Differentiations of neonatal mice spermatogonial stem cells in presence of different concentration of alpha-Ketoglutarate

Abstract For the first time, alpha-ketoglutarate (AKG) was used to differentiate Spermatogonial stem cells (SSCs) in the presence of BMP-4 and Retinoic acid. So, SSCs were isolated from testis of 3-6 day-old mice by enzymatic digestion by collagenase and trypsin. The Cell suspension was cultured for one week in DMEM/F12 and 20% FBS in presence of GDNF growth factor. The proliferated cells were divided between control and treatment groups. In the control group, the cells were cultured for three weeks in DMEM/F12 containing 10% FBS in presence of 10-6 M retinoic acid and 40 ng/ml of BMP-4. A dose of 0.1 M AKG was added to the treatment group. The presence of Sertoli cells in culture system was confirmed by positive reaction of vimentin immunocytochemistry. The colonies that appeared on Sertoli cells also showed positive alkaline phosphatase activity and Oct4 immunocytochemistry reaction. qRT-PCR studies showed that the expression of Acrosin and Sycp3 genes was low in two groups after 7 days of culture. 21 days after culture, in the treatment group, the expression of Acrosin and Sycp3 genes was significantly increased rather than control group (p≤0.05). Large number of early spermatids were observed in the treatment group based on TEM studies.