Skip to main content
SpringerLink
Log in

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

  • Research article
  • Published:
Journal of Assisted Reproduction and Genetics Aims and scope Submit manuscript

Abstract

Purpose

To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat.

Methods

The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR).

Results

The viability of isolated testicular cells was > 90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers.

Conclusion

Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats. Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Phillips BT, Gassei K, Orwig KE. Spermatogonial stem cell regulation and spermatogenesis. Philos Trans R Soc Lond B Biol Sci. 2010;365:1663–78.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Caires K, Broady J, Mclean D. Maintaining the male germline: regulation of spermatogonial stem cells. J Endocrinol. 2010;205:133–45.

    Article  CAS  PubMed  Google Scholar 

  3. Goossens E, Van Saen D, Tournaye H. Spermatogonial stem cell preservation and transplantation: from research to clinic. Hum Reprod. 2013;28:897–907.

    Article  CAS  PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  5. Kanatsu-Shinohara M, Kato M, Takehashi M, Morimoto H, Takashima S, Chuma S, et al. Production of transgenic rats via lentiviral transduction and xenogeneic transplantation of spermatogonial stem cells. Biol Reprod. 2008;79:1121–8.

    Article  CAS  PubMed  Google Scholar 

  6. Aoshima K, Baba A, Makino Y, Okada Y. Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement. PLoS ONE. 2013;8:e77715.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Hamra FK, Gatlin J, Chapman KM, Grellhesl DM, Garcia JV, Hammer RE, et al. Production of transgenic rats by lentiviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A. 2002;99:14931–6.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Kanatsu-Shinohara M, Lee J, Inoue K, Ogonuki N, Miki H, Toyokuni S, et al. Pluripotency of a single spermatogonial stem cell in mice. Biol Reprod. 2008;78:681–7.

    Article  CAS  PubMed  Google Scholar 

  9. He Z, Kokkinaki M, Jiang J, Dobrinski I, Dym M. Isolation, characterization, and culture of human spermatogonia. Biol Reprod. 2010;82:363–72.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Bahadorani M, Hosseini SM, Abedi P, Hajian M, Hosseini SE, Vahdati A, et al. Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations. J Assist Reprod Genet. 2012;29:39–46.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Nasiri Z, Hosseini SM, Hajian M, Abedi P, Bahadorani M, Baharvand H, et al. Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro. Theriogenology. 2012;77:1519–28.

    Article  CAS  PubMed  Google Scholar 

  12. Goharbakhsh L, Mohazzab A, Salehkhou S, Heidari M, Zarnani AH, Parivar K, et al. Isolation and culture of human spermatogonial stem cells derived from testis biopsy. Avicenna J Med Biotech. 2013;5:54–61.

    Google Scholar 

  13. Liu S, Tang Z, Xiong T, Tang W. Isolation and characterization of human spermatogonial stem cells. Reprod Biol Endocrinol. 2011;9 (141).

  14. Mahla RS, Reddy N, Goel S. Spermatogonial stem cells (SSCs) in buffalo (Bubalus bubalis) testis. PLoS One. 2012;7e36020.

  15. van Pelt AMM, Morena AR, van Dissel-Emiliani FMF, 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 

  16. 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;124:85–94.

    Article  CAS  PubMed  Google Scholar 

  17. Shinohara T, Orwig KE, Avarbock MR, Brinster RL. Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells. Proc Natl Acad Sci. 2000;97:8346–51.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Buageaw A, Sukhwani M, Ben-Yehudah A, Ehmcke J, Rawe VY, Orwig PC, et al. GDNF family receptor alpha1 phenotype of spermatogonial stem cells in immature mouse testes. Biol Reprod. 2005;73:1011–6.

    Article  CAS  PubMed  Google Scholar 

  19. Ryu BY, Orwig KE, Kubota H, Avarbock MR, Brinster RL. Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev Biol. 2004;274:158–70.

    Article  CAS  PubMed  Google Scholar 

  20. Bruno AH, Giassetti MI, de Barros FRO, Siqueira AF, Mendes CM, Lopes E, et al. Expression of molecular markers for bovine spermatogonial stem cells in prepubertal and adults Nelore. Anim Reprod. 2013;10:602.

    Google Scholar 

  21. Piravar Z, Jeddi-Tehrani M, Sadeghi MR, Mohazzab A, Eidi A, Akhondi MM. In vitro culture of human testicular stem cells on feeder-free condition. J Reprod Infertil. 2013;14:17–22.

