SIRT1 mediates KU70 to maintain genomic stability in spermatogonial stem cells via the NHEJ repair pathway

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. In this study, human testicular single-cell RNA sequencing datasets were reanalyzed to characterize SSC transcriptional programs in non-obstructive azoospermia (NOA) patients. Clinical validation was performed on testicular sections from obstructive azoospermia controls and NOA patients. X-ray irradiation and hydroxyurea-based DNA damage models were applied to interrogate SSC DNA damage responses in vivo and in vitro. Immunofluorescence, western blotting, co-immunoprecipitation, growth and survival assays, flow cytometry, a GFP-based NHEJ reporter, and acetylation analyses were used to define SIRT1-associated pathways. Single-cell analysis revealed an overall attenuation of NHEJ-related signatures and reduced SIRT1 expression in SSCs from NOA compared with controls. In clinical specimens, confocal immunofluorescence confirmed a reduced SSC pool and decreased SIRT1 and 53BP1 signals within PLZF-positive SSCs, while KU70 levels were not significantly changed. In experimental models, acute DNA damage induced a rapid SIRT1 response in SSCs. Functional assays showed that SIRT1 supports SSC homeostasis by promoting proliferative capacity and influencing apoptosis and survival under hydroxyurea-induced DNA damage. Mechanistically, SIRT1 co-localized and physically interacted with KU70, with enhanced association 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. Collectively, these findings identify SIRT1 as a stress-responsive regulator of SSC genome maintenance that functionally cooperates with KU70 to support NHEJ-associated repair and limit DNA damage-driven SSC loss. The SIRT1-KU70 axis represents a potential target to mitigate genotoxic injury-associated germline stem cell attrition and preserve male fertility.