Study On GSK-3β Modulating Oocytes Meiotic Prophase Ⅰ And Primordial Folliculogenesis In Mice

Due to the lack of germline stem cell in adult ovary,female mammalians have a limited reproductive lifespan,which is determined by the size of primordial follicle pool formed perinatally.In fetal ovary,immature oocytes progress through the meiotic prophase I and become arrested in the dictyate stage before surrounded by flattened pre-granulosa cells to form primordial follicle.Meiotic defects in fetal oocytes lead to primordial follicle assemble failure in ovary.Thus,the studies focused on the faithful regulation network during early oogenesis help us better understanding the establishment of female ovarian reserve in mammalians.Glycogen synthase kinase-3 beta(GSK-3β)is a highly evolutionary conserved serine-threonine kinase,which was found to be a multifunctional enzyme.GSK-3P phosphorylates multiple substrates and is involved in regulation of protein synthesis,cell proliferation and differentiation,microtubule dynamics and apoptosis.However,role of GSK-3β in early oogenesis and folliculogenesis remained unclear.In this study,we demonstrated that GSK-3β was widely expressed in fetal and perinatal ovary in mice and primarily located within the cytoplasm of both somatic and germ cells.Further,we found that the kinase activity of GSK-3P in oocytes decreased during fetal ovarian development.To examine the potential function of GSK-3β in fetal ovary,we used specific GSK-3P inhibitor BIO to block the GSK-3β activity in an in vitro culture system.Our results showed that inhibition of GSK-3β activity leads to dramatic fetal oocytes loss via apoptosis pathway.In addition,inhibition of GSK-3β impeded meiotic progression in fetal oocytes and induced DSBs repair deficiency.By detecting the DNA damage checkpoint signaling,we revealed that p63,one of the major genome guardians of female germline,was prematurely upregulated and induced pro-apoptotic genes expression in fetal ovaries following GSK-3βinhibition.To explore the underlying mechanism,we found that the nuclear translocation and transcriptional activation of β-catenin was responsible for the disrupted p63 expression pattern and fetal oocytes attrition following GSK-3β inhibition.Meanwhile,our results proved that P-catenin display increasing transcriptional activity in pace with meiotic progression,which was in accordance with p63 expression within fetal oocytes.To validate the physiological role of GSK-3P during early oogenesis in vivo,we generated a mouse model with germ cell-specific deletion of Gsk-3β and referred as Gsk-3β cKO mice.According to our results,Gsk-3β cKO ovaries contained significantly fewer follicle reserves than the control ovary on 7 dpp.Exceeding cell apoptosis was detected in Gsk-3β cKO ovary.Moreover,we detected incomplete DSBs repair in oocytes after GSK-3P deletion in fetal ovary.In sum,GSK-3P is essential for early oogenesis and folliculogenesis via regulating the cytoplasm-nuclear translocation of P-catenin,which modulated timely p63 expression during meiotic prophase I in mice.Our study provided perspective that concerning the regulatory role of DNA damage checkpoint signaling in fetal germ cell guardianship and female fertility preservation in mammalians.