Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

In vitro production of functional sperm in cultured neonatal mouse testes

Nature volume 471, pages 504–507 (2011)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

References

  1. Clermont, Y. Kinetics of spermatogenesis in mammals: seminiferous epithelial cycle and spermatogonial renewal. Physiol. Rev. 52, 198–236 (1972)

    Article  CAS  Google Scholar 

  2. Russell, L. D., Ettlin, R. A., SinhaHikim, A. P. & Clegg, E. D. in Histological and Histopathological Evaluation of the Testis 1–40 (Cache River, 1990)

    Google Scholar 

  3. Staub, C. A century of research on mammalian male germ cell meiotic differentiation in vitro . J. Androl. 22, 911–926 (2001)

    Article  CAS  Google Scholar 

  4. Parks, J. E., Lee, D. R., Huang, S. & Kaproth, M. T. Prospects for spermatogenesis in vitro . Theriogenology 59, 73–86 (2003)

    Article  CAS  Google Scholar 

  5. La Salle, S., Sun, F. & Handel, M. A. Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. Methods Mol. Biol. 558, 279–297 (2009)

    Article  Google Scholar 

  6. Miura, T., Yamauchi, K., Takahashi, H. & Nagahama, Y. Hormonal induction of all stages of spermatogenesis in vitro in the male Japanese eel (Anguilla japonica). Proc. Natl Acad. Sci. USA 88, 5774–5778 (1991)

    Article  ADS  CAS  Google Scholar 

  7. Sakai, N. Transmeiotic differentiation of zebrafish germ cells into functional sperm in culture. Development 129, 3359–3365 (2002)

    CAS  PubMed  Google Scholar 

  8. Champy, C. Quelques résultats de la méthode de culture des tissues. Arch. Zool. Exp. Gen. 60, 461–500 (1920)

    Google Scholar 

  9. Martinovitch, P. N. Development in vitro of the mammalian gonad. Nature 139, 413 (1937)

    Article  ADS  Google Scholar 

  10. Steinberger, A., Steinberger, E. & Perloff, W. H. Mammalian testes in organ culture. Exp. Cell Res. 36, 19–27 (1964)

    Article  CAS  Google Scholar 

  11. Steinberger, A. & Steinberger, E. Factors affecting spermatogenesis in organ cultures of mammalian testes. J. Reprod. Fertil., Suppl. 2, 117–124 (1967)

    Google Scholar 

  12. Feng, L. X. et al. Generation and in vitro differentiation of a spermatogonial cell line. Science 297, 392–395 (2002)

    Article  ADS  CAS  Google Scholar 

  13. Rassoulzadegan, M. et al. Transmeiotic differentiation of male germ cells in culture. Cell 75, 997–1006 (1993)

    Article  CAS  Google Scholar 

  14. Staub, C. et al. The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Exp. Cell Res. 260, 85–95 (2000)

    Article  CAS  Google Scholar 

  15. Trowell, O. A. The culture of mature organs in a synthetic medium. Exp. Cell Res. 16, 118–147 (1959)

    Article  CAS  Google Scholar 

  16. Tanaka, H. et al. Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells. J. Biol. Chem. 274, 17049–17057 (1999)

    Article  CAS  Google Scholar 

  17. Nakanishi, T. et al. Real-time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein. FEBS Lett. 449, 277–283 (1999)

    Article  CAS  Google Scholar 

  18. Ventelä, S. et al. Regulation of acrosome formation in mice expressing green fluorescent protein as a marker. Tissue Cell 32, 501–507 (2000)

    Article  Google Scholar 

  19. Gohbara, A. et al. In vitro murine spermatogenesis in an organ culture system. Biol. Reprod. 83, 261–267 (2010)

    Article  CAS  Google Scholar 

  20. Goetz, P., Chandley, A. C. & Speed, R. M. Morphological and temporal sequence of meiotic prophase development at puberty in the male mouse. J. Cell Sci. 65, 249–263 (1984)

    CAS  PubMed  Google Scholar 

  21. Goldsborough, M. D. et al. Serum-free culture of murine embryonic stem (ES) cells. Focus 20, 8–12 (1998)

    Google Scholar 

  22. Price, P. J., Goldsborough, M. D. & Tilkins, M. L. Embryonic stem cell serum replacement. PCT/US98/00467: WO 98/30679 (1998)

  23. Wachs, F. P. et al. High efficacy of clonal growth and expansion of adult neural stem cells. Lab. Invest. 83, 949–962 (2003)

    Article  Google Scholar 

  24. Tres, L. L. & Kierszenbaum, A. L. Viability of rat spermatogenic cells in vitro is facilitated by their coculture with Sertoli cells in serum-free hormone-supplemented medium. Proc. Natl Acad. Sci. USA 80, 3377–3381 (1983)

    Article  ADS  CAS  Google Scholar 

  25. Sharpe, R. M. in Sertoli Cell Biology (eds Skinner, M. K. & Griswold, M. D. ) 199–216 (Elsevier Academic Press, 2006)

    Google Scholar 

  26. 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)

    Article  ADS  Google Scholar 

  27. Ogura, A., Inoue, K. & Matsuda, J. Mouse spermatid nuclei can support full term development after premature chromosome condensation within mature oocytes. Hum. Reprod. 14, 1294–1298 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.).

Author information

Authors and Affiliations

  1. Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama 236-0004, Japan

    Takuya Sato, Kumiko Katagiri, Ayako Gohbara, Yoshinobu Kubota & Takehiko Ogawa

  2. RIKEN, Bioresource Center, Ibaraki 305-0074, Japan

    Kimiko Inoue, Narumi Ogonuki & Atsuo Ogura

  3. Project Leader of Advanced Medical Research Center, Yokohama City University, Yokohama 236-0004, Japan

    Takehiko Ogawa

Authors
  1. Takuya Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kumiko Katagiri
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ayako Gohbara
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kimiko Inoue
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Narumi Ogonuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Atsuo Ogura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yoshinobu Kubota
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takehiko Ogawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Takehiko Ogawa.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-11 with legends and Supplementary Tables 1-2. (PDF 1252 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sato, T., Katagiri, K., Gohbara, A. et al. In vitro production of functional sperm in cultured neonatal mouse testes. Nature 471, 504–507 (2011). https://doi.org/10.1038/nature09850

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09850

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing