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 (BTB) 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, LH and FSH 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.