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New insights into the GDF9-Hedgehog-GLI signaling pathway in human ovaries: from fetus to postmenopause

  • Reproductive Physiology and Disease
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Research question

Are glioma-associated oncogene homolog 1, 2, and 3 (GLI1, 2, and 3) and protein patched homolog 1 (PTCH1) specific markers for precursor theca cells in human ovaries as in mouse ovaries?

Design

To study the GDF9-HH-GLI pathway and assess whether GLI1 and 3 and PTCH1 are specific markers for precursor theca cells in the human ovary, growth differentiation factor 9 (GDF9), Indian Hedgehog (IHH), Desert Hedgehog (DHH), Sonic Hedgehog (SHH), PTCH1 and GLI1, 2 and 3 were investigated in fetal (n=9), prepubertal (n=9), reproductive-age (n=15), and postmenopausal (n=8) human ovarian tissue. Immunohistochemistry against GDF9, IHH, DHH, SHH, PTCH1, GLI1, GLI2, and GLI3 was performed on human ovarian tissue sections fixed in 4% formaldehyde and embedded in paraffin. Western blotting was carried out on extracted proteins from the same samples used in the previous step to prove the antibodies’ specificity. The quantitative real-time polymerase chain reaction was performed to identify mRNA levels for Gdf9, Ihh, Gli1, Gli2, and Gli3 in menopausal ovaries.

Results

Our results showed that, in contrast to mice, all studied proteins were expressed in primordial follicles of fetal, prepubertal, and reproductive-age human ovaries and stromal cells of reproductive-age and postmenopausal ovaries. Intriguingly, Gdf9, Ihh, and Gli3 mRNA, but not Gli1 and 2, was detected in postmenopausal ovaries. Moreover, GLI1, GLI3, and PTCH1 are not limited to a specific population of cells. They were spread throughout the organ, which means they are not specific markers for precursor theca cells in human ovaries.

Conclusion

These results could provide a basis for understanding how this pathway modulates follicle development and ovarian cell steroidogenesis in human ovaries.

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References

  1. Edson MA, Nagaraja AK, Matzuk MM. The mammalian ovary from genesis to revelation. Endocr Rev. 2009;30(6):624–712.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Matzuk MM, Lamb DJ. The biology of infertility: research advances and clinical challenges. Nat Med. 2008;14(11):1197–213.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Liu C, Peng J, Matzuk MM, Yao HH-C. Lineage specification of ovarian theca cells requires multicellular interactions via oocyte and granulosa cells. Nat Commun. 2015;6(1):1–11.

    CAS  Google Scholar 

  4. Lapointe E, Boerboom D. WNT signaling and the regulation of ovarian steroidogenesis. Front Biosci (Scholar ed). 2011;3:276–85.

    Google Scholar 

  5. Honda A, Hirose M, Hara K, Matoba S, Inoue K, Miki H, et al. Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells. PNAS. 2007;104(30):12389–94.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dong J, Albertini DF, Nishimori K, Kumar TR, Lu N, Matzuk MM. Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature. 1996;383(6600):531–5.

    Article  CAS  PubMed  Google Scholar 

  7. Varjosalo M, Taipale J. Hedgehog: functions and mechanisms. Genes Dev. 2008;22(18):2454–72.

    Article  CAS  PubMed  Google Scholar 

  8. Liu C-F, Liu C, Yao HH-C. Building pathways for ovary organogenesis in the mouse embryo. In: Current topics in developmental biology, vol. 90. Amsterdam: Elsevier; 2010. p. 263–90.

    Google Scholar 

  9. Wijgerde M, Ooms M, Hoogerbrugge JW, Grootegoed JA. Hedgehog signaling in mouse ovary: Indian hedgehog and desert hedgehog from granulosa cells induce target gene expression in developing theca cells. Endocrinology. 2005;146(8):3558–66.

    Article  CAS  PubMed  Google Scholar 

  10. Yao HH-C, Whoriskey W, Capel B. Desert hedgehog/patched 1 signaling specifies fetal Leydig cell fate in testis organogenesis. Genes Dev. 2002;16(11):1433–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Richards JS, Ren YA, Candelaria N, Adams JE, Rajkovic A. Ovarian follicular theca cell recruitment, differentiation, and impact on fertility: 2017 update. Endocr Rev. 2017;39(1):1–20.

