Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Lgr5 marks stem/progenitor cells in ovary and tubal epithelia

Nature Cell Biology volume 16, pages 745–757 (2014)Cite this article

Subjects

Abstract

The ovary surface epithelium (OSE) undergoes ovulatory tear and remodelling throughout life. Resident stem cells drive such tissue homeostasis in many adult epithelia, but their existence in the ovary has not been definitively proven. Lgr5 marks stem cells in multiple epithelia. Here we use reporter mice and single-molecule fluorescent in situ hybridization to document candidate Lgr5+ stem cells in the mouse ovary and associated structures. Lgr5 is broadly expressed during ovary organogenesis, but becomes limited to the OSE in neonate life. In adults, Lgr5 expression is predominantly restricted to proliferative regions of the OSE and mesovarian–fimbria junctional epithelia. Using in vivo lineage tracing, we identify embryonic and neonate Lgr5+ populations as stem/progenitor cells contributing to the development of the OSE cell lineage, as well as epithelia of the mesovarian ligament and oviduct/fimbria. Adult Lgr5+ populations maintain OSE homeostasis and ovulatory regenerative repair in vivo. Thus, Lgr5 marks stem/progenitor cells of the ovary and tubal epithelia.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Lgr5 expression in the developing ovary and ovary-associated structures.
Figure 2: Lgr5 expression in the adult mouse and human ovary and ovary-associated structures.
Figure 3: The adult Lgr5+ OSE transcriptome.
Figure 4: Embryonic and neonate Lgr5+ populations are early stem/progenitor cells of adult OSE lineages in vivo.
Figure 5: Clonal output and characterization of embryonic Lgr5+ cell progeny in vivo.
Figure 6: Adult Lgr5+ OSE populations are capable of epithelial regenerative repair.
Figure 7: Embryonic and neonate Lgr5+ populations are early stem/progenitor cells of the epithelial cell lineage of the oviduct/fimbria and mesovarian ligament in vivo.
Figure 8: Proposed model of Lgr5+ stem/progenitor cell activity during ovary development and adult homeostasis/regenerative repair.

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Auersperg, N., Wong, A. S., Choi, K. C., Kang, S. K. & Leung, P. C. Ovarian surface epithelium: biology, endocrinology, and pathology. Endocr. Rev. 22, 255–288 (2001).

    CAS  PubMed  Google Scholar 

  2. Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Barker, N. et al. Lgr5+ve stem/progenitor cells contribute to nephron formation during kidney development. Cell Rep. 2, 540–552 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. Jaks, V. et al. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat. Genet. 40, 1291–1299 (2008).

    Article  CAS  PubMed  Google Scholar 

  6. Barker, N. et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457, 608–611 (2009).

    Article  CAS  PubMed  Google Scholar 

  7. Szotek, P. P. et al. Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics. Proc. Natl Acad. Sci. USA 105, 12469–12473 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Paik, D. Y. et al. Stem-like epithelial cells are concentrated in the distal end of the fallopian tube: a site for injury and serous cancer initiation. Stem Cells 30, 2487–2497 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gamwell, L. F., Collins, O. & Vanderhyden, B. C. The mouse ovarian surface epithelium contains a population of LY6A (SCA-1) expressing progenitor cells that are regulated by ovulation-associated factors. Biol. Reprod. 87, 80 (2012).

    Article  PubMed  Google Scholar 

  10. Wang, Y. et al. Identification of quiescent, stem-like cells in the distal female reproductive tract. PLoS ONE 7, e40691 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Flesken-Nikitin, A. et al. Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche. Nature 495, 241–245 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Itzkovitz, S. et al. Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat. Cell Biol. 14, 106–114 (2012).

