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However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis\u2010enriched genes: lipocalin family genes (<jats:italic>Lcn5<\/jats:italic>, <jats:italic>Lcn6<\/jats:italic>, <jats:italic>Lcn8<\/jats:italic>, <jats:italic>Lcn9<\/jats:italic>, and <jats:italic>Lcn10<\/jats:italic>) and Ly6 family genes (<jats:italic>Ly6g5b<\/jats:italic> and <jats:italic>Ly6g5c<\/jats:italic>). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively.<\/jats:p><\/jats:sec><jats:sec><jats:title>Objective<\/jats:title><jats:p>To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR\/Cas9 system and analyzed their phenotype.<\/jats:p><\/jats:sec><jats:sec><jats:title>Materials and methods<\/jats:title><jats:p>We generated four lines of KO mice: <jats:italic>Lcn9<\/jats:italic> single KO, the lipocalin family quadruple KO (<jats:italic>Lcn5<\/jats:italic>, <jats:italic>Lcn6<\/jats:italic>, <jats:italic>Lcn8<\/jats:italic>, and <jats:italic>Lcn10<\/jats:italic>), quintuple KO (<jats:italic>Lcn5<\/jats:italic>, <jats:italic>Lcn6<\/jats:italic>, <jats:italic>Lcn8<\/jats:italic>, <jats:italic>Lcn10<\/jats:italic>, and <jats:italic>Lcn9<\/jats:italic>), and double KO of Ly6 family genes (<jats:italic>Ly6g5b<\/jats:italic> and <jats:italic>Ly6g5c<\/jats:italic>).<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Although the <jats:italic>Lcn9<\/jats:italic> single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A\/B that are required for ADAM3 maturation. Intriguingly, <jats:italic>Ly6g5b<\/jats:italic> and <jats:italic>Ly6g5c<\/jats:italic> double KO male mice also showed subfertility with reduced sperm ADAM3.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability.<\/jats:p><\/jats:sec>","DOI":"10.1111\/andr.13350","type":"journal-article","created":{"date-parts":[[2022,11,26]],"date-time":"2022-11-26T02:39:33Z","timestamp":1669430373000},"page":"981-990","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["CRISPR\/Cas9\u2010mediated disruption of <i>lipocalins<\/i>, <i>Ly6g5b<\/i>, and <i>Ly6g5c<\/i> causes male subfertility in mice"],"prefix":"10.1111","volume":"12","author":[{"given":"Nobuyuki","family":"Sakurai","sequence":"first","affiliation":[{"name":"Department of Bioscience and Genetics National Cerebral and Cardiovascular Center  Suita Osaka Japan"},{"name":"Research Institute for Microbial Diseases Osaka University  Suita Osaka Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8332-3507","authenticated-orcid":false,"given":"Yoshitaka","family":"Fujihara","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Genetics National Cerebral and Cardiovascular Center  Suita Osaka Japan"},{"name":"Research Institute for Microbial Diseases Osaka University  Suita Osaka Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kiyonori","family":"Kobayashi","sequence":"additional","affiliation":[{"name":"Research Institute for Microbial Diseases Osaka University  Suita Osaka Japan"},{"name":"Graduate School of Frontier Biosciences Osaka University  Suita Osaka Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9859-6217","authenticated-orcid":false,"given":"Masahito","family":"Ikawa","sequence":"additional","affiliation":[{"name":"Research Institute for Microbial Diseases Osaka University  Suita Osaka Japan"},{"name":"Graduate School of Pharmaceutical Sciences Osaka University  Suita Osaka Japan"},{"name":"Graduate School of Medicine Osaka University  Suita Osaka Japan"},{"name":"The Institute of Medical Science The University of Tokyo  Minato\u2010ku Tokyo 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