The Function Of Bora During Mouse Oocyte Meiosis

Bora, as the binding partner of Aurora A, is required for regulating the activation of Aurora A, promoting the phosphorylation of Polo like kinase1(PLK1), regulating the spindle assembly and the progress of cell cycle during mitosis. However, its mechanism of action during mouse oocyte meiosis has remained elusive. The objective of this study was to show the function of Bora in mouse oocyte meiosis with a series of techniques such as parthenogentic activation, western blot, immunofluorescence, confocal microscopy and microinjection. We examined the expression and subcellular localization of Bora and revealed the relationship between Bora and microtubule formation along with spindle assembly. Then we investigated the effects of Bora on spindle assembly, the first polar body extrusion and cell cycle during mouse oocyte meiotic maturation in order to explore the regulation mechanism of mouse oocyte meiosis. It’s significant for the research on the mechanism of oocyte maturation.1. To detect the changes in Bora expression during the meiotic maturation, the eggs were collected at different stages of the first meiosis (GV, GVBD, MⅠ,AⅠ/TⅠ,MⅡ) and for Western blot analysis. The result showed that Bora was stably expressed at all stages of oocyte maturation.2. To investigate the possible roles of Bora during mouse oocyte meiosis, mouse oocytes were collected at different stages of the first meiosis by means of cell culture in vitro and MⅡ oocytes were collected after superovulation. The subcellular localization of Bora was observed by confocal microscopy. The results showed that the subcellular localization pattern of Bora is similar to that of spindle during mouse oocyte maturation: the localization of Bora can’t be detected in GV oocytes. When GVBD occurred, Bora concentrated around the condensed chromatin. With the formation of the spindle, Bora gradually accumulated two parts around chromosomes as the chromosomes arranged to the middle of the spindle. At anaphase/telophase I, Bora began to migrate to the inner sides of chromosomes. At metaphase Ⅱ, Bora distributed as two parts around chromosomes again. After parthenogenetic activation, the localization of Bora during anaphase/telophase Ⅱ is similar to that during anaphase/telophase Ⅰ. With pronuclear formation, Bora can’t be detected again. 3. To explore the co-localization of Bora and microtubule organization during meiotic maturation, eggs were collected at different stages of the first meiosis to determine the co-localization of Bora and a-tubulin. We proved co-localization of Bora and a-tubulin in mouse oocyte meiosis: when GVBD occurred, Bora and a-tubulin both concentrated around the condensed chromatin. At metaphase I, the subcellular localization pattern of Bora is similar to that of spindle, however Bora wasn’t localized to the middle of the spindle. At anaphase I, Bora was still accumulated at the spindle as the chromosomes arranged to the two poles of the spindle. At telophase I, the subcellular localization of Bora began to varied as the spindle rotation.4. To further reveal the relationship between Bora and microtubule protein, MⅡ eggs were treated with colchicine, staurosporine, Taxol respectively. We found that the subcellular localization of Bora can’t be examined after the destruction of microtubule polymerization.5. The find out the possible function of Bora in mouse oocyte meiosis after antibody microinjection, Bora antibody was injected into the GV oocytes. The GVBD and PB1were recorded1.5and14h after culture. In oocytes injected with Bora antibody,70.67%of them underwent GVBD, but90.63%of the oocytes injected with IgG underwent GVBD. In Bora antibody microinjected group, PB1rate was23.89%, but the PB1rate of control group was41.80%. The data suggested that the progress of oocyte meiosis was significantly inhibited after antibody microinjection. In virtue of immunofluorescence and confocal microscopy to examine the spindle structure, we observed the oocytes injected with Bora antibody can form abnormal spindles, including one pole and unformed spindles. Meanwhile, some oocytes injected with Bora antibody were arrested at metaphase I, namely they couldn’t entry into anaphase I. These results implied that Bora regulated the spindle assembly and metaphase-anaphase I transition during mouse meiosis.The conclusion of this study is that the expression of Bora was relatively stable during mouse oocyte meiosis and co-localization with the spindle. Bora is also responsible for the regulation of spindle assembly and the process of mouse oocyte meiosis.