{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T13:02:45Z","timestamp":1772456565596,"version":"3.50.1"},"reference-count":22,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T00:00:00Z","timestamp":1769472000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T00:00:00Z","timestamp":1772409600000},"content-version":"vor","delay-in-days":34,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["24K21284"],"award-info":[{"award-number":["24K21284"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["25K02455"],"award-info":[{"award-number":["25K02455"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["24H02055"],"award-info":[{"award-number":["24H02055"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["18H05546"],"award-info":[{"award-number":["18H05546"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["22H00485"],"award-info":[{"award-number":["22H00485"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["23H04956"],"award-info":[{"award-number":["23H04956"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"MEXT | Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["19H05758"],"award-info":[{"award-number":["19H05758"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP24mk0121304"],"award-info":[{"award-number":["JP24mk0121304"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP24gn0110086"],"award-info":[{"award-number":["JP24gn0110086"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Grant for Strategic Research Promotion of Yokohama City Universit"},{"DOI":"10.13039\/501100003382","name":"MEXT | JST | Core Research for Evolutional Science and Technology","doi-asserted-by":"publisher","award":["JPMJCR21N1"],"award-info":[{"award-number":["JPMJCR21N1"]}],"id":[{"id":"10.13039\/501100003382","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Commun Biol"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Organ culture systems enabling in vitro spermatogenesis from neonatal mouse testes exist, but differentiation from fetal testes shortly after sex determination remains unsuccessful. Here, we report the in vitro generation of fertile haploid cells from E12.5 fetal testes. While optimizing in vitro spermatogenesis protocols for neonatal testes, we find that supplementing the culture medium with reverse transcriptase inhibitors (RTIs) significantly improves the efficiency of spermatogenesis, by suppressing retrotransposon activity and protecting genomic integrity. Applying this approach, we successfully induce spermatogenesis through to the elongating spermatid by culturing E12.5 fetal testes under hypoxic conditions in RTI-supplemented medium. Notably, microinsemination using in vitro-derived spermatids produces healthy and fertile offspring, confirming their functional competence. These findings demonstrate the faithful in vitro recapitulation of testicular development and complete spermatogenesis from an early fetal stage, providing a valuable platform for investigating early germ cell development and reproductive biology.<\/jats:p>","DOI":"10.1038\/s42003-026-09613-y","type":"journal-article","created":{"date-parts":[[2026,1,27]],"date-time":"2026-01-27T17:36:10Z","timestamp":1769535370000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Reverse transcriptase inhibitors enable the generation of fertile spermatids from fetal mouse testes in vitro"],"prefix":"10.1038","volume":"9","author":[{"given":"Mayuka","family":"Nishida","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yukina","family":"Ono-Sunagare","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sayuri","family":"Kato","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-2438-619X","authenticated-orcid":false,"given":"Yu","family":"Ishikawa-Yamauchi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takafumi","family":"Matsumura","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mitsuru","family":"Komeya","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0474-232X","authenticated-orcid":false,"given":"Shogo","family":"Matoba","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7344-4786","authenticated-orcid":false,"given":"Kimiko","family":"Inoue","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Narumi","family":"Ogonuki","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Atsuo","family":"Ogura","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1422-0317","authenticated-orcid":false,"given":"Takehiko","family":"Ogawa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6198-2234","authenticated-orcid":false,"given":"Takuya","family":"Sato","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,1,27]]},"reference":[{"key":"9613_CR1","doi-asserted-by":"publisher","first-page":"504","DOI":"10.1038\/nature09850","volume":"471","author":"T