Non-invasive assessment of stem cell DNA integrity in response to environmental stressors.

The maintenance of DNA integrity in spermatogonial stem cells (SSCs) is essential for ensuring genetic stability and proper function in male germline cells. Here, we established a non-invasive approach to evaluate SSC DNA integrity and investigated the impact of detrimental environmental stressors on genomic stability. SSCs were isolated and cultured from mouse testes. SSCs identity was confirmed through immunofluorescence staining and flow cytometric analysis of specific molecular markers.DNA strand breaks were detected using a novel TdT enzyme-Endo IV-fluorescent probe biosensor, confirming the methodological feasibility. Heat stress and cryopreservation models were established, with the biosensor quantifying stress-induced DNA damage in SSCs. Additionally, extracellular free DNA fragments in culture supernatants were quantified as a non-invasive measure of SSCs DNA integrity.