{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T07:02:42Z","timestamp":1779951762330,"version":"3.53.1"},"reference-count":46,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T00:00:00Z","timestamp":1779926400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>Tissue-restricted gene expression serves as a key indicator of functional importance within specific organs. Despite advances in high-throughput sequencing and genome editing over the past decade, this principle remains the gold standard for identifying candidate genes for functional studies. In reproductive biology, testis-restricted expression is anticipated to indicate genes crucial for gametogenesis, given the conserved features shared between spermatogenesis and oogenesis. Concurrently, rapid evolution of spermatogenesis-related genes is well documented and thought to arise through duplication and subsequent mutation of ubiquitously expressed genes. In this study, we established 14 knockout (KO) mouse lines for male reproductive organ-enriched genes, all of which exhibited normal growth and fertility in natural mating assays. These genes share sequence similarity with previously characterized gene families or functionally defined paralogs. These observations indicate that while these genes acquired enriched expressions in male reproductive organ following their evolutionary origin, they may represent: (1) currently non-functional loci, (2) genes with redundant functions compensated by paralogs, or (3) genes undergoing loss of function.<\/jats:p>","DOI":"10.3389\/fcell.2026.1851315","type":"journal-article","created":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T06:18:08Z","timestamp":1779949088000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional analysis of genes enriched in male reproductive organs: validation via fertility assessment of 14 knockout mouse lines"],"prefix":"10.3389","volume":"14","author":[{"given":"Kazumasa","family":"Takemoto","sequence":"first","affiliation":[{"name":"Department of Genome Editing, Institute of Biomedical Science, Kansai Medical University","place":["Hirakata, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katarzyna","family":"Kent","sequence":"additional","affiliation":[{"name":"Department of Pathology and Immunology, Baylor College of Medicine, Center for Drug Discovery","place":["Houston, TX, United States"]},{"name":"Department of Pathology and Immunology, Baylor College of Medicine","place":["Houston, TX, United States"]},{"name":"Department of Molecular and Human Genetics, Baylor College of Medicine","place":["Houston, TX, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kaori","family":"Nozawa","sequence":"additional","affiliation":[{"name":"Department of Pathology and Immunology, Baylor College of Medicine, Center for Drug Discovery","place":["Houston, TX, United States"]},{"name":"Department of Pathology and Immunology, Baylor College of Medicine","place":["Houston, TX, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shingo","family":"Tonai","sequence":"additional","affiliation":[{"name":"Department of Experimental Genome Research, Research Institute for Microbial Diseases, The University of Osaka","place":["Suita, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ahn H.","family":"Pham","sequence":"additional","affiliation":[{"name":"Department of Experimental Genome Research, Research Institute for Microbial Diseases, The University of Osaka","place":["Suita, Japan"]},{"name":"Graduate School of Pharmaceutical Sciences, The University of Osaka","place":["Suita, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Terumi","family":"Fujimoto","sequence":"additional","affiliation":[{"name":"Department of Genome Editing, Institute of Biomedical Science, Kansai Medical University","place":["Hirakata, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas X.","family":"Garcia","sequence":"additional","affiliation":[{"name":"Department of Pathology and Immunology, Baylor College of Medicine, Center for Drug Discovery","place":["Houston, TX, United States"]},{"name":"Department of Pathology and Immunology, Baylor College of Medicine","place":["Houston, TX, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin M.","family":"Matzuk","sequence":"additional","affiliation":[{"name":"Department of Pathology and Immunology, Baylor College of Medicine, Center for Drug Discovery","place":["Houston, TX, United States"]},{"name":"Department of Pathology and Immunology, Baylor College of Medicine","place":["Houston, TX, United States"]},{"name":"Department of Molecular and Human Genetics, Baylor College of