Establishment of Oxidative Stress Modeling during Spermatogonial Stem Cells Cultivation Treated with Different Doses of H2O2

Abstract

Introduction: Nowadays, spermatogonial stem cells (SSCs) cultivation has been used by many researchers as an effective tool for infertility treatments. Oxidative conditions can be effective on cell proliferation and differentiation of these cells. So, the aim of this study was to establish oxidative stress model for antioxidant activity of some drugs investigation during SSCs in vitro culture. Methods: Neonatal NMRI male mice (3-5 day) were used for isolation of SSCs. The cell suspension was prepared by twice enzymatic digestion. The cell suspension contents were spermatogonial and sertoli cells and treated by different doses of H2O2 logarithmic concentrations from 0-100 ;muM after 24 hours. To access the optimal dose, extra doses from 10-100;muM was evaluated. After 2 hours of H2O2 treatment, viability was determined by Trypan blue assay. The data were analyzed using SPSS software and One-way ANOVA test. Results: Our data showed that spermatogonial stem cells colonies appeared after 4 days of isolation. These cells expressed OCT4 and PLZF proteins. Many of spermatogonial stem cells were removed after using higher doses of H2O2. The results showed that 30 ;muM concentration of H2O2 could induce oxidative stress in spermatogonial stem cell during in vitro culture. Conclusion: According to this study, 30 ;muM concentration of H2O2 can cause cell death lower than 50% of total number of cells and can increase oxidative stress in cultivation of SSCs. This model is a suitable tool for studying some new antioxidant drugs.

Keywords

• spermatogonial stem cells
• oxidative stress
• hydrogen peroxide