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.

  • Article
  • Published:

Reconstitution of spermatogenesis from frozen spermatogonial stem cells

Abstract

Spermatozoa from a number of species can be cryopreserved and then subsequently used to fertilize eggs1. However, this technique has several limitations. First, the freezing protocol varies for each species and must be determined empirically, and for some species appropriate methods have not yet been identified1,2. Second, because these cells are fully differentiated, they will not undergo replication when thawed, and recombination of genetic information cannot occur. We now demonstrate, by using the recently developed spermatogonial transplantation technique3,4, that male germline stem cells can be successfully cryopreserved. Donor testis cells isolated from prepubertal or adult mice and frozen from 4 to 156 days at −196 °C were able to generate spermatogenesis in recipient seminiferous tubules. Relatively standard preservation techniques were used, suggesting that male germ cells from other species can also be stored for long periods. Because transplanted testis stem cells will ultimately undergo replication and meiotic recombination during spermatogenesis, one might consider these preserved male germ lines as biologically immortal.

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

Access options

Buy this article

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

Similar content being viewed by others

References

  1. Hafez, E.S.E. Preservation and cryopreservation of gametes and embryos. in Reproduction in Farm Animals, 6th edn. (ed. Hafez, E.S.E.) 503–525 (Lea & Febiger, Philadelphia, 1993).

    Google Scholar 

  2. Tada, N. et al. Cryopreservation of mouse spermatozoa in the presence of raffi-nose and glycerol. Reprod. Fert. 89, 511–516 (1990).

    Article  CAS  Google Scholar 

  3. 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 

  4. Brinster, R.L. & Avarbock, M.R. Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci. USA 91, 11303–11307 (1994).

    Article  CAS  Google Scholar 

  5. Baker, T.G. Oogenesis and ovarian development. in Reproductive Biology (eds. Balin, H. & Glasser, S.) 398–437 (Excerpta Medica, Amsterdam, 1972).

    Google Scholar 

  6. Russell, L.D., Ettlin, R.A., Hikim, A.P. & Clegg, E.D. Mammalian spermatogenesis. in Histological and Histopathological Evaluation of the Testis 1–40 (Cache River Press, Clearwater, Florida, 1990).

    Google Scholar 

  7. Ewing, L.L., Davis, J.C. & Zirkin, B.R. Regulation of testicular function: A spatial and temporal view. in International Review of Physiology (ed. Creep, R.O.) 41–115 (University Park Press, Baltimore, 1980).

    Google Scholar 

  8. De Rooij, D.G., Van Dissel-Emiliani, F.M.F. & Van Pelt, A.M.M. Regulation of spermatogonial proliferation. Annu. NY Acad. Sci. 364, 140–153 (1992).

    Google Scholar 

  9. Gosden, R.G. Transplantation of ovaries and testes. in Fetal Tissue Transplants in Medicine (ed. Edwards, R.G.) 253–280 (Cambridge Univ. Press, New York, 1992).

    Google Scholar 

  10. Deanesly, R.J. Spermatogenesis and endocrine activity in grafts of frozen and thawed rat testis. Endocrinology 11, 201–206 (1954).

    Article  CAS  Google Scholar 

  11. Zambrowicz, B.P. et al. Expression of a mouse Zfy-1/lacZ transgene in the somatic cells of the embryonic gonad and germ cells of the adult testis. Development 120, 1549–1559 (1994).

    CAS  PubMed  Google Scholar 

  12. Freshney, R.I. Instability, variation, and preservation. in Culture of Animal Cells, 3rd edn. (ed. Freshney, R.I.) 253–266 (Wiley-Liss, New York, 1994).

    Google Scholar 

  13. Bucci, L.R. & Meistrich, M.L. Effects of busulfan on murine Spermatogenesis: Cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations. Mutat. Res. 176, 259–268 (1987).

    Article  CAS  Google Scholar 

  14. Dym, M. The male reproductive system. in Histology Cell and Tissue Biology, 5th edn. (ed. Weiss, L.) 1000–1053 (Elsevier Science Pub. Co., New York, 1993).

    Google Scholar 

  15. Meistrich, M.L. Nuclear morphogenesis during spermiogenesis. in Molecular Biology of the Male Reproductive System (ed. de Kretser, D.) 67–97 (Academic Press, New York, 1993).

    Google Scholar 

  16. Clermont, Y., Oko, R. & Hermo, L. Cell biology of mammalian spermiogenesis. in Cell and Molecular Biology of the Testis (eds. Desjardins, C. & Ewing, L.L.) 332–376 (Oxford Univ. Press, New York, 1993).

    Google Scholar 

  17. Setchell, B.P. Spermatogenesis and spermatozoa. in Reproduction in Mammals: Germ Cells and Fertilization, 2nd edn. (eds. Austin, C.R. & Short, R.V.) 63–101 (Cambridge Univ. Press, New York, 1982).

    Google Scholar 

  18. Clouthier, D.E., Avarbock, M.R., Maika, S.D., Hammer, R.E. & Brinster, R.L. Rat Spermatogenesis. Nature 381 (in the press).

  19. Kimura, Y. & Yanagimachi, R. Mouse oocytes injected with testicular spermatozoa or round spermatids can develop into normal offspring. Development 121, 2397–2405 (1995).

    CAS  Google Scholar 

  20. Aitken, R.J. & Irvine, D.S. Fertilization without sperm. Nature 379, 493–495 (1996).

    Article  CAS  Google Scholar 

  21. Fishel, S. et al. Pregnancy after intracytoplasmic injection of spermatid. Lancet 345, 1641–1642 (1995).

    Article  CAS  Google Scholar 

  22. Tesarik, J., Mendoza, C. & Testart, J. Viable embryos from injection of round spermatids into oocytes. N. Engl. J. Med. 333, 525 (1995).

    Article  CAS  Google Scholar 

  23. Bellvé, A.R. et al. Spermatogenic cells of the prepubertal mouse. Cell Biol. 74, 68–85 (1977).

    Article  Google Scholar 

  24. Bellvé, A.R. Purification, culture, and fractionation of Spermatogenic cells. Methods Enzymol. 225, 84–113 (1993).

    Article  Google Scholar 

  25. Robertson, E.J. Embryo-derived stem cell lines. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (ed. Robertson, E.J.) 71–112 (IRL Press, Oxford, 1987).

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Avarbock, M., Brinster, C. & Brinster, R. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nat Med 2, 693–696 (1996). https://doi.org/10.1038/nm0696-693

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm0696-693

This article is cited by

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