    Article  CAS  Google Scholar 

  13. Lustig, B. et al. Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol. Cell. Biol. 22, 1184–1193 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tan, O. L. & Fleming, J. S. Proliferating cell nuclear antigen immunoreactivity in the ovarian surface epithelium of mice of varying ages and total lifetime ovulation number following ovulation. Biol. Reprod. 71, 1501–1507 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Burdette, J. E., Kurley, S. J., Kilen, S. M., Mayo, K. E. & Woodruff, T. K. Gonadotropin-induced superovulation drives ovarian surface epithelia proliferation in CD1 mice. Endocrinology 147, 2338–2345 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2011).

    Article  CAS  PubMed  Google Scholar 

  17. Chassot, A. A. et al. RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary. PLoS ONE 6, e25641 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Chassot, A. A. et al. Activation of β-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Hum. Mol. Genet. 17, 1264–1277 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Usongo, M. & Farookhi, R. β-catenin/Tcf-signaling appears to establish the murine ovarian surface epithelium (OSE) and remains active in selected postnatal OSE cells. BMC Dev. Biol. 12, 17–31 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fafilek, B. et al. Troy, a tumor necrosis factor receptor family member, interacts with lgr5 to inhibit wnt signaling in intestinal stem cells. Gastroenterology 144, 381–391 (2013).

    Article  CAS  PubMed  Google Scholar 

  21. Stange, D. E. et al. Differentiated Troy+ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 155, 357–368 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schepers, A. G. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730–735 (2012).

    Article  CAS  PubMed  Google Scholar 

  23. Auersperg, N. The origin of ovarian carcinomas: a unifying hypothesis. Int. J. Gynecol. Pathol. 30, 12–21 (2011).

    Article  PubMed  Google Scholar 

  24. Auersperg, N. The origin of ovarian cancers–hypotheses and controversies. Front. Biosci. (Schol. Ed.) 5, 709–719 (2013).

    Article  Google Scholar 

  25. Piek, J. M. et al. Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer. J. Pathol. 195, 451–456 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Carlson, J. W. et al. Serous tubal intraepithelial carcinoma: its potential role in primary peritoneal serous carcinoma and serous cancer prevention. J. Clin. Oncol. 26, 4160–4165 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Snippert, H. J., Schepers, A. G., Delconte, G., Siersema, P. D. & Clevers, H. Slide preparation for single-cell-resolution imaging of fluorescent proteins in their three-dimensional near-native environment. Nat. Protoc. 6, 1221–1228 (2011).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Ong, A. Wong and A. Tan for technical assistance, G. Wright and J. Lim for imaging assistance, S. Mustafah for FACS experiments, and D. Tan for technical expertise and manuscript proofreading. We also thank M. Choolani and B. Pang for providing human material and critical discussions before publication. N.B. is supported by the Agency for Science, Technology and Research (A*STAR), Singapore.

Author information

Authors and Affiliations

  1. A-STAR Institute of Medical Biology, 8A Biomedical Grove, 06-06 Immunos, 138648, Singapore

    Annie Ng, Shawna Tan, Gurmit Singh, Pamela Rizk, Yada Swathi, Marc Leushacke & Nick Barker

  2. Cancer Science Institute of Singapore, National University of Singapore, 117599, Singapore

    Tuan Zea Tan & Ruby Yun-Ju Huang

  3. Department of Obstetrics & Gynaecology, National University Hospital, 119228, Singapore

    Ruby Yun-Ju Huang

  4. Centre for Regenerative Medicine, 47 Little France Crescent, University of Edinburgh, EH16 4TJ, UK

    Nick Barker

  5. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 117596, Singapore

    Nick Barker

Authors
  1. Annie Ng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shawna Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gurmit Singh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pamela Rizk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yada Swathi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tuan Zea Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ruby Yun-Ju Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marc Leushacke
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nick Barker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.N. designed and performed experiments, analysed data and wrote the manuscript. S.T., P.R. and Y.S. performed microarray experiments and bioinformatics analyses. G.S. performed mouse injections. T.Z.T. and R.Y-J.H. conducted the gene set enrichment analyses. M.L. performed proliferation experiments and provided technical expertise. N.B. supervised the project, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Nick Barker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Lgr5 expression in the Lgr5–LacZ reporter knock-in mouse.

