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Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women

Nature Medicine volume 18, pages 413421 (2012) | Download Citation

Abstract

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

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Acknowledgements

The authors thank L. Prickett-Rice and K. Folz-Donahue of the Harvard Stem Cell Institute Flow Cytometry Core Facility and J. Groeneweg for expert technical assistance. We also thank J.R. Mann and K.J. MacLaughlin for the provision of TgOG2 transgenic mice. This work was supported by a Method to Extend Research in Time (MERIT) Award from the US National Institute on Aging (NIH R37-AG012279), the Henry and Vivian Rosenberg Philanthropic Fund, the Sea Breeze Foundation and Vincent Memorial Hospital Research Funds. This work was conducted while D.C.W. was supported in part by a Ruth L. Kirschstein National Research Service Award (NIH F32-AG034809).

Author information

Author notes

    • Yvonne A R White
    •  & Dori C Woods

    These authors contributed equally to this work.

Affiliations

  1. Vincent Center for Reproductive Biology, Massachusetts General Hospital Vincent Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts, USA.

    • Yvonne A R White
    • , Dori C Woods
    •  & Jonathan L Tilly
  2. Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, Massachusetts, USA.

    • Yvonne A R White
    • , Dori C Woods
    •  & Jonathan L Tilly
  3. Department of Obstetrics and Gynecology, Saitama Medical Center, Saitama Medical University, Saitama, Japan.

    • Yasushi Takai
    • , Osamu Ishihara
    •  & Hiroyuki Seki

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Contributions

Y.A.R.W., D.C.W. and J.L.T. designed the experiments, analyzed the data and wrote the manuscript. Y.A.R.W. and D.C.W. conducted the experiments. Y.T., O.I. and H.S. collected, cryopreserved and provided human ovarian cortical tissue. J.L.T. directed the project. All authors reviewed and approved the final manuscript for submission.

Competing interests

J.L.T. declares interest in intellectual property described in US Patent 7,955,846 and is a co-founder of OvaScience, Inc., and Y.A.R.W. and D.C.W. are scientific consultants for OvaScience, Inc.

Corresponding author

Correspondence to Jonathan L Tilly.

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    Supplementary Methods, Supplementary Tables 1 and 2 and Supplementary Figures 1–5

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DOI

https://doi.org/10.1038/nm.2669

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