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

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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Figure 1: FACS-based protocol for OSC isolation.
Figure 2: Isolation of OSCs from adult mouse and human ovaries.
Figure 3: Mouse OSCs generate functional eggs after intraovarian transplantation.
Figure 4: Evaluation of mouse- and human-ovary–derived OSCs in defined cultures.
Figure 5: Spontaneous oocyte generation by cultured mouse and human OSCs.
Figure 6: Human OSCs generate oocytes in human ovary tissue.

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References

  1. Zuckerman, S. The number of oocytes in the mature ovary. Recent Prog. Horm. Res. 6, 63–108 (1951).

    Google Scholar 

  2. Johnson, J., Canning, J., Kaneko, T., Pru, J.K. & Tilly, J.L. Germline stem cells and follicular renewal in the postnatal mammalian ovary. Nature 428, 145–150 (2004).

    Article  CAS  Google Scholar 

  3. Brinster, R.L. Male germline stem cells: from mice to men. Science 316, 404–405 (2007).

    Article  CAS  Google Scholar 

  4. Tilly, J.L., Niikura, Y. & Rueda, B.R. The current status of evidence for and against postnatal oogenesis in mammals: a case of ovarian optimism versus pessimism? Biol. Reprod. 80, 2–12 (2009).

    Article  CAS  Google Scholar 

  5. Zou, K. et al. Production of offspring from a germline stem cell line derived from neonatal ovaries. Nat. Cell Biol. 11, 631–636 (2009).

    Article  CAS  Google Scholar 

  6. Pacchiarotti, J. et al. Differentiation potential of germ line stem cells derived from the postnatal mouse ovary. Differentiation 79, 159–170 (2010).

    Article  CAS  Google Scholar 

  7. Johnson, J. et al. Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. Cell 122, 303–315 (2005).

    Article  CAS  Google Scholar 

  8. Wang, N. & Tilly, J.L. Epigenetic status determines germ cell meiotic commitment in embryonic and postnatal mammalian gonads. Cell Cycle 9, 339–349 (2010).

    Article  Google Scholar 

  9. Niikura, Y., Niikura, T., Wang, N., Satirapod, C. & Tilly, J.L. Systemic signals in aged males exert potent rejuvenating effects on the ovarian follicle reserve in mammalian females. Aging 2, 999–1003 (2010).

    Article  CAS  Google Scholar 

  10. Tilly, J.L. & Telfer, E.E. Purification of germline stem cells from adult mammalian ovaries: a step closer towards control of the female biological clock? Mol. Hum. Reprod. 15, 393–398 (2009).

    Article  Google Scholar 

  11. Niikura, Y., Niikura, T. & Tilly, J.L. Aged mouse ovaries possess rare premeiotic germ cells that can generate oocytes following transplantation into a young host environment. Aging 1, 971–978 (2009).

    Article  CAS  Google Scholar 

  12. Massasa, E., Costa, X.S. & Taylor, H.S. Failure of the stem cell niche rather than loss of oocyte stem cells in the aging ovary. Aging 2, 1–2 (2010).

    Article  Google Scholar 

  13. Fujiwara, Y. et al. Isolation of a DEAD-family protein gene that encodes a murine homolog of Drosophila vasa and its specific expression in germ cell lineage. Proc. Natl. Acad. Sci. USA 91, 12258–12262 (1994).

    Article  CAS  Google Scholar 

  14. Castrillon, D.H., Quade, B.J., Wang, T.Y., Quigley, C. & Crum, C.P. The human VASA gene is specifically expressed in the germ lineage. Proc. Natl. Acad. Sci. USA 97, 9585–9590 (2000).

    Article  CAS  Google Scholar 

  15. Noce, T., Okamoto-Ito, S. & Tsunekawa, N. Vasa homolog genes in mammalian germ cell development. Cell Struct. Funct. 26, 131–136 (2001).

    Article  CAS  Google Scholar 

  16. Normile, D. Reproductive biology. Study suggests a renewable source of eggs and stirs more controversy. Science 324, 320 (2009).

    Article  CAS  Google Scholar 

  17. 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  CAS  Google Scholar 

  18. Ohinata, Y. et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436, 207–213 (2005).

    Article  CAS  Google Scholar 

  19. Dolci, S. et al. Stem cell factor activates telomerase in mouse mitotic spermatogonia and in primordial germ cells. J. Cell Sci. 115, 1643–1649 (2002).

    CAS  PubMed  Google Scholar 

  20. Gong, S.P. et al. Embryonic stem cell-like cells established by culture of adult ovarian cells in mice. Fertil. Steril. 93, 2594–2601 (2010).

    Article  Google Scholar 

  21. Zou, K., Hou, L., Sun, K., Xie, W. & Wu, J. Improved efficiency of female germline stem cell purification using Fragilis-based magnetic bead sorting. Stem Cells Dev. 20, 2197–2204 (2011).

    Article  CAS  Google Scholar 

  22. Suzumori, N., Yan, C., Matzuk, M.M. & Rajkovic, A. Nobox is a homeobox-encoding gene preferentially expressed in primordial and growing oocytes. Mech. Dev. 111, 137–141 (2002).

    Article  CAS  Google Scholar 

  23. Rajkovic, A., Pangas, S.A., Ballow, D., Suzumori, N. & Matzuk, M.M. NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression. Science 305, 1157–1159 (2004).

    Article  CAS  Google Scholar 

  24. Pangas, S.A. et al. Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8. Proc. Natl. Acad. Sci. USA 103, 8090–8095 (2006).

