Journal of Reproduction & Infertility

Journal of Reproduction & Infertility

Ovarian Stem Cells Differentiation into Primary Oocytes Using Follicle Stimulating Hormone, Basic Fibroblast Growth Factor, and Neurotrophin 3

Authors
Sara Tanbakooei 1
Seyed Mohammad Amin Haramshahi 2
Gelareh Vahabzadeh 3
Mahmood Barati 4
Majid Katebi 5
Fereshteh Golab 1
Qazal Shetbi 6
Narges Niknam 6
Leila Roudbari 1
Motahareh Rajabi Fomeshi 7
Soheila Amini Moghadam 8
1 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran
2 Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
3 Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
4 Department of Biotechnology, School of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
5 Department of Anatomy, Hormozgan University of Medical Sciences, Bandar Abbas, Iran
6 Department of Biology, Islamic Azad University, Science and Research Branch, Tehran, Iran
7 Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran; Department of Tissue Engineering and Regenerative Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Scienc
8 Department of Gynecology, Firoozgar Hospital, Iran University of Medical Sciences, Tehran, Iran
10.18502/jri.v22i4.7649
Abstract
Background: In vitro obtaining oocytes can be an appropriate alternative for patients with gonadal insufficiency or cancer survivors. The purpose of the current research was isolating stem cells from ovarian cortical tissue as well as evaluating the effectiveness of follicle stimulating hormone (FSH), basic fibroblast growth factor (bFGF), and neurotrophin 3 (NT3) in differentiating to oocyte-like cells. Methods: A human ovary was dissected and cortical tissue pieces were cultured for cell isolation. Isolated cells were divided into 8 groups (3 cases in each group) of control, FSH, NT3, bFGF, FSH+NT3, FSH+bFGF, NT3+bFGF, and FSH+NT3+ bFGF. Pluripotency specific gene (OCT4-A and Nanog), initial germ cells (c-KIT and VASA) and PF growth initiators (GDF-9 and Lhx-8) were evaluated by qRT-PCR. Experiments were performed in triplicate and there were 3 samples in each group. The results were analyzed using one-way ANOVA and p-value less than 0.05 was considered statistically significant. Results: Flow cytometry results showed that cells isolated from the ovarian cortex expressed markers of pluripotency. The results showed that the expression of Nanog, OCT4, GDF-9 and VASA was significantly increased in FSH+NT3 group, while treatment with bFGF caused significant expression of c-KIT and Lhx-8 (p<0.05). Also, according to the results, isolated cells treated with NT3 significantly increased c-KIT expression. Conclusion: According to our results, the ovarian cortex cells could be differentiated into primordial follicles if treated with the proper combination of FSH, bFGF, and NT3. These findings provided a new perspective for the future of in vitro gamete proudest.
Keywords
cell differentiation
Growth Factors
Oogenesis
Ovarian tissue
stem cells
  1. Feigin E, Freud E, Fisch B, Orvieto R, Kravarusic D, Avrahami G, et al. Fertility preservation in female adolescents with malignancies. Cancer Female Adol. 2008:38:101.
  2. Abir R, Feinmesser M, Yaniv I, Fisch B, Cohen IJ, Ben-Haroush A, et al. Occasional involvement of the ovary in Ewing sarcoma. Hum Reprod. 2010; 25(7):1708-12.
  3. Oron G, Ao A, Friedman O, Fisch B, Zhang XY, Ben-Haroush A, et al. Expression of neurotrophin 3 and its tropomyosin-related kinase receptor C in human preantral follicles. Fertil Steril. 2011;95(6): 2056-62.
  4. Brännström M, Milenkovic M. Advances in fertility preservation for female cancer survivors. Nat Med. 2008;14(11):1182-4.
  5. Telfer EE, McLaughlin M, Ding C, Thong KJ. A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin. Hum Reprod. 2008;23(5):1151-8.
  6. Parte S, Bhartiya D, Manjramkar DD, Chauhan A, Joshi A. Stimulation of ovarian stem cells by follicle stimulating hormone and basic fibroblast growth factor during cortical tissue culture. J Ovarian Res. 2013;6(1):20.
  7. Hardy K, Fenwick M, Mora J, Laird M, Thomson K, Franks S. Onset and heterogeneity of responsiveness to FSH in mouse preantral follicles in culture. Endocrinology. 2017;158(1):134-47.
  8. Matos MH, Lima-Verde IB, Bruno JB, Lopes CA, Martins FS, Santos KD, et al. Follicle stimulating hormone and fibroblast growth factor-2 interact and promote goat primordial follicle development in vitro. Reprod Fertil Dev. 2007;19(5):677-84.
