assemble around chromosomes during the pro-metaphase Ⅰ stage, and then chromosomes migrate to the central plate of the bipolar spindle at the MI stage. Subsequently the spindle migrates to the cortex, and the oocyte emits the first polar body, followed by the formation of the metaphase Ⅱ (MII) spindle located beneath the plasma membrane. The unfertilized mouse oocyte contains over80small microtubule-organizing centers (MTOCs) within the cytoplasm. In mitosis, the centrosomes form the two poles of the mitotic spindle and contribute to the nucleation and organization of the highly dynamic spindle microtubules (MTs). MTOCs vary in shape and size, but all contain the pericentriolar material (PCM) components:y-tubulin and pericentrin, a critical protein that anchors γ-tubulin at the PCM in somatic cells. At prometaphase, MTOCs begin to accumulate to form spindle poles. While oocyte MTOC function is similar to that of centrosomes and MTOCs are essential for meiotic spindle assembly, the underlying mechanisms that regulate the recruitment of pericentriolar material proteins for the biogenesis and organization of MTOCs and the regulation of microtubule nucleation and assembly are not well understood. NIMA-like kinases are named for the Aspergillus nidulans protein kinase encoded by the NIMA (never in mitosis Aspergillus) gene that participates in a broad array of mitotic processes. The mammalian genome encodes11protein kinases. Available reports indicate that the NIMA-family kinases are the essential molecules in mitosis progression and are involved in many processes such as centrosome separation, chromosome condensation in prophase, nuclear envelope breakdown and spindle assembly in pro-metaphase, as well as in exit from mitosis and cytokinesis. Nek9, one of the NIMA kinases, is a107-kDa polypeptide whose amino-terminal catalytic domain is followed by a domain homologous to RCC1, the exchange factor for the small G protein Ran. The RCC1domain of Nercc1acts as an auto-inhibitory domain through the direct binding to Nek9protein kinase domain during interphase. Mammalian Nek9coimmunoprecipitates with y-tubulin and the activated Nek9polypeptides localize to the centrosomes and spindle poles during early cell division, suggesting that active Nek9has important functions at the microtubule organizing center during m itosis. Nek6and Nek7can bind strongly to the carboxy-terminal tail of Nek9distal to the RCC domain.Moreover, Nek6, which is itself a mitotic kinase, can be directly phosphorylated and activated by Nek9in vivo and in vitro. Both Nek6and Nek7kinases activation contributes to mitotic progression downstream of Nek9, and interfering with their activity by either knockdown or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Nek9activation by PLK1contributes to the phosphorylation of the mitotic kinesin Eg5at Ser1033, which together with the CDK1, is necessary for subsequent centrosome separation and timely mitosis. The mitogen-activated protein kinases (MAPKs) are a superfamily of serine/threonine protein kinases that are highly conserved and whose members have been implicated in many critical cellular processes including cell proliferation, differentiation, apoptosis, and stress responses. ERK8is one of the most recently described members in ERK family which shares69%amino acid sequence identity with ERK7. Fluorescence in situ hybridization localized the ERK8gene to chromosome8, band q24.3.ERK8contains two SH3-binding motifs in its C-terminal region, and can be activated by Src-dependent signaling pathway, by serum and by RET/PTC3, an activated form of the RET proto-oncogene. ERK8is required for the stability of proliferating cell nuclear antigen (PCNA) protein which acts as a scaffold, coordinator, and stimulator of numerous processes required for faithful transmission of genetic information. Human double minute2(HDM2) mediated PCNA destruction can be prevented by ERK8through the way of inhibiting the association of PCNA with HDM2. ERK8also controls the cell cycle. ERK8in Trypanosoma brucei strongly affected growth phenotypes, and appeared to be essential for normal growth and proliferation. Silencing of ERK8affected normal cell proliferation. ERK8is also a limiting factor in regulating the proliferation rate of MCF-10A cells and controls entry into S phase. Loss of ERK8resulted in increase in cyclin D1, cyclin E, and p27and decrease in cyclin A levels. Knockdown of ERK8decreased cell proliferation approximately two folds. However, ERK8is not well studied and information about its upstream activators, downstream effectors, and functions is scarce, and very little is known about the relationship between ERK8and meiosis.As precise regulation of these events is important for the production of fertilizable eggs and health offsprings. At present, whether Nek9or ERK8participates in acentriolar meiotic