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Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid

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Abstract

Purpose

The aim of this study was to develop and optimize a strategy for long-term cultivation of luteinizing human granulosa cells (GCs).

Methods

GCs were cultivated in DMEM/F12 medium supplemented with 2% fetal calf serum. In vitro proliferation of GCs was supported by follicular fluid as well as FSH and growth factors.

Results

The cultured GCs were maintained for 45 days with a doubling time of 159 ± 24 h. GCs initiated by the addition of follicular fluid and cultivated under low serum conditions reached 10 ± 0.7 population doublings. GCs maintain the typical phenotypic expression and the telomere length according to specific culture conditions.

Conclusion

Our present study has demonstrated that GCs can be maintained in vitro for at least 45 days and this cell model can be beneficial when studying hormonal regulation associated with follicular maturation and preparation of oocytes for fertilization.

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Abbreviations

bFGF:

basic fibroblast growth factor

COV434:

cell line derived from a human granulosa cell tumor (Sigma-Aldrich)

DMEM/F12:

dulbecco’s modified eagle’s medium

DT:

doubling time

EGF:

epidermal growth factor

FCS:

fetal calf serum

FF:

follicular fluid

FSH:

follicle stimulating hormone

GCs:

luteinizing human granulosa cells

GC8:

one of the isolated GCs from patient with different characters

IVF:

in vitro fertilization

LH:

luteinizing hormone

PD:

the number of population doublings

References

  1. Figenschau Y, Sundsfjord JA, Yousef MI, Fuskevag OM, Sveinbjornsson B, Bertheussen K. A simplified serum-free method for preparation and cultivation of human granulosa-luteal cells. Hum Reprod. 1997;12:523–31.

    Article  PubMed  CAS  Google Scholar 

  2. Quinn MCJ, McGregor SB, Stanton JL, Hessian PA, Gillett WR, Gren DPL. Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates. Reprod Fertil Dev. 2006;18:501–8.

    Article  PubMed  CAS  Google Scholar 

  3. Lavranos TC, Mathis JM, Latham SE, Kalionis B, Shay JW, Rodgers RJ. Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles. Biol Reprod. 1999;61:358–66.

    Article  PubMed  CAS  Google Scholar 

  4. Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev. 1996;17:121–55.

    PubMed  CAS  Google Scholar 

  5. Niswender GD, Juengel JL, Silva PJ, Rollyson MK, McIntush EW. Mechanisms controlling the function and the life span of the corpus luteum. Physiol Rev. 2000;80:1–24.

    PubMed  CAS  Google Scholar 

  6. Wen X, Li D, Tozer AJ, Docherty SM, Iles RK. Estradiol, progesterone, testosterone profiles in human follicular fluid and cultured granulosa cells from luteinized pre-ovulatory follicles. Reprod Biol Endocrinol. 2010;8:117–27.

    Article  PubMed  Google Scholar 

  7. Gómez E, Tarín JJ, Pellicer A. Oocytes maturation in humans: the role of gonadotrophins and growth factors. Fertil Steril. 1993;60:40–6.

    PubMed  Google Scholar 

  8. Muttukrishna S, Groome N, Ledger W. Gonadotropic control of secretion of dimeric inhibins and activin A by human granulosa-luteal cells in vitro. J Assist Reprod Genet. 1997;14:566–74.

    Article  PubMed  CAS  Google Scholar 

  9. Lee HC, Lee SW, Lee KW, Cha KY, Kim KH, Lee S. Identification of new proteins in follicular fluid from mature human follicles by direct sample rehydration method of two-dimensional polyacrylamide gel electrophoresis. J Korean Med Sci. 2005;20:456–60.

    Article  PubMed  CAS  Google Scholar 

  10. Rodgers RJ, Irving-Rodgers HF. Formation of the ovarian follicular antrum and follicular fluid. Biol Reprod. 2010;82:1021–9.

    Article  PubMed  CAS  Google Scholar 

  11. Shay JW, Wright WE. Hayflick, his limit and cellular ageing. Nat Rev Mol Cell Biol. 2000;1:72–6.

    Article  PubMed  CAS  Google Scholar 

  12. Van den Berg-Bakker CA, Hagemeijer A, Franken EM. Establishment and characterization of 7 ovarian carcinoma cell lines and one granulosa tumor cell line: growth features and cytogenetics. Int J Cancer. 1993;53:613–20.

