To investigate the mechanism by which peripubertal exposure to di-2-ethylhexyl phthalate （DEHP） induces reproductive injury in adult male mice through impairment of Sertoli cell function and the blood-testis barrier.

A total of 180 male C57BL/6J mice （3 weeks old） were randomly divided into corn oil group （control group），and 5，25，125，250 and 500 mg/kg DEHP exposure groups. All groups received daily oral gavage for 5 consecutive weeks. Ten mice from each group were sacrificed for first material collection when they were 9 weeks old. The remaining F0 generation mice were mated with unexposed adult female mice at a male-to-female ratio of 1∶2 at 14 weeks of age for a period of 2 weeks. The second material collection was completed at 16 weeks of age，with 20 mice taken from each group. A computer-assisted analysis system was used to detect sperm density and motility in 9-week-old and 16-week-old male mice. The average birth body weight of F1 generation pups，survival rate of pups at 4 d after birth per litter，number of pups per litter，and male-to-female sex ratio within the litter were recorded. ELISA was used to detect hormone levels in the serum. Sperm abnormalities were observed using eosin staining. HE staining was used to observe pathological changes in testicular tissue. Immunofluorescence assay was utilized to detect the protein expression of tight junction protein 1 （zonula occludens-1，ZO-1） and gap junction protein 43 （connexin43，Cx43） related to the blood-testis barrier in testicular tissue，as well as the protein expression of apoptosis-related molecule Cleaved-Caspase3. Immunohistochemistry was used to detect the expression of Sertoli cell marker SOX9 protein in testicular tissue. TUNEL was used to detect apoptosis in testicular tissue. Comparative toxicogenomics database （CTD） was used to screen for mono-2-ethylhexyl phthalate （MEHP） induced reproductive damage-associated genes and combined with GO/KEGG and Reactome enrichment analysis to predict possible signaling pathways. An in vitro exposure model was established by treating TM4 cells with MEHP at concentrations of 0，50，100，200 and 400 μmol/L，respectively. CCK-8 assay，flow cytometry and Western blotting were applied to detect the cell viability，apoptosis，and protein expression of apoptosis-related proteins B-cell lymphoma 2 （Bcl-2） and Bcl-2-associated X protein （Bax），and blood-testis barrier-related proteins ZO-1 and Cx43.

Compared with the control group，the pregnancy rate was decreased from 80.0% to 52.5%，and the total number of pups was decreased from 212 to 125 in the female mice mating with the male from the 500 mg/kg group. Compared with the control group，the sperm density and motility were significantly reduced，and the total rate of sperm abnormalities was obviously increased in the 9-week-old DEHP-exposed mice （P<0.05），and even in the 16-week-old mice，the sperm motility was still in a dose-dependent downward trend （P<0.05）. In the 25，125，250 and 500 mg/kg DEHP exposure groups，the thickness of the seminiferous epithelium was obviously decreased，with more cellular vacuolization and notable epithelial atrophy and degeneration. Cell apoptosis in the testicular tissue was enhanced，while the expression of ZO-1 and Cx43 were decreased in the 250 and 500 mg/kg exposure groups. The levels of androgen-binding protein，gonadotropin-releasing hormone，and testosterone in the serum were decreased in a dose-dependent manner （P<0.05），while those of luteinizing hormone and follicle-stimulating hormone showed a dose-dependent increase （P<0.05）. Bioinformatics analysis showed that 153 genes related to male system diseases were screened out by CTD for MEHP. GO enrichment，KEGG pathway，and Reactome enrichment analyses displayed that the apoptosis signaling pathway was significantly enriched. CCK-8 assay showed that TM4 cell viability was decreased in a dose-dependent manner after MEHP treatment when compared with the control group （P<0.05）. Western blotting indicated that MEHP treatment reduced the expression levels of Bcl-2，ZO-1 and Cx43 （P<0.05），and enhanced that of Bax in comparison with the control group （P<0.05）.

Peripubertal DEHP exposure may lead to reduced sperm quality and reproductive damage in adult male mice by disrupting the blood-testis barrier and activating Sertoli cell apoptosis.