    CAS  PubMed Central  PubMed  Google Scholar 

  22. Kanatsu-Shinohara M, Inoue K, Ogonuki N, Morimoto H, Ogura A, Shinohara T. Serum- and feeder-free culture of mouse germline stem cells. Biol Reprod. 2011;84:97–105.

    Article  CAS  PubMed  Google Scholar 

  23. Rastegar T, Minaee MB, Roudkenar MH, Kashani IR, Amidi F, Abolhasani F, et al. Improvement of expression of α6 and β1 integrins by the Co-culture of adult mouse spermatogonial stem cells with SIM mouse embryonic fibroblast cells (STO) and growth factors. Iran J Basic Med Sci. 2013;16:134–9.

    PubMed Central  PubMed  Google Scholar 

  24. Oatley J, de Avila DM, McLean DJ, Griswold MD, Reeves JJ. Transplantation of bovine germinal cells into mouse testes. J Anim Sci. 2002;80:1925–31.

    CAS  PubMed  Google Scholar 

  25. Oatley JM, de Avila DM, Reeves JJ, McLean DJ. Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue. Biol Reprod. 2004;71:494–501.

    Article  CAS  PubMed  Google Scholar 

  26. 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 

  27. Koruji M, Movahedin M, Mowla SJ, Gourabi H, Arfaee AJ. Efficiency of adult mouse spermatogonial stem cell colony formation under several culture conditions. In Vitro Cell Dev-An. 2009;45:281–9.

    CAS  Google Scholar 

  28. Olive V, Cuzin F. The spermatogonial stem cell: from basic knowledge to transgenic technology. Int J Biochem Cell Biol. 2005;37:246–50.

    CAS  PubMed  Google Scholar 

  29. Zhao Y, Yu M, Wang L, Li Y, Fan J, Yang Q, et al. Spontaneous uptake of exogenous DNA by goat spermatozoa and selection of donor bucks for sperm-mediated gene transfer. Mol Biol Rep. 2012;39:2659–64.

    Article  CAS  PubMed  Google Scholar 

  30. 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 

  31. Hamra FK, Chapman KM, Nguyen DM, Williams AA, Hammer RE, Garbers DL. Self renewal, expansion, and transfection of rat spermatogonial stem cells in culture. Proc Natl Acad Sci U S A. 2005;102:17430–5.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  32. Hamra FK, Chapman KM, Wu Z, Garbers DL. Isolating highly pure rat spermatogonial stem cells in culture. Methods Mol Biol. 2008;450:163–79.

    Article  CAS  PubMed  Google Scholar 

  33. 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;136:543–57.

    Article  CAS  PubMed  Google Scholar 

  34. Kala S, Kaushik R, Singh KP, Kadam PH, Singh MK, Manik RS, et al. In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell. J Assist Reprod Genet. 2012;29:1335–42.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Kadam PH, Kala S, Agrawal H, Singh KP, Singh MK, Chauhan MS, et al. Effects of glial cell line-derived neurotrophic factor, fibroblast growth factor 2 and epidermal growth factor on proliferation and the expression of some genes in buffalo (Bubalus bubalis) spermatogonial cells. Reprod Fert Develop. 2013;25:1149–57.

    Article  CAS  Google Scholar 

  36. Izadyar F, Wong J, Maki C, Pacchiarotti J, Ramos T, Howerton K, et al. Identification and characterization of repopulating spermatogonial stem cells from the adult human testis. Hum Reprod. 2011;26:1296–306.

    Article  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. Pramod RK, Sharma SK, Singhi A, Pan S, Mitra A. Differential ovarian morphometry and follicular expression of BMP15, GDF9 and BMPR1B influence the prolificacy in goat. Reprod Domest Anim. 2013;48:803–9.

    Article  CAS  PubMed  Google Scholar 

  39. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCTCt method. Methods. 2001;25:402–8.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Yang Y, Yarahmadi M, Honaramooz A. Development of novel strategies for the isolation of piglet testis cells with a high proportion of gonocytes. Reprod Fert Develop. 2010;22:1057–65.

    Article  CAS  Google Scholar 

  42. Chang Y, Jennifer S. Lee-Chang, Panneerdoss S, MacLeanII JA, Rao MK. Isolation of sertoli, Leydig, and spermatogenic cells from the mouse testis. Biotechniques. 2011;51:341–4.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  43. 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 

  44. Creemers LB, den Ouden K, van Pelt AMM, de Rooij DG. Maintenance of adult mouse type a spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture. Reproduction. 2002;124:791–9.