  9. O'shaughnessy PJ, Dudley K, Rajapaksha WR. Expression of follicle stimulating hormone-receptor mRNA during gonadal development. Mol Cell Endocrinol. 1996;125(1-2):169-75.
  10. Van Wezel IL, Umapathysivam K, Tilley WD, Rodgers RJ. Immunohistochemical localization of basic fibroblast growth factor in bovine ovarian follicles. Mol Cell Endocrinol. 1995;115(2):133-40.
  11. Quennell JH, Stanton JA, Hurst PR. Basic fibroblast growth factor expression in isolated small human ovarian follicles. Mol Hum Reprod. 2004;10(9):623-8.
  12. Tang K, Yang WC, Li X, Wu CJ, Sang L, Yang LG. GDF-9 and bFGF enhance the effect of FSH on the survival, activation, and growth of cattle primordial follicles. Anim Reprod Sci. 2012;131(3-4):129-34.
  13. Bukovsky A, Svetlikova M, Caudle MR. Oogenesis in cultures derived from adult human ovaries. Reprod Biol Endocrinol. 2005;3:17.
  14. Pereira RC, Economides AN, Canalis E. Bone morphogenetic proteins induce gremlin, a protein that limits their activity in osteoblasts. Endocrinology. 2000;141(12):4558-63.
  15. Liu Y, Song Z, Zhao Y, Qin H, Cai J, Zhang H, et al. A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem Biophys Res Commun. 2006;346(1):131-9.
  16. Levenstein ME, Ludwig TE, Xu RH, Llanas RA, VanDenHeuvel‐Kramer K, Manning D, et al. Basic fibroblast growth factor support of human embryonic stem cell self‐renewal. Stem Cells. 2006;24(3):568-74.
  17. Virant-Klun I, Stimpfel M, Skutella T. Ovarian pluripotent/multipotent stem cells and in vitro oogenesis in mammals. Histol Histopathol. 2011;26(8):1071-82.
  18. Wandji SA, Srsen V, Nathanielsz PW, Eppig JJ, Fortune JE. Initiation of growth of baboon primordial follicles in vitro. Hum Reprod. 1997;12(9):1993-2001.
  19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.
  20. Esmaielzadeh F, Hosseini SM, Nasiri Z, Hajian M, Chamani M, Gourabi H, et al. Kit ligand and glial-derived neurotrophic factor as alternative supplements for activation and development of ovine preantral follicles in vitro. Mol Reprod Dev. 2013;80(1):35-47.
  21. Nilsson E, Parrott JA, Skinner MK. Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol. 2001;175(1-2):123-30.
  22. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals. Reproduction. 2001;122(6):829-38.
  23. Van den Hurk R, Abir R, Telfer E, Bevers M. Primate and bovine immature oocytes and follicles as sources of fertilizable oocytes. Hum Reprod Update. 2000;6(5):457-74.
  24. Dissen GA, Romero C, Hirshfield AN, Ojeda SR. Nerve growth factor is required for early follicular development in the mammalian ovary. Endocrinology. 2001;142(5):2078-86.
  25. Andrade GM, Da Silveira JC, Perrini C, Del Collado M, Gebremedhn S, Tesfaye D, et al. The role of the PI3K-Akt signaling pathway in the developmental competence of bovine oocytes. PLoS One. 2017;12(9):e0185045.
  26. Bayne RA, Kinnell HL, Coutts SM, He J, Childs AJ, Anderson RA. GDF9 is transiently expressed in oocytes before follicle formation in the human fetal ovary and is regulated by a novel NOBOX transcript. PLoS One. 2015;10(3):e0119819.
  27. 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.
  28. Roy S, Gandra D, Seger C, Biswas A, Kushnir VA, Gleicher N, et al. Oocyte-derived factors (GDF9 and BMP15) and FSH regulate AMH expression via modulation of H3K27AC in granulosa cells. Endocrinology. 2018;159(9):3433-45.
  29. El-Hayek S, Clarke HJ. Control of oocyte growth and development by intercellular communication within the follicular niche. Results Probl Cell Differ. 2016;58:191-224.
  30. Ren Y, Suzuki H, Jagarlamudi K, Golnoski K, McGuire M, Lopes R, et al. Lhx8 regulates primordial follicle activation and postnatal folliculogenesis. BMC Biol. 2015;13:39.
  31. Takai H, Smogorzewska A, de Lange T. DNA damage foci at dysfunctional telomeres. Curr Biol. 2003;13(17):1549-56.
  32. Clark AT, Bodnar MS, Fox M, Rodriquez RT, Abeyta MJ, Firpo MT, et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum Mol Genet. 2004;13(7):727-39.