    Article  PubMed  Google Scholar 

  13. Zhang H, Vollmer M, De Geyter M, Litzistorf Y, Ladewig A, Dürrenberger M, et al. Characterization of an immortalized human granulosa cells cell line (COV434). Mol Hum Reprod. 2000;6:146–53.

    Article  PubMed  CAS  Google Scholar 

  14. Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30:e47.

    Article  PubMed  Google Scholar 

  15. Asai A, Oshima Y, Yamamoto Y, Uochi TA, Kusaka H, Akinaga S, et al. A novel telomerase template antagonist (GRN163) as a potential anticancer agent. Cancer Res. 2003;63:3931–9.

    PubMed  CAS  Google Scholar 

  16. Bruckova L, Soukup T, Moos J, Moosova M, Pavelkova J, Rezabek K, et al. The cultivation of human granulosa cells. Acta Medica (Hradec Kralove). 2008;51:165–72.

    Google Scholar 

  17. Sasson R, Dantes A, Tajima K, Amsterdam A. Novel genes modulated by FSH in normal and immortalized FSH responsive cells: new insights into the mechanism of FSH action. FASEB J. 2003;17:1256–66.

    Article  PubMed  CAS  Google Scholar 

  18. Lima-Verde IB, Matos MH, Saraiva MV, Bruno JB, Tenorio SB, Martins FS, et al. Interaction between estradiol and follicle-stimulating hormone promotes in vitro survival and development of caprine preantral follicles. Cells Tissues Organs. 2010;191:240–7.

    Article  PubMed  CAS  Google Scholar 

  19. Parborell F, Pecci A, Gonzalez O, Vitale A, Tesone A. Effects of a gonadotropin releasing hormone agonist on rat ovarian follicle apoptosis: regulation by epidermal growth factor and the expression of Bcl-2-related genes. Biol Repro. 2002;67:481–6.

    Article  CAS  Google Scholar 

  20. Matos MH, van den Hurk R, Lima-Verde IB, Luque MC, Santos KD, Martins FS, et al. Effects of fibroblast growth factor-2 on the in vitro culture of caprine preantral follicles. Cells Tissues Organs. 2007;186:112–20.

    Article  PubMed  CAS  Google Scholar 

  21. Adachi T, Iwashita M, Kuroshima A, Takeda Y. Regulation of IGF binding proteins by FSH in human luteinizing granulosa cells. J Assist Reprod Genet. 1995;12:639–43.

    Article  PubMed  CAS  Google Scholar 

  22. Wakim AN, Polizotto SL, Burholt DR. Influence of thyroxine on human granulosa cell steroidogenesis in vitro. J Assist Reprod Genet. 1995;12:274–7.

    Article  PubMed  CAS  Google Scholar 

  23. Lobb DK, Younglai EV. A simplified method for preparing IVF granulosa cells for culture. J Assist Reprod Genet. 2006;23:93–5.

    Article  PubMed  Google Scholar 

  24. Hwang DH, Kee SH, Kim K, Cheong KS, Yoo YB, Lee BL. Role of reconstituted basement membrane in human granulosa cell culture. Endocr J. 2000;47:177–83.

    Article  PubMed  CAS  Google Scholar 

  25. Woodruff TK, Shea LD. The role of the extracellular matrix in ovarian follicle development. Reprod Sci. 2007;4:6–10.

    Article  Google Scholar 

  26. Holst N, Bertheussen K, Burhol PG, Forsdahl F. Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells and isolated from patients undergoing in-vitro fertilization. Hum Reprod. 1991;6:1343–8.

    PubMed  CAS  Google Scholar 

  27. Hanrieder J, Nyakas A, Naessén T, Bergquist J. Proteomic analysis of human follicular fluid using an alternative bottom-op approach. J Proteome Res. 2008;7:443–9.

    Article  PubMed  CAS  Google Scholar 

  28. Zachariae F. Studies on the mechanism of ovulation. Permeability of the blood-liquir barrier. Acta Endocrinol. 1958;27:339–42.

    PubMed  CAS  Google Scholar 

  29. Salustri A, Camaioni A, Di Giacomo M, Fulop C, Hascall VC. Hyaluronan and proteoglycans in ovarian follicles. Hum Reprod. 1999;4:293–301.

