CRISPR-Cas9-mediated mutation revealed BSPH2 protein is dispensable for male fertility.

Members of the Binder of SPerm (BSP) superfamily have been identified in both human and mouse epididymis. These proteins are known to bind sperm membrane and promote sperm capacitation. Studies suggest that BSPH2 might play a different role in sperm functions from its counterparts; however, the role of BSPH2 remains mainly unexplored. To investigate whether the absence of one member of the BSP family could affect fertility, mice lacking Bsph2 expression were generated using clustered regularly interspaced short palindromic repeats (CRISPR) associated 9 (Cas9) technology. Knockout (KO) male mice were mated with wild-type (WT) females, and the number and weight of the pups were determined. Sperm motility in WT and KO was assessed using sperm class analyzer (SCA). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for protein identification. Fertility analysis of null Bsph2 mice did not reveal any phenotype. No differences were noticed on average litter size or average pup weight. Normal testis weight and morphology were observed in Bsph2 and Bsph2 compared to the WT. Quantitative polymerase chain reaction analyses revealed that Bsph1 messenger RNA expression was increased in mutant mice, whereas LC-MS/MS analysis displayed no increase in protein expression level. Taken together, we show the existence of redundant function for murine BSPH2 and the lack of BSPH2 itself does not lead to sterility.