The Function And Mechanism Of MFN2 In Mouse Germ Cell Development

Part 1 The localization of MFN2 in mouse germ cells.[Purpose] To study the expression and localization of MFN2 in mouse testis.[Methods] The expression patterns of MFN2 in different organs and germ cells were analyzed by RT-q PCR,Western blot and immunofluorescence.[Results] MFN2 was highly expressed in heart,brain,and ovarian tissues,and predominantly expressed in testes.MFN2 was expressed in all types of spermatogenic cells,highly expressed in pachytene spermatocytes and followed by round spermatids.[Conclusions] MFN2 was predominantly expressed in the testis;it was localized in all spermatogenic cells,highly expressed in pachytene spermatocytes and followed by round spermatids.Part 2 The function of MFN2 on spermatogenesis and male fertility in mice[Purpose] To study the function of MFN2 in mouse germ cells.[Methods] We generated Mfn2 germline-specific knockout mice by using Stra8-Cre transgenic mice,and the spermatogenesis was analyzed through analying testicular morphology and histology.[Results] The germ cell MFN2 knockout mouse model was successfully constructed,and the test found that the germ cell MFN2 knockout mice were male sterile,the number of empty seminiferous tubules in the testes of adult mice increased with aging.In addition,the number of sperm in the epididymis decreased and the percentage of abnormal sperm increased.[Conclusions] MFN2 plays an important function in mice spermatogenesis and the deletion can lead to abnormal spermatogenesis and male sterility.Part 3 The mechanism of MFN2 in mouse spermatogenesis[Purpose] To investigate the mechanism and function of MFN2 in spermatogenesis in mice[Methods] Using a transmission electron microscope(TEM)analysis,we observed the structure of mitochondria,IMC and MAM in mouse spermatogenic cells.We examined the interaction between Nuage associated proteins and MFN2 in vitro and in vivo using immunoprecipitation.In addition,we examined the abundance of pi RNA distributions through small RNA sequencing and detected the expression of transposon by RT-q PCR and immunofluorescence.[Results] In MFN2 deficient germ cells,mitochondria exhibited swelling and fragmentation and the thickness of IMC and MAM increased.MFN2 protein interacted with Nuage associated proteins MIWI,DDX4,GASZ,and TDRKH both in vivo and in vitro.When MFN2 is absent,the expression of MIWI,DDX4,GASZ in germ cells decreased;the amount of pi RNA in germ cells of Mfn2 c KO mice decreased compared with the control group,but the expression of transposon didn't change.[Conclusions] MFN2 affected the morphology of mitochondrial related structures,including MAM and IMC,and decreased the expression of Nuage associated proteins MIWI,DDX4,and GASZ,and pi RNA decreased by nearly 50%.