    Google Scholar 

  30. Ben-Ze’ev A, Amsterdam A. Regulation of cytoskeletal proteins involved in cell contact formation during differentiation of granulosa cells on extracellular matrix. Proc Natl Acad Sci U SA. 1986;83:2894–8.

    Article  Google Scholar 

  31. Asem EK, Feng S, Stingley-Salazar SR, Turek JJ, Peter AT, Robinson JP. Basal lamina of avian ovarian follicle: influence on morphology of granulose cells in-vitro. Comp Biochem Physiol Part C. 2000;125:189–201.

    CAS  Google Scholar 

  32. Kossowska-Tomaszczuk K, De Geyter C, De Geyter M, Martin I, Holzgreve W, Scherberich A, et al. The multipotency of luteinizing granulosa cells collected from mature ovarian follicles. Stem Cells. 2009;27:210–9.

    Article  PubMed  CAS  Google Scholar 

  33. Gutiérrez CG, Glazyrin AN, Robertson GW, Campbell BK, Gong JG, Bramley TA, et al. Ultra-structural characteristics of bovine granulosa cells associated with maintenance of oestradiol production in vitro. Mol Cell Endocrinol. 1997;134:51–8.

    Article  PubMed  Google Scholar 

  34. Wright WE, Piatyszek MA, Rainey WE, Byrd W, Shay JW. Telomerase activity in human germline and embryonic tissues and cells. Dev Genet. 1996;18:173–9.

    Article  PubMed  CAS  Google Scholar 

  35. Kyo S, Takakura M, Kohama T, Inoue M. Telomerase activity in human endometrium. Cancer Res. 1997;57:610–4.

    PubMed  CAS  Google Scholar 

  36. Broccoli D, Young JW, De Lange T. Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci USA. 1995;92:9082–6.

    Article  PubMed  CAS  Google Scholar 

  37. Burger AM, Bibby MC, Double JA. Telomerase activity in normal and malignant mammalian tissues: feasibility of telomerase as a target for cancer chemotherapy. Br J Cancer. 1997;75:516–22.

    Article  PubMed  CAS  Google Scholar 

  38. Harle-Bachor C, Boukamp P. Telomerase activity in the regenerative basal layer of the epidermis in human skin and in immortal and carcinoma-derived skin keratinocytes. Proc Natl Acad Sci USA. 1996;93:6476–81.

    Article  PubMed  CAS  Google Scholar 

  39. Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, et al. Extension of life-span by introduction of telomerase into normal human cells. Science. 1998;279:349–52.

    Article  PubMed  CAS  Google Scholar 

  40. Camp TA, Rahal JO, Mayo KE. Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. Mol Endocrinol. 1991;5:1405–17.

    Article  PubMed  CAS  Google Scholar 

  41. Lei ZM, Rao CV. Novel presence of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and the downregulating action of hCG on gonadotropin releasing hormone gene expression in immortalized hypothalamic GT1–7 neurons. Mol Endocrinol. 1994;8:1111–21.

    Article  PubMed  CAS  Google Scholar 

  42. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11–25.

    Article  PubMed  CAS  Google Scholar 

  43. Yang JT, Rayburn H, Hynes RO. Cell adhesion events mediated by a4 integrins are essential in placental and cardiac development. Development. 1995;121:549–60.

    PubMed  CAS  Google Scholar 

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Acknowledgments

The authors wish to acknowledge MSMT 0021620820, MSMT 0021627502 and IGA MZ NS/9781-3 for financial support of research program. The authors would like to acknowledge Ms. Rückerova for help in the tissue cultures laboratory.

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Correspondence to Tomas Soukup.

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Capsule

This paper is especially focused on the improvement of the cultivation protocol enabling granulosa cells to proliferate and maintain the typical phenotypic expression.

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Bruckova, L., Soukup, T., Visek, B. et al. Proliferative potential and phenotypic analysis of long-term cultivated human granulosa cells initiated by addition of follicular fluid. J Assist Reprod Genet 28, 939–950 (2011). https://doi.org/10.1007/s10815-011-9617-6

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  • DOI: https://doi.org/10.1007/s10815-011-9617-6

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