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Morphologic and proliferative characteristics of goat type a spermatogonia in the presence of different sets of growth factors

  • Gamete Biology
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Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

The present study by using different growth factors was aimed to develop the best practical culture condition for purification of goat undifferentiated SSCs and their colonization under in vitro and in vivo conditions.

Methods

The enzymatically isolated SSCs obtained from one month old goat testes were cultured in DMEM plus FCS supplemented with different sets of growth factors (GDNF, LIF, bFGF, and EGF) for 2 weeks. At the end of each week, the morphological characteristics of cells and colonies alongside with purification rate of undifferentiated type A spermatogonia were evaluated by immunocytochemical staining and flow cytometry.

Results

The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in control group. In immunocytochemical evaluation, the proportion of KIT and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively.

Conclusions

The culture medium comprising all four growth factors, especially the one supplemented with the higher concentration of GDNF, was superior to the other groups with respect to the population of undifferentiated type A spermatogonia and its propagation in culture system. Additionally, goat SSCs could colonize within the mouse testis following xenotransplantation.

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References

  1. Franca LR, Becker-Silva SC, Crhiarini-Garcia H. The length of the cycle of seminiferous epithelium in goats (Capra hircus). Tissue Cell. 1999;31:274–80.

    Article  CAS  PubMed  Google Scholar 

  2. Aponte PM, Soda T, Teerds KJ, Mizrak SC, van de Kant HJG, de Rooij DG. Propagation of bovine spermatogonial stem cells in vitro. Reproduction. 2008;136:543–57.

    Article  CAS  PubMed  Google Scholar 

  3. de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask. J Atmos Ocean Technol. 2000;21:776–98.

    Google Scholar 

  4. Lee J, Kanatsu-Shinohara M, Inoue K, Ogonuki N, Miki H, Toyokuni S, et al. Akt mediates self-renewal division of mouse spermatogonial stem cells. Development. 2007;134:1853–9.

    Article  CAS  PubMed  Google Scholar 

  5. Dobrinski I. Advances and applications of germ cell transplantation. Hum Fertil. 2006;9:9–14.

    Article  Google Scholar 

  6. Hong Y, Liu T, Zhao H, Xu H, Wang W, Liu R, et al. Establishment of normal medaka fish spermatogonial cell line capable of sperm production in vitro. PNAS. 2004;101:8011–6.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Guan K, Nayernia K, Maier LS, Wagner S, Dressel R, Lee JH, et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature. 2006;440:1190–203.

    Article  Google Scholar 

  8. Han SY, Gupta MK, Uhm SJ, Lee HT. Isolation and in vitro culture of pig spermatogonial stem cell. Asian-Australas J Anim Sci. 2009;22:187–93.

    Article  CAS  Google Scholar 

  9. Dobrinski I, Travis AJ. Germ cell transplantation for the propagation of companion animals, non-domestic and endangered species. Reprod Fertil Dev. 2007;19:732–9.

    Article  CAS  PubMed  Google Scholar 

  10. Yoshida S, Nabeshima Y, Nakagawa T. Stem cell heterogeneity: actual and potential stem cell compartments in mouse spermatogenesis. Ann NY Acad Sci. 2007;1120:47–58.

    Article  PubMed  Google Scholar 

  11. Oatley MJ, Racicot KE, Oatley JM. Sertoli cells dictate spermatogonial stem cell niches in the mouse testis. Biol Reprod. 2011;84:639–45.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Oatley JM, Brinster RL. The germline stem cell nich unit in mammalian testes. Physiol Rev. 2012;92:577–95.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. de Barros FRO, Giassetti MI, Visintin JA. Spermatogonial stem cells and animal transgenesis. In: Agbo EC, editor. Innovations in biotechnology. Rijeka: InTech; 2012. p. 303–18. ISBN 978-953-51-0096-6.

