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In vitro production of functional sperm in cultured neonatal mouse testes


Spermatogenesis is one of the most complex and longest processes of sequential cell proliferation and differentiation in the body, taking more than a month from spermatogonial stem cells, through meiosis, to sperm formation1,2. The whole process, therefore, has never been reproduced in vitro in mammals3,4,5, nor in any other species with a very few exceptions in some particular types of fish6,7. Here we show that neonatal mouse testes which contain only gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media. Spermatogenesis was maintained over 2 months in tissue fragments positioned at the gas–liquid interphase. The obtained spermatids and sperm resulted in healthy and reproductively competent offspring through microinsemination. In addition, neonatal testis tissues were cryopreserved and, after thawing, showed complete spermatogenesis in vitro. Our organ culture method could be applicable through further refinements to a variety of mammalian species, which will serve as a platform for future clinical application as well as mechanistic understanding of spermatogenesis.

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Figure 1: Effect of FBS and serum replacement on pup testis tissues.
Figure 2: Effect of KSR on neonatal testis tissues.
Figure 3: Formation of spermatids.
Figure 4: Fertility of sperm and spermatids produced in vitro.


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We thank S. Yoshida and G. Yoshizaki for comments and pre-submission review. We thank A. Tanaka and Y. W. Zheng for their technical help in flow cytometric analysis. T.O. is grateful to his mentor R. L. Brinster for his advice on devising the experimental strategies and for his encouragements. We would like to thank A. Steinberger and the late E. Steinberger whose work in the study of in vitro spermatogenesis became the ground work for our present study. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, “Regulatory Mechanism of Gamete Stem Cells” (#20116005); a Grant-in-Aid for Scientific Research (C) (#21592080) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; a grant from the Yokohama Foundation for Advancement of Medical Science; and a grant for Research and Development Project II (No.S2116) of Yokohama City University, Japan (to T.O.).

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Authors and Affiliations



T.S. performed the experiments, interpreted the results, and prepared the manuscript. K.K. performed all culture experiments. A.G. contributed to the culture experiments. K.I. and N.O. performed microinsemination experiments. A.O. performed microinsemination experiments and discussed the results. Y.K. supervised the project and discussed the results. T.O. designed and performed the experiments and wrote the manuscript.

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Correspondence to Takehiko Ogawa.

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The authors declare no competing financial interests.

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Sato, T., Katagiri, K., Gohbara, A. et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471, 504–507 (2011).

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