| Objective:The infertility rate in China is about 12.5%,and more than 40 million adults are having trouble conceiving.However,only 30%of infertile couples became pregnant following fertility treatments.50%of infertility cases can be attributed solely to the male.Male infertility is a complicated disease with high-heterogeneity phenotypes,in which 15%cases are caused by genetic factors including chromosome mutations and gene alterations.Teratozoospermia and asthenozoospermia are common clinical manifestations,and semen analysis is the major way for diagnosing them,but little is known about the pathogenesis.Therefore,it is necessary to further develop the technique for the etiological diagnosis of male infertility.Studies have shown that intraflagellar transport(IFT)is essential for spermatogenesis and male fertility in mice.Deletion of IFT subunits would damage sperm structure and disrupt functions,which will result in the male infertility.It has been reported that IFT139,IFT140 and IFT144 mutations could cause male infertility.However,the working mechanisms of IFTs remain unclear.In this study,protein expressions of IFT20,IFT52 and IFT88 were detected in the testis and sperm of mouse and human.To explore potential etiological biomarkers for male teratozoospermia and asthenozoospermia,and provide new ideas for treatment of male infertility,the differences in the expressions of IFTs between normal sperms and abnormal sperms were determined.Methods:RT-PCR was performed to detect the mRNA expressions of Ift20,Ift52 and Ift88 in different tissues of adult mice and testes of mice of different ages.Western Blot was performed to determine the protein expressions of IFT20,IFT52 and IFT88 in different tissues of adult mice and testes of mice of different ages.Immunofluorescence(IF)staining was performed to detect the localization of IFT20,IFT52 and IFT88 in adult mouse testes and mature sperms.Meanwhile,the protein expressions of IFT20,IFT52 and IFT88 in normal adult testes and mature sperms were detected by Western Blot.IF staining was performed to determine the localization of IFT20,IFT52 and IFT88 in normal mature sperms.Western Blot was also performed to evaluate the protein expressions of IFT20,IFT52 and IFT88 in sperms of patients with teratozoospermia and asthenozoospermia.Statistical analyses were performed to analyze the differences of IFT20,IFT52 and IFT88 in sperms among healthy people,patients with teratozoospermia and patients with asthenozoospermia.Results:1.The gene expression of Ift20 was detected in various tissues including heart,liver,spleen,lung,kidney,brain,epididymis and testis.The protein of IFT20 is mainly expressed in epididymis and testis,and weakly expressed in liver,spleen,lung,kidney,brain and stomach.The expression of IFT20 has been detected in mouse testes at 7 days postpartum(dpp),and maintained at a high level in adulthood.IF staining showed that IFT20 was located in the cytoplasm of germ cells after spermatocytes in seminiferous tubules.Higher expressions of IFT20 in phases Ⅱ-Ⅲ and Ⅴ-Ⅺ,and lower expressions in phases Ⅰ,Ⅳ and XII were found in mouse testes.In the mature sperms of mice and adult,IFT20 was located in the posterior ring of sperm nuclei and the full length of sperm tails.The expression of IFT20 was also detected in the sperm of patients with teratozoospermia and asthenozoospermia.The average relative content of IFT20 in the sperm of 62 normal adults and 48 patients with teratozoospermia were 1.09±0.62 and 1.52±1.73,respectively.The average relative content of IFT20 in the sperm of 32 patients with asthenozoospermia was 1.23±0.83.No significant changes of IFT20 expressions of sperms were found in patients with teratozoospermia and asthenozoospermia.2.The gene expression of Ift52 was mainly detected in liver,lung,brain and epididymis,with weak expression in heart,spleen,kidney and testis.The protein expression of IFT52 was mainly determined in mouse testes,and was relatively weak in liver,spleen,lung,kidney,brain,stomach,and epididymis.The expression of IFT52 has been detected in mouse testes at 7 days postpartum(dpp),and maintained at a high level in adulthood.IF staining showed that IFT52 was expressed in both seminiferous tubules and testicular interstitium of mice,and located in the cytoplasm of all germ cells and Sertoli cells.The expression of IFT52 maintains high in the whole phase I-XII.In the mature sperms of mice and adult,IFT52 was located in the posterior ring of sperm nuclei and the full length of sperm tails.The expression of IFT52 was also detected in the sperm of patients with teratozoospermia and asthenozoospermia.The average relative content of IFT52 in the sperm of 62 normal adults and 48 patients with teratozoospermia were 0.99±0.43 and 1.38±0.48,respectively.The average relative content of IFT52 in the sperm of 32 patients with asthenozoospermia was 1.40±0.61.A significant difference was found in the expression of IFT52 of sperms between healthy adults and patients with teratozoospermia or asthenozoospermia.3.The gene expression of Ift88 was mainly detected in brain,epididymis and testis,and weakly detected in heart,liver,spleen,lung and kidney.The protein expression of IFT88 was mainly determined in mouse testes,and was relatively weak in liver,spleen,lung,kidney,brain,stomach,and epididymis.IFT88 has been detected in mouse testes at 7 days postpartum(dpp),and maintained at a high level in adulthood.IF staining showed that IFT88 was located in the cytoplasm of germ cells that after spermatocytes in seminiferous tubules.IFT88 was expressed remarkably in stages Ⅰ-Ⅹ of spermatogenesis.Highest expressions of IFT88 in phases Ⅳ-Ⅸ and lowest expressions in phases Ⅺ-Ⅻ were detected in mouse testes.In the mature sperms of mice and adult,IFT88 was located in the posterior ring of sperm nuclei and the full length of sperm tails.The expression of IFT88 was also detected in the sperm of patients with teratozoospermia and asthenozoospermia.The average relative content of IFT88 in the sperm of 62 normal adults and 48 patients with teratozoospermia were 1.39±0.71 and 2.05±1.58,respectively.The average relative content of IFT88 in the sperm of 32 patients with asthenozoospermia was 1.68±0.71.The expression of IFT88 was increased significantly in sperms of patients with teratozoospermia,while no significant changes were found in patients with asthenozoospermia.Conclusion:In mice,IFT20 is a testis dominant molecule that expressed in the cytoplasm of germ cells after spermatocytes in seminiferous tubules.Its human homologous gene is also expressed in testis and sperm,and its localization in human sperm is similar to that in mouse sperm,which is located in the posterior ring of sperm nuclei and the full length of sperm tails.Its relative content in normal adult sperm were not associated with teratozoospermia or asthenozoospermia.In mice,IFT52 is also predominantly expressed in testis,in which it is present in the cytoplasm of all germ cells and Sertoli cells.Its human homologous gene is also expressed in testis and sperm,and its localization in human sperm is similar to that in mouse sperm,which is located in the posterior ring of sperm nuclei and the full length of sperm tails.Its relative content in normal adult sperm were associated with teratozoospermia and asthenozoospermia.The expression pattern of ift88 is similar to that of ift20,and it is also a testis dominant molecule expressed in testis and expressed in the cytoplasm of spermatogenic cells at all levels after spermatocyte.Its relative content in normal adult sperm was correlated with teratozoospermia,but not with asthenozoospermia. |
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