Effect On Sperm Number,Motility,and Male Fertility By Intraflagellar Transport Protein IFT172 In Mice

Objective:To investigate the role of Intraflagellar transport 172?IFT172?in male germ cells of mice,its function in the formation of sperm flagellar of mice and effect on male fertility of mice.Method:1.The adult floxed Ift172 female mice were bred with Stra8-iCre mice so that the Ift172 gene was disrupted in spermatocytes/spermatids and used wildtype mice as the control.2.To test the male fertility of Ift172 conditional knockout male mice,10 couple of 6weeks or older Ift172 conditional knockout male mice and wildtype male mice were crossed with adult wildtype female mice for more than 2 months.3.The sperm were collected from epididymides of wildtype and Ift172 conditional knockout mice.Test the sperm number,motility and their morphology under the light microscope.4.HE staining of the testis and epididymides from wildtype and Ift172 conditional knockout mice were conducted to observe all the phases of spermatogenesis.5.Observed the ultrastructural of sperm from wildtype and Ift172 conditional knockout mice with TEM.6.Tested the expression level of some associated protein in the testises from wildtype and Ift172conditional knockout mice.7.Isolated germ cells from wildtype and Ift172 conditional knockout mice testises,to investigate the localization of IFT172 and the knockout efficiency of IFT172 in Ift172 conditional knockout mice.Observe the localization of AKAP4,a fiber sheath protein,and ODF2,a outer dense fiber protein,in the sperm of wildtype and Ift172conditional knockout mice.Result:The Ift172^flox/flox:Stra8-iCre mutant mice did not show any gross abnormalities,and all of the mutant mice survived to adulthood.1.There was no difference between testis weight/body weight between controls and mutant mice.About half of the adult homozygous mutant males examined were infertile.2.Histological examination of the testes revealed abnormally developed germ cells at spermiogenesis phase.3.The cauda epididymides contained less developed sperm,and sperm number was reduced about ten folds compared to the controls.Sperm collected from cauda epididymides showed significantly reduced motility in the mutant mice.Examination of epididymal sperm by light microscopy revealed bent tails in the mutant mice.4.In the conditional Ift172 mutant testis,expression levels of other complex B components,IFT20,IFT25,IFT27,IFT81,and an complex A component,IFT140 were not changed compared to the controls.Expression level of a central apparatus protein,SPAG16L was also not changed.However,expression levels of ODF2,a component of the outer dense fiber,and AKAP4,a component of fibrous sheath,were dramatically reduced.5.In the germ cells of wildtype mice,IFT172 localize on the manchette of elongating spermatid.And the single of IFT172 in the conditional Ift172mutant mice significantly reduced.Compared with wildtype,the localization of AkAP4 and ODF2 in the sperm of Ift172 mutant mice have no change.Conclusion:Reduced sperm number,motility and male fertility in mice with reduced expression of intraflagellar transport protein IFT172 in male germ cell.IFT172 is essential for male fertility and spermiogenesis in mice.Given that ye conditional mutant mice using Stra8-iCre still express residual IFT172 protein in the germ cells,which is about one tenth of the controls,we expect more severe abnormalities if Ift172 gene is completely disrupt.