Gene-environment interaction in hypospadias: DEHP exposure aggravates DNAH8-related disruption of testicular steroidogenesis

Hypospadias is a prevalent congenital malformation of the male external genitalia with ill-defined pathogenesis. The rising incidence of isolated cases cannot be explained by genetic factors alone, and its multifactorial etiology suggests that genetic susceptibility is often exacerbated by environmental factors, aligning with 'two-hit' hypothesis. Here, we investigated the combined effects of the hypospadias risk gene DNAH8 and prenatal exposure to the endocrine-disrupting chemical di-(2-ethylhexyl) phthalate (DEHP). Fetal wild-type and Dnah8-knockout (KO) mice were exposed to DEHP (0, 250, or 500 mg/kg/day) to evaluate urethral morphology, androgen dynamics, and testicular phenotypes. Dnah8 KO fetuses exposed to low-dose DEHP (250 mg/kg/day) exposure exhibited aggravated urethral fusion defects at E16.5. By E18.5, this co-exposure abolished the compensatory developmental mechanisms typically observed in Dnah8 KO mice, resulting in a 35.0% incidence of hypospadias, comparable to the 36.6% incidence induced by high-dose DEHP (500 mg/kg/day) alone. This combined exposure induced persistent testicular steroidogenic dysfunction, characterized by declining testosterone levels and significant reductions in Sertoli and fetal Leydig cells (FLCs). Proteomic analysis confirmed marked disruptions in oxidative stress and steroid biosynthesis pathways. Mechanistically, DEHP and its active metabolite, mono-(2-ethylhexyl) phthalate, induced oxidative stress, reduced cell viability, and inhibited the migration of mesenchymal progenitor cells (MPCs), compounding DNAH8 deficiency-induced MPCs differentiation delay. This impaired MPC trajectory led to deficient FLCs, irreversible disruption of testosterone synthesis, and hypospadias. These findings elucidate a novel DNAH8-DEHP interaction, establish a physiologically relevant in vivo model, and highlight early oxidative stress in progenitor cells as a critical node in hypospadias pathogenesis.