    Article  CAS  Google Scholar 

  25. Elvin, J.A., Clark, A.T., Wang, P., Wolfman, N.M. & Matzuk, M.M. Paracrine actions of growth differentiation factor-9 in the mammalian ovary. Mol. Endocrinol. 13, 1035–1048 (1999).

    Article  CAS  Google Scholar 

  26. Zheng, P. & Dean, J. Oocyte-specific genes affect folliculogenesis, fertilization, and early development. Semin. Reprod. Med. 25, 243–251 (2007).

    Article  CAS  Google Scholar 

  27. Gu, W. et al. Mammalian male and female germ cells express a germ cell-specific Y-Box protein, MSY2. Biol. Reprod. 59, 1266–1274 (1998).

    Article  CAS  Google Scholar 

  28. Yang, J. et al. Absence of the DNA-/RNA-binding protein MSY2 results in male and female infertility. Proc. Natl. Acad. Sci. USA 102, 5755–5760 (2005).

    Article  CAS  Google Scholar 

  29. Page, S.L. & Hawley, R.S. The genetics and molecular biology of the synaptonemal complex. Annu. Rev. Cell Dev. Biol. 20, 525–558 (2004).

    Article  CAS  Google Scholar 

  30. Yuan, L. et al. Female germ cell aneuploidy and embryo death in mice lacking the meiosis-specific protein SCP3. Science 296, 1115–1118 (2002).

    Article  CAS  Google Scholar 

  31. Kagawa, W. & Kurumizaka, H. From meiosis to postmeiotic events: uncovering the molecular roles of the meiosis-specific recombinase Dmc1. FEBS J. 277, 590–598 (2010).

    Article  CAS  Google Scholar 

  32. West, F.D., Mumaw, J.L., Gallegos-Cardenas, A., Young, A. & Stice, S.L. Human haploid cells differentiated from meiotic competent clonal germ cell lines that originated from embryonic stem cells. Stem Cells Dev. 20, 1079–1088 (2011).

    Article  CAS  Google Scholar 

  33. Abban, G. & Johnson, J. Stem cell support of oogenesis in the human. Hum. Reprod. 24, 2974–2978 (2009).

    Article  CAS  Google Scholar 

  34. 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 

  35. Brinster, C.J. et al. Restoration of fertility by germ cell transplantation requires effective recipient preparation. Biol. Reprod. 69, 412–420 (2003).

    Article  CAS  Google Scholar 

  36. Oktay, K. & Karlikaya, G. Ovarian function after transplantation of frozen, banked autologous ovarian tissue. N. Engl. J. Med. 342, 1919 (2000).

    Article  CAS  Google Scholar 

  37. Sönmezer, M. & Oktay, K. Orthotopic and heterotopic ovarian tissue transplantation. Best Pract. Res. Clin. Obstet. Gynaecol. 24, 113–126 (2010).

    Article  Google Scholar 

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

    Article  Google Scholar 

  39. Ko, K. & Schöler, H.R. Embryonic stem cells as a potential source of gametes. Semin. Reprod. Med. 24, 322–329 (2006).

    Article  CAS  Google Scholar 

  40. Nicholas, C.R., Haston, K.M., Grewall, A.K., Longacre, T.A. & Reijo Pera, R.A. Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility. Hum. Mol. Genet. 18, 4376–4389 (2009).

    Article  CAS  Google Scholar 

  41. Kee, K., Angeles, V.T., Flores, M., Nguyen, H.N. & Reijo Pera, R.A. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature 462, 222–225 (2009).

    Article  CAS  Google Scholar 

  42. Nicholas, C.R., Chavez, S.L., Baker, V.L. & Reijo Pera, R.A. Instructing an embryonic stem cell-derived oocyte fate: lessons from endogenous oogenesis. Endocr. Rev. 30, 264–283 (2009).

    Article  CAS  Google Scholar 

  43. Yeom, Y.I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

    CAS  Google Scholar 

  44. Yoshimizu, T. et al. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684 (1999).

    Article  CAS  Google Scholar 

  45. Szabó, P.E. et al. Allele specific expression of imprinted genes in mouse migratory primordial germ cells. Mech. Dev. 115, 157–160 (2002).

    Article  Google Scholar 

  46. Lee, H.-J. et al. Bone marrow transplantation generates immature oocytes and rescues long-term fertility in a preclinical mouse model of chemotherapy-induced premature ovarian failure. J. Clin. Oncol. 25, 3198–3204 (2007).

    Article  CAS  Google Scholar 

  47. Kagawa, N., Silber, S. & Kuwayama, M. Successful vitrification of bovine and human ovarian tissue. Reprod. Biomed. Online 18, 568–577 (2009).

    Article  Google Scholar 

  48. Selesniemi, K., Lee, H.-J., Muhlhauser, A. & Tilly, J.L. Prevention of maternal aging-associated oocyte aneuploidy and meiotic spindle defects in mice by dietary and genetic strategies. Proc. Natl. Acad. Sci. USA 108, 12319–12324 (2011).

    Article  CAS  Google Scholar 

  49. Weissman, A. et al. Preliminary experience with subcutaneous human ovarian cortex transplantation in the NOD-SCID mouse. Biol. Reprod. 60, 1462–1467 (1999).

    Article  CAS  Google Scholar 

  50. Matikainen, T. et al. Aromatic hydrocarbon receptor-driven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals. Nat. Genet. 28, 355–360 (2001).

    Article  CAS  Google Scholar 

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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).

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Authors

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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.

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Correspondence to Jonathan L Tilly.

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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.

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White, Y., Woods, D., Takai, Y. et al. Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women. Nat Med 18, 413–421 (2012). https://doi.org/10.1038/nm.2669

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