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  • Protocol Extension
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Reconstitution of mouse oogenesis in a dish from pluripotent stem cells

Nature Protocols volume 12, pages 1733–1744 (2017)Cite this article

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Abstract

This protocol is an extension to: Nat. Protoc. 8, 1513–1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013

Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

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Figure 1: Overview of the culture system.
Figure 2: Representative results of IVDi culture.
Figure 3: Manual isolation of single follicles in the rOvary (representative isolation process).
Figure 4: Representative results of IVG culture.

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References

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

    Article  Google Scholar 

  2. Qing, T. et al. Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation 75, 902–911 (2007).

    Article  CAS  Google Scholar 

  3. Salvador, L.M., Silva, C.P., Kostetskii, I., Radice, G.L. & Strauss, J.F. III. The promoter of the oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells with an oocyte-like phenotype. Methods 45, 172–181 (2008).

    Article  CAS  Google Scholar 

  4. Yu, Z. et al. Dazl promotes germ cell differentiation from embryonic stem cells. J. Mol. Cell Biol. 1, 93–103 (2009).

    Article  CAS  Google Scholar 

  5. Hikabe, O. et al. Reconstitution in vitro of the entire cycle of the mouse female germ line. Nature 539, 299–303 (2016).

    Article  Google Scholar 

  6. Zhang, H. & Liu, K. Cellular and molecular regulation of the activation of mammalian primordial follicles: somatic cells initiate follicle activation in adulthood. Hum. Reprod. Update 21, 779–786 (2015).

    Article  CAS  Google Scholar 

  7. Edson, M.A., Nagaraja, A.K. & Matzuk, M.M. The mammalian ovary from genesis to revelation. Endocr. Rev. 30, 624–712 (2009).

    Article  CAS  Google Scholar 

  8. Hayashi, K. et al. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Science 338, 971–975 (2012).

    Article  CAS  Google Scholar 

  9. Hayashi, K., Ohta, H., Kurimoto, K., Aramaki, S. & Saitou, M. Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011).

    Article  CAS  Google Scholar 

  10. Hayashi, K. & Saitou, M. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat. Protoc. 8, 1513–1524 (2013).

    Article  CAS  Google Scholar 

  11. Eppig, J.J. & O'Brien, M.J. Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod. 54, 197–207 (1996).

    Article  CAS  Google Scholar 

  12. O'Brien, M.J., Pendola, J.K. & Eppig, J.J. A revised protocol for in vitro development of mouse oocytes from primordial follicles dramatically improves their developmental competence. Biol. Reprod. 68, 1682–1686 (2003).

    Article  CAS  Google Scholar 

  13. Morohaku, K. et al. Complete in vitro generation of fertile oocytes from mouse primordial germ cells. Proc. Natl. Acad. Sci. USA 113, 9021–9026 (2016).

    Article  CAS  Google Scholar 

  14. Morohaku, K., Hirao, Y. & Obata, Y. Development of fertile mouse oocytes from mitotic germ cells in vitro. Nat. Protoc. http://dx.doi.org/10.1038/nprot.2017.069 (2017).

  15. Jefferson, W., Newbold, R., Padilla-Banks, E. & Pepling, M. Neonatal genistein treatment alters ovarian differentiation in the mouse: inhibition of oocyte nest breakdown and increased oocyte survival. Biol. Reprod. 74, 161–168 (2006).

    Article  CAS  Google Scholar 

  16. Chen, Y., Breen, K. & Pepling, M.E. Estrogen can signal through multiple pathways to regulate oocyte cyst breakdown and primordial follicle assembly in the neonatal mouse ovary. J. Endocrinol. 202, 407–417 (2009).

    Article  CAS  Google Scholar 

  17. Dong, J. et al. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531–535 (1996).

    Article  CAS  Google Scholar 

  18. Yan, C. et al. Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function. Mol. Endocrinol. 15, 854–866 (2001).

    Article  CAS  Google Scholar 

  19. Hirao, Y. et al. In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium. Biol. Reprod. 70, 83–91 (2004).

    Article  CAS  Google Scholar 

  20. Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253–268 (2015).

    Article  CAS  Google Scholar 

  21. Sasaki, K. et al. Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 17, 178–194 (2015).

    Article  CAS  Google Scholar 

  22. Xu, J. et al. Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture. Hum. Reprod. 28, 2187–2200 (2013).

    Article  CAS  Google Scholar 

  23. Tingen, C.M. et al. A macrophage and theca cell-enriched stromal cell population influences growth and survival of immature murine follicles in vitro. Reproduction 141, 809–820 (2011).

    Article  CAS  Google Scholar 

  24. Xu, M., Kreeger, P.K., Shea, L.D. & Woodruff, T.K. Tissue-engineered follicles produce live, fertile offspring. Tissue Eng. 12, 2739–2746 (2006).

    Article  CAS  Google Scholar 

  25. Yin, H., Kristensen, S.G., Jiang, H., Rasmussen, A. & Andersen, C.Y. Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture. Hum. Reprod. 31, 1531–1539 (2016).

    Article  CAS  Google Scholar 

  26. Matoba, S. & Ogura, A. Generation of functional oocytes and spermatids from fetal primordial germ cells after ectopic transplantation in adult mice. Biol. Reprod. 84, 631–638 (2011).

    Article  CAS  Google Scholar 

  27. Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual 3rd edn. (Cold Spring Harbor Laboratory Press, 2003).

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Acknowledgements

We thank N. Hamada, S. Shomamoto, N. Hamazaki and G. Nagamatsu for providing technical details of the protocol. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI nos. 25114006, 15K21736, 25290033 and 17H01395); by JST-PRESTO; by the Uehara Memorial Foundation; and by the Takeda Science Foundation.

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Authors and Affiliations

  1. Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan

    Katsuhiko Hayashi & Orie Hikabe

  2. PRESTO, Japan Science and Technology Agency (JST), Fukuoka, Japan

    Katsuhiko Hayashi

  3. Department of Bioscience, Tokyo University of Agriculture, Tokyo, Japan

    Yayoi Obata

  4. NARO Institute of Livestock and Grassland Science, Tsukuba, Japan

    Yuji Hirao

Authors
  1. Katsuhiko Hayashi
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  2. Orie Hikabe
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  3. Yayoi Obata
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  4. Yuji Hirao
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Contributions

K.H., O.H., Y.O. and Y.H. developed the techniques. K.H. wrote the manuscript.

Corresponding author

Correspondence to Katsuhiko Hayashi.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Evaluation of 11 media for IVD culture

Representative images of E12.5 gonads cultured with the media and on the days indicated are shown. Note that the number of oocytes is highest in the culture with SptemPro34. Further evaluation revealed that the combination of αMEM and StemPro34 media was suitable for IVD culture of reconstituted ovaries5. Scale bars, 500 μm. These experiments were performed under the ethical guidelines of Kyushu University.

Supplementary Figure 2 IVD culture with fresh or frozen-thawed gonadal somatic cells

Two representative images of IVDi culture with fresh or frozen-thawed gonadal somatic cells are shown. BF, bright field; SC, stella-ECFP. Scale bars, 200 μm. These experiments were performed under the ethical guidelines of Kyushu University.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2. (PDF 399 kb)

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Hayashi, K., Hikabe, O., Obata, Y. et al. Reconstitution of mouse oogenesis in a dish from pluripotent stem cells. Nat Protoc 12, 1733–1744 (2017). https://doi.org/10.1038/nprot.2017.070

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