Mechanism Study Of Chromosome Cohesion Proteins Sororin And Wapl In Regulation Of Mammalian Oocyte Meiotic Maturation

Low quality of oocytes is one of the core factors that impair the fecundity of female animals,limiting the levels of livestock production and economic benefits.Oocyte maturation disorder and aneuploidy are the main manifestations of low oocyte quality.Due to nuclear or cytoplasmic defects,oocytes with maturation disorders cannot be fertilized;although aneuploid eggs can be fertilized,the instability of the genome will eventually lead to embryonic development failure,embryonic death,miscarriage or birth defects.However,the underlying mechanisms of low quality of oocytes have not been fully elucidated,and the molecular basis is still poorly understood.Identification of regulators that affect oocyte quality will contribute to develop the strategy to improve the oocyte quality.Euploidy and genome stability require dynamic changes of chromosome cohesion which is mediated by chromosome cohesion proteins.As oocyte meiosis is a special way of cell division that differs from mitosis,the functions and regulatory mechanisms of chromosome cohesion proteins during mammalian oocyte maturation is still unclear.Therefore,investigation of the roles of chromosome cohesion proteins in oocyte maturation and quality control will provide important theoretical guidances for the maintenance of livestock fertility and production.The roles of chromosome cohesion proteins Sororin and Wapl in somatic mitosis have been extensively studied.They take important part in driving the dynamic changes of chromosome cohesion.However,the functions and regulatory mechanisms of Sororin and Wapl in mammalian oocyte meiosis remain unclear.Therefore,in this study,we used mouse and porcine oocytes as the model,and applied the methods of targeted morpholino or si RNA gene silencing,microinjection,immunofluorescence,immunoblotting,mass spectrometry as well as immunoprecipitation to investigate the functional mechanisms of Sororin and Wapl during mouse oocyte maturation and their conservation in porcine oocytes.The study is divided into three parts.The specific research content and results are shown as follows:Experiment 1.The cohesin stabilizer Sororin drives G2/M transition and spindle assembly in mammalian oocytes.Cohesin complex mediates sister chromatid cohesion to guarantee faithful chromosome segregation.In eukaryotic cells,cohesin is composed of Smc1,Smc3,Scc1 and Scc3 subunits and regulated by a number of accessory factors.It is acetylated on Smc3 subunit by the acetyltransferases Esco1 and Esco2 to establish sister chromatid cohesion during S phase.Then,cohesin stabilizer Sororin is recruited to cohesin complex by acetylated Smc3 to stabilize sister chromatid cohesion through counteracting Wapl’s interaction with Pds5.During prophase,phosphorylation of Sororin results in formation of Wapl-Pds5 heterodimers and thus removes cohesin from chromosome arms.After that Sororin is released to the cytoplasm and its functions are largely unknown.In this study,we report that Sororin acts as a regulator of meiotic G2/M transition and spindle assembly in mammalian oocytes.Sororin is localized in the nucleus of germinal vesicle（GV）oocytes and translocated to the spindle apparatus during meiosis I in mice.Depletion of Sororin results in the failure of germinal vesicle breakdown（GVBD）due to the inactivation of cyclin-dependent kinase 1（Cdk1）caused by the reduced level of Cyclin B2.Futhermore,Sororin depletion results in the defective spindle assembly by showing the spherical morphology.Expression of exogenous Cyclin B2 recovers the spindle length and area to the normal levels in Sororin-depleted oocytes.We further demonstate that Sororin interacts with Cyclin B2 and protects it from destruction by APC^Cdh1,consequently driving M phase entry and biplolar spindle formation.Specially,these meiotic functions of Sororin are conserved among species.In short,our findings provide novel insights into the noncanonical role of Sororin in governing meiotic resumption and progression through meiosis I in mammalian oocytes.Experiment 2.The cohesin release factor Wapl interacts with Bub3 to govern SAC activity in female meiosis IDuring mitotic prophase,a mass of cohesins is removed from chromosome arms by Wapl,a pivotal cohesin release factor,to ensure sister chromatid separation.However,during mammalian oocyte meiosis I,homologs separate from each other and the resolution of chiasmata requires proteolytic cleavage along chromosome arms of cohesin’s Rec8 subunit by Separase.Thus,whether Wapl is involved in the resolution of chiasmata as a cohesin release factor or it has other unique functions during oocyte meiosis I are still elusive.In this study,we show that Wapl functions as a regulator of spindle assembly checkpoint（SAC）during mouse oocyte meiosis.Depletion of Wapl accelerates meiotic progression and inactivates SAC in oocytes.Wapl depletion also causes meiotic defects such as aberrant spindle assembly,improper chromosome alignment and incorrect kinetochore-microtubule（K-MT）attachment,consequently leading to aneuploid eggs.Specially,we identify Bub3,a key SAC component,as a new binding partner of Wapl by immunoprecipitation coupled with mass spectrometry analysis.We further substantiate that the involvement of Wapl in SAC control is mediated by its role in maintenance of Bub3 protein level.Expression of exogenous Bub3 could recover the meiotic defects in Wapl-depleted oocytes.In brief,our findings uncover a noncanonical role of Wapl in mediating SAC control in mouse oocyte meiosis I.Experiment 3.Wapl orchestrates porcine oocyte meiotic progression via control of SAC activityAs a cohesin release factor,Wapl acts to remove cohesin complexes from chromosome arms during mitosis prophase to allow the accurate chromosome segregation in anaphase.However,we have recently verified that Wapl exerts a unique meiotic function in the SAC control through maintaining Bub3 stability during mouse oocyte meiosis I.Whether this noncanonical function is conserved among species is still unknown.In this study,we applied porcine oocytes as a model to validate that Wapl performs conserved roles in the regulation of SAC activity during mammalian oocyte maturation.We found that depletion of Wapl via RNAi-based gene silencing approach resulted in the accelerated meiotic progression by displaying the precocious polar body extrusion and compromised spindle assembly and chromosome alignment.Notably,we observed that the protein level of Bub3 was substantially reduced in Wapl-depleted oocytes,especially at kinetochores.Taken together,our data demonstrate that Wapl participates in the porcine oocyte meiotic progression through maintenance of Bub3 protein levels and SAC activity.This meiotic function of Wapl in oocytes is highly conserved between pigs and mice.To sum up,our study uncovered the noncanonical functions of cohesin regulatory factors Sororin and Wapl during oocyte meiosis using mouse and porcine oocytes as the research objects.By using a mouse model,we performed mechanism-based research and discovered that Sororin associates with Cyclin B2 and protects it from destruction by APCCdh1 to driving G2/M transition and biplolar spindle formation.Moreover,Wapl interacts with Bub3 to govern SAC activity and maintain the euploidy in mouse oocyte meiosis I.Particularly,we verified the unique roles of Sororin and Wapl in porcine oocytes,indicating that their functions are highly conserved among mammals.Thus,our findings reveal the unique roles of chromosome cohesion proteins Sororin and Wapl during mammalian oocyte maturation and expand the understanding of the oocyte meiotic mechanisms,providing the theoretical basis for defining chromosome cohesion proteins as the molecular marker of oocyte quality.