Summary
Spermatogonial stem cells (SSCs) produce sperm throughout the post-pubertal life of a male. Transgenic loss- and gain-of-function mouse models have shown that their self-renewal and differentiation are controlled in vivo by glial-cell-line-derived neurotrophic factor (GDNF) in a dose-dependent manner. After this in vivo observation, the culture conditions for mouse SSCs were rapidly developed. GDNF together with other growth factors, hormones, and vitamins maintain proliferation and self-renewal of SSCs for years in vitro. Both serum-supplemented and serum-free culture methods have been described. The cells are cultivated either on feeder layer or laminin-coated dishes. First reports from random and targeted mutagenesis of SSCs have been published. Some cells in the spermatogonial stem cell culture transform to embryonic stem cell-like cells and form teratomas in nude mice. In general, the spermatogonial stem cells maintain their germline identity in long-term culture. The mechanism for transformation to embryonic stem cell-like cells is not known, but the data suggest that germline and embryonic stem cells are closely related. We describe in detail the culture system of SSCs developed by Dr. Takashi Shinohara in 2003.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
1. Tegelenbosch, R. A. J., and de Rooij, D. G. (1993) A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse. Mut. Res. 290, 193–200.
2. Meng, X., Lindahl, M., Hyvonen, M. E., et al. (2000) Regulation of cell fate decision of undif-ferentiated spermatogonia by GDNF. Science. 287, 1489–1493.
3. Sariola, H., and Saarma, M. (2003) Novel functions and signalling pathways for GDNF. J. Cell Sci. 116, 3855–3862.
4. Oatley, J.M., Avarbock, M.R., Telaranta, A.I., Fearon, D.T., and Brinster, R.L. (2006) Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc. Natl. Acad. Sci. U. S. A. 103, 9524–9529.
5. de Rooij, D. G., and Russell, L. D. (2000) All you wanted to know about spermatogonia but were afraid to ask. J. Androl. 21, 776–798.
6. de Rooij, D. G. (2006) Rapid expansion of the spermatogonial stem cell toolbox. Proc. Natl. Acad. Sci. U. S. A. 103, 7939–7940.
7. Aponte, P. M., van Bragt, M. P. A, de Rooij, D. G., and van Pelt, A. M. M. (2005) Spermatogonial stem cells: characteristics and experimental possibilities. APMIS. 113, 727–742.
8. Kanatsu-Shinohara, M., Ogonuki, N., Inoue, K., et al. (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol. Reprod. 69, 612–616.
9. Kanatsu-Shinohara, M., Inoue, K., Lee, J., et al. (2004) Generation of pluripotent stem cells from neonatal mouse testis. Cell. 119, 1001–1012.
10. Guan, K., Nayernia, K., Maier, L. S., et al. (2006) Pluripotency of spermatogonial stem cells from adult mouse testis. Nature. 440, 1199–1203.
11. Kanatsu-Shinohara, M., Ogonuki, N., Iwano, T., et al. (2005) Genetic and epigenetic properties of mouse male germline stem cells during long-term culture. Development. 132, 4155–4163.
12. Nagano, M., Brinster, C. J., Orwig, K. E., Ryu, B. Y., Avarbock, M. R., and Brinster, R. L. (2001) Transgenic ice produced by retroviral transduction of male germ-line stem cells. Proc. Natl. Acad. Sci. U. S. A. 98, 13090–13095.
13. Kanatsu-Shinohara, M., Ikawa, M., Takehashi, M., et al. (2006) Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc. Natl. Acad. Sci. U. S. A. 103, 8018–8023.
14. Jeong, D., McLean, D. J., and Griswold, M. D. (2003) Long-term culture and transplantation of murine testicular germ cells. J. Androl. 24, 661–669.
15. Kubota, H., Avarbock, M. R., and Brinster, R. L. (2004) Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol. Reprod. 71, 722–731.
16. Kubota, H., Avarbock, M. R., and Brinster, R.L. (2004) Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. Proc. Natl. Acad. Sci. U. S. A. 101, 16489–16494.
17. Kanatsu-Shinohara, M., Miki, H., Inoue, K., et al. (2005) Long-term culture of mouse male germline stem cells under serum-or feeder-free conditions. Biol. Reprod. 72, 985–991.
18. Hofmann M. C., Braydich-Stolle L., and Dym, M. (2005) Isolation of male germ-line stem cells; influence of GDNF. Dev. Biol. 279, 114–124.
19. Creemers, L. B., den Ouden, K., van Pelt, A. M. M., and de Rooij, D. G. (2002) Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture. Reproduction. 124, 791–799.
20. Kanatsu-Shinohara, M., Inoue, K., Lee, J., et al. (2006) Anchorage-independent growth of mouse male germline stem cells in vitro. Biol. Reprod. 74, 522–529.
21. Brinster, R. L., and Avarbock, M. R. (1994) Germline transmission of donor haplotype following spermatogonial transplantation. Proc. Natl. Acad. Sci. U. S. A. 91, 11303–11307.
22. Brinster, R. L., and Zimmermann. J. W. (1994) Spermatogenesis following male germ-cell transplantation. Proc. Natl. Acad. Sci. U. S. A. 91, 11298–11302.
23. Clouthier, D. E., Avarbock, M. R., Maika, S. D., Hammer, R. E., and Brinster, R. L. (1996) Rat spermatogenesis in mouse testis. Nature. 381, 418–421.
24. Shinohara, T., Kato, M., Takehashi, M., et al. (2006) Rats produced by interspecies spermato-gonial transplantation in mice and in vitro microinsemination. Proc. Natl. Acad. Sci. U. S. A. 103, 13624–13628.
25. Spector, D. L., Goldman, R. D., and Leinwand, L. A. (eds.). (1998) Cells: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Acknowledgments
Special thanks are given to Dr. Takashi Shinohara, who was kindly ready to share his invaluable hands-on knowledge and the protocol details of this method.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Sariola, H., Immonen, T. (2008). GDNF Maintains Mouse Spermatogonial Stem Cells In Vivo and In Vitro. In: Hou, S.X., Singh, S.R. (eds) Germline Stem Cells. Methods in Molecular Biology™, vol 450. Humana Press. https://doi.org/10.1007/978-1-60327-214-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-60327-214-8_9
Publisher Name: Humana Press
Print ISBN: 978-1-60327-213-1
Online ISBN: 978-1-60327-214-8
eBook Packages: Springer Protocols