AIMTo characterize mouse capping protein alpha3 (CPalpha3) during spermatogenesis and sperm maturation.

METHODSWe produced rat anti-CPalpha3 antiserum and examined the expression of CPalpha3 in various mouse tissues using Western blot analysis and the localization of CPalpha3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPalpha3 and beta-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPalpha3 antiserum and anti-actin antibody.

RESULTSWestern blot analysis using specific antiserum revealed that CPalpha3 was expressed specifically in testes. Interestingly, the molecular weight of CPalpha3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPalpha3 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 CPalpha3 and ACTB localization was virtually identical to the localization before the acrosomal reaction.

CONCLUSIONCPalpha3 might play an important role in sperm morphogenesis and/or sperm function.

Acrosome Reaction ; physiology ; Actins ; metabolism ; Animals ; Blotting, Western ; CapZ Actin Capping Protein ; metabolism ; Cells, Cultured ; Epididymis ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Sperm Head ; metabolism ; Sperm Tail ; metabolism ; Spermatogenesis ; physiology ; Spermatozoa ; cytology ; metabolism ; Testis ; cytology ; metabolism

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.
Animals ; CRISPR-Cas Systems/genetics* ; Fertility/genetics* ; Gene Editing ; Humans ; Male ; Mice ; Mice, Knockout ; Testis/metabolism*