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Reconstitution of mouse oogenesis in a dish from pluripotent stem cells

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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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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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

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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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