Multiple genes in the Pate5-13 genomic region contribute to ADAM3 processing

Sperm proteins undergo post-translational modifications during sperm transit through the epididymis to acquire fertilizing ability. We previously reported that the genomic region coding Pate family genes is key to the proteolytic processing of the sperm membrane protein ADAM3 and male fertility. This region contains nine Pate family genes (Pate5-13), and two protein-coding genes (Gm27235 and Gm5916), with a domain structure similar to Pate family genes. Therefore, in this study, we aimed to identify key factors by narrowing the genomic region. We generated three knockout (KO) mouse lines using CRISPR/Cas9: single KO mice of Pate10 expressed in the caput epididymis; deletion KO mice of six caput epididymis-enriched genes (Pate5-7, 13, Gm27235, and Gm5916) (Pate7-Gm5916 KO); and deletion KO mice of four genes expressed in the placenta and epididymis (Pate8, 9, 11, and 12) (Pate8-12 KO). We observed that the fertility of only Pate7-Gm5916 KO males was reduced, whereas the rest remained unaffected. Furthermore, when the caput epididymis-enriched genes, Pate8 and Pate10 remained in Pate7-Gm5916 KO mice were independently deleted, both KO males displayed more severe subfertility due to a decrease in mature ADAM3 and a defect in sperm migration to the oviduct. Thus, our data showed that multiple caput epididymis-enriched genes within the region coding Pate5-13 cooperatively function to ensure male fertility in mice.