Medicine","place":["Houston, TX, United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masahito","family":"Ikawa","sequence":"additional","affiliation":[{"name":"Department of Experimental Genome Research, Research Institute for Microbial Diseases, The University of Osaka","place":["Suita, Japan"]},{"name":"Graduate School of Pharmaceutical Sciences, The University of Osaka","place":["Suita, Japan"]},{"name":"Division of Microbiology and Immunology, Center for Infectious Disease Education and Research, The University of Osaka","place":["Suita, Japan"]},{"name":"Laboratory of Reproductive Systems Biology, The Institute of Medical Science, The University of Tokyo","place":["Tokyo, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Keizo","family":"Tokuhiro","sequence":"additional","affiliation":[{"name":"Department of Genome Editing, Institute of Biomedical Science, Kansai Medical University","place":["Hirakata, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,5,28]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"jcs221648","DOI":"10.1242\/jcs.221648","article-title":"RSPH6A is required for sperm flagellum formation and male fertility in mice","volume":"131","author":"Abbasi","year":"2018","journal-title":"J. Cell Sci."},{"key":"B2","doi-asserted-by":"publisher","first-page":"1430","DOI":"10.1038\/ng1919","article-title":"In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication","volume":"38","author":"Baltus","year":"2006","journal-title":"Nat. Genet."},{"key":"B3","doi-asserted-by":"publisher","first-page":"505","DOI":"10.1038\/s41586-019-1338-5","article-title":"Gene expression across mammalian organ development","volume":"571","author":"Cardoso-Moreira","year":"2019","journal-title":"Nature"},{"key":"B4","doi-asserted-by":"publisher","first-page":"703","DOI":"10.1016\/S0092-8674(00)80706-0","article-title":"T2Rs function as bitter taste receptors","volume":"100","author":"Chandrashekar","year":"2000","journal-title":"Cell"},{"key":"B5","doi-asserted-by":"publisher","first-page":"833","DOI":"10.1111\/andr.70088","article-title":"Mouse genome engineering uncovers 18 genes dispensable for male reproduction","volume":"14","author":"Chang","year":"2026","journal-title":"Andrology"},{"key":"B6","first-page":"e33111","article-title":"Structural insights into the assembly and polyA signal recognition mechanism of the human CPSF complex","author":"Clerici","year":"2017"},{"key":"B7","doi-asserted-by":"publisher","first-page":"2157","DOI":"10.1093\/molbev\/msn162","article-title":"Recent origins of sperm genes in drosophila","volume":"25","author":"Dorus","year":"2008","journal-title":"Mol. Biol. Evol."},{"key":"B8","doi-asserted-by":"publisher","first-page":"15579883231182673","DOI":"10.1177\/15579883231182673","article-title":"Roles of different \u03b2-Defensins in the human reproductive system: a review study","volume":"17","author":"Fesahat","year":"2023","journal-title":"Am. J. Mens. Health"},{"key":"B9","doi-asserted-by":"publisher","first-page":"1513","DOI":"10.1101\/gad.5.9.1513","article-title":"Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice","volume":"5","author":"Friedrich","year":"1991","journal-title":"Genes Dev."},{"key":"B10","doi-asserted-by":"publisher","first-page":"eabd4914","DOI":"10.1126\/science.abd4914","article-title":"Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility","volume":"371","author":"Gadadhar","year":"2021","journal-title":"Science"},{"key":"B11","doi-asserted-by":"publisher","first-page":"1112","DOI":"10.1016\/j.cell.2015.04.003","article-title":"Multivalent microtubule recognition by tubulin tyrosine ligase-like family glutamylases","volume":"161","author":"Garnham","year":"2015","journal-title":"Cell"},{"key":"B12","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1093\/molbev\/msx057","article-title":"The Goddard and Saturn Genes Are Essential for Drosophila Male Fertility and may Have Arisen de novo","volume":"34","author":"Gubala","year":"2017","journal-title":"Mol. Biol. Evol."},{"key":"B13","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1093\/biolre\/ioz158","article-title":"The testis-specific serine proteases PRSS44, PRSS46, and PRSS54 are dispensable for Male mouse fertility","volume":"102","author":"Holcomb","year":"2020","journal-title":"Biol. Reprod."},{"key":"B14","doi-asserted-by":"publisher","first-page":"110022","DOI":"10.1016\/j.fsi.2024.110022","article-title":"The antibacterial defence role of \u03b2-defensin in the seahorse testis","volume":"155","author":"Huang","year":"2024","journal-title":"Fish & Shellfish Immunol."