SIRT1 haploinsufficiency recapitulates age-associated subfertility through sperm α-tubulin hyperacetylation.

Advanced paternal age compromises male fertility and correlates to a decline of the deacetylase SIRT1 activity, a central regulator of germline homeostasis and chromatin dynamics. Acetylation imbalance has been pointed out as a driver of testicular aging. We therefore asked whether SIRT1 insufficiency reproduces aging-associated shifts in the acetylation landscape and how these might propagate to sperm function, fertilization capacity and embryo development. To address this, we combined acetylomic profiling of Sirt1+/− and naturally aged mice with functional assays of sperm quality and in vitro fertilization (IVF). Both Sirt1+/− and aged wild type (WT) testes shared a distinct acetylation signature absent in young WT controls, including nuclear regulators (ZNF638, MORC4), proteins involved in sperm structure and motility (Rootletin, Kinectin, CFAP58), and phosphoinositide signaling mediators such as PLCη1, which regulate intracellular Ca2 + release. Conversely, 22 proteins displayed acetylation exclusively in WT controls but were absent in Sirt1+/− and aged testes, encompassing modules related to flagellar organization (CEP170, CEP350, AKAP13), meiotic control (ANAPC7, PDS5B, ESCO1, CENPE), signaling and metabolism (SHIP1, GAK, ABCD4), chromatin regulation (BRD4, MAGEB4), and testicular architecture (MFAP2). This differential acetylomic profile persisted in mature sperm, with Sirt1+/− males showing elevated acLys levels and, more specifically, midpiece-restricted α-tubulin hyperacetylation, a pattern particularly shared with aged cohorts. Notably, this tubulin hyperacetylation remained after capacitation and correlated with mitochondrial dysfunction, elevated reactive oxygen species, reduced acrosome responsiveness, and diminished fertilization capacity. IVF assays further revealed decreased cleavage and blastocyst developmental rates, indicating defective paternal support of early embryogenesis despite preserved blastocyst quality. Together, these findings indicate that testicular SIRT1 contributes to germline acetylation patterns and that midpiece-restricted α-tubulin hyperacetylation is a shared feature of SIRT1 insufficiency and natural aging, correlating with the mitochondrial dysfunction and impaired sperm performance. Overall, our work broadens current understanding by integrating acetylomic and functional evidence within a model that reflects the physiological, age-related reduction of SIRT1.