An interplay of NOX1-derived ROS and oxygen determines the spermatogonial stem cell self-renewal efficiency under hypoxia

Abstract

Reactive oxygen species (ROS) produced by NADPH1 oxidase 1 (NOX1) are thought to drive spermatogonial stem cell (SSC) self-renewal through feed-forward production of ROS by the ROS-BCL6B-NOX1 pathway. Here we report the critical role of oxygen on ROS-induced self-renewal. Cultured SSCs proliferated poorly and lacked BCL6B expression under hypoxia despite increase in mitochondria-derived ROS. Due to lack of ROS amplification under hypoxia, NOX1-derived ROS were significantly reduced, and Nox1-deficient SSCs proliferated poorly under hypoxia but normally under normoxia. NOX1-derived ROS also influenced hypoxic response in vivo because Nox1-deficient undifferentiated spermatogonia showed significantly reduced expression of HIF1A, a master transcription factor for hypoxic response. Hypoxia-induced poor proliferation occurred despite activation of MYC and suppression of CDKN1A by HIF1A, whose deficiency exacerbated self-renewal efficiency. Impaired proliferation of Nox1- or Hif1adeficient SSCs under hypoxia was rescued by Cdkn1a depletion. Consistent with these observations, Cdkn1adeficient SSCs proliferated actively only under hypoxia but not under normoxia. On the other hand, chemical suppression of mitochondria-derived ROS or Top1mt mitochondria-specific topoisomerase deficiency did not influence SSC fate, suggesting that NOX1-derived ROS play a more important role in SSCs than mitochondria-derived ROS. These results underscore the importance of ROS origin and oxygen tension on SSC self-renewal.