Function Of Centromere Protein F During Mouse Oocyte Meiosis And Early Embryo Development

Centromere protein F(CENPF)is a kinetochore protein belongs to centromere protein family,which containing multiple leucine zipper and coiled-coil motifs.CENPF plays multiple function through its association with the microtubule(MT)network in cell mitosis,including regulating cell proliferation,vesicular transport and cell shape.Till now,little is known about its role in mouse oocyte meiosis and early embryo development.This study will focus on the detailed function of CENPF during mouse oocyte meiosis and early embryo development.In part 1(chapter 2),we described the CENPF function during mouse oocyte meiosis maturation.Firstly,CENPF protein expression and its subcellular localization during mouse oocyte meiosis were investigated,and the possible mechanism of CENPF localization was discussed.Secondly,by using the Cenpf specific siRNA and the Morpholino(MO),detailed CENPF function during mouse oocyte meiosis was investigated through knocking down the expression of CENPF.At last,the potential mechanism of CENPF regulating mouse oocyte meiosis were investigated.In the study of chapter 2,GV,GVBD,MI,AI-TI and M? stage oocytes were collected to examine CENPF protein expression and the subcellular localization by employing western blot and immunofluorescence.Western blot results showed that CENPF express in each stage of oocyte meiosis.The expression level of CENPF was low in GV stage and increased in metaphase(MI and M? stages).Immunostaining results showed that CENPF localized on nuclear membrane at GV stage.After GVBD,CENPF localized on the spindle and centromere till to the M? stage(except vanish at centromere in T? stage).To investigate the mechanism of CENPF localization,two kinds of MT disturbing drugs nocodazole(induces MTs depolymerization)and taxol(prevents MTs depolymerization)were used to treat M? stage oocytes.The results showed that the localization of CENPF on spindle but not centromere depend on the stabilization of MT.Furthermore,by using farnesyl transferase inhibitor(FTI)SCH66336,we found that localization of CENPF in GV and MI stage oocytes is independent of self farnesylation,but its localization in M? stage oocytes depend on its self farnesylation.The results of Cenpf-siRNA and CENPF-MO knock down showed that depletion of CENPF lead to failure of GVBD and first polar body extrusion(PBE).Immunofluorescent staining for TUBB and DNA displayed that CENPF depletion lead to the defects of spindle assembly and chromosome congression.And these results were confirmed by time-lapse imaging of oocytes labled with eGFP-TUBB and mCherry-H2B.We further confirmed that depletion of CENPF induced failure of spindle assembly as well as disordered chromosomes alignment at metaphase plates,which leading to the subsequent meiosis failure.Chromosome spreads results showed that loss of CENPF resulted in increase of aneuploidy in M? stage oocytes.For those oocytes arrest at MI stage and failed to complete meiosis,although the chromosome stability was not affected,the increased inter-sister kinetochore distance induced by CENPF knockdown will increase the risk of aneuploidy in later development.Finally,according to the results of immunofluorescent staining for CREST and TUBB,we found that loss of CENPF resulted in the defects of MT-kinetochore attachment and failure of grabbing chromosomes by MTs.Meanwhile,loss of CENPF led to activation of spindle assembly checkpoint(SAC)related proteins Mad1 and Mad2,which resulted in the metaphase-anaphase transition block.In sum,loss of CENPF led to the failures of MT assembly and kinetochores capture,which resulted in the continuous SAC activation.This will lead to oocytes failed to complete the metaphase-anaphase transition and oocyte maturation.Conversly,even a portion of oocytes "escaped" from the SAC monitor and complete the meiosis,incorrect spindle assembly and MT-kinetochores instability will lead to abnormal chromosomes separation,which resulted in the increased incidence of aneuploidy of M? stage oocytes.In part 2(chapter 3),we studied CENPF function during mouse early embryo development.Firstly,the CENPF protein expression and its subcellular localization during mouse early embryo development were investigated.The mechanism of CENPF localization in early embryos was also investigated.Secondly,CENPF-MO was injected into M? stage oocytes,PN and 2-cell stage embryos to further study the effect of CENPF knock down on early embryonic development.Embryos at A?-T?,PN,Late 1-cell,2-cell,4-cell,8-cell,morula and blastocyst stages were collected to analyze CENPF expression and subcellular localization by employing western blot and immunofluorescence methods.The western blot results showed that CENPF expressed at all stages of early embryos,and decreased from 8-cell stage.The immunofluorescence results showed that during the mitotic interphase,CENPF localized on the nuclear membrane and intracellular bridges.At metaphase,CENPF localized on the spindle and centromere.FTI SCH66336 was employed to treat PN stage embryos.The results showed that CENPF localization disappeared in morula and blastocyst embryos,which indicated that CENPF farnesylation plays key role during its localization in early embryos.M? stage oocytes were injected by CENPF-MO to record the early embryonic development after parthenogenetic activation.The results showed that CENPF knockdown led to the failure of embryonic development,and the embryos were arrested at 4-cell or 8-cell with almost no blastocyst formation.To futher detect the effect of CENPF knock down on early embryo development,2-cell stage embryos with one blastomere injected with Ctrl-MO labelled with red fluorophore and one with CENPF-MO labelled with green fluorophore were also assessed using time-lapse imaging.The results showed that blastomere injected with Ctrl-MO can developed to blastocyst stage whereas blastomere injected with CENPF-MO failed to form blastocyst.In conclusion,our data suggest CENPF play importent roles during mouse oocyte meiosis and early embryonic development.