    Google Scholar 

  14. Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL. Identifying genes important for spermatogonial stem cell self-renewal and survival. PNAS. 2006;103:9524–9.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Kubota H, Avarbock MR, Brinster RL. Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells. PNAS. 2004;10:16489–94.

    Article  Google Scholar 

  16. Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod. 2004;71:722–31.

    Article  CAS  PubMed  Google Scholar 

  17. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod. 2003;69:612–6.

    Article  CAS  PubMed  Google Scholar 

  18. Kanatsu-Shinohara M, Miki H, Inoue K, Ogonuki N, Toyokuni S, Ogura A, et al. Long-term culture of mouse male germline stem cells under serum- or feeder-free conditions. Biol Reprod. 2005;72:985–91.

    Article  CAS  PubMed  Google Scholar 

  19. Kubota H, Brinster RL. Technology insight: in vitro culture of spermatogonial stem cells and their potential therapeutic uses. Nat Clin Pract Endocrinol Metab. 2006;2:99–108.

    Article  CAS  PubMed  Google Scholar 

  20. de Rooij DG, Mizrak SC. Deriving multipotent stem cells from mouse spermatogonial stem cells: a new tool for developmental and clinical research. Development. 2008;135:2207–13.

    Article  PubMed  Google Scholar 

  21. 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;287:1489–93.

    Article  CAS  PubMed  Google Scholar 

  22. Kubota H, Wu X, Goodyear SM, Avarbock MR, Brinster RL. Glial cell line-derived neurotrophic factor and endothelial cells promote self-renewal of rabbit germ cells with spermatogonial stem cell properties. FASEB J. 2011;25:2604–14.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Ryu BY, Kubota H, Avarbock MR, Brinster RL. Conservation of spermatogonial stem cell self-renewal signaling between mouse and rat. Proc Natl Acad Sci U S A. 2005;102:14302–7.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Kanatsu-Shinohara M, Muneto T, Lee J, Takenaka M, Chuma S, Nakatsuji N, et al. Long-term culture of male germline stem cells from hamster testes. Biol Reprod. 2008;78:611–7.

    Article  CAS  PubMed  Google Scholar 

  25. Oatley JM, Oatley MJ, Avarbock MR, Tobias JW, Brinster RL. Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development. 2009;136:1191–9.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Heidari B, Gifani M, Shirazi A, Akhondi MM, Zarnani AH, Behzadi B, et al. Enrichment of undifferentiated type A spermatogonia from goat testis using discontinuous percoll density gradient and differential plating. AJMB. 2014;6:94–103.

    PubMed Central  PubMed  Google Scholar 

  27. Schrans-Stassen BH, van de Kant HJ, de Rooij DG, van Pelt AM. Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology. 1999;140:5894–900.

    Article  CAS  PubMed  Google Scholar 

  28. 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;29:1029–38.

    Article  PubMed Central  PubMed  Google Scholar 

  29. Yasuhiro K, Daiji E, Toshihiko I. 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;54:333–41.

    Article  Google Scholar 

  30. 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;29:418–30.

    Article  PubMed  Google Scholar 

  31. Rodriguez-Sosa JR, Dobson H, Hahnel A. Isolation and transplantation of spermatogonia in sheep. Theriogenology. 2006;66:2091–103.

    Article  PubMed  Google Scholar 

  32. 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;25:1104–12.

    Article  Google Scholar 

  33. Zeng W, Snedaker AK, Megee S, Rathi R, Chen F, Honaramooz A, et al. Preservation and transplantation of porcine testis tissue. Reprod Fertil Dev. 2009;21:4.

    Article  Google Scholar 

  34. Wrobel KH, Bickel D, Kujat R, Schimmel M. Configuration and distribution of bovine spermatogonia. Cell Tissue Res. 1995;279:277–89.

    Article  CAS  PubMed  Google Scholar 

  35. Lok D, Weenk D, de Rooij DG. Morphology, proliferation, and differentiation of undifferentiated spermatogonia in the Chinese hamster and the ram. Anat Rec. 1982;203:83–99.