    Article  PubMed Central  Google Scholar 

  12. Marion G, Gier H, Choudary J. Micromorphology of the bovine ovarian follicular system. J Anim Sci. 1968;27(2):451–65.

    Article  CAS  PubMed  Google Scholar 

  13. Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature. 1996;384(6605):129–34.

    Article  CAS  PubMed  Google Scholar 

  14. Fuse N, Maiti T, Wang B, Porter JA, Hall TMT, Leahy DJ, et al. Sonic hedgehog protein signals not as a hydrolytic enzyme but as an apparent ligand for patched. Proc Natl Acad Sci. 1999;96(20):10992–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ingham P, Taylor A, Nakano Y. Role of the Drosophila patched gene in positional signalling. Nature. 1991;353(6340):184–7.

    Article  CAS  PubMed  Google Scholar 

  16. Taipale J, Cooper M, Maiti T, Beachy P. Patched acts catalytically to suppress the activity of Smoothened. Nature. 2002;418(6900):892–6.

    Article  CAS  PubMed  Google Scholar 

  17. Ingham PW, McMahon AP. Hedgehog signaling in animal development: paradigms and principles. Genes Dev. 2001;15(23):3059–87.

    Article  CAS  PubMed  Google Scholar 

  18. Gulino A, Di Marcotullio L, Ferretti E, De Smaele E, Screpanti I. Hedgehog signaling pathway in neural development and disease. Psychoneuroendocrinology. 2007;32:S52–S6.

    Article  CAS  PubMed  Google Scholar 

  19. Di Marcotullio L, Ferretti E, Greco A, De Smaele E, Screpanti I, Gulino A. Multiple ubiquitin-dependent processing pathways regulate hedgehog/gli signaling: implications for cell development and tumorigenesis. Cell Cycle. 2007;6(4):390–3.

    Article  PubMed  Google Scholar 

  20. Lum L, Beachy PA. The Hedgehog response network: sensors, switches, and routers. Science. 2004;304(5678):1755–9.

    Article  CAS  PubMed  Google Scholar 

  21. Russell MC, Cowan RG, Harman RM, Walker AL, Quirk SM. The hedgehog signaling pathway in the mouse ovary. Biol Reprod. 2007;77(2):226–36.

    Article  CAS  PubMed  Google Scholar 

  22. Asiabi P, Ambroise J, Giachini C, Coccia ME, Bearzatto B, Chiti MC, et al. Assessing and validating housekeeping genes in normal, cancerous, and polycystic human ovaries. J Assist Reprod Genet. 2020. https://doi.org/10.1007/s10815-020-01901-8.

  23. Young J, McNeilly AS. Theca: the forgotten cell of the ovarian follicle. Reproduction. 2010;140(4):489–504.

    Article  CAS  PubMed  Google Scholar 

  24. Magoffin DA, Magarelli PC. Preantral follicles stimulate luteinizing hormone independent differentiation of ovarian theca-interstitial cells by an intrafollicular paracrine mechanism. Endocr Rev. 1995;3(2):107–12.

    Article  CAS  Google Scholar 

  25. Magoffin DA. Ovarian theca cell. Int J Biochem Cell Biol. 2005;37(7):1344–9.

    Article  CAS  PubMed  Google Scholar 

  26. Orisaka M, Tajima K, Mizutani T, Miyamoto K, Tsang BK, Fukuda S, et al. Granulosa cells promote differentiation of cortical stromal cells into theca cells in the bovine ovary. Biol Reprod. 2006;75(5):734–40.

    Article  CAS  PubMed  Google Scholar 

  27. Qiu M, Quan F, Han C, Wu B, Liu J, Yang Z, et al. Effects of granulosa cells on steroidogenesis, proliferation and apoptosis of stromal cells and theca cells derived from the goat ovary. J Steroid Biochem Mol Biol. 2013;138:325–33.

    Article  CAS  PubMed  Google Scholar 

  28. Asiabi P, Dolmans M, Ambroise J, Camboni A, Amorim C. In vitro differentiation of theca cells from ovarian cells isolated from postmenopausal women. Hum Reprod. 2020;35(12):2793–807.

