Study Of Function And Regulatory Mechanisms Of Calcineurin On Mouse Follicle Oocyte Development

Calcineurin is an important serine / threonine protein phosphatase,composed of catalytic subunit A and regulatory subunit B,subunit A contains ?,? and ? iosforms,B subunit contains 1 and 2 iosfotms.They are widely distributed in various tissues and organs of animals and are directly regulated by Ca2 + / calmodulin(Ca M)during the process of cell signaling.They are involved in the regulation of many important processes in vivo by acting with dephosphorylation function,such as cell cycle,cell growth and differentiation.However,it has little reports of calcineurin in the role and regulatory mechanisms of calcineurin in mouse oocytes and granulosa cell development.In this study,we used immunohistochemistry(IHC),immunofluorescence(IF),western blot(WB),real-Time PCR,ELISA,flow cytometry technics and calcineurin inhibitor FK506,cultured mouse oocytes,granulocytes,cumulus cells and follicles to to reveal the expression pattern of calcineurin in mouse male and female reproductive system and clarify the fuctions of calcineurin on mouse oocytes development and related regulation mechanisms.The expression and location of calcineurin: calcineurin was mainly located in ovarian follicles and corpus luteum,oviduct epithelium and uterus,and it expressioned from primary follicles to big antral follicles.Calcineurin expressed in the testis and epididymal head,epididymal body and epididymal tail and mainly located in leydig cells,pseudostratified columnar epithelium cells.The expression pattern of calcineurin subunit in oocytes and granulosa cells were that the three subtypes of calcineurin A subunit(?,? and ?)were detected in oocytes and granulosa cells,the m RNA levels of calcineurin A? were significantly higher than calcineurin ? and ?.And it decreased significantly after GVBD.Both of the calcineurin B 1 and B2 were detected in granulosa cells and the m RNA expression of type B1 m RNA was significantly higher than that of B2.While only the m RNA of type B1 was detected in oocytes,and the protein expression level of B1 in GV,GVBD and MII had no significant difference,displaying a stable expression pattern.Further study of subcellular localization of calcineurin A subunit in oocytes and granulosa cells showed that calcineurin A subunit is localized in germinal vesicle of GV stage oocytes,which diffuse into the cytoplasm when oocytes undergo GVBD,and the protein levels of calcineurin A subunit in GVBD stage oocytes were significantly lower than GV and MII stage oocytes.While,calcineurin A subunit was localized in the nucleus of granulosa cells.After culturing in medium contains FK506,the GVBD occurs in oocytes from Dos,COCs and Fos and the GVBD rate were significant higher than control group.But FK506 did not affect the expansion of COCs cumulus.Calcieurin inhibitor FK506 could promote the phosphorylation of MAPK in oocytes and granulosa cells,and inhibit the proliferation of granulosa cells in low concentration and high concentration.However,only high concentration group can promote the apoptosis of granulosa cells.In addition,FK506 could inhibit the secretion of progesterone in corpus luteum cells,furthermore,the results of gavage treatment of calcineurin inhibitior on female mouse showed that there are a large number of large ovarian follicles without ovulation were exisited in ovary of low concentration group.The results of this study showed that calcineurin could regulate oocyte development through regulating the development and hormone secretion of granulosa cells and corpus luteum cells and MPF and MAPK signaling pathways in female mouse and calcineurin A? may play an important role in this process.These results provided a basic according for the study of calcineurin in mammalian ovaries.