Effects Of IFT74 On Spermatogenesis And Fertility In Male Mice

Objective:To investigate the effects of intraflagellar transport protein 74（IFT74）on spermatogenesis and fertility in male mice and provide scientific clues for a more comprehensive understanding of male reproductive dysfunction.Method:Adult Ift74^flox/flox female mice were crossed with Stra8-iCre male mice to establish Ift74 gene conditional knockout mice.The genotypes of the mice were analyzed by the conventional PCR method.Stra8-iCre⁺/Ift74^flox/flox male mice were selected as the experimental group,and Stra8-iCre^-male mice were used as the control group.The hearts,brain,spleen,lung,liver,kidney,muscle,testis and epididymis of each group were collected.Western blot was used to detect the expression levels of IFT74 in various tissues of the control mice and different stages in the first wave of spermatogenesis in the testis.Immunofluorescence staining was used to detect the localization of IFT74 in spermatogenic cells of testicular tissue in the experimental group and the control group.HE staining was used to detect the morphological changes in various stages of spermatogenesis in testicle and epididymis.Scanning electron microscopy was used to observe the morphological changes of sperm morphology in epididymis and the ultrastructural changes of mouse testis were observed by transmission electron microscopy.Western blot was performed to detect the expression levels of spermatogenesis related genes such as IFT20,IFT25,IFT27,IFT81,IFT88,IFT140,ODF2,SPAG16L and AKAP4 in the testis of each group.Sperm in the epididymis of male mice in each group were collected to detect sperm count and sperm motility.At the same time,6 pairs of 6-week-old Ift74 gene-exclusive male mice and control male mice were mated with adult wild-type female mice for more than two months to evaluate their fertility and reproductive capacity,respectively.Result:IFT74 protein was expressed in the brain,lung,kidney and testis of male mice,and has the highest expression in testis tissue.During the first wave of spermatogenesis in male mice after birth,IFT74 protein was expressed from day 12,and its expression level was significantly up-regulated on day 20 and reached the highest level on day 35.The IFT74protein is mainly localized in the vesicles of spermatocytes and round sperm cells,the acrosome and centrosome of prolonged sperm cells,and the developing sperm tail during the spermatogenesis stage.The results of HE staining showed that the flagella of sperms were released into the lumen of the testis in the control group.The cytoplasmic residual of germ cells was phagocytized by the supporting cells.The spermatogenesis was normal,and the epididymis was filled with sperm containing normal head and tail.However,in the testicular tissue of Ift74 gene conditional knockout mice,the spermatozoa of the VIII stage was abnormal,and no normal residual body was formed.The cytoplasm remained in the lumen,and the abnormal sperm cells at the 16th stage were phagocytosed.Abnormal sperm cells with a deformed head and no tail were observed at the XI stage.Although there were normal round spermatids in the seminiferous tubules of stage XIII,there was an abnormal13th elongating spermatid of which tails were missing.And excessive cytoplasm of sperm cells seems to fall into the lumen.In the epididymis,almost no sperm with the normal head and tail were observed,and it was found to contain a large number of abnormal cytoplasmic residues,detached round cells,and abnormal sperm with abnormal head and short tail or no tail.Scanning electron microscopy showed that the sperm in the epididymis of the control mice had a normal shape head and a long and smooth tail,while the Ift74 gene conditional knockout male mice had abnormal sperm structure and a round head,short tail or no tail.At the same time,transmission electron microscopy showed that there were a large number of normal"9+2"double microtubule structures and normal heads in the testicular seminiferous tubules of the control group.However,in the testes of Ift74 gene conditional knockout male mice,the axons and microtubules of sperm in the seminiferous tubules exhibit diverse abnormalities such as disordered microtubules,microtubules with no core axis structure and mitochondrial clusters,or the absence of central microtubules to form abnormal axial filaments.The expression level of the other components of IFT-B complex such as IFT27,IFT57,IFT81,IFT88 and IFT140 and the main component of the fiber sheath AKAP4 was significantly lower in testes from the testes of Ift74 gene conditional knockout male mice than that of the control mice（P<0.05）.Moreover,compared with the control group,the sperm count and motility from the Ift74 gene conditional knockout male mice were significantly decreased,and the movement ability of the sperm was also decreased（P<0.05）.Although Ift74 gene conditional knockout male mice had normal mating behavior,they were completely infertile.Conclusion:IFT74 is an essential protein for spermatogenesis and fertility of male mice.It may be involved in the process of spermatogenesis by regulating axoneme and microtubule assembly and affect male fertility.Therefore,it is suggested that IFT74 may be a potential genetic factor affecting male reproduction.