MEHP exposure disrupts the self-renewal and differentiation of GC-1 cells by up-regulating m6A levels, leading to reproductive damage in immature rats.

Di-(2-ethylhexyl) phthalate (DEHP) and its active metabolite mono-(2-ethylhexyl) phthalate (MEHP) are environmental endocrine disruptors associated with male reproductive toxicity, but the underlying mechanisms remain unclear. This study investigated whether N6-methyladenosine (m6A) modification is involved in DEHP/MEHP-induced reproductive damage using both an immature rat DEHP exposure model in vivo and MEHP-treated GC-1 cells in vitro. In vivo, DEHP exposure induced obvious testicular injury in immature rats, including structural disorder of seminiferous tubules and decreased seminiferous epithelium height and tubule diameter. In vitro, MEHP increased global m6A levels, altered cell cycle distribution, and disrupted the expression of self-renewal- and differentiation-related markers in GC-1 cells. MeRIP-seq further revealed increased m6A peaks and enrichment of cell cycle-related pathways after MEHP exposure. Among the examined m6A regulators, YTHDF1 showed significant upregulation after MEHP treatment. Moreover, YTHDF1 knockdown reversed the abnormal expression of self-renewal- and differentiation-related proteins induced by MEHP. These findings suggest that dysregulated m6A modification, potentially involving YTHDF1, participates in DEHP/MEHP-induced male reproductive damage.