DNMT1 Regulates Testosterone Synthesis in Aged Rat Leydig Cells via FTO ‐Mediated HSP90AA1 m 6 A Levels

ABSTRACT Testosterone levels decline progressively with age from the third decade of life, impairing spermatogenesis and contributing to age‐related disorders, underscoring the importance of enhancing endogenous testosterone production. DNA methyltransferase 1 (DNMT1) promotes testosterone synthesis in Leydig cells (LCs), yet the underlying mechanism remains unclear. Here, we found that DNMT1 expression is downregulated in aging rat testes and senescent LCs in vitro, correlating with reduced testosterone levels. Gain‐ and loss‐of‐function experiments confirmed that DNMT1 positively regulates testosterone synthesis and alleviates LC senescence, and that DNMT1 overexpression reverses the senescent phenotype and restores testosterone production. Mechanistically, RNA‐seq of DNMT1‐knockdown LCs identified FTO as a downstream target; DNMT1 suppresses FTO expression by promoting Fto promoter methylation. catRAPID prediction and RIP‐qPCR validated Hsp90aa1 mRNA as a direct FTO substrate; FTO regulates HSP90AA1 expression through m 6 A modification, with DNMT1 indirectly modulating HSP90AA1 via FTO. Using HSP90AA1 overexpression, pharmacological inhibition, and rescue experiments, we confirmed that the DNMT1–FTO axis inhibits LC apoptosis through HSP90AA1‐mediated AKT activation, thereby promoting testosterone synthesis. Expression patterns of this axis in aging rat testes and senescent LCs further corroborated its involvement in age‐related testosterone dysregulation. In vivo AAV‐mediated testicular DNMT1 knockdown recapitulated these findings. Collectively, our study elucidates the physiological role of DNMT1 in age‐related testosterone decline and delineates the mechanism by which DNMT1 governs LC senescence and steroidogenic function, providing a potential therapeutic target for reproductive system aging.