},{"key":"B15","doi-asserted-by":"publisher","first-page":"429","DOI":"10.1016\/j.devcel.2020.01.010","article-title":"MEIOSIN directs the switch from mitosis to meiosis in mammalian germ cells","volume":"52","author":"Ishiguro","year":"2020","journal-title":"Dev. Cell"},{"key":"B16","doi-asserted-by":"publisher","first-page":"6382","DOI":"10.1038\/srep06382","article-title":"Simple knockout by electroporation of engineered endonucleases into intact rat embryos","volume":"4","author":"Kaneko","year":"2014","journal-title":"Sci. Rep."},{"key":"B17","first-page":"564","article-title":"Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice","volume":"54","author":"Kito","year":"2004","journal-title":"Comp. Med."},{"key":"B18","first-page":"257","article-title":"Chapter eight - gametogenesis: a journey from inception to conception","author":"Larose","year":"2019"},{"key":"B19","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1016\/0076-6879(93)25012-Q","article-title":"Culture of preimplantation embryos","volume":"225","author":"Lawitts","year":"1993","journal-title":"Methods Enzym."},{"key":"B20","doi-asserted-by":"publisher","first-page":"100825","DOI":"10.1016\/j.xcrm.2022.100825","article-title":"\u03b2-Defensin 19\/119 mediates sperm chemotaxis and is associated with idiopathic infertility","volume":"3","author":"Li","year":"2022","journal-title":"Cell Rep. Med."},{"key":"B21","doi-asserted-by":"publisher","first-page":"3233","DOI":"10.1242\/jcs.035071","article-title":"STRA8-deficient spermatocytes initiate, but fail to complete, meiosis and undergo premature chromosome condensation","volume":"121","author":"Mark","year":"2008","journal-title":"J. Cell Sci."},{"key":"B22","doi-asserted-by":"publisher","first-page":"7704","DOI":"10.1073\/pnas.1608458113","article-title":"Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for Male fertility in mice","volume":"113","author":"Miyata","year":"2016","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B23","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1016\/S0925-4773(99)00021-0","article-title":"Ten years of gene targeting: targeted mouse mutants, from vector design to phenotype analysis","volume":"82","author":"M\u00fcller","year":"1999","journal-title":"Mech. Dev."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1120","DOI":"10.1093\/bioinformatics\/btu743","article-title":"CRISPRdirect: software for designing CRISPR\/cas guide RNA with reduced off-target sites","volume":"31","author":"Naito","year":"2015","journal-title":"Bioinformatics"},{"key":"B25","doi-asserted-by":"publisher","first-page":"635","DOI":"10.1038\/nature12943","article-title":"The evolution of lncRNA repertoires and expression patterns in tetrapods","volume":"505","author":"Necsulea","year":"2014","journal-title":"Nature"},{"key":"B26","doi-asserted-by":"publisher","first-page":"1411162","DOI":"10.3389\/fcell.2024.1411162","article-title":"Gene-deficient mouse model established by CRISPR\/Cas9 system reveals 15 reproductive organ-enriched genes dispensable for male fertility","volume":"12","author":"Nguyen","year":"2024","journal-title":"Front. Cell Dev. Biol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"644","DOI":"10.1111\/andr.12621","article-title":"Nine genes abundantly expressed in the epididymis are not essential for male fecundity in mice","volume":"7","author":"Noda","year":"2019","journal-title":"Andrology"},{"key":"B28","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1016\/S0015-0282(16)48918-1","article-title":"Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid","volume":"44","author":"Quinn","year":"1985","journal-title":"Fertil. Steril."},{"key":"B29","doi-asserted-by":"publisher","first-page":"103","DOI":"10.1186\/s12915-020-00826-z","article-title":"Large-scale discovery of male reproductive tract-specific genes through analysis of RNA-seq datasets","volume":"18","author":"Robertson","year":"2020","journal-title":"BMC Biol."},{"key":"B30","doi-asserted-by":"publisher","first-page":"2684","DOI":"10.1101\/gad.9.21.2684","article-title":"Roles for two RecA homologs in promoting meiotic chromosome synapsis","volume":"9","author":"Rockmill","year":"1995","journal-title":"Genes Dev."},{"key":"B31","doi-asserted-by":"publisher","first-page":"dev175950","DOI":"10.1242\/dev.175950","article-title":"The RNA-binding protein DND1 acts sequentially as a negative regulator of pluripotency and a positive regulator of epigenetic modifiers required for germ cell reprogramming","volume":"146","author":"Ruthig","year":"2019","journal-title":"Development"},{"key":"B32","doi-asserted-by":"publisher","first-page":"12201","DOI":"10.1073\/pnas.1635054100","article-title":"A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets","volume":"100","author":"Schultz","year":"2003","journal-title":"Proc. Natl. Acad. Sci."