Functional Study Of CMTM6 In Spermatogenesis In Mice

Objective: By constructing CKLF-like MARVEL transmembrane domain-containing protein 6(Cmtm6)knockout mice,the effects of Cmtm6 knockout on the expression of related functional proteins in testis and epididymis and the sperm function of Cmtm6 knockout mice were detected,and the effects of CMTM6 knockout mice on spermatogenesis,sperm maturation and sperm function were further analyzed.It lays a foundation for exploring the role of CMTM6 in male infertility and as a suitable marker of sperm quality.Methods:(1)Cmtm6 KO and Cmtm6 WT mice were bred and identified.The localization and expression of CMTM6 in testis,epididymis and sperm were detected by immunofluorescence technique,and the expression of Cmtm6 gene in main tissues of mice was detected by q PCR.(2)the testis,epididymis and spermatozoa of appropriate age mice were collected,and the morphology of testis,epididymis and sperm of Cmtm6 KO and Cmtm6 WT mice were detected by HE staining and Coomassie brilliant blue staining,respectively.(3)the expression and localization of marker functional proteins in testis,epididymis and spermatozoa of Cmtm6 KO and Cmtm6 WT mice were detected by Westernblotting and immunohistochemistry.(4)the sperm motility of Cmtm6 KO and Cmtm6 WT mice was detected by CASA.The in vivo and in vitro functions of spermatozoa of Cmtm6 KO and Cmtm6 WT mice were detected by closed cage and IVF techniques,respectively.(5)the acrosome reaction rate of Cmtm6 KO and Cmtm6 WT mice was detected by capacitation and induced acrosome reaction in vitro,and the expression of CMTM6 before and after acrosome reaction was detected by immunofluorescence technique.The above indexes of Cmtm6 KO mice and Cmtm6 WT mice were compared and evaluated to find out the effects of Cmtm6 gene knockout on fertility and spermatogenesis in mice,and to study the possible role of CMTM6 in spermatogenesis.(6)the obesity models induced by high fat diet were established in Cmtm6 KO mice and Cmtm6 WT mice respectively.The sperm motility was detected by CASA,and the morphology of testis and sperm was detected by HE staining or Coomassie brilliant blue staining.The above indexes of Cmtm6 KO mice and Cmtm6 WT mice were compared and analyzed to study the protective role of CMTM6 in the process of impaired fertility in mice caused by obesity,and to further explore the function of CMTM6 in mouse spermatogenesis and maturation.Results: in this study,it was found that CMTM6 was widely distributed in mouse testis and epididymis,located in acrosome in spermatozoa and no longer expressed after acrosome reaction.Cmtm6 knockout can promote the expression of androgen synthesis related enzymes CYP11A1 and HSD3 β,promote the expression of Sertoli cell related protein VIM,thus affect the level of spermatogenesis in mouse testis;Cmtm6 knockout can promote the expression of aquaporin AQP9 in epididymis and affect the further maturation of sperm in epididymis;Cmtm6 knockout can promote sperm acrosome reaction and promote mouse embryonic development,thus affecting mouse sperm function.Cmtm6 knockout can improve the reproductive damage induced by high-fat diet in male mice.Conclusion: 1.CMTM6 is related to the androgen synthesis of mouse Leydig cells and the function of Sertoli cells.2.CMTM6 is related to the expression of aquaporin AQP9 in mouse epididymis,which is involved in the regulation of epididymal microenvironment.3.CMTM6 is related to the occurrence of mouse sperm acrosome reaction and affects the development of mouse blastocyst and the number of offspring.4.CMTM6 is related to reproductive damage caused by obesity induced by high fat.