  33. Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, et al. Derivation of oocytes from mouse embryonic stem cells. Science. 2003;300(5623):1251-6.
  34. Qing T, Shi Y, Qin H, Ye X, Wei W, Liu H, et al. Induction of oocyte-like cells from mouse embryonic stem cells by co-culture with ovarian granulosa cells. Differentiation. 2007;75(10):902-11.
  35. Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, et al. Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem. 2005;280(26):24731-7.
  36. Mohamed JS, Gaughwin PM, Lim B, Robson P, Lipovich L. Conserved long noncoding RNAs transcriptionally regulated by Oct4 and Nanog modulate pluripotency in mouse embryonic stem cells. RNA. 2010;16(2):324-37.
  37. Heng D, Wang Q, Ma X, Tian Y, Xu K, Weng X, et al. Role of OCT4 in the regulation of FSH-induced granulosa cells growth in female mice. Front Endocrinol (Lausanne). 2020;10:915.
  38. Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science. 2003;301(5630):215-8.
  39. Dyce PW, Shen W, Huynh E, Shao H, Villagómez DA, Kidder GM, et al. Analysis of oocyte-like cells differentiated from porcine fetal skin-derived stem cells. Stem Cells Dev. 2010;20(5):809-19.
  40. Kee K, Angeles VT, Flores M, Nguyen HN, Pera RAR. Human DAZL, DAZ and BOULE genes modulate primordial germ-cell and haploid gamete formation. Nature. 2009;462(7270):222-5.
  41. Linher K, Dyce P, Li J. Primordial germ cell-like cells differentiated in vitro from skin-derived stem cells. PLoS One. 2009;4(12):e8263.
  42. Silva JR, van den Hurk R, de Matos MH, dos Santos RR, Pessoa C, de Moraes MO, et al. Influences of FSH and EGF on primordial follicles during in vitro culture of caprine ovarian cortical tissue. Theriogenology. 2004;61(9):1691-704.
  43. Oktay K, Briggs D, Gosden RG. Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J Clin Endocrinol Metab. 1997;82(11):3748-51.
  44. Méduri G, Charnaux N, Driancourt MA, Combettes L, Granet P, Vannier B, et al. Follicle-stimulating hormone receptors in oocytes? J Clin Endocrinol Metab. 2002;87(5):2266-76.
  45. Roy SK, Albee L. Requirement for follicle-stimulating hormone action in the formation of primordial follicles during perinatal ovarian development in the hamster. Endocrinology. 2000;141(12):4449-56.
  46. Packer AI, Hsu YC, Besmer P, Bachvarova RF. The ligand of the c-kit receptor promotes oocyte growth. Dev Biol. 1994;161(1):194-205.
  47. Parte S, Bhartiya D, Telang J, Daithankar V, Salvi V, Zaveri K, et al. Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary. Stem Cells Dev. 2011;20(8):1451-64.
  48. Nilsson E, Dole G, Skinner MK. Neurotrophin NT3 promotes ovarian primordial to primary follicle transition. Reproduction. 2009;138(4):697-707.
  49. Kezele PR, Nilsson EE, Skinner MK. Insulin but not insulin-like growth factor-1 promotes the primordial to primary follicle transition. Mol Cell Endocrinol. 2002;192(1-2):37-43.
  50. Garor R, Abir R, Erman A, Felz C, Nitke S, Fisch B. Effects of basic fibroblast growth factor on in vitro development of human ovarian primordial follicles. Fertil Steril. 2009;91(5 Suppl):1967-75.
  51. Nilsson EE, Skinner MK. Kit ligand and basic fibroblast growth factor interactions in the induction of ovarian primordial to primary follicle transition. Mol Cell Endocrinol. 2004;214(1-2):19-25.
  52. Derrar N, Price CA, Sirard MA. Effect of growth factors and co-culture with ovarian medulla on the activation of primordial follicles in explants of bovine ovarian cortex. Theriogenology. 2000;54 (4):587-98.
  53. Waraksa JA, Lindsay RM, Ip NY, Hutz RJ. Neurotrophin-3 augments steroid secretion by hamster ovarian follicles in vitro. Zoolog Sci. 1995;12(4):499-502.
  54. Dissen GA, Parrott JA, Skinner MK, Hill DF, Costa ME, Ojeda SR. Direct effects of nerve growth factor on thecal cells from antral ovarian follicles. Endocrinology. 2000;141(12):4736-50.
  55. Dissen GA, Romero C, Paredes A, Ojeda SR. Neurotrophic control of ovarian development. Microsc Res Tech. 2002;59(6):509-15.
Journal of Reproduction & Infertility
Volume 22, Issue 4
Autumn 2021
Pages 241-250