    Article  CAS  PubMed  Google Scholar 

  29. Bitgood MJ, McMahon AP. Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo. Develop Biol. 1995;172:126.

    Article  CAS  PubMed  Google Scholar 

  30. Huang CCJ, Yao HHC. Diverse functions of Hedgehog signaling in formation and physiology of steroidogenic organs. Molecul Reprod Dev Incorp Gamete Res. 2010;77(6):489–96.

    Article  CAS  Google Scholar 

  31. Spicer LJ, Sudo S, Aad PY, Wang LS, Chun S-Y, Ben-Shlomo I, et al. The hedgehog-patched signaling pathway and function in the mammalian ovary: a novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells. Reproduction. 2009;138(2):329–39.

    Article  CAS  PubMed  Google Scholar 

  32. Otsuka F, McTavish KJ, Shimasaki S. Integral role of GDF-9 and BMP-15 in ovarian function. Mol Reprod Dev. 2011;78(1):9–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sun R, Lei L, Cheng L, Jin Z, Zu S, Shan Z, et al. Expression of GDF-9, BMP-15 and their receptors in mammalian ovary follicles. J Mol Histol. 2010;41(6):325–32.

    Article  CAS  PubMed  Google Scholar 

  34. Riepsamen AH, Chan K, Lien S, Sweeten P, Donoghoe MW, Walker G, et al. Serum concentrations of oocyte-secreted factors BMP15 and GDF9 during IVF and in women with reproductive pathologies. Endocrinology. 2019;160(10):2298–313.

    Article  CAS  PubMed  Google Scholar 

  35. Echelard Y, Epstein DJ, St-Jacques B, Shen L, Mohler J, McMahon JA, et al. Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell. 1993;75(7):1417–30.

    Article  CAS  PubMed  Google Scholar 

  36. Bitgood MJ, Shen L, McMahon AP. Sertoli cell signaling by Desert hedgehog regulates the male germline. Curr Biol. 1996;6(3):298–304.

    Article  CAS  PubMed  Google Scholar 

  37. Van Den Brink GR, Bleuming SA, Hardwick JC, Schepman BL, Offerhaus GJ, Keller JJ, et al. Indian Hedgehog is an antagonist of Wnt signaling in colonic epithelial cell differentiation. Nat Genet. 2004;36(3):277–82.

    Article  PubMed  Google Scholar 

  38. Parikh G, Varadinova M, Suwandhi P, Araki T, Rosenwaks Z, Poretsky L, et al. Vitamin D regulates steroidogenesis and insulin-like growth factor binding protein-1 (IGFBP-1) production in human ovarian cells. Horm Metab Res. 2010;42(10):754–7.

    Article  CAS  PubMed  Google Scholar 

  39. Channing CP. Steroidogenesis and morphology of human ovarian cell types in tissue culture. J Endocrinol. 1969;45(2):297–NP.

    Article  CAS  PubMed  Google Scholar 

  40. Qiu M, Liu J, Han C, Wu B, Yang Z, Su F, et al. The influence of ovarian stromal/theca cells during in vitro culture on steroidogenesis, proliferation and apoptosis of granulosa cells derived from the goat ovary. Reprod Domest Anim. 2014;49(1):170–6.

    Article  CAS  PubMed  Google Scholar 

  41. Fogle RH, Stanczyk FZ, Zhang X, Paulson RJ. Ovarian androgen production in postmenopausal women. J Clin Endocrinol Metab. 2007;92(8):3040–3.

    Article  CAS  PubMed  Google Scholar 

  42. Couzinet B, Meduri G, Lecce MG, Young J, Brailly S, Loosfelt H, et al. The postmenopausal ovary is not a major androgen-producing gland. J Clin Endocrinol Metab. 2001;86(10):5060–6.

    Article  CAS  PubMed  Google Scholar 

  43. Dupont E, Labrie F, Luu-The V, Pelletier G. Immunocytochemical localization of 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase in human ovary. J Clin Endocrinol Metab. 1992;74(5):994–8.

    CAS  PubMed  Google Scholar 

  44. Havelock JC, Rainey WE, Bradshaw KD, Carr BR. The post-menopausal ovary displays a unique pattern of steroidogenic enzyme expression. Hum Reprod. 2005;21(1):309–17.

    Article  PubMed  Google Scholar 

  45. Hammerschmidt M, Brook A, McMahon AP. The world according to bedgebog. Trends Genet. 1997;13(1):14–21.

    Article  CAS  PubMed  Google Scholar 

  46. Walterhouse DO, Lamm ML, Villavicencio E, Iannaccone PM. Emerging roles for hedgehog-patched-Gli signal transduction in reproduction. Biol Reprod. 2003;69(1):8–14.

