Intraflagellar Transport Protein25 (IFT25) Is Essential For Spermiogenesis And Male Fertility In Mice

Objective: To explore the role of intraflagellar transport 25(Ift25)in mouse sperm flagellar formation and male fertility.Method: 1.Ift25flox/flox females crossed with Stra8-i Cre males to generate Ift25 conditional knock out males.2.RT-PCR to detect the m RNA expression of IFT25 in multiple mouse tissues.3.Western blot to detect the different proteins expression in different mouse tissues and Ift25 conditional knock out testis.4.HE staining to detect the histology of Ift25 conditional knock out male testis and epidydimis.5.Germ cell slides preparation to detect the ODF2 and AKAP4 expression in sperm apparatus.6.Transmission electronic microscopy(TEM)and scaning Electronic Microscopy(SEM)to examine the utra-structur of sperm,testis and epidydimis.7.Direct yeast two hybrid assay and co-immunoprecipitation to detect the protein-protein interactions.8.statistic analysis of sperm count?sperm motility and percentage of motile sperm.Results : In mice,the gene is highly expressed in the testis,and its expression is up-regulated during the final phase when sperm flagella are formed.To investigate the role of IFT25 in sperm flagella formation,the gene was specifically disrupted in male germ cells.All conditional knockout mice survived to adulthood and did not show any gross abnormalities.However,all homozygous knockout males were completely infertile.Sperm numbers were reduced and these sperm were completely immotile.Multiple morphological abnormalities were observed,including round heads,short and bent tails,with some tails showing branched flagella and others with frequent abnormal thicknesses,as well as swollen tips of the tail.TEM revealed that flagellaraccessory structures,including the fibrous sheath and outer dense fibers,were disorganized,and most sperm had also lost the “9+2” microtubule structure.In the testis,IFT25 forms a complex with other IFT proteins.In Ift25 knockout testes,IFT27,an IFT25 binding partner,was missing,and IFT20 and IFT81 levels were also reduced.Conclusion:IFT25 is indispensable for sperm flagellum formation and male fertility in mice.