Impact of the hypoxic microenvironment on spermatogonial stem cells in culture

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O₂ supply by blood and local O₂ tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O₂ tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O₂ and 0.1% O₂) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O₂ tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O₂ conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O₂. In 3.5% O₂ conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O₂ at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.