:: Volume 20, Issue 3 (Bimonthly 2016) ::
Feyz 2016, 20(3): 205-213 Back to browse issues page
Effect of testosterone on of ovine spermatogonial colony formation in-vitro
Ataollah Zandi, Peyman Rahimi-Feyli *, Ali Asghar Moghaddam
Department of Clinical Sciences, Faculty of Veterinary Medicine, Razi University, Kermanshah, I. R. Iran. , peymanrahimi@razi.ac.ir
Abstract:   (2813 Views)

Background: Spermatogonial stem cells (SSCs) are able to establish balance between self-renewal and differentiation, thereby maintain the spermatogenesis. The aim of this study was to investigate effects of different doses of Testosterone on SSCs colony formation.

Materials and Methods: The cells were isolated from testes of prepubertal lambs by two-step enzymatic digestion and then purified by differential platting. The cells were cultured for 13 days in 4 groups: Control [DMEM containing antibiotic (1%) and FBS (5%)]; and Treatment groups 1, 2 and 3 (DMEM+Testosterone 60, 120 and 240 µg/ml, respectively). The culture media was changed every 72 h. Identification of SSCs was performed by immunocytochemistry staining against PGP9.5. Finally, following the evaluation of percentage for viability rate of SSCs immediately after isolation and also the number and surface areas of the colonies on days 5, 9 and 13 after the beginning of culturing by invert microscopy, the analyses were made using statistical tests.

Results: The percentage for viability rate of SSCs after isolation was 86.77±3.63%. The decrease in the number and surface areas of spermatogonial colonies in Treatment groups 2 and 3 were significant compared to Control and Treatment group1 (P<0.05). Furthermore, colony number and surface area were significantly increased at day 13 compared to those of the days 5 and 9 (P<0.05).

Conclusion: The results of this study showed that Testosterone has no effect on colony induction of SSCs in vitro. Increasing the Testosterone dose decreases the colony number and surface area of SSCs.