P1: Lgr5–LacZ expression on the ovary surface (black arrow) and sub-surface (arrowheads), as well as oviduct and fimbria (red arrow). P7: Lgr5–LacZ expression in the OSE (arrow). Adult: Lgr5–LacZ expression in the OSE (arrow), fimbrial base (black arrowhead), mesovarian ligament (white arrows) and mesovarian-fimbria junction (white arrowhead). WT insets show wild-type littermate ovaries. Neutral red counter-stain. Scale bars: 100 μm (wholemount images); 10 μm (sections).

Supplementary Figure 2 Active Wnt signaling in P7 and adult Lgr5–EGFP+ cells.

(Top) P7 Lgr5-KI/Axin-LacZ ovary section stained for LacZ activity (left) then co-immunostained with EGFP and K8 (right). Arrows point to Axin2+ Lgr5–EGFPhi OSE cells. (Bottom) An adult Lgr5-KI/Axin-LacZ ovary section stained for LacZ activity (left) then co-immunostained with EGFP–HRP (middle). Black and white arrowheads point to Axin2+Lgr5–EGFPhi and Axin2+Lgr5–EGFPlo/neg OSE cells respectively. Neutral red counter-stain. qPCR with Axin2 (right) confirming expression in sorted Lgr5–EGFPhi, Lgr5–EGFPlo and Lgr5–EGFPneg OSE cells, but not in OSE-stripped ovary cells. Histograms represent fold-change relative to Lgr5–EGFPneg cells set as 1. Error bars indicate s.e.m. Gapdh was used as endogenous reference. Scale bars: 10 μm.

Supplementary Figure 3 Spatial distribution of Lgr5–EGFP+ and Aldh1+ cells in the adult ovary.

(Top) EGFP immunostaining of adult Lgr5-KI ovary sections, revealing Lgr5–EGFP+ cells concentrated at cleft regions between growing follicles (a,b) and corpora lutea (c,d). Note OSE cells at surface of corpora lutea were devoid of Lgr5 expression (arrowhead in d) except at ovulating follicles (that is, prospective corpora lutea) comprising stigma-associated OSE/tunica albuginea (arrows in e,f) and rupturing OSE (g,h). F, follicle; CL, corpus luteum; Ov, ovulating follicle; R, rupture site. (Bottom) EGFP/Aldh1 (detecting a1 subunit) co-immunostaining of adult Lgr5-KI ovary sections, revealing Aldh1 expression within OSE cells at the ovary hilum (1) and anterior regions (2,3). Note Aldh1 expression in both Lgr5–EGFPlo (arrows) and Lgr5–EGFPhi (arrowheads) OSE cells. Also note Aldh1 expression in non-OSE cells within the ovary interior. Scale bars: 10 μm.

Supplementary Figure 4 Lgr5 and Aldh1a1/a2 co-FISH in the adult ovary and gene set enrichment analysis (GSEA).

Co-FISH of Lgr5 and Aldh1a1/a2 on adult wild-type ovary sections, confirming Aldh1a1/a2 transcripts within OSE cells at the ovary hilum (1,5) and anterior regions (2,3,6,7). Bright white dots represent positive FISH signals. OSE cells are denoted above yellow dashed lines. Note presence of Aldh1a1/a2 transcripts in Lgr5+ (arrowheads) and Lgr5− (arrows) cells. RNase A-treated control sections revealed specificity of Lgr5 and Aldh1a1/a2 transcripts (4,8). GSEA reveals no significant gene set enrichment of the published Aldh1+ signature (Flesken-Nikitin et al., 2013) in Lgr5–EGFPhi OSE cells (P = 0.24). ES, enrichment score. Scale bars: 10 μm.

Supplementary Figure 5 Lgr5 and Troy co-FISH in the adult ovary.

