Impact of melatonin on the colonies diameter, apoptosis status and apoptosis related genes expression in sheep’s spermatogonial stem cells

The aim of this research was to assess the effect of melatonin on the diameter of colonies, apoptosis status and apoptosis related genes expression in sheep’s spermatogonial stem cells (SSCs). SSCs at the basal membrane of seminiferous tubules were isolated from testes of Afshari sheep using enzymatic digestion steps. The samples assigned into four groups. The control group received basic medium and the other groups contained H2O2 (30 µM), melatonin (1 nmol) and melatonin+H2O2 (30 µM H2O2 along with 1 nmol melatonin), respectively. The cells were cultured for 3 weeks and the colonies’ diameter were evaluated on the 5th, 14th and 21th days of culture. At the end of culturing period, apoptosis status and apoptosis related genes expression (BAX and BCL2) were evaluated. On the 5th and 14th days of culture, the diameter of colonies were higher (P≤0.05) in the control and melatonin groups compared to the other groups. On the 21th day of culture, the highest and the least (P≤0.05) diameter of colonies were observed in melatonin and H2O2 groups, respectively. The least and the highest (P≤0.05) rate of apoptotic SSCs, BAX expression and BAX/BCL2 ratio were observed in melatonin and H2O2 groups, respectively. Moreover, the highest (P≤0.05) expression of BCL2 gene was found in melatonin group. In conclusion, using melatonin in culture medium could be an effective way to improve the quality and decrease apoptosis status in sheep’s SSCs.