Keywords: Stem cell, Spermatogonia, Colony formation, Testosterone, PGP9.5
Full-Text [PDF 320 kb]   (743 Downloads)    
Type of Study: Research | Subject: General
Received: 2016/08/8 | Accepted: 2016/08/8 | Published: 2016/08/8
1. Dann CT, Alvardo AL, Molyneux LA, Denard BS, Garbers DL, Porteus MH. Spermatogonial stem cell self-renewal requires OCT4., a factor downregulated during retinoic acid-induced differentiation. Stem Cells 2008: 26(11): 2928-37.
2. Hil JR, Dobrinski I. Male germ cell transplantation in livestock. Reprod Fertil Dev 2006: 18(1-2): 13-8.
3. Heidari B, Rahmati-Ahmadabadi M, Akhondi MM, Zarnani AH, Jeddi-Tehrani M, Shirazi A, et al. Isolation., identification., and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers. J Assist Reprod Genet 2012: 29(10): 1029-38.
4. Aponte PM, Soda T, Teerds KJ, Mizrak SC, Van de Kant HJ, De Rooij DG. Propagation of bovine spermatogonial stem cells in vitro. Reproduction 2008: 136(5): 543-57.
5. Anjamrooz SH, Movahedin M, Tiraihi T, Mowla SJ. In vitro effects of epidermal growth factor., follicle stimulating hormone and testosterone on mouse spermatogonial cell colony formation. Reprod Fertil Dev 2006; 18(6): 709-20.
6. Izadyar F, Den Ouden K, Creemers LB, Posthuma G, Parvinen M, De Rooij DG. Proliferation and differentiation of bovine type A spermatogonia during long-term culture. Biol Reprod 2003: 68(1): 272-81.
7. Shen F, Zhang C, Zheng H, Xiong Y, Wang X, Liao W, et al. Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice. Cells Tissues Organs 2010: 191(5): 372-81.
8. Meng X, Lindahl M, Hyvönen ME, Parvinen M, De Rooij DG, Hess MW, et al. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287(5457): 1489-93.
9. Jegou B. The Sertoli-germ cell communication network in mammals. Int Rev Cytol 1993: 147: 25-96.
10. Walker WH. Testosterone signaling and the regulation of spermatogenesis. Spermatogenesis 2011; 1(2): 116-20.
11. Ruwanpura SM, McLachlan RI, Stanton PG, Meachem SJ. Follicle-stimulating hormone affects spermatogonial survival byregulating the intrinsic apoptotic pathway in adult rats. Biol Reprod 2008; 78(4): 705-13.
12. Vera Y, Erkkila K, Wang C, Nunez C, Kyttanen S, Lue Y, et al. Involvement of p38 mitogen-activated proteinkinase and inducible nitric oxide synthase in apoptotic signalling of murineand human male germ cells after hormone deprivation. Mol Endocrinol 2006; 20(7): 1597-609.
13. Tesarik J, Greco E, Mendoza C. Assisted reproduction with in-vitro-cultured testicular spermatozoa in cases of severe germ cell apoptosis: a pilot study. Hum Reprod 2001; 16(12): 2640-5.
14. Zhou XC, Wei P, Hu ZY, Gao F, Zhou RJ, Liu YX. Role of Fas/FasL genes in azoospermia or oligozoospermia induced by testosteroneundecanoate in rhesus monkey. Acta Pharmacol Sin 2001; 22(11): 1028–33.
15. Zhang ZH, Zhou XC, Wei P, Hu ZY, Liu YX. Expression of Bcl-2 andBax in rhesus monkey testis during germ cell apoptosis induced bytestosterone undecanoate. Arch Androl 2003; 49(6): 439–47.
16. Franca LR, Avelar GF, Almeida FF. Spermatogenesis and sperm transit through the epididymis in mammals with emphasis on pigs. Theriogenology 2005: 63(2): 300-18.
17. Rodriguez-Sosa JR, Dobson H, Hahnel A. Isolation and transplantation of spermatogonia in sheep. Theriogenology 2006: 66(9): 2091-103.
18. Van Pelt AM, Morena AR, Van Dissel-Emiliani FM, Boitani C, Gaemers IC, De Rooij DG, et al. Isolation of the synchronized a spermatogonia from adult vitamin A-deficient rat testes. Biol Reprod 1996: 55(2): 439-44.
19. Ruwanpura SM, McLachlan RL, Meachem SJ. Hormonal regulation of male germ cell development. J Endocrinol 2010; 205(2): 117-31.
20. Niu Z, Goodyear SM, Rao S, Wu X, Tobias JW, Avarbock MR, et al. MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 2011; 108(31): 12740-5.
21. Izadyar F, Spierenberg GT, Creemers LB, Den Ouden K, De Rooij DG. Isolation and purification of type A spermatogonia from the bovine testis. Reproduction 2002; 124(1): 85–94.
22. Tajik P, Narenji-Sani R, Moezifar M, Yousefi MH, Movahedian M, Qasemi-Panahi B, et al. Effect of follicle-stimulating hormone and testosterone on colony formation of bovine spermatogonial stem cell. Comp Clin Path 2014; 23(4): 901-6.
23. Kon Y, Endoh D, Iwanaga T. Expression of protein gene product 9.5., a neuronal ubiquitin C-terminal hydrolase., and its developing change in sertoli cells of mouse testis. Mol Reprod Dev 1999; 54(4): 333-41.
24. Zhang Z, Hill J, Holland M, Kurihara Y, Loveland KL. Bovine sertoli cells colonize and form tubules in murine hosts following transplantation and grafting procedures. J Androl 2008; 29(4): 418-30.
25. Von Kopylow K, Kirchhoff C, Jezek D, Schulze W, Feig C, Primig M, et al. Screening for biomarkers of spermatogonia within the human testis: a whole genome approach. Hum Reprod 2010; 25(5): 1104-12.
26. Asadi MH, Javanmardi S, Movahedin M. Isolation, Expansion and Purification of Mouse Spermatogonial Stem Cells in an Autologous Sertoli Cell Co-culture System. MJMS 2013; 15(4): 21-33. [in Persian]
27. Hasthorpe S. Clonogenic culture of normal spermatogonia: in vitro regulation of postnatal germ cell proliferation. Biol Reprod 2003; 68(4): 1354-60.
28. Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 2004; 71(3): 722-31.
29. Koruji SM, Movahedin M, Mowla SJ, Gourabi H. Colony formation ability of frozen thawed spermatogonial stem cell from adult mouse. Iran J Reprod Med 2007; 5(3): 109–15.
30. Aponte PM, Van Bragt MP, De Rooij DG, Van Pelt AM. Spermatogonial stem cells: characteristics and experimental possibilities. APMIS 2005; 113(11-12): 727-42.
31. Singh J, Handelsman DJ. The effects of recombinant FSH on testosterone-induced spermatogenesis in gonadotrophin-deficient (hpg) mice. J Androl 1996; 17(4): 382–93.
32. Holmes SD, Spotts G, Smith RG. Rat Sertoli cells secrete agrowth factor that blocks epidermal growth factor (EGF) bindingto its receptor. J Biol Chem 1986; 261(9): 4076-80.
33. Nagano M, Ryu BY, Brinster CJ, Avarbock MR, Brinster RL. Maintenance of mouse male germ line stem cells in vitro. Biol Reprod 2003; 68(6): 2207-14.
34. Salarvandian M, Tajik P, Barin A. Effects of gonadotropin releasing hormone (GnRH) on sheep spermatogonial stem cells proliferation co-cultured with Sertoli cells. Res Opin Anim Vet Sci 2015: 4(7): 314-19.
35. Vigier M, Weiss M, Perrard MH, Godet M, Durand P. The effects of FSH and of testosterone on the completion of meiosis and the very early steps of spermiogenesis of the rat: an in vitro study. J Mol Endocrinol 2004; 33(3): 729-42.

XML   Persian Abstract   Print

Volume 20, Issue 3 (Bimonthly 2016) Back to browse issues page