Function And Mechanism Analysis Of TMEM232 In Mouse Sperm Development

Male infertility has become an increasingly serious global problem.There are many causes of male infertility,including genetic factors,external mechanical damage,environment and bad living habits.The main clinical manifestations are reduced sperm number,decreased sperm motility and abnormal sperm structure and morphology,collectively known as Oligoasthenospermia（OAT）.Although many genes and related mutations causing oligospermia have been identified in recent years,the pathogenesis remains unclear.TMEM232 Protein is a member of TMEM Transmembrane Protein（TMEMs）family,and it has been shown to be highly specifically expressed in testis.It has been reported that TMEM232 gene is a susceptibility gene to atopic dermatitis（AD）in Chinese Han population.In addition,there are few studies and reports on TMEM232 protein,and no diseases caused by abnormal expression of TMEM232 protein have been found clinically.In the previous observation of TMEM232 knockout(Tmem232^-/-)mouse model constructed by CRISPR/Cas9 gene editing technology,our laboratory found that male Tmem232^-/-mice were sterile,while female mice were able to get pregnant and give birth normally.The appearance and testicular weight and size of Tmem232^+/+and Tmem232^-/-mice were not significantly different,but there were defects in sperm structure and motility in male Tmem232^-/-mice,suggesting that Tmem232 may play an important role in spermatogenesis and maturation.The results of H＆E staining showed that TMEM232deficiency did not affect the normal development of ovary in female mice.Scanning electron microscopy showed that the nasal epithelial cilia of Tmem232^-/-mice were shorter than those of Tmem232^+/+,but their axial filament microtubules were normal.In order to further explore the effects of TMEM232 on spermatogenesis and maturation,we prepared TMEM232-specific rabbit antibodies.After verification and identification,the prepared polyantibodies can specifically recognize human and mouse TMEM232 proteins.Using this antibody,we detected the spatiotemporal expression of TMEM232 protein during sperm development,and the results showed that TMEM232 began to be highly expressed during the round sperm stage.We also identified the localization of TMEM232 to the Golgi apparatus in tumor cells and demonstrated it using an exogenous expression system,and it is mainly located in the acrosome and flagella in sperm.Analysis and validation of proteomic results in knockout mice and wild-type mice suggest that deletion of TMEM232 may hinder sperm maturation in male mice and lead to reduced sperm count through changes in multiple protein expression levels.In sperm structure,TMEM232interacts with septins,HDAC6,SIRT2 and other proteins to participate in the formation and maintenance of normal sperm structure and the occurrence of sperm capacitation.In conclusion,TMEM232 may be involved in spermatogenesis,and regulate spermatogenesis,especially the correct assembly of flagellum structure and the smooth occurrence of fertilization.These abnormalities may be the potential pathogenic factors leading to TMEM232 knockout infertility in male mice.These results can provide reference for future research on human male infertility,and also provide us with a further understanding of the function of TMEM232 protein.