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.

  • Protocol
  • Published:

In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method

Nature Protocols volume 8, pages 2098–2104 (2013)Cite this article

Subjects

Abstract

The in vitro propagation of mouse spermatogonial stem cells (SSCs) became possible in 2003; these cultured SSCs were named germ-line stem (GS) cells. To date, however, it has not been possible to induce spermatogenesis from GS cells in vitro. Recently, we succeeded in producing functional sperm from primitive spermatogonia in explanted neonatal mouse testis tissues. Here we describe a protocol that can support spermatogenesis from GS cells up to sperm formation in vitro using an organ culture method. GS cells transplanted in the extracted testis form colonies in the tissue fragments and differentiate into sperm under the described in vitro organ culture conditions. It takes about 6 weeks to obtain sperm from GS cells. The sperm are viable, resulting in healthy offspring through micro-insemination. Thus, this protocol should be a valuable tool for the study of mammalian spermatogenesis.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Schematic diagram of the experimental procedure, from the establishment of the GS cells to in vitro spermatogenesis using the organ culture method.
Figure 2: Injection procedure.
Figure 3: Morphological features of GS cells.
Figure 4: Process of cell injection and culture of the testis tissues.
Figure 5: Spermatogenesis from GS cells in vitro.

Similar content being viewed by others

References

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

    Article  Google Scholar 

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

    Article  CAS  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. Griswold, M.D. & Oatley, J.M. Concise review: defining characteristics of mammalian spermatogenic stem cells. Stem Cells 31, 8–11 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Sato, T. et al. Testis tissue explantation cures spermatogenic failure in c-Kit ligand mutant mice. Proc. Natl. Acad. Sci. USA 109, 16934–16938 (2012).

    Article  CAS  Google Scholar 

  8. Kanatsu-Shinohara, M. et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–616 (2003).

    Article  CAS  Google Scholar 

  9. Kanatsu-Shinohara, M. et al. Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 103, 8018–8023 (2006).

    Article  CAS  Google Scholar 

  10. Takehashi, M. et al. Adenovirus-mediated gene delivery into mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 104, 2596–2601 (2007).

    Article  CAS  Google Scholar 

  11. Brinster, R.L. & Zimmermann, J.W. Spermatogenesis following male germ-cell transplantation. Proc. Natl. Acad. Sci. USA 91, 11298–11302 (1994).

    Article  CAS  Google Scholar 

  12. Ogawa, T., Arechaga, J.M., Avarbock, M.R. & Brinster, R.L. Transplantation of testis germinal cells into mouse seminiferous tubules. Int. J. Dev. Biol. 41, 111–122 (1997).

    CAS  PubMed  Google Scholar 

  13. Sato, T. et al. In vitro production of fertile sperm from murine spermatogonial stem cell lines. Nat. Commun. 2, 472 (2011).

    Article  Google Scholar 

  14. Ogawa, T. et al. Derivation and morphological characterization of mouse spermatogonial stem cell lines. Arch. Histol. Cytol. 67, 297–306 (2004).

    Article  Google Scholar 

  15. Kita, K. et al. Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules. Biol. Reprod. 76, 211–217 (2007).

    Article  CAS  Google Scholar 

  16. 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 

  17. Kubota, H., Avarbock, M.R. & Brinster, R.L. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. USA 101, 16489–16494 (2004).

    Article  CAS  Google Scholar 

  18. Kim, J., Seandel, M., Falciatori, I., Wen, D. & Rafii, S. CD34+ testicular stromal cells support long-term expansion of embryonic and adult stem and progenitor cells. Stem Cells 26, 2516–2522 (2008).

    Article  Google Scholar 

  19. Yokonishi, T., Sato, T., Katagiri, K. & Ogawa, T. In vitro spermatogenesis using an organ culture technique. Methods Mol. Biol. 927, 479–488 (2013).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, 'Regulatory Mechanism of Gamete Stem Cells' 20116005 (to T.O.); a Grant-in-Aid for Scientific Research (B) 24390371 (to T.O.); a Grant-in-Aid for Young Scientists (B) 24770216 (to T.S.); a grant for Research and Development Project II of Yokohama City University (to T.O.); and a grant for Establishment of Research Center for Clinical Proteomics of Post-translational Modifications (to T.O.).

Author information

Authors and Affiliations

  1. Laboratory of Proteomics, Institute of Molecular Medicine and Life Science, Yokohama City University Association of Medical Science, Yokohama, Japan

    Takuya Sato, Kumiko Katagiri & Takehiko Ogawa

  2. Department of Urology, Yokohama City University Graduate School of Medicine, Yokohama, Japan

    Yoshinobu Kubota & 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. Yoshinobu Kubota
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takehiko Ogawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.S., K.K. and T.O. wrote the manuscript. Y.K. supervised the research.

Corresponding authors

Correspondence to Takuya Sato or Takehiko Ogawa.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

In vitro transplantation procedure. Preparation of testis, dissociation of efferent ducts, insertion of glass needle, and injection of dye solution into the rete cavity, which spread into the lumen of the seminiferous tubules. (MOV 10100 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sato, T., Katagiri, K., Kubota, Y. et al. In vitro sperm production from mouse spermatogonial stem cell lines using an organ culture method. Nat Protoc 8, 2098–2104 (2013). https://doi.org/10.1038/nprot.2013.138

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.138

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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