The role of quercetin in primary culture of ovine spermatogonial stem cells

Abstract

The aim of the present study was to examine the effect of quercetin on the survival and primary culture of ovine spermatogonial stem cells (SSCs). The two-time enzymatic digestion process was employed to obtain SSCs from lamb testes. In the next step, the use of filtration and differential plating methods caused an increase in the number of SSCs in the cell suspension resulting from enzymatic and mechanical digestions. Mitomycin-C-treated Sertoli cells were used to prepare the feeder layer. The stem cells were then cultured on the Sertoli cell feeder layer. The identification of the colonies was done through alkaline phosphatase staining methods and specific gene expression of ram’s SSCs (nanog and Plzf). The results of methylthiazolyldiphenyl-tetrazolium bromide assay on SSCs 72 h after culture with different treatments of quercetin demonstrated that the highest percentage of survival was for 5 μM and 10 μM concentrations, respectively; however, compared to the control, no significant difference was observed. In comparison with the control, the concentration equal to and greater than 20 μM quercetin caused a significant decrease in the survival of SSCs (P < 0.05). Seven days after culture, 40 μM quercetin caused a substantial reduction in the mean number of colonies, compared to the control (P < 0.05). The results demonstrated that compared to the control, 5 μM to 40 μM of quercetin significantly reduced Plzf gene expression. Furthermore, the concentration equal to and higher than 10 μM quercetin significantly decreased bcl-2 gene expression in the cells under study (P < 0.05). Based on the findings of the present study, the use of quercetin for the primary culture of ovine SSCs is not recommended. It is suggested that the function of this antioxidant should be investigated on the differentiation of SSCs.