Co-FISH of Lgr5 and Troy on adult wild-type ovary sections, demonstrating enriched Troy mRNA expression in Lgr5+ OSE cells. Bright white dots represent positive FISH signals. OSE cells are denoted above yellow dashed lines. Note similar expression profile of Troy to that of Lgr5, with abundant transcripts present in OSE cells around a follicle but absent atop a corpus luteum. RNase A-treated sections revealed specificity of Lgr5 and Troy transcripts. F, follicle; CL, corpus luteum. Scale bars: 10 μm.

Supplementary Figure 6 Lgr5–EGFP/Wnt4 and Lgr5–EGFP/Id2 co-IHC.

(Top) EGFP/Wnt4 co-immunostaining of adult Lgr5-KI ovary sections, demonstrating enriched Wnt4 expression in Lgr5–EGFPhi OSE cells (arrowheads). (Bottom) EGFP/Id2 co-immunostaining of adult Lgr5-KI ovary sections, demonstrating down-regulation of Id2 expression in Lgr5–EGFPhi OSE cells. Arrows point to Id2−Lgr5–EGFPhi OSE cells. Arrowheads point to Id2+Lgr5–EGFPneg/lo OSE cells. Scale bars: 10 μm.

Supplementary Figure 7 Neonate Lgr5+ populations and their progeny are lifelong contributors of the adult OSE lineage in vivo.

(Top) Wholemount and histological analyses of LacZ activity in ovary at various times post P1-induction. A single limiting dose of 4-OHT induced LacZ activity at clonal density. Sparse isolated LacZ+ cells were first observed 2 days (2d) p.i. Uniform rate of expansion pre-puberty led to similar-sized LacZ+ clones 1 week (1w) and 4 weeks (4w) p.i., whilst variable rate of expansion post-puberty led to heterogenous-sized LacZ+ clones at 3 months (3m) p.i. Frequency of lineage tracing was maintained up to 16 months (16m). Note larger-sized clones adjacent to corpora lutea (red arrowheads) compared to follicles (white arrowheads). Dot plot shows LacZ+ OSE clone size quantification post P1-induction. n = 5 females/time-point, total of 461 clones counted. Each dot represents a disparate LacZ+ OSE clone. Red bars indicate s.d. ranges. Green circles denote mean clone size, which steadily increased over time (green line). F, follicle; CL, corpus luteum. (Bottom) Confocal z-stack images of Lgr5-KI/tdTomato ovary treated with an increased dose of 4-OHT at various times post P1-induction, revealing steady increase in the number of tdTomato+ progeny over time. Scale bars: 100 μm (wholemount images); 10 μm (sections).

Supplementary Figure 8 Adult Lgr5+ cells beyond the hilum are capable of OSE generation.

Confocal z-stack images of near-native Lgr5-KI/Rosa26-tdTomato ovary slices 2 days (2d), 2 weeks (2w) and 4 months (4m) post adult-induction (p.i.). At 2 days p.i., 29.5% of Lgr5–EGFPhi cells have activated the tdTomato reporter (432 tdTomato+ cells out of 1463 Lgr5-GFPhi OSE cells counted). These cells rapidly expanded to form tdTomato+ clones throughout the ovary surface encompassing the ovary hilum and anterior ovary 2w and 4m p.i. Arrowheads point to larger-sized clones at the edges or atop corpora lutea. White dashed line demarcates ovary-mesovarian junction. Yellow dashed lines demarcate follicles. Scale bars: 10 μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2507 kb)

Supplementary Table 1

Supplementary Information (XLSX 481 kb)

Supplementary Table 2

Supplementary Information (XLSX 38 kb)

Supplementary Table 3

Supplementary Information (XLSX 9 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ng, A., Tan, S., Singh, G. et al. Lgr5 marks stem/progenitor cells in ovary and tubal epithelia. Nat Cell Biol 16, 745–757 (2014). https://doi.org/10.1038/ncb3000

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3000

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing