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:

Derivation of embryonic germ cells and male gametes from embryonic stem cells

Abstract

Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac1. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo2,3. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Expression of germ-cell-specific genes during EB development.
Figure 2: Development of primordial germ cells in the differentiating EB.
Figure 3: RT–PCR detection of germ-cell-specific genes in SSEA1+ cells isolated from developing EBs.
Figure 4: EBs support differentiation of haploid male germ cells that support fertilization of oocytes.

Similar content being viewed by others

References

  1. Lawson, K. A. & Hage, W. J. Clonal analysis of the origin of primordial germ cells in the mouse. Ciba Found. Symp. 182, 68–84, 84–91 (1994)

    CAS  PubMed  Google Scholar 

  2. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. & Kemler, R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27–45 (1985)

    CAS  PubMed  Google Scholar 

  3. Leahy, A., Xiong, J. W., Kuhnert, F. & Stuhlmann, H. Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J. Exp. Zool. 284, 67–81 (1999)

    Article  CAS  PubMed  Google Scholar 

  4. Kyba, M., Perlingeiro, R. C. & Daley, G. Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109, 29–37 (2002)

    Article  CAS  PubMed  Google Scholar 

  5. Saitou, M., Barton, S. C. & Surani, M. A. A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300 (2002)

    Article  ADS  CAS  PubMed  Google Scholar 

  6. Wang, P. J., McCarrey, J. R., Yang, F. & Page, D. C. An abundance of X-linked genes expressed in spermatogonia. Nature Genet. 27, 422–426 (2001)

    Article  PubMed  Google Scholar 

  7. Cooke, H. J., Lee, M., Kerr, S. & Ruggiu, M. A murine homologue of the human DAZ gene is autosomal and expressed only in male and female gonads. Hum. Mol. Genet. 5, 513–516 (1996)

    Article  CAS  PubMed  Google Scholar 

  8. Seboun, E. et al. Gene sequence, localization, and evolutionary conservation of DAZLA, a candidate male sterility gene. Genomics 41, 227–235 (1997)

    Article  CAS  PubMed  Google Scholar 

  9. Moore, F. L. et al. Human Pumilio-2 is expressed in embryonic stem cells and germ cells and interacts with DAZ (Deleted in AZoospermia) and DAZ-like proteins. Proc. Natl Acad. Sci. USA 100, 538–543 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Zandstra, P. W., Le, H. V., Daley, G. Q., Griffith, L. G. & Lauffenburger, D. A. Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation. Biotechnol. Bioeng. 69, 607–617 (2000)

    Article  CAS  PubMed  Google Scholar 

  11. Bortvin, A. et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 130, 1673–1680 (2003)

    Article  CAS  PubMed  Google Scholar 

  12. Koshimizu, U., Watanabe, M. & Nakatsuji, N. Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro. Dev. Biol. 168, 683–685 (1995)

    Article  CAS  PubMed  Google Scholar 

  13. Labosky, P. A., Barlow, D. P. & Hogan, B. L. Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 120, 3197–3204 (1994)

    CAS  PubMed  Google Scholar 

  14. Barlow, D. P., Stoger, R., Herrmann, B. G., Saito, K. & Schweifer, N. The mouse insulin-like growth factor type-2 receptor is imprinted and closely linked to the Tme locus. Nature 349, 84–87 (1991)

    Article  ADS  CAS  PubMed  Google Scholar 

  15. Stoger, R. et al. Maternal-specific methylation of the imprinted mouse Igf2r locus identifies the expressed locus as carrying the imprinting signal. Cell 73, 61–71 (1993)

    Article  CAS  PubMed  Google Scholar 

  16. Matsui, Y., Zsebo, K. & Hogan, B. L. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 70, 841–847 (1992)

    Article  CAS  PubMed  Google Scholar 

  17. Resnick, J. L., Bixler, L. S., Cheng, L. & Donovan, P. J. Long-term proliferation of mouse primordial germ cells in culture. Nature 359, 550–551 (1992)

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D. & Orkin, S. H. Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101, 508–516 (2003)

    Article  CAS  PubMed  Google Scholar 

  19. Fuhrmann, G. et al. Mouse germline restriction of Oct4 expression by germ cell nuclear factor. Dev. Cell 1, 377–387 (2001)

    Article  CAS  PubMed  Google Scholar 

  20. Wassarman, P. M. Mouse gamete adhesion molecules. Biol. Reprod. 46, 186–191 (1992)

    Article  CAS  PubMed  Google Scholar 

  21. Cao, T., Shannon, M., Handel, M. A. & Etkin, L. D. Mouse ret finger protein (rfp) proto-oncogene is expressed at specific stages of mouse spermatogenesis. Dev. Genet. 19, 309–320 (1996)

    Article  CAS  PubMed  Google Scholar 

  22. Ogawa, S. et al. Molecular cloning of a novel RING finger-B box-coiled coil (RBCC) protein, terf, expressed in the testis. Biochem. Biophys. Res. Commun. 251, 515–519 (1998)

    Article  CAS  PubMed  Google Scholar 

  23. Tezel, G., Nagasaka, T., Shimono, Y. & Takahashi, M. Differential expression of RET finger protein in testicular germ cell tumors. Pathol. Int. 52, 623–627 (2002)

    Article  CAS  PubMed  Google Scholar 

  24. Fenderson, B. A., O'Brien, D. A., Millette, C. F. & Eddy, E. M. Stage-specific expression of three cell surface carbohydrate antigens during murine spermatogenesis detected with monoclonal antibodies. Dev. Biol. 103, 117–128 (1984)

    Article  CAS  PubMed  Google Scholar 

  25. Hubner, K. et al. Derivation of oocytes from mouse embryonic stem cells. Science 300, 1251–1256 (2003)

    Article  ADS  PubMed  Google Scholar 

  26. Toyooka, Y., Tsunekawa, N., Akasu, R. & Noce, T. Embryonic stem cells can form germ cells in vitro. Proc. Natl Acad. Sci. USA 100, 11457–11462 (2003)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett. 407, 313–319 (1997)

    Article  CAS  PubMed  Google Scholar 

  28. Selig, S., Okumura, K., Ward, D. C. & Cedar, H. Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J. 11, 1217–1225 (1992)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Reijo, R. et al. Mouse autosomal homolog of DAZ, a candidate male sterility gene in humans, is expressed in male germ cells before and after puberty. Genomics 35, 346–352 (1996)

    Article  CAS  PubMed  Google Scholar 

  30. Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank S. L. Opitz, N. Watson and E. Dikovskaia for technical support; K. Hochedlinger and T. Holm for assistance with intracytoplasmic oocyte injection; and W. Lensch for critical revisions of the manuscript. This work was supported by grants from the National Institutes of Health, the National Science Foundation Biotechnology Process Engineering Center, and the Dutch Cancer Society ‘Koningin Wilhelmina fonds’. G.Q.D. is a Birnbaum Scholar of the Leukemia and Lymphoma Society of America. J.G. was sponsored by the Human Frontiers Science Foundation. K.E. is a Junior Fellow in the Harvard Society of Fellows.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to George Q. Daley.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Geijsen, N., Horoschak, M., Kim, K. et al. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 427, 148–154 (2004). https://doi.org/10.1038/nature02247

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature02247

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

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