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Effect of different serum concentrations on short term in vitro culture of goat testicular cells

Bahadorani, Mehrnoosh, Hosseini, Morteza S., Abbasi, Hassan, Abedi, Parvaneh, Nasr-Esfahani, Mohammad H.
Russian journal of developmental biology 2016 v.47 no.5 pp. 269-277
alkaline phosphatase, blood serum, cell viability, epithelial cells, gene expression, goats, immunocytochemistry, in vitro culture, staining, stem cells, testes, vimentin
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.