The Function Of PRC1 In Oocyte Meiosis And CHK2 In Early Embryo Development

Early embryo development in mammals begins with the fertilization of oocyte and sperm.After the first meiosis,the oocyte retains half of nuclear genetic material and most of cytoplasm,and arrested at metaphase of the second meiosis before fertilization.The early process of embryo development is controlled by the maternal DNA,and oocytes also provide the material basis for early embryo development.Therefore,high quality mature oocytes are essential for early embryo development.Meiotic of oocyte is the most important process of oogenesis.After DNA replication,the oocyte is stimulated by gonadotropin to enter the first meiosis.After germinal vesicle breakdown,microtubules assemble spindles in the center of oocytes,and chromosomes are uniformly arranged on the equatorial plate.At anaphase/telophase I,the microfilaments migrate the spindle to the oocyte cortex,and homologous chromosomes are separated and moved toward the spindle poles.Contraction ring is formed in oocyte,which together with actin promote continuous contraction of cleavage furrow,and oocyte extrudes the first polar body to complete the first meiosis.After fertilization,the oocyte completes the second meiosis to form a zygote.The zygote completes first cleavage and produces two cells under the regulation of the maternal genome.Subsequently,the embryonic genome is activated,and the embryo continues to cleavage,developing into 4cell,8-cell,morula,and blastocyst.Any errors in oocyte development events may lead to aneuploidy and fertilization failure,affecting the development of early embryos.Errors in early embryo development can also lead to embryo death or implantation failure.Therefore,improving the quality of oocytes and early embryos is essential to ensure the genetic stability of the embryonic genome and to treat infertility.In this study,female ICR mice were used as the research object,and the function of PRC1 in the maturation process of mouse oocytes and the function of CHK2 in the early embryo development of mouse were respectively explored by using siRNA interference and inhibitor treatment.The results are as follows:Experiment 1:The function of PRC1 in mouse oocyte meiosisPRC1(Protein regulating cytokinesis 1),which is a microtubule bundling protein,has been reported to involve in the regulation of the central spindle bundle and spindle orientation for cytokinesis in mitosis.However,the functions of PRC 1 in meiosis are rarely studied.In this study,we explored the roles of PRC1 in meiosis using oocyte model,and we found that PRC1 expressed at all stages during mouse oocyte meiosis,and PRC1 accumulated to the midzone/midbody during anaphase/telophase I.Depletion of PRC1 caused the defects of polar body extrusion during mouse oocyte maturation.Further analysis showed that PRC1 knockdown did not affect meiotic spindle formation and chromosome segregation,however,its deletion prevented the formation of midzone at the anaphase stage of meiosis I,which caused the cytokinesis defects and further induced two spindles in the oocytes.And the knock down of PRC1 caused the increased level of tubulin acetylation,indicating that the microtubule stability was affected.Furthermore,we found that KIF4A and PRC1 showed similar localization in the midzone/midbody at anaphase/telophase I in oocytes,while the depletion of KIF4 A affected the expression and localization of PRC1.In summary,our results suggested that PRC 1 was critical for midzone/midbody formation and cytokinesis under the regulation of KIF4A in mouse oocytes.Experiment 2:The function of CHK2 in mouse early embryo developmentCHK2(Checkpoint kinase 2),as a cell cycle checkpoint,which is involved in monitoring DNA replication,cell cycle arrest and spindle assemble in mitosis or meiosis.However,functions of CHK2 during mouse early embryo development remain uncertain.In this study,we found that CHK2 located at spindle poles at metaphase in first cleavage.CHK2 inhibition led to failure of cleavage in early mouse embryos.Further analysis indicated that inhibition of CHK2 caused abnormal spindle assemble and misaligned chromosomes.These results demonstrated CHK2 regulated cleavage of early embryos through affected spindle assemble.Moreover,interference with CHK2 activity induced increased DNA damage,which then resulted in oxidative stress.Finally,irreparable DNA damage and oxidative stress activated apoptosis and autophagy.These results showed that CHK2 was involved in regulating repair of interphase DNA damage.In summary,our results suggested that CHK2 was a critical regulator for spindle assembly and DNA repair during mouse early embryo development.In this study,we explored the important regulatory factors in oocyte meiosis or early embryo development,PRC 1 or CHK2 is necessary for oocyte meiosis or early embryo development.It provides a theoretical basis for providing high quality oocytes/early embryos and improving fertility of livestock.