    Article  CAS  PubMed  Google Scholar 

  36. Izadyar F, Spierenberg G, Creemers L, Ouden K, de Rooij DG. Isolation and purification of type A spermatogonia from the bovine testis. Reproduction. 2002;124:85–94.

    Article  CAS  PubMed  Google Scholar 

  37. Dirami G, Ravindranath N, Pursel V, Dym M. Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol Reprod. 1999;61:225–30.

    Article  CAS  PubMed  Google Scholar 

  38. Sugiyama N, Obinata M, Matsui Y. BCL-2 inhibits apoptosis of spermatogonia and growth of spermatogenic stem cells in a cell-intrinsic manner. Mol Reprod Dev. 2001; 5830–38.

  39. Creemers LB, Meng X, den Ouden K, van Pelt AM, Izadyar F, Santoro M, et al. Transplantation of germ cells from glial cell line-derived neurotrophic factor-overexpressing mice to host testes depleted of endogenous spermatogenesis by fractionated irradiation. Biol Reprod. 2002;66:1579–84.

    Article  CAS  PubMed  Google Scholar 

  40. Kaul G, Kumar S, Kumari S. Enrichment of CD9+ spermatogonial stem cells from goat (Capra aegagrus hircus) testis using magnetic microbeads. Stem Cell Disc. 2012;2:92–9.

    Article  CAS  Google Scholar 

  41. 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;55:439–44.

    Article  PubMed  Google Scholar 

  42. Marret C, Durand P. Culture of porcine spermatogonia: effects of purification of the germ cells, extracellular matrix, and fetal calf serum on their survival and multiplication. J Reprod Dev. 2000;40:305–19.

    CAS  Google Scholar 

  43. Liu S, Tang Z, Xiong T, Tang W. Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol. 2011;9:141–50.

    Article  PubMed Central  PubMed  Google Scholar 

  44. Bellvé AR, Cavicchia JC, Millete CF, O’Brien DA, Bhatnagar YM, Dym M. Spermatogenic cells of the prepuberal mouse. J cell boil. 1977;74:68–85.

    Article  Google Scholar 

  45. Morena AR, Boitani C, Pesce M, De Felici M, Stefanini M. Isolation of highly purified type A spermatogonia from prepubertal rat testis. J Atmos Ocean Technol. 1990;17:708–17.

    Google Scholar 

  46. Izadyar F, DenOuden 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;68:272–81.

    Article  CAS  PubMed  Google Scholar 

  47. Aponte PM, Soda T, van de Kant HJ, de Rooij DG. Basic features of bovine spermatogonial culture and effects of glial cell line-derived neurotrophic factor. Theriogenology. 2006;65:1828–47.

    Article  CAS  PubMed  Google Scholar 

  48. van Dissel-Emiliani FM, De Boer-Brouwer M, De Rooij DG. Effect of fibroblast growth factor-2 on Sertoli cells and gonocytes in coculture during the perinatal period. Endocrinology. 1996;37:647–54.

    Google Scholar 

  49. Jeong D, McLean DJ, Griswold MD. Long-term culture and transplantation of murine testicular germ cells. J Atmos Ocean Technol. 2003;24:661–9.

    Google Scholar 

  50. Wahab-Wahlgren A, Martinelle N, Holst M, Jahnukainen K, Parvinen M, Soder O. EGF stimulates rat spermatogonial DNA synthesis in seminiferous tubule segments in vitro. Mol Cell Endocrinol. 2003;201:39–46.

    Article  CAS  PubMed  Google Scholar 

  51. Shinohara MK, Inoue M, Takashima S, Takehashi M, Ogonuki N, Morimoto H, et al. Reconstitution of mouse spermatogonial stem cell niches in culture. Cell Stem Cell. 2011;11:567–78.

    Article  Google Scholar 

  52. Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J. Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol Reprod. 2006;74:314–21.