Sato","year":"2011","unstructured":"Sato, T. et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471, 504\u2013507 (2011).","journal-title":"Nature"},{"key":"9613_CR2","doi-asserted-by":"publisher","first-page":"e0130171","DOI":"10.1371\/journal.pone.0130171","volume":"10","author":"T Sato","year":"2015","unstructured":"Sato, T. et al. In vitro spermatogenesis in explanted adult mouse testis tissues. PLoS ONE 10, e0130171 (2015).","journal-title":"PLoS ONE"},{"key":"9613_CR3","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms5320","volume":"5","author":"T Yokonishi","year":"2014","unstructured":"Yokonishi, T. et al. Offspring production with sperm grown in vitro from cryopreserved testis tissues. Nat. Commun. 5, 4320 (2014).","journal-title":"Nat. Commun."},{"key":"9613_CR4","doi-asserted-by":"publisher","first-page":"63","DOI":"10.1095\/biolreprod.116.140277","volume":"95","author":"K Kojima","year":"2016","unstructured":"Kojima, K., Sato, T., Naruse, Y. & Ogawa, T. Spermatogenesis in explanted fetal mouse testis tissues. Biol. Reprod. 95, 63 (2016).","journal-title":"Biol. Reprod."},{"key":"9613_CR5","doi-asserted-by":"publisher","first-page":"2409","DOI":"10.1101\/gad.228080.113","volume":"27","author":"T Svingen","year":"2013","unstructured":"Svingen, T. & Koopman, P. Building the mammalian testis: origins, differentiation, and assembly of the component cell populations. Genes Dev. 27, 2409\u20132426 (2013).","journal-title":"Genes Dev."},{"key":"9613_CR6","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1159\/000338612","volume":"7","author":"EK Ungewitter","year":"2013","unstructured":"Ungewitter, E. K. & Yao, H. H. How to make a gonad: cellular mechanisms governing formation of the testes and ovaries. Sex. Dev. 7, 7\u201320 (2013).","journal-title":"Sex. Dev."},{"key":"9613_CR7","doi-asserted-by":"publisher","first-page":"849","DOI":"10.1016\/j.molcel.2012.11.001","volume":"48","author":"S Seisenberger","year":"2012","unstructured":"Seisenberger, S. et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol. Cell 48, 849\u2013862 (2012).","journal-title":"Mol. Cell"},{"key":"9613_CR8","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-82792-2","volume":"11","author":"T Matsumura","year":"2021","unstructured":"Matsumura, T. et al. Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control. Sci. Rep. 11, 3458 (2021).","journal-title":"Sci. Rep."},{"key":"9613_CR9","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-023-39323-y","volume":"13","author":"K Hashimoto","year":"2023","unstructured":"Hashimoto, K. et al. Culture-space control is effective in promoting haploid cell formation and spermiogenesis in vitro in neonatal mice. Sci. Rep. 13, 12354 (2023).","journal-title":"Sci. Rep."},{"key":"9613_CR10","doi-asserted-by":"publisher","first-page":"e0283773","DOI":"10.1371\/journal.pone.0283773","volume":"18","author":"X Feng","year":"2023","unstructured":"Feng, X. et al. In vitro spermatogenesis in isolated seminiferous tubules of immature mice. PLoS ONE 18, e0283773 (2023).","journal-title":"PLoS ONE"},{"key":"9613_CR11","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2091-12-18","volume":"12","author":"L Dai","year":"2011","unstructured":"Dai, L., Huang, Q. & Boeke, J. D. Effect of reverse transcriptase inhibitors on LINE-1 and Ty1 reverse transcriptase activities and on LINE-1 retrotransposition. BMC Biochem. 12, 18 (2011).","journal-title":"BMC Biochem."},{"key":"9613_CR12","doi-asserted-by":"publisher","first-page":"20110338","DOI":"10.1098\/rstb.2011.0338","volume":"368","author":"S Chuma","year":"2013","unstructured":"Chuma, S. & Nakano, T. piRNA and spermatogenesis in mice. Philos. Trans. R. Soc. Lond. B Biol. Sci. 368, 20110338 (2013).","journal-title":"Philos. Trans. R. Soc. Lond. B Biol. Sci."