},{"key":"B33","doi-asserted-by":"publisher","first-page":"2179","DOI":"10.1016\/j.celrep.2013.05.031","article-title":"Cellular source and mechanisms of high transcriptome complexity in the mammalian testis","volume":"3","author":"Soumillon","year":"2013","journal-title":"Cell Rep."},{"key":"B34","doi-asserted-by":"publisher","first-page":"152","DOI":"10.1262\/jrd1955.16.152","article-title":"Studies on the fertilization of mouse eggs in vitro","volume":"16","author":"Toyoda","year":"1971","journal-title":"Jpn. Journal Animal Reproduction"},{"key":"B35","doi-asserted-by":"publisher","first-page":"1239","DOI":"10.1126\/science.1085222","article-title":"Conserved role of Nanos proteins in germ Cell development","volume":"301","author":"Tsuda","year":"2003","journal-title":"Science"},{"key":"B36","doi-asserted-by":"publisher","first-page":"1465","DOI":"10.1098\/rstb.2009.0323","article-title":"Evolution and spermatogenesis","volume":"365","author":"White-Cooper","year":"2010","journal-title":"Philos. Trans. R. Soc. Lond B Biol. Sci."},{"key":"B37","doi-asserted-by":"publisher","first-page":"D1133","DOI":"10.1093\/nar\/gkaf1148","article-title":"International mouse phenotyping consortium portal: facilitating investigation of gene function and providing insights into human disease","volume":"54","author":"Wilson","year":"2026","journal-title":"Nucleic Acids Res."},{"key":"B38","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1093\/molehr\/gas040","article-title":"Functional characterization of bitter-taste receptors expressed in Mammalian testis","volume":"19","author":"Xu","year":"2013","journal-title":"Mol. Hum. Reprod."},{"key":"B39","doi-asserted-by":"publisher","first-page":"1712","DOI":"10.1136\/ard-2022-222795","article-title":"Loss-of-function variants in SAT1 cause X-linked childhood-onset systemic lupus erythematosus","volume":"81","author":"Xu","year":"2022","journal-title":"Ann. Rheumatic Dis."},{"key":"B40","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1016\/S0925-4773(02)00144-2","article-title":"Testis-specific expression of a novel mouse defensin-like gene, tdl","volume":"116","author":"Yamamoto","year":"2002","journal-title":"Mech. Dev."},{"key":"B41","doi-asserted-by":"publisher","first-page":"1071","DOI":"10.1093\/biolre\/ioae119","article-title":"Eighteen genes primarily expressed in the testis are not required for male fertility in mice","volume":"111","author":"Yamamoto","year":"2024","journal-title":"Biol. Reprod."},{"key":"B42","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1095\/biolreprod.112.106328","article-title":"Three testis-specific paralogous serine proteases play different roles in murine spermatogenesis and are involved in germ cell survival during Meiosis","volume":"88","author":"Yoneda","year":"2013","journal-title":"Biol. Reprod."},{"key":"B43","doi-asserted-by":"publisher","first-page":"360","DOI":"10.1038\/nature03595","article-title":"The Ter mutation in the dead end gene causes germ cell loss and testicular germ cell tumours","volume":"435","author":"Youngren","year":"2005","journal-title":"Nature"},{"key":"B44","doi-asserted-by":"publisher","first-page":"298","DOI":"10.1016\/j.metabol.2018.04.007","article-title":"Spermidine\/spermine N1-acetyltransferase-mediated polyamine catabolism regulates beige adipocyte biogenesis","volume":"85","author":"Yuan","year":"2018","journal-title":"Metabolism"},{"key":"B45","doi-asserted-by":"publisher","first-page":"267","DOI":"10.1186\/s12964-024-01637-3","article-title":"Single-cell and spatial transcriptomic investigation reveals the spatiotemporal specificity of the beta-defensin gene family during mouse sperm maturation","volume":"22","author":"Zhang","year":"2024","journal-title":"Cell Commun. Signal"},{"key":"B46","doi-asserted-by":"publisher","first-page":"e1003826","DOI":"10.1371\/journal.pgen.1003826","article-title":"Partial deletion of chromosome 8 \u03b2-defensin cluster confers sperm dysfunction and infertility in Male mice","volume":"9","author":"Zhou","year":"2013","journal-title":"PLOS Genet."}],"container-title":["Frontiers in Cell and Developmental Biology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2026.1851315\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,28]],"date-time":"2026-05-28T06:18:09Z","timestamp":1779949089000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2026.1851315\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,28]]},"references-count":46,"alternative-id":["10.3389\/fcell.2026.1851315"],"URL":"https:\/\/doi.org\/10.3389\/fcell.2026.1851315","relation":{},"ISSN":["2296-634X"],"issn-type":[{"value":"2296-634X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,28]]},"article-number":"1851315"}}