    Article  CAS  PubMed  Google Scholar 

  53. Kassab M, Abd-Elmaksoud A, Ali MA. Localization of the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the bovine testis. J Mol Hist. 2007;38:207–14.

    Article  CAS  Google Scholar 

  54. Kuijk EW, Colenbrander B, Roelen BAJ. The effects of growth factors on in vitro-cultured porcine testicular cells. Reproduction. 2009;138:721–31.

    Article  CAS  PubMed  Google Scholar 

  55. Matsui Y, Toksoz D, Nishikawa S, Nishikawa S, Williams D, Zsebo K, et al. Effect of steel factor and leukaemia inhibitory factor on murine primordial germ cells in culture. Nature. 1991;353:750–2.

    Article  CAS  PubMed  Google Scholar 

  56. Dorval-Coiffec I, Delcros JG, Hakovirta H, Toppari J, Jegou B, Piquet-Pellorce C. Identification of the leukemia inhibitory factor cell targets within the rat testis. Biol Reprod. 2005;72:602–11.

    Article  CAS  PubMed  Google Scholar 

  57. Dobrinski I, Avarbock MR, Brinster RL. Transplantation of germ cells from rabbits and dogs into mouse testes. Biol Reprod. 1999;61(5):1331–9.

    Article  CAS  PubMed  Google Scholar 

  58. Dobrinski I, Avarbock MR, Brinster RL. Germ cell transplantation from large domestic animals into mouse testes. Mol Reprod Dev. 2000;5:270–9.

    Article  Google Scholar 

  59. Nagano M, McCarrey JR, Brinster RL. Primate spermatogonial stem cells colonize mouse testes. Biol Reprod. 2001;64:1409–16.

    Article  CAS  PubMed  Google Scholar 

  60. Nagano M, Patrizio P, Brinster RL. Long-term survival of human spermatogonial stem cells in mouse testes. Fertil Steril. 2002;78:1225–123.

    Article  PubMed  Google Scholar 

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Acknowledgments

The authors would like to thank the Avicenna Research Institute for technical and financial supports, ACECR, Tehran.

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Authors and Affiliations

  1. Reproductive Biotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

    Mohammad Sadra Shirazi, Banafsheh Heidari, Abolfazl Shirazi, Maryam Rahmati-Ahmadabadi, Mohammad Mehdi Naderi, Bahareh Behzadi, Moretza Farab, Ali Sarvari, Sara Borjian-Boroujeni & Mohammad Mehdi Akhondi

  2. Research Institute of Animal Embryo Technology, Shahrekord University, P.O. Box: 19615–1177, 1936773493, Shahrekord, Iran

    Abolfazl Shirazi

  3. Nanobiotechnology Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

    Amir Hassan Zarnani

  4. Monoclonal Antibody Research Center, Avicenna Research Institute (ACECR), Tehran, Iran

    Mahmood Jeddi-Tehrani

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  1. Mohammad Sadra Shirazi
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  2. Banafsheh Heidari
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Corresponding author

Correspondence to Abolfazl Shirazi.

Additional information

Mohammad Sadra Shirazi and Banafsheh Heidari these authors contributed equally to the work.

Capsule Spermatogonial stem cells are the foundation of spermatogenesis. Addition of growth factors to the culture medium could effectively retain the undifferentiation status of goat SSCs. The culture medium containing higher concentration of GDNF was superior to the other groups comprising either the lower concentration of GDNF or other combinations of growth factors. The colonization of goat SSCs within the mouse testis following xenotransplantation, was promising in male fertility preservation and production of transgenic animals.

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Shirazi, M.S., Heidari, B., Shirazi, A. et al. Morphologic and proliferative characteristics of goat type a spermatogonia in the presence of different sets of growth factors. J Assist Reprod Genet 31, 1519–1531 (2014). https://doi.org/10.1007/s10815-014-0301-5

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  • DOI: https://doi.org/10.1007/s10815-014-0301-5

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