Inhibition of FTO expression by thiodiphenol impairs Leydig cell maturation in pubertal male rats

This study investigated the multifaceted effects of 4,4'-thiodiphenol (TDP) on male reproductive health, focusing on its toxicity, hormonal modulation, and impact on steroidogenesis. Male rats were exposed to TDP at varying doses (0, 1, 10, and 100 mg/kg) from day 35-56 postpartum. Overt toxicity was evident as TDP significantly reduced inhibited spermatogenesis at 10 and 100 mg/kg. TDP exhibited anti-androgenic properties by inhibiting testosterone biosynthesis at 10 and 100 mg/kg, correlating with increased LH levels at 10 mg/kg and altered T/LH ratios at ≥ 1 mg/kg. Immunohistochemical analysis revealed a dose-dependent increase in Leydig cell (LC) numbers, particularly at 100 mg/kg, attributed to enhanced LC proliferation as evidenced by elevated PCNA labeling. RNA-seq and qPCR analyses demonstrated the impact of TDP on LC gene expression, notably downregulating steroidogenesis-related genes such as Lhcgr, Star, Cyp11a1, and Hsd3b1, and inducing oxidative stress pathway genes. Protein expression studies confirmed reduced levels of key steroidogenic proteins and increased oxidative stress, evidenced by elevated malondialdehyde levels and reduced SOD1 expression. In vitro studies further elucidated the inhibition of TDP on steroidogenesis and induction of reactive oxygen species (ROS), independent of cytotoxicity. Additionally, the modulation of m6A RNA methylation was explored, showing an increase in m6A levels and a reduction in FTO and ALKBH5 expression and overexpression of Fto reversed TDP-mediated suppression of testosterone and steroidogenic and antioxidative genes, suggesting a novel regulatory mechanism involving RNA modification. Collectively, these findings underscore the potential of TDP as an endocrine disruptor with significant implications for male reproductive health.