    Article  CAS  PubMed  Google Scholar 

  47. Nüsslein-Volhard C, Wieschaus E. Mutations affecting segment number and polarity in Drosophila. Nature. 1980;287(5785):795–801.

    Article  PubMed  Google Scholar 

  48. McMahon AP, Ingham PW, Tabin CJ. 1 Developmental roles and clinical significance of Hedgehog signaling. Curr Top Dev Biol. 2003;53:1–114.

    Article  CAS  PubMed  Google Scholar 

  49. Mara H. Rendi, Atis Muehlenbachs, Rochelle L. Garcia, Kelli L. Boyd. 17 - Female Reproductive System. In: Piper M. Treuting, Dintzis SM, editors. Comparative Anatomy and Histology Cambridge: Academic Press; 2012. p. 253-284.

  50. Jimenez R. Ovarian organogenesis in mammals: mice cannot tell us everything. Sex Dev. 2009;3(6):291–301.

    Article  CAS  PubMed  Google Scholar 

  51. Griffin J, Emery BR, Huang I, Peterson CM, Carrell DT. Comparative analysis of follicle morphology and oocyte diameter in four mammalian species (mouse, hamster, pig, and human). J Exper Clin Assist Reprod. 2006;3(1):1–9.

    Article  Google Scholar 

  52. Fan X, Bialecka M, Moustakas I, Lam E, Torrens-Juaneda V, Borggreven N, et al. Single-cell reconstruction of follicular remodeling in the human adult ovary. Nat Commun. 2019;10(1):1–13.

    Article  Google Scholar 

  53. Wagner M, Yoshihara M, Douagi I, Damdimopoulos A, Panula S, Petropoulos S, et al. Single-cell analysis of human ovarian cortex identifies distinct cell populations but no oogonial stem cells. Nat Commun. 2020;11(1):1147–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Man L, Lustgarten-Guahmich N, Kallinos E, Redhead-Laconte Z, Liu S, Schattman B, et al. Comparison of human antral follicles of xenograft versus ovarian origin reveals disparate molecular signatures. Cell Rep. 2020;32(6):108027.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to Mira Hryniuk, BA, for reviewing the English language of the manuscript and Dolores Gonzalez and Olivier Van Kerk for their technical assistance.

Funding

This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C. A. Amorim is an FRS-FNRS Research Associate; grant MIS #F4535 16 awarded to C. A. Amorim; grant 5/4/150/5 awarded to M. M. Dolmans; grant ASP-RE314 awarded to P. Asiabi) and the Fondation Contre le Cancer (grant 2018-042 awarded to A. Camboni).

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Authors and Affiliations

  1. Pôle de Recherche en Gynécologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Avenue Mounier 52, bte B1.52.02, 1200, Brussels, Belgium

    Parinaz Asiabi, Clara David, Alessandra Camboni, Marie-Madeleine Dolmans & Christiani A. Amorim

  2. Gynecology and Andrology Department, Cliniques Universitaires Saint-Luc, 1200, Brussels, Belgium

    Etienne Marbaix

  3. Pathology Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium

    Marie-Madeleine Dolmans

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  1. Parinaz Asiabi
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  2. Clara David
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  4. Etienne Marbaix
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Contributions

P. A.: study design, experimental procedures, analysis, interpretation of data, and manuscript preparation. C. D.: experimental procedures and analysis. M. M. D.: manuscript revision. E. M: tissue supply, analysis of IHC staining, and manuscript revision. A. C.: tissue supply. C. A. A.: experimental design, experimental procedures, interpretation of results, and manuscript revision.

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Correspondence to Christiani A. Amorim.

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Supplementary information

Supplementary Table 1

Summary of immunohistochemical protocols applied in this study (DOCX 17 kb)

Supplementary Table 2

Primary antibodies used in Western blot and their dilutions (DOCX 14 kb)

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Asiabi, P., David, C., Camboni, A. et al. New insights into the GDF9-Hedgehog-GLI signaling pathway in human ovaries: from fetus to postmenopause. J Assist Reprod Genet 38, 1387–1403 (2021). https://doi.org/10.1007/s10815-021-02161-w

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