Age-dependent testicular autophagy disruption mediates juvenile susceptibility to dibutyl phthalate-induced reproductive toxicity†

Dibutyl phthalate (DBP) is a known reproductive toxicant, but the mechanisms underlying age-dependent susceptibility in the testis remain poorly defined. Here, we investigated how DBP exposure differentially affects testicular homeostasis in juvenile (3-week-old) versus adult (12-week-old) male mice, with a focus on autophagy regulation and its functional consequences for spermatogenesis. Juvenile and adult mice were orally administered DBP at 100 or 500 mg/kg/day for 35 days. We found that juvenile mice exhibited significantly greater testicular injury than adults, characterized by severe disruption of the seminiferous epithelium, Sertoli cell vacuolization, and compromised blood-testis barrier integrity. Mechanistically, DBP suppressed testicular autophagy-evidenced by reduced LC3-II/I ratio and nuclear translocation of TFEB-particularly in juvenile animals, leading to impaired mitochondrial quality control and accumulation of damaged organelles. Autophagy impairment was accompanied by robust NLRP3 inflammasome activation, elevated NF-κB phosphorylation, and diminished antioxidant capacity. Functionally, these molecular alterations were associated with decreased serum testosterone, luteinizing hormone and follicle-stimulating hormone levels, germ cell loss, and disrupted spermatogenic progression. Our findings demonstrate that autophagy serves as a critical protective mechanism in the developing testis, and its disruption underlies the heightened vulnerability of juvenile males to DBP-induced reproductive toxicity. These results advance our understanding of how environmental stressors perturb testicular physiology during critical windows of postnatal development.