spindle assembly and subsequent accurate chromosome segregation remains unknown. We therefore asked whether Nek9or ERK8contribute to the meiotic maturation and early embryo cleavage. This study contains two chapters.Chapterl Nek9regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosisObjective:whether Nek9participates in acentriolar meiotic spindle assembly and subsequent accurate chromosome segregation remains unknown. We explored the expression, location and the function of Nek9during mouse oocyte meiotic maturation and also explored its location in early embryo cleavage.Method:With the methods of Western blot, Immunofluorescence and confocal microscopy, spindle perturbing drugs treatment, Chromosome spreading, Microinjection of morpholino and Time-lapse live imaging experiments, we explored the roles of Nek9in Meiotic Maturation of Mouse Oocytes and its location in early embryo mitosis.Result:Experiments results showed that Nek9protein was expressed from GV to MII stages, without detectable changes.Immuno-fluorescent staining show that Nek9was mainly distributed in the germinal vesicle at the GV stage. After GV break down, Nek9began to accumulate in the vicinity of condensed chromosomes. At the pro-metaphase Ⅰ stage, metaphase Ⅰ, Nek9was stably presented at the spindle poles. At the stage of anaphase Ⅰ/telophase Ⅰ, Nek9was stained in the region between the separating homologous chromosomes, and associated with the midbody between the first polar body and the oocyte. At the MII stage, Nek9was again localized at the spindle poles.Co-localization of Nek9with γ-tubulin further confirmed that the localization of Nek9was at the two spindle poles. Subcellular localization of Nek9during fertilization and early embryo cleavage showed that In oocytes at telophase II, Nek9moved to the midbody. In the fertilized eggs, many Nek9dots were distributed within the pronuclei. After nuclear envelope breakdown, Nek9began to form around the periphery of chromosomes until the MI spindle was formed. At prophase and M phase, Nek9was stably stained at the first mitotic spindle poles.When the zygote progressed into Ana and Tel stages, Nek9moved to the middle area between the separated chromatids or midbody. By the completion of the first mitotic cell cycle, two blastomeres formed and both of them entered interphase. At this stage, again Nek9signals were stained in the nucleus, similar to the stage of2PN.Next,Taxol and nocodazole were employed. When mouse oocytes were treated with taxol to promote non-spindle microtubule asters in the cytoplasm, Nek9foci were detected in the center of these cytoplasmic asters, in addition to those at the spindle poles. In addition, when oocytes were exposed to nocodazole to completely disassemble microtubule, the Nek9foci dispersed into the cytoplasm. When the treated oocytes were thoroughly washed and cultured to allow microtubule reassembly, the Nek9signals again appeared at the poles of the reformed spindle apparatus. These results further indicate the involvement of Nek9in microtubule organization. Further more,We we knocked down Nek9by its specific morpholino (MO) injection. Western blotting analysis showed that the expression level of Nek9was notably reduced, which revealed the efficiency of the Nek9depletion. In the Nek9-MO injection group, the oocytes exhibited different kinds of morphologically defective spindles and misaligned chromosomes. The major spindle defects were abnormal spindle poles (52%, n=94), including spindles with no pole, one pole, multi-poles. Others defects were elongated and malformed spindles with astral microtubules. Chromosome misalignment was also observed.The rate of abnormal spindles in the Nek9-MO injection group (62.2±1.6%, n=94) was significantly higher than that in the control-MO injection group (26.1±1.3%, n=92)(p<0.05). Furthermore, the rate of misaligned chromosomes in Nek9MO (69.2±0.6%, n=94) and control groups (28.1±2.3%, n=92) were significantly different (p<0.05). Since Nek9co-localized with γ-tubulin during meiotic maturation, we further investigated the effect of Nek9depletion on y-tubulin localization. γ-tubulin was localized to the spindle poles in control-MO injected oocytes at the MI stage, while in Nek9-MO injected oocytes, γ-tubulin was no longer accumulated at the spindle poles, being irregularly dispersed to the spindle fibers or distributed into the cytoplasm. Since Nek9depletion disrupted the spindle assembly with the majority of oocytes blocked in Pro-MI/MI arrest, we asked whether the chromosomes could undergo correct segregation. Oocytes in both Nek9MO and control groups were cultured for12h. Chromosome spreading experiments confirmed the failed chromosome separation. Chromosome spreading showed that chromosomes were still in the bivalent state in Nek9depleted oocytes (10/10); in contrast, univalent chromosomes