Abstract
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.
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Acknowledgements
We would like to thank the members of the Department of Regenerative Medicine for their experimental assistance and discussion. We thank Dr. Alex Bortvin of Carnegie Institution for Science for kindly providing the anti-LINE1-ORF1p antibody, which was essential for the initial stages of this study. This work was supported by KAKENHI grants-in-aid from MEXT, Japan (24H02055 to Y.I.-Y.; 23H04956 to K.I.; 19H05758 to A.O.; 18H05546 and 22H00485 to T.O.; 20K21657, 24K21284 and 25K02455 to T.S.); by an AMED (JP24gn0110086 to T.O.; JP24mk0121304 to T.S.); by JST CREST (JPMJCR21N1 to T.O.) and by a Grant for Strategic Research Promotion of Yokohama City University (SK202403 to T.S.).
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M.N., Y.O.-S., S.K. and T.S. conceived, designed, and conducted the experiments, and performed data analyses; M.N., Y.O.-S., S.K., Y.I.-Y., T.M., M.K. and T.S. performed organ culture experiments; S.M., K.I., N.O. and A.O. performed round spermatid injection experiments; M.N., T.O. and T.S. wrote the manuscript, incorporating feedback from all the authors.
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Nishida, M., Ono-Sunagare, Y., Kato, S. et al. Reverse transcriptase inhibitors enable the generation of fertile spermatids from fetal mouse testes in vitro. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09613-y
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DOI: https://doi.org/10.1038/s42003-026-09613-y