    Article  CAS  PubMed  Google Scholar 

  45. 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 

  46. Sousa M, Cremades N, Alves C, Silva J, Barros A. Developmental potential of human spermatogenic cells co-cultured with Sertoli cells. Hum Reprod. 2002;17:161–72.

    Article  PubMed  Google Scholar 

  47. van der Wee K, Johnson EW, Dirami G, Dym M, Hofmann MC. Immunomagnetic isolation and long term culture of mouse type a spermatogonia. J Androl. 2001;22:696–704.

    PubMed  Google Scholar 

  48. Zhang DY, He DW, Wei GH, Song XF, Li XL, In T. Long-term coculture of spermatogonial stem cells on sertoli cells feeder layer in vitro, Sichuan Da Xue Xue Bao Yi Xue Ban. 2008;39:6-9

  49. Kurita K, Sakai N. Functionally distinctive testicular cell lines of zebra fish to support male germ cell development. Mol Reprod Dev. 2004;67:430–8.

    Article  CAS  PubMed  Google Scholar 

  50. Bahadorani M, Hosseini SM, Abedi P, Hajian M, Afrough M, Azhdari TZ, Azizi H, Hosseini SE, Vahdati A, Baharvand H, Nasr-Esfahani M.H. Comparative Immunohistochemical Analysis of VASA, PLZF and THY1 in Goats and Sheep Suggests that these Markers are also Conserved in these Species. J Cytol Histol. 2011;2.

  51. Shinohara T, Avarbock MR, Brinster RL. beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA. 1999;96:5504-5509.

  52. He Z, Kokkinaki M, Dym M. Signaling molecules and pathways regulating the fate of spermatogonial stem cells. Microsc Res Tech. 2009;72:586–95.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  53. Franke WW, Schmid E, Schiller DL, Winter S, Jarasch ED, Moll R, et al. Differentiation-related patterns of expression of proteins of intermediate-size filaments in tissues and cultured cells. Cold Spring Harb Symp Quant Biol. 1982;46:431–53.

    Article  PubMed  Google Scholar 

  54. Dann CT, Alvarado 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;26:2928–37.

    Article  CAS  PubMed  Google Scholar 

  55. Kanatsu-Shinohara M, Takehashi M, Takashima S, Lee J, Morimoto H, Chuma S, et al. Homing of mouse spermatogonial stem cells to germline niche depends on β1 integrin. Cell Stem Cell. 2008;3:533–42.

    Article  CAS  PubMed  Google Scholar 

  56. Kanatsu-Shinohara M, Inoue K, Lee J, Yoshimoto M, Ogonuki N, Miki H, et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell. 2004;119:1001–12.

    Article  CAS  PubMed  Google Scholar 

  57. 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;440:1199–203.

    Article  CAS  PubMed  Google Scholar 

  58. Conrad S, Renninger M, Hennenlotter J, Wiesner T, Just L, Bonin M, et al. Generation of pluripotent stem cells from adult human testis. Nature. 2008;456:344–9.

    Article  CAS  PubMed  Google Scholar 

  59. Kossack N, Meneses J, Shefi S, Nguyen HN, Chavez S, Nicholas C, et al. Isolation and characterization of pluripotent human spermatogonial stem cell-derived cells. Stem Cells. 2009;27:138–49.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

This work is supported by National Fellow project, Indian Council of Agricultural Research (ICAR), Government of India. Authors duly acknowledge ICAR-Senior Research Fellowship to RKP during his PhD programme and Dr. G.K. Das, Principal Scientist, Animal Reproduction Division, IVRI for critical reading of the manuscript.

Author information

Authors and Affiliations

  1. Genome Analysis Laboratory, Animal Genetics Division, Indian Veterinary Research Institute, Izatnagar, Bareilly, U.P., India

    R. Kumar Pramod & Abhijit Mitra

Authors
  1. R. Kumar Pramod
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abhijit Mitra
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Abhijit Mitra.

Additional information

Capsule

The results of this study demonstrated a simple three-step enzymatic digestion method for isolation, a Percoll density gradient method for enrichment and the use of Sertoli cell feeder layer for long term culture of SSC in goat.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pramod, R.K., Mitra, A. In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus). J Assist Reprod Genet 31, 993–1001 (2014). https://doi.org/10.1007/s10815-014-0277-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10815-014-0277-1

Keywords

Search

Navigation