could be seen in control oocytes (9/9), indicating completion of anaphase. Next, we analyzed the localization of Bub3in oocytes from the Nek9-MO group to explore the activation of SAC proteins. Specific signals for Bub3were detected in the Pro-MI/MI-arrested oocytes in the Nek9-MO group even after10h culture. In contrast, the control oocytes entered anaphase and showed no signals of Bub3. Detection of Bub3signal indicates activation of the spindle assembly checkpoint.Last Live cell imaging showed that in the control group, the meiotic spindle was visible about4hr of GVBD and slowly migrated toward the oocyte cortex followed by rapid polar bodyl extrusion at about9h of culture following GVBD. In contrast, in the Nek9-MO injection group, various morphologically defective spindles were seen and chromosomes failed to separate and remained at the Pro-MI/MI stage even at12h of GVBD. Additionally, no first polar body extrusion was observed in the Nek9-MO injection group.Conclusion:Our results suggest that Nek9may 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.Chapter2The distribution and possible role of ERK8in mouse oocyte meiotic maturation and early embryo cleavageObjective:whether ERK8participates in acentriolar meiotic spindle assembly and subsequent accurate chromosome segregation remains unknown. We explored the expression,location and function of ERK8during mouse oocyte meiotic maturation and also explored its location in early embryo cleavage.Method:With the methods of Western blot, Immunofluorescence and confocal microscopy, spindle perturbing drugs treatment, Microinjection of siRNA or Antibody, we explored the roles of ERK8in Meiotic Maturation of Mouse Oocytes and its location in early embryo mitosis.Result:Results of experiments on ERK8showed that ERK8was expressed at all stages with no detectable difference.Immunofluorescent staining show that no specific staining of ERK8was found in GV oocytes. After GVBD, ERK8began to migrate to the periphery of chromosomes. At ProI, MI, Anal, Tell and MII stages, ERK8was stably distributed on the entire length of the meiotic spindle. Immunofluorescent staining in mouse zygote and2-cell embryo showed that at two pronuclei zygote stage, no specific ERK8signal was observed. After nuclear envelope breakdown (NEBD), ERK8began to aggregate to the periphery of chromosomes and then on the mitotic spindle. When the zygote progressed into anaphase, ERK8still showed the same location with a-tubulin. When two blastomeres entered interphase, again no specific ERK8signal was observed. Next, mouse oocytes at Metaphase were treated with taxol or nocodazole.In taxol-treated oocytes, ERK8signal was detected on the abnormal spindles as well as cytoplasmic asters. After treatment with nocodazole for10min to disassemble microtubules completely, ERK8became distributed into the cytoplasm. To explore the roles of ERK8in mouse oocyte meiosis, we first disrupted the ERK8protein activity by antibody injection. In ERK8antibody injection group, the oocytes exhibited various morphologically defective spindles and chromosome misalignment phenotypes. The major defect was no poles, and others include spindles with one pole, multi poles, small spindles, long spindles and unformed spindles with astral microtubules. Severely impaired spindle in ERK8antibody injection group also accompanied chromosome misalignment phenotypes which include minor and severe misalignment. Compared to the control group (23.2%±2.7%, n=217), an obvious increase in abnormal spindle formation was observed in the ERK8antibody injection group (57.1%±4.9%, n=185,p<0.05). ERK8antibody injection group(60.4%±3.8%, n=185) also showed higher chromosome misalignment than that of the control group (20.6%±1.8%, n=217,p<0.05). Further more, ERK8specific siRNA microinjection was used to deplete ERK8expression.The rate of abnormal spindles in the ERK8-siRNA injection group (59.3±3.5%, n=159) was significantly higher than that of the control group (25.3±1.5%, n=155, p<0.05)(Fig.5C). ERK8siRNA knockdown group (60.5±3.8%, n=159) also showed higher chromosome misalignment than that of the scrambled siRNA-injected group (30.6±3.4%, n=155, p <0.05) at the MI stage. Last, the PB1extrusion rate in the ERK8siRNA group (31.1±2.8%, n=144) was significantly lower than that of control siRNA group(63.4±4.2%, n=157, p<0.05).The rate of PB1extrusion (35.3±5.7%, n=114) in the ERK8antibody injection group was also significantly lower than that of the control IgG injection group (72.7±2.8%, n=125,p<0.05).Conclusion:Taken together, our results suggest that ERK8may play important roles in the microtubule organization and meiotic cell cycle progression in mouse oocytes, fertilized eggs, and early embryos. Further studies are necessary to identify the upstream kinases and the downstream substrates of the ERK8family in mammalian meiosis.