Letter | Published:

Reconstitution in vitro of the entire cycle of the mouse female germ line

Nature volume 539, pages 299303 (10 November 2016) | Download Citation

Abstract

The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg1,2. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

Data deposits

The RNA-seq data have been deposited at Gene Expression Omnibus (GEO) database under accession number GSE79729.

References

  1. 1.

    , & Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762 (2007)

  2. 2.

    , & The mammalian ovary from genesis to revelation. Endocr. Rev. 30, 624–712 (2009)

  3. 3.

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

  4. 4.

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

  5. 5.

    , , , & . 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)

  6. 6.

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

  7. 7.

    & Entry of mouse embryonic germ cells into meiosis. Dev. Biol. 187, 107–113 (1997)

  8. 8.

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

  9. 9.

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

  10. 10.

    , , , & A comprehensive, non-invasive visualization of primordial germ cell development in mice by the Prdm1-mVenus and Dppa3-ECFP double transgenic reporter. Reproduction 136, 503–514 (2008)

  11. 11.

    , , , & Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011)

  12. 12.

    , & Continuous loss of oocytes throughout meiotic prophase in the normal mouse ovary. Dev. Biol. 258, 334–348 (2003)

  13. 13.

    , , & Gene-specific timing and epigenetic memory in oocyte imprinting. Hum. Mol. Genet. 13, 839–849 (2004)

  14. 14.

    et al. Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos. Dev. Cell 7, 597–606 (2004)

  15. 15.

    et al. Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape. Genome Biol. 16, 209 (2015)

  16. 16.

    et al. Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer. Biol. Reprod. 65, 1813–1821 (2001)

  17. 17.

    et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008)

  18. 18.

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

  19. 19.

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

  20. 20.

    & Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. Nat. Protocols 8, 1513–1524 (2013)

  21. 21.

    & Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)

  22. 22.

    et al. A germ cell-specific gene, Prmt5, works in somatic cell reprogramming. J. Biol. Chem. 286, 10641–10648 (2011)

  23. 23.

    , , , & Gene activation-associated long noncoding RNAs function in mouse preimplantation development. Development 142, 910–920 (2015)

  24. 24.

    & NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7, e30619 (2012)

  25. 25.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  26. 26.

    et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protocols 7, 562–578 (2012)

  27. 27.

    , , & QuasR: quantification and annotation of short reads in R. Bioinformatics 31, 1130–1132 (2015)

  28. 28.

    et al. A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints. Dev. Cell 15, 547–557 (2008)

  29. 29.

    et al. Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells. Development 129, 1807–1817 (2002)

  30. 30.

    , & QUMA: quantification tool for methylation analysis. Nucleic Acids Res. 36, W170–W175 (2008)

  31. 31.

    et al. HP1γ links histone methylation marks to meiotic synapsis in mice. Development 138, 4207–4217 (2011)

  32. 32.

    & Simultaneous analysis of chromosomes and chromosome-associated proteins in mammalian oocytes and embryos. Chromosoma 111, 165–169 (2002)

Download references

Acknowledgements

We thank Y. Takada for technical support, H. Ohta for technical advice on the iPSCs and embryo transfer, Y. Ohkawa for technical assistance on the RNA-seq, K. Kitajima and C. Meno for providing microscopes, F. Arai for providing FACSAriaII, and H. Leitch for proofreading the manuscript. We also thank the Research Support Center, Research Center for Human Disease Modeling, Kyushu University Graduate School of Medical Sciences for technical assistance. N.H. was a JSPS Research Fellow. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI #25114006); by JST-PRESTO; by the Uehara Memorial Foundation; and by the Takeda Science Foundation.

Author information

Author notes

    • Orie Hikabe
    •  & Katsuhiko Hayashi

    These authors contributed equally to this work.

Affiliations

  1. Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

    • Orie Hikabe
    • , Nobuhiko Hamazaki
    • , Go Nagamatsu
    • , Norio Hamada
    • , So Shimamoto
    • , Takuya Imamura
    • , Kinichi Nakashima
    •  & Katsuhiko Hayashi
  2. Department of Bioscience, Tokyo University of Agriculture, 1-1-1, Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

    • Yayoi Obata
  3. NARO Institute of Livestock and Grassland Science, Ikenodai 2, Tsukuba 305-0901, Japan

    • Yuji Hirao
  4. Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

    • Norio Hamada
  5. Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

    • Mitinori Saitou
  6. Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan

    • Mitinori Saitou
  7. Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan

    • Mitinori Saitou
  8. JST, ERATO, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan

    • Mitinori Saitou
  9. JST, PRESTO, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan

    • Katsuhiko Hayashi

Authors

  1. Search for Orie Hikabe in:

  2. Search for Nobuhiko Hamazaki in:

  3. Search for Go Nagamatsu in:

  4. Search for Yayoi Obata in:

  5. Search for Yuji Hirao in:

  6. Search for Norio Hamada in:

  7. Search for So Shimamoto in:

  8. Search for Takuya Imamura in:

  9. Search for Kinichi Nakashima in:

  10. Search for Mitinori Saitou in:

  11. Search for Katsuhiko Hayashi in:

Contributions

O.H., N.H., S.S. and K.H. performed the culture, embryo transfer, immunofluorescence, PCR analysis and chimaera analysis; O.H., N.H., S.S. and K.H. performed the differentiation to mature oocytes and are able to replicate the entire process, independently. N.H., T.I., and K.N. performed the RNA-seq analysis. G.N. generated the iPSC lines. Y.O and Y.H. conducted the assessment of the culture conditions. M.S. contributed to the inception of the study. K.H. designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Katsuhiko Hayashi.

Reviewer Information Nature thanks D. Egli, S. Mitalipov and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1 (gel source data) and Supplementary Tables 1-2.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature20104

Further reading

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.