Aim： To characterize mouse capping protein α3 （CPα3） during spermatogenesis and sperm maturation. Methods： We produced rat anti-CPα3 antiserum and examined the expression of CPα3 in various mouse tissues using...Aim： To characterize mouse capping protein α3 （CPα3） during spermatogenesis and sperm maturation. Methods： We produced rat anti-CPα3 antiserum and examined the expression of CPα3 in various mouse tissues using Western blot analysis and the localization of CPα3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPα3 and β-actin （ACTB） in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPα3 antiserum and anti-actin antibody. Results： Western blot analysis using specific antiserum revealed that CPα3 was expressed specifically in testes. Interestingly, the molecular weight of CPα3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPα3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPα3 and ACTB localization was virtually identical to the localization before the acrosomal reaction. Conclusion： CPα3 might play an important role in sperm morphogenesis and/or sperm function.展开更多
Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes.Although functional analyses of hundreds of these genes have been performed,there are still many t...Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes.Although functional analyses of hundreds of these genes have been performed,there are still many testis-enriched genes whose functions remain unexplored.Analyzing gene function using knockout(KO)mice is a powerful tool to discern if the gene of interest is essential for sperm formation,function,and male fertility in vivo.In this study,we generated KO mice for 12 testis-enriched genes,1700057G04Rik,4921539E11Rik,4930558C23Rik,Cby2,Ldhal6b,Rasef,Slc25a2,Slc25a41,Smim8,Smim9,Tmem210,and Tomm20l,using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation.Mating tests of KO mice reveal that these 12 genes are not essential for male fertility,at least when individually ablated,and not together with other potentially compensatory paralogous genes.Our results could prevent other laboratories from expending duplicative effort generating KO mice,for which no apparent phenotype exists.展开更多
文摘Aim： To characterize mouse capping protein α3 （CPα3） during spermatogenesis and sperm maturation. Methods： We produced rat anti-CPα3 antiserum and examined the expression of CPα3 in various mouse tissues using Western blot analysis and the localization of CPα3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPα3 and β-actin （ACTB） in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPα3 antiserum and anti-actin antibody. Results： Western blot analysis using specific antiserum revealed that CPα3 was expressed specifically in testes. Interestingly, the molecular weight of CPα3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPα3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPα3 and ACTB localization was virtually identical to the localization before the acrosomal reaction. Conclusion： CPα3 might play an important role in sperm morphogenesis and/or sperm function.
文摘Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes.Although functional analyses of hundreds of these genes have been performed,there are still many testis-enriched genes whose functions remain unexplored.Analyzing gene function using knockout(KO)mice is a powerful tool to discern if the gene of interest is essential for sperm formation,function,and male fertility in vivo.In this study,we generated KO mice for 12 testis-enriched genes,1700057G04Rik,4921539E11Rik,4930558C23Rik,Cby2,Ldhal6b,Rasef,Slc25a2,Slc25a41,Smim8,Smim9,Tmem210,and Tomm20l,using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9(CRISPR/Cas9)system.We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation.Mating tests of KO mice reveal that these 12 genes are not essential for male fertility,at least when individually ablated,and not together with other potentially compensatory paralogous genes.Our results could prevent other laboratories from expending duplicative effort generating KO mice,for which no apparent phenotype exists.
