The Function And Mechanism Of WDR62 In Mouse Oocyte Meiotic Maturation And Granulosa Cells

Compromised meiosis or abnormal chromosome segregation induced by disturbed oocyte growth and development can affect fertilization and embryonic development,thereby result in abortion and fetal malformation.Notwithstanding,oocyte development is influenced by endogenously complex regulation of meiosis and exogenously multiple factors including granulosa cells,therefore deciphering the regulatory molecular mechanism of endogenous and exogenous factors is substantial for improving livestock reproduction and promoting its fecundity.MCPH proteins play important roles in mitosis and meiosis.As one of the MCPH protein,WDR62(WD40-repeat protein 62)participates in the manipulation of mitotic spindle assembly and cell cycle progression by involving in centriole replication and maintaining centrosome integrity.Meanwhile,the recent study revealed that Wdr62-/female mice were completely infertile,coupled with defective meiotic initiation and absence of ovarian follicles and germ cells,indicative of the unrivaled significance of WDR62 in female reproduction;nonetheless,the functions of WDR62 during meiotic maturation as well as in granule cells remains unclear.Herein,with the employment of small RNA interference,we knocked down WDR62 expression in oocytes and granulosa cells,and combined with real-time PCR,immunoblotting,immunofluorescence and confocal,fluorescence intensity measurement,chromosome spread,to investigate the contribution of WDR62 in oocyte meiotic maturation and granule cells.The main results in this study are as follow:1.The function and mechanism of WDR62 in meiotic maturation of mouse oocytes(1)WDR62 was localized in oocyte germinal vesicle at GV stage,and after meiotic resumption(marked by GVBD),WDR62 was mainly co-localized with chromosomes in the whole meiotic maturation stage.In addition,WDR62 was expressed in oocyte meiotic progression,with the highest expression in GV stage and the lowest expression in metaphase Ⅱ(MⅡ)stage.(2)Depletion of WDR62 did not affect the GVBD(P>0.05)but blocked the first polar body extrusion(PBE)(P<0.05).Meanwhile,loss of WDR62 affected asymmetric cytokinesis and led to the generation of large polar body(P<0.01).(3)Depletion of WDR62 caused aberrant localization(P<0.001)and decreased expression(P<0.05)of p-JNK as well as caused an elevation(P<0.0001)of H3K9me3 level,which damaged spindle assembly(P<0.01),chromosome arrangement(P<0.01)and kinetochore-microtubule attachment(K-MT)structure(P<0.01),thus resulting in the continuous activation of spindle assembly checkpoint(SAC)and ultimately disturbed meiotic progression.Repressing p-JNK expression with the employment of SP600125(specific inhibitor of p-JNK)could cause the similar abnormal phenotypes:increased level of H3K9me3(P<0.0001),coupled with a higher incidence of spindle abnormality(P<0.001)and chromosome misalignment(P<0.001),as well as the impaired meiotic progression.These results revealed that that WDR62 participated in regulating meiotic maturation through the JNK signaling pathway.(4)Depletion of WDR62 resulted in abnormal localization(P<0.01)and increased expression(P<0.05)of Arp2/3 complex,which led to actin cap formation failure(P<0.05),blocking spindle migration to oocyte cortex(P<0.01),consequently impairing asymmetric cytokinesis.These data indicated that WDR62 regulated spindle migration-mediated asymmetric cytokinesis by involving in the regulation of the Arp2/3 complex manipulating the dynamics of actin.2.The function and mechanism of WDR62 in mouse granulosa cells(1)WDR62 was expressed in both oocytes and granulosa cells of the mouse ovarian tissue.(2)Depletion of WDR62 resulted in the decrease of PCNA expression(P<0.0001),thus reducing the number of granule cells(P<0.001)and viability(P<0.001).(3)Depletion of WDR62 led to an elevation(P<0.05)of the proapoptotic Bax protein and induced the apoptosis of granulosa cells(P<0.05).(4)Depletion of WDR62 intervened the expression of mitotic spindle regulators AURKA(P<0.01)and p-JNK(P<0.001),which perturbed cell cycle progression,displaying the decrease of S phase(P<0.05)and increase of G2 phase(P<0.05).(5)Depletion of WDR62 resulted in the decreased expression of the granulosa cell-related hormone receptors FSHR(P<0.0001)and ER(P<0.05),which affected the function of the granulosa cell.The above results demonstrated that WDR62 regulates granule cell physiological state and function via operating the expression of proliferation,apoptosis and cell cycle-related proteins as well as the expression of granulosa cell-related hormone receptors.In summary,our study documented depletion of WDR62 compromised oocyte maturation through affecting JNK signaling and H3K9me3 status;meanwhile,loss of WDR62 disrupted the cytoskeletal dynamics via perturbing Arp2/3 complex localization,resultant to the abnormal asymmetric cytokinesis.In addition,decline of WDR62 inhibited the proliferation and induced the apoptosis of granulosa cells,as well as disturbed its cell cycle progression and hormone receptor expression.