SIRT1 mediates KU70 to maintain Genomic Stability in Spermatogonial Stem Cells via NHEJ Repair pathway

Background Male infertility is closely related to DNA double-strand breaks in spermatogonial stem cells (SSCs); however, the precise mechanism still remains to be fully elucidated. While SIRT1 is a key regulator of DNA damage response and cellular senescence in other contexts, its role in SSCs is still poorly understood. Methods Single-cell RNA sequencing (scRNA-seq) datasets from human testes were analyzed to identify transcriptional changes in SSCs from non-obstructive azoospermia (NOA) patients. X-ray irradiation in vivo and SSC in vitro models were used to assess DNA damage responses by immunofluorescence, western blotting, co-immunoprecipitation, proliferation assays, GFP-based non-homologous end joining (NHEJ) reporter analysis, and acetylation assays. Results scRNA-seq of human testis identified NHEJ pathway gene involved and marked downregulation of SIRT1 in SSCs from NOA patients while compared with control group (obstructive azoospermia patients). Acute DNA damage triggered rapid SIRT1 activation both in vivo and in vitro. Functional assays demonstrated that SIRT1 regulates SSC self-renewal and DNA-damage susceptibility by modulating sensitivity to hydroxyurea-induced replication stress and altering KU70 and p53 signaling. Immunofluorescence confirmed dynamic changes in KU70 expression after X-ray irradiation, while co-localization and co-immunoprecipitation established a direct SIRT1–KU70 interaction that was enhanced under genotoxic stress. NHEJ reporter assays showed reduced repair efficiency following Sirt1 knockdown. Moreover, Sirt1 overexpression may down-regulate KU70 acetylation, indicating a deacetylation-dependent mechanism in NHEJ regulation. Conclusions SIRT1 safeguards SSC genomic stability by modulating KU70 expression and post-translational regulation to enhance NHEJ repair, highlighting the SIRT1–KU70 axis as a potential target for preserving male fertility under genotoxic stress.