Testicular m⁶A-RNA hypomethylation in a high-fat, AGE-rich dietary mouse model of male reproductive dysfunction

Advanced glycation end products (AGEs), prevalent in Western thermally processed diets, contribute to metabolic dysfunction, oxidative stress, and impaired male reproduction; however, their effects on testicular RNA methylation remain unclear. This study investigated the effects of a 20-week high-fat, AGE-rich diet compared with a standard diet on global m⁶A RNA levels and m⁶A regulatory proteins in testicular tissue of male C57BL/6 mice (n = 6/group). Molecular analyses were performed exploratorily in a randomly selected subset of three animals per group using archived testicular tissue from the original experiment. Phenotypic outcomes, including metabolic indices, organ weights, sperm parameters, oxidative stress markers, and sperm chromatin/DNA integrity, were obtained from the primary study. Global m⁶A RNA content was measured in total RNA, and METTL14, ALKBH5, FTO, and YTHDF2 protein levels were assessed in the same exploratory subset. Statistical analyses were conducted using GraphPad Prism 10.4.0, with appropriate parametric or nonparametric tests and significance set at P < 0.05. The high-fat, AGE-rich diet induced metabolic disturbance, impaired sperm quality, increased oxidative stress, histone retention, DNA damage, and altered testicular redox balance (P < 0.05). It also significantly reduced global testicular m⁶A RNA levels and METTL14 expression while increasing ALKBH5 and YTHDF2 expression (P < 0.05). FTO reduction was not statistically significant (P > 0.05). Overall, high-fat, AGE-rich dietary exposure was associated with reproductive dysfunction and disrupted testicular m⁶A RNA methylation homeostasis, suggesting that altered m⁶A regulation may be a potential molecular mechanism underlying male reproductive impairment.