In Vitro Culture of Ovine Spermatogonial Stem Cells: Effects of Grape Seed Extracts and Vitamin C

Abstract

Background: Spermatogonial stem cells (SSCs) transmit genetic information to the next generation. For the successful transplantation of these cells, the culture medium of these cells must be optimized. Objectives: This study investigated the natural antioxidant of grape seed extract (GSE) in comparison to vitamin C in the culture medium of SSCs. Methods: A Soxhlet extractor was used to prepare ethanolic and acetonic extracts, and the Clevenger apparatus was used to prepare aqueous extracts of grape seeds. Then, their antioxidant capacities were determined using the DPPH method. The SSCs were extracted from lamb testicular tissue by a two-step digestion method, and different concentrations of GSE and vitamin C were investigated for survival, colony formation, and expression of apoptotic-related genes. Results: The results showed that the acetonic extract in the concentration of 400 µg/mL showed about 90% antioxidant properties based on the DPPH test; nevertheless, the aqueous and ethanolic extracts had only 50% of their antioxidant properties at this concentration. The acetonic extracts significantly decreased the viability of SSCs without any colony formation when used in a culture medium. The highest survival rate was obtained from the ethanolic extracts of grape seeds at the concentration of 50 µg/mL, and a significant difference was obtained with ethanolic extracts (100 to 1000 µg/mL) (P < 0.05). The viability of SSCs treated with vitamin C (50 µg/mL) was significantly higher (P < 0.05) than the control and 5 - 25 µg/mL of vitamin C groups. Conclusions: Aqueous, ethanolic, and acetonic extracts of grape seeds should not be used in the culture of SSCs. However, the use of 25 - 50 µg/mL of vitamin C is recommended in the culture of SSCs.