Investigation Of Mechanisms Underlying The Oocyte Quality Regulated By Oocyte Maturation And Fertilization Associated Proteins

Mammalian oocyte quality is one of the most important factors in livestock fecundity and animal hunsbandy development,which involves two key biological processes.1)Oocytes maturation,implicated in many proteins regulating the correct spindle formation and chromosomes separation;2)Fertilization,implicated in a number of important molecules regulating the sperm-egg interaction.Any errors in this series of processes are likely to result in abnormal oocyte maturation,generation of aneuploid eggs and loss of fertilization ability.Fertilization of aneuploid eggs is a major cause of pregnancy loss,and if there is survival to term,it will result in developmental disabilities.Although various molecules have been proposed to contribute to oocyte meiosis and fertilization,the mechanisms of modulate remain to be discovered in this process.Therefore,it is important to exploreg and discovere the molecular regulatory mechanisms for better understanding the oocytes quality,meanwhile,establishing the theoretical foundation for improving livestock reproductive.In these experiments,ICR mice were employed as the experimental animal model.Taking advantage of in vitro culture,siRNA injection,morpholino injection,vector construction,in vitro transcription,drug treatment,immunofluorescent staining and quantification,as well as western blot,we investigated the molecules and their possible regulating mechanisms involved in the oocyte maturation and fertilization.This study is divided into four parts,and the main results are shown as follows:Experiment 1.Zfp207 is a Bub3 binding protein regulating meiotic chromosome alignment in mouse oocytesZinc finger proteins are a massive,diverse family of proteins that serve a wide variety of biological functions.However,the roles of them during meiosis are not yet clearly defined.Here,we report that Zfp207 localizes at the kinetochores during mouse oocyte meiotic maturation.Depletion of Zfp207 leads to a significantly higher proportion of impaired spindle organization and misaligned chromosomes in oocytes.This is coupled with the defective kinetochore-microtubule attachments,and resultantly increasing incidence of aneuploid metaphase ? eggs.The precocious polar body extrusion and escape of metaphase ? arrest induced by nocodazole treatment in Zfp207-depleted oocytes indicates that Zfp207 is essential for activation of SAC(Spindle Assembly Checkpoint)activity.Notably,we find that Zfp207 binds to Bub3 to form a complex and maintains its protein level in oocytes,and that overexpression of Bub3 is able to partially rescue the occurrence of aneuploid eggs in Zfp207-depleted oocytes.Collectively,we identify Zfp207 as a novel Bub3 binding protein in oocytes which plays an important role in controlling meiotic chromosome alignment and SAC function.Experiment 2.Cullin9 protects mouse eggs from aneuploidy by controlling microtubule dynamics via SurvivinThe Cullin9 gene encodes a putative E3 ligase that serves a wide variety of biological functions in mitosis,whereas its roles in meiosis have not yet clearly defined.Here,we report that Cullin9 accumulates on the spindle apparatus and colocalizes with the microtubule fibers during mouse oocyte meiotic maturation.Depletion of Cullin9 by morpholino microinjection results in a remarkably higher rate of disorganized spindles and misaligned chromosomes in oocytes,which is coupled with the impaired kinetochore-microtubule attachments.Resultantly,the incidence of aneuploid eggs significantly increases in Cullin9-depleted oocytes.Moreover,we show that Cullin9 controls Survivin's protein level during meiotic maturation,and thus regulates microtubule stability in oocytes.Thus,our study assigns a new meiotic function to Cullin9 and reveals that it prevents mouse eggs from aneuploidy by regulating microtubule dynamics via Survivin.Experiment 3.Chk2 regulates cell cycle progression during mouse oocyte maturationAs a tumor suppressor homologue during mitosis,Chk2 is involved in replication checkpoints,DNA repair,and cell cycle arrest,although its functions during mouse oocyte meiosis and early embryo development remain uncertain.We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development.Chk2 exhibited a dynamic localization pattern;Chk2 expression was restricted to germinal vesicles at the germinal vesicle(GV)stage,was associated with centromeres at pro-metaphase I(Pro-MI),and localized to spindle poles at metaphase I(MI).Disrupting Chk2 activity resulted in cell cycle progression defects.First,inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown(GVBD);this could be rescued after Chk2 inhibition release.Second,Chk2 inhibition after oocyte GVBD caused MI arrest.Third,the first cleavage of early embryo development was disrupted by Chk2 inhibition.Additionally,in inhibitor-treated oocytes,checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage,which indicated that the spindle assembly checkpoint(SAC)was activated.Moreover,disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption.In inhibitortreated oocytes,centrosome protein y-tubulin and Pololike kinase 1(Plkl)were dissociated from spindle poles.These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.Experiment 4.Melatonin improves the fertilization potential of postovulatory aged eggs by maintaining Ovastacin and Juno in the mouseThe quality of mammalian metaphase ? eggs undergoes an inreversible deterioration with increasing time following ovulation,which thereby results in the severe fertilization failure.However,the underlying mechanisms of this process and how to prevent or ameliorate it have not yet been clearly defined.Here,we investigate the key regulators and events during fertilization in postovulatory aged mouse eggs.We find that the number of sperm binding to zona pellucida surrounding aged eggs is remarkably reduced compared to controls,which is consistent with the cleavage status of N-terminus of Zp2,the sperm binding site in the zona pellucida.In addition,ovastacin is shown to be mislocalized and prematurely exocytosed to cleave Zp2 in aged eggs,leading to the defective sperm binding ability.Coupled with this,actin cap formation is disrupted following postovulatory aging,showing the disappearance of cortical granule free domain.Notably,we also find that the localization and protein level of Juno,the sperm receptor on the egg membrane,are strikingly impaired in aged eggs.Finally,we show that melatonin exposure to postovulatory aged eggs significantly elevates the in vitro fertilization rate by rescuing above aging-associated defects of fertilization participants and events,which might be through reducing ROS levels and inhibiting apoptosis.Taken together,our data reveal that both zona pellucida and egg membrane in postovulatory aged eggs are compromised in interaction with sperm,and melatonin is able to improve the fertilization potential of these aged eggs by maintaining the localization and protein level of two key fertilization players ovastacin and Juno.