CRISPR/Cas9-mediated genome editing reveals seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice.

Background: Mammalian fertilization is mediated by multiple sperm acrosomal proteins, many of which are testis-enriched transmembrane glycoproteins expressed during spermiogenesis (e.g., Izumo sperm-egg fusion 1, Sperm acrosome associated 6, and Transmembrane protein 95).

Methods: We hypothesized that proteins with these features might have a role in sperm-egg interaction and thus carried out an in-silico screen based on multiple public databases. We generated knockout mouse lines lacking seven candidate proteins by the CRISPR/Cas9 system and conducted detailed analyses on the fecundity of the knockout males, as well as their testis appearance and weight, testis and epididymis histology, and sperm motility and morphology.

Results: Through the in-silico screen, we identified 4932438H23Rik, A disintegrin and metalloproteinase domain-containing protein 29, SAYSvFN domain-containing protein 1, Sel-1 suppressor of lin-12-like 2 (C. elegans), Testis-expressed protein 2, Transmembrane and immunoglobulin domain-containing 3, and Zinc and ring finger 4. Phenotypic analyses unveiled that the knockout males showed normal testis gross appearance, normal testis and epididymis histology, and normal sperm morphology and motility. Fertility tests further indicated that the knockout male mice could sire pups with normal litter sizes when paired with wild-type females.

Discussion And Conclusion: These findings suggest that these seven proteins are individually dispensable for male reproduction and fertilization. Future studies are warranted to devise advanced in-silico screening approaches that permit effective identification of gamete fusion-required sperm proteins.