Abstract
Proper in vitro culture and proliferation of spermatogonial stem cells (SSCs) are key tools to achieve functional potentials of SSCs. The fresh serum extracted has been successfully used to stimulate growth of spermatogonial stem cells in vitro. However, there is no report on the optimal concentration of serum for in vitro culture of goat SSCs. This study investigated this issue.
Material and methods
Retrieved testicular cells were cultured in DMEM in presence of different 0 to 15% serum for 7 days and examined on 3rd and 7th day of culture for colony formation, cell viability, immunocytochemistry and quantitative gene expression analyses.
Results
The maximum numbers of colonies were observed in presence of 1% serum on days 3 and 7 of culture. Differential viable staining revealed that most cells were non-viable in complete absence of serum (0%). Immunocytochemistry showed that cells positive for pan cytokeratin were observed in colonies developed in presence of 1, 5, 10 and 15% serum but not 0%, suggesting the presence of epithelial cells within the developing colonies. The colonies developed in the presence of 1 and 5% serum had remarkable alkaline phosphatase activity. The maximum expressions of Plzf, Bcl6b and Vasa were observed in colonies developed in presence of 1% serum, with a significant difference to all the other groups. No significant difference was observed for the expression of Vimentin between different treatment groups.
Conclusion
The results of this study indicate that for maintenance of SSCs, 1% serum required for maintaining viability while allowing proliferation and maintenance of SSCs like colonies.
Similar content being viewed by others
Abbreviations
- SSC:
-
spermatogonial stem cell
- PBS:
-
phosphate buffer saline
- DMEM:
-
Dulbecco’s modified eagle medium
- FBS:
-
fetal bovine serum
References
Abbasi, H., Hosseini, S.M., Hajian, M., Nasiri, Z., Bahadorani, M., Tahmoorespur, M., et al., Lentiviral vectormediated transduction of goat undifferentiated spermatogonia, Anim. Reprod. Sci., 2015, vol. 163, pp. 10–17.
Arora, M., Cell culture media: a review, MATER. METHOD., 2013, vol. 3, p. 175.
Bahadorani, M., Hosseini, S.M., Abedi, P., Abbasi, H., and Nasr-Esfahani, M.H., Glial cell line-derived neurotrophic factor in combination with insulin-like growth factor 1 and basic fibroblast growth factor promote in vitro culture of goat spermatogonial stem cells, Growth Factors, 2015, vol. 33, no. 3, pp. 181–191.
Belloc, F., Dumain, P., Boisseau, M.R., Jalloustre, C., Reiffers, J., Bernard, P., et al., A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells, Cytometry, vol. 1994, vol. 17, no. 1, pp. 59–65.
Cheng, C.Y. and Mruk, D.D., Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development, Physiol. Rev., 2002, vol. 82, pp. 825–874.
Ciancio, G., Pollack, A., Taupier, M.A., Block, N.L., and Irvin, G.L., Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation, J. Histochem. Cytochem., 1988, vol. 36, no. 9, pp. 1147–1152.
Freshney, R., Culture of Animal Cells: A Manual of Basic Technique, New York: Alan R. Liss, 1987, p. 117.
Goel, S., Sugimoto, M., Minami, N., Yamada, M., Kume, S., and Imai, H., Identification, isolation, and in vitro culture of porcine gonocytes, Biol. Reprod., 2007, vol. 77, pp. 127–137.
Guan, K., Nayernia, K., Maier, L.S., Wagner, S., Dressel, R., Lee, J.H., et al., Pluripotency of spermatogonial stem cells from adult mouse testis, Nature, 2006, vol. 440, pp. 1199–1203.
Izadyar, F., Den Ouden, K., Creemers, L.B., Posthuma, G., Parvinen, M., and De Rooij, D.G., Proliferation and differentiation of bovine type A spermatogonia during long-term culture, Biol. Reprod., 2003, vol. 68, pp. 272–281.
Kanatsu-Shinohara, M., Muneto, T., Lee, J., Takenaka, M., Chuma, S., Nakatsuji, N., Horiuchi, T., and Shinohara, T., Long-term culture of male germline stem cells from hamster testes, Biol. Reprod., 2008, vol. 78, no. 4, pp. 611–617.
Kanatsu-Shinohara, M., Inoue, K., Ogonuki, N., Morimoto, H., Ogura, A., and Shinohara, T., Serum- and feeder-free culture of mouse germline stem cells, Biol. Reprod., 2011, vol. 84, no. 1, pp. 97–105.
Kubota, H., Avarbock, M.R., and Brinster, R.L., Growth factors essential for self-renewal and expansion of mouse spermatogonial stem cells, Proc. Natl. Acad. Sci. U. S. A., 2004, vol. 101, pp. 16489–6494.
Leal, M.C., Becker-Silva, S.C., Chiarini-Garcia, H., and Franca, L.R., Sertoli cell efficiency and daily sperm production in goats (Capra hircus), Anim. Reprod., 2004, vol. 1, pp. 122–128.
Livak, K.J. and Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(_Delta Delta C(T)), Methods, 2001, vol. 25, pp. 402–408.
Nasr-Esfahani, M.H., Hosseini, S.M., Hajian, M., Forouzanfar, M., Ostadhosseini, S., Abedi, P., et al., Development of an optimized zona-free method of somatic cell nuclear transfer in the goat, Cell Reprogram., 2011, vol. 13, pp. 157–170.
Oatley, J.M. and Brinster, R.L. Regulation of spermatogonial stem cell self-renewal in mammals, Annu. Rev. Cell Dev. Biol., 2008, vol. 24, pp. 263–286.
Sadri-Ardekani, H. and Atala, A., Testicular tissue cryopreservation and spermatogonial stem cell transplantation to restore fertility: from bench to bedside, Stem Cell Res. Ther., 2014, vol. 28, no. 5 (3), p. 68.
Tegelenbosch, R.A.J. and de Rooij, D.G., A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse, Mutat. Res., 1993, vol. 290, pp. 193–200.
Triglia, R. and Linscott, W., Titers of nine complement components, conglutinin and C3b-inactivator in adult and fetal bovine sera, Mol. Immunol., 1980, vol. 17, pp. 741–748.
Wyns, C., Male fertility preservation before gonadotoxic therapies, Facts Views Vis. Obgyn., 2010, vol. 2, no. 2, pp. 88–108.
Author information
Authors and Affiliations
Corresponding author
Additional information
The article is published in the original.
Rights and permissions
About this article
Cite this article
Bahadorani, M., Hosseini, M.S., Abbasi, H. et al. Effect of different serum concentrations on short term in vitro culture of goat testicular cells. Russ J Dev Biol 47, 269–277 (2016). https://doi.org/10.1134/S1062360416050064
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S1062360416050064