},{"key":"9613_CR13","doi-asserted-by":"publisher","first-page":"504","DOI":"10.1038\/s42003-022-03449-y","volume":"5","author":"K Hirano","year":"2022","unstructured":"Hirano, K. et al. Temperature sensitivity of DNA double-strand break repair underpins heat-induced meiotic failure in mouse spermatogenesis. Commun. Biol. 5, 504 (2022).","journal-title":"Commun. Biol."},{"key":"9613_CR14","doi-asserted-by":"publisher","first-page":"E5635","DOI":"10.1073\/pnas.1701069114","volume":"114","author":"SJ Newkirk","year":"2017","unstructured":"Newkirk, S. J. et al. Intact piRNA pathway prevents L1 mobilization in male meiosis. Proc. Natl. Acad. Sci. USA 114, E5635\u2013E5644 (2017).","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"9613_CR15","doi-asserted-by":"publisher","first-page":"521","DOI":"10.1016\/j.devcel.2014.04.027","volume":"29","author":"S Malki","year":"2014","unstructured":"Malki, S., van der Heijden, G. W., O\u2019Donnell, K. A., Martin, S. L. & Bortvin, A. A role for retrotransposon LINE-1 in fetal oocyte attrition in mice. Dev. Cell 29, 521\u2013533 (2014).","journal-title":"Dev. Cell"},{"key":"9613_CR16","doi-asserted-by":"publisher","first-page":"404","DOI":"10.1159\/000520683","volume":"16","author":"S Zhou","year":"2022","unstructured":"Zhou, S., Sakashita, A., Yuan, S. & Namekawa, S. H. Retrotransposons in the mammalian male germline. Sex. Dev. 16, 404\u2013422 (2022).","journal-title":"Sex. Dev."},{"key":"9613_CR17","doi-asserted-by":"publisher","first-page":"2899","DOI":"10.1089\/scd.2015.0196","volume":"24","author":"K Hogg","year":"2015","unstructured":"Hogg, K. & Western, P. S. Differentiation of fetal male germline and gonadal progenitor cells is disrupted in organ cultures containing knockout serum replacement. Stem Cells Dev. 24, 2899\u20132911 (2015).","journal-title":"Stem Cells Dev."},{"key":"9613_CR18","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1016\/S0014-5793(99)00433-0","volume":"449","author":"T Nakanishi","year":"1999","unstructured":"Nakanishi, T. et al. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett. 449, 277\u2013283 (1999).","journal-title":"FEBS Lett."},{"key":"9613_CR19","doi-asserted-by":"publisher","first-page":"934","DOI":"10.1093\/biolre\/ioab104","volume":"105","author":"K Higuchi","year":"2021","unstructured":"Higuchi, K. et al. Sertoli cell replacement in explanted mouse testis tissue supporting host spermatogenesis. Biol. Reprod. 105, 934\u2013943 (2021).","journal-title":"Biol. Reprod."},{"key":"9613_CR20","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-023-39304-1","volume":"13","author":"T Matsumura","year":"2023","unstructured":"Matsumura, T. et al. Generation of rat offspring using spermatids produced through in vitro spermatogenesis. Sci. Rep. 13, 12105 (2023).","journal-title":"Sci. Rep."},{"key":"9613_CR21","doi-asserted-by":"publisher","first-page":"e11062","DOI":"10.1371\/journal.pone.0011062","volume":"5","author":"N Ogonuki","year":"2010","unstructured":"Ogonuki, N. et al. The effect on intracytoplasmic sperm injection outcome of genotype, male germ cell stage and freeze-thawing in mice. PLoS ONE 5, e11062 (2010).","journal-title":"PLoS ONE"},{"key":"9613_CR22","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms6011","volume":"5","author":"M Van Meter","year":"2014","unstructured":"Van Meter, M. et al. SIRT6 represses LINE1 retrotransposons by ribosylating KAP1 but this repression fails with stress and age. Nat. Commun. 5, 5011 (2014).","journal-title":"Nat. Commun."}],"container-title":["Communications Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09613-y","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09613-y.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09613-y.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T12:13:26Z","timestamp":1772453606000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s42003-026-09613-y"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,27]]},"references-count":22,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,12]]}},"alternative-id":["9613"],"URL":"https:\/\/doi.org\/10.1038\/s42003-026-09613-y","relation":{},"ISSN":["2399-3642"],"issn-type":[{"value":"2399-3642","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,27]]},"assertion":[{"value":"1 July 2025","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"19 January 2026","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"27 January 2026","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"329"}}