Mechanism Investigation Of Kinesin Eg5 During Oocyte Maturation

The proper development of mammalian oocytes is a prerequisite for the normal oogenesis,fertilization and embryo development,which is the basis for the initiation of life.The maturation of oocytes must undergo a delicate and complex process of meiosis.The DNA duplicates once and divides twice,producing a haploid gamete and two polar bodies.In this series of processes,there are many events involved,including microtubule assembly and dynamics,kinetochore-microtubule attachment,chromosome alignment and separation,etc.The precise distribution of genetic material can only be guaranteed when all events are completed without errors.Any error in these events will cause the meiosis failure.Eg5,as a famuly member of kinesin proteins,participates in the formation and maintenance of mitotic bipolar spindles,playing an important role in mitosis.However,whether Eg5 is involved in oocyte meiosis and what is the underlying mechanism are still unclear.Because porcine oocytes are developmentally and physiologically similar to humans,the present study is aimed to explore the regulatory mechanism of Eg5 in the process of porcine oocyte meiotic progression,providing theoretical basis for the oocyte maturation and quality control.In the present study,porcine oocytes were selected as the research subject to investigate the influencing factors and regulatory mechanisms of oocyte quality under different conditions via in vitro culture system,immunofluorescent staining and western blot analysis.The main results are shown as follows:In this study,different concentrations of Eg5 inhibitor monastrol were supplemented into the culture medium to observe and record the maturation rate and morphological changes of porcine oocytes.We found that different concentrations of monastrol impaired the oocyte development,showing that the poor expansion status of granulosa cells with the increase of doses of the inhibitor.Meanwhile,the effects of different concentration of monastrol on the oocyte meiotic progression were observed and recorded.The result showed that different dosage had different effects on the maturation of oocytes,and the polar body extrusion rate of oocyte decreased with increasing dosage.The subsequent experiments were carried out with a concentration of 100 ?M of monastrol which not only compromised oocyte maturation,but also allowed some oocytes to develop to M? stage for subsequent experiments.We then assessed the proportion of oocytes arrested at different developmental stages when Eg5 was inhibited,and found that inhibition of Eg5 had no effect on the GVBD rate,but significantly reduced the polar body extrusion compared to the controls.Among of them,most of arrested oocytes were found at MI stage by nuclear staining.In addition,inhibition of Eg5 disrupted the chromosome alignment and spindle assembly,as well as the microtubule stability as judged by staining of acetylated tubulin,thereby disturbing the nuclear maturation of oocytes.We also found that inhibition of Eg5 perturbed the localization pattern of atcin filament and mitochondria,as well as the distribution and release of cortical granulesand ovastacin,which in turn,perturbs the cytoplasmic maturation of oocytes.In conclusion,monastrol treatment inhibits the activity of Eg5,which has an adverse impact on both nuclear and cytoplasmic maturation of porcine oocytes,thus affecting the meiotic progression and development of oocytes.