Enhancing spermatogonial stem cell differentiation: The role of alpha-ketoglutarate in in-Vitro cultures.

Spermatogonial stem cells (SSCs) have the unique ability to self-renew and differentiate into mature spermatozoa. In vitro culture of SSCs, however, presents several challenges, particularly in promoting efficient differentiation. This study investigates the role of metabolic intermediates, such as alpha-ketoglutarate (AKG), on the differentiation of SSCs isolated from the testes of 3-6 day-old mice. SSCs and sertoli cells were extracted using collagenase ІV and trypsin and co-cultured in DMEM/F12 supplemented with 20 % fetal bovine serum (FBS) and glial cell-derived neurotrophic factor (GDNF) for one week. The survival rate of cells was evaluated under influence of different dosages of AKG (0.04, 0.1, 0.4, 4, 10 mM) after 1 and 7 days of culture using the MTT test. The cell viability was significantly increased at the 0.1 mM dose of AKG rather than other groups. This dosage was selected for adding to culture system. In the control group, the cells cultured for three weeks in DMEM/F12 with 10 % FBS, 10⁻6 M retinoic acid, and 40 ng/mL bone morphogenetic protein-4 (BMP-4). The treatment group received the same medium with the addition of 0.1 mM AKG. The presence of Sertoli cell in the culture system was confirmed by SOX9-positive immunocytochemistry. The Colonies that formed on the Sertoli cells exhibited positive alkaline phosphatase activity and reacted positively for Oct4 and GFRa1 immunocytochemistry. The expression of testicular-specific genes (Acrosin and Sycp3) and anti-apoptosis-related genes (Nrf2 and Bcl2) was evaluated after 7 days (as the zero group) and again after 21 days of culture, in treatment (0.1 mM AKG) and control groups. A high expression of Acrosin and Sycp3 expression was observed in AKG-treated group compared to control and zero groups (p ≤ 0.05). The expression of Nrf2 and Bcl2 genes was also significantly increased in the treatment group (p ≤ 0.05). These findings suggest that AKG activates mechanisms of cellular antioxidant response and subsequently increase the expression of differentiation genes in SSCs.