To analyze the effect of subchronic exposure of polystyrene nanoplastics（PS-NPs）on reproductive function in adult male mice, and to explore the preliminary molecular mechanism of its impact on testosterone synthesis.

Sixty 8-week-old C57BL/6 male mice were fully randomly divided into control group（deionized water）and PS-NPs low（10 mg/kg, 10 PS）and high exposure group（40 mg/kg, 40 PS）, with 20 mice in each group. The mice were treated once per day for 60 d by gavage. The weights and organ coefficient of the testes, epididymis, brain, liver, spleen, lung and kidney of each group of mice were weighed and calculated at 24 h after the last exposure. HE staining was used to analyze the histopathological differences of testicular tissue, and transmission electron microscopy was used to observe the ultrastructural changes of testicular cells. The computer assisted sperm assay system was used to analyze semen parameters, the contents of serum testosterone（T）, luteinizing hormone（LH）and follicle-stimulating hormone（FSH）were detected by enzyme-linked immunosorbent assay（ELISA）, and the expression levels of key genes for T synthesis, which had been screened by transcriptome sequencing, were determined in testicular tissues by real-time PCR.

Compared with the control group, the weight of the epididymis in the 40 PS group was increased significantly（P<0.05）, the testicular organ coefficients of mice in the 10 PS group and the epididymis organs coefficients of mice in the 40 PS group were increased obviously（P<0.05）, and the weight and organ coefficients of the liver and spleen of the mice in the 10 PS and 40 PS groups were decreased remarkably（P<0.05）. The pathological results showed that compared with the testis of the control group, the lumen of the semininogenic tubule in the 10 PS and 40 PS groups were enlarged, the semimatogenic cells were loose, and the vacuoleization was increased. Transmission electron microscopy displayed that mitochondrial swelling and endoplasmic reticulum expansion were observed in the cytoplasm of Leydig cells in 10 PS and 40 PS groups. The sperm motility of mice in the 10 PS group was decreased significantly compared with the control group（P<0.05）. The serum T level of mice in the 10 PS and 40 PS groups was decreased, with that of the 40 PS group more significant（P<0.05）. The serum LH and FSH levels of mice in the 10 PS and 40 PS groups were increased notably. Compared with the control group, the mRNA levels of CYP11A1 in the testes of mice were significantly down-regulated in both 10 PS and 40 PS groups（P<0.05）.

PS-NPs can cause damage to the testes pathologically and functionally in mice, and may reduce T synthesis by inducing structural abnormalities of Leydig cells and down-regulating CYP11A1 expression.

To establish a damage model of mouse spermatocyte GC-2spd cells induced by benzo[k] fluoranthene (BkF), a representative compound of high-molecular weight polycyclic aromatic hydrocarbons, so as to investigate the effect and mechanism of BkF on GC-2 cells.

Related genes of male reproductive diseases associated with BkF were screened in Comparative Toxicogenomics Database (CTD), and functional and pathway enrichment analysis is conducted subsequently to explore the possible pathway mechanism. Moreover, GC-2 cells were treated with BkF at a final concentration of 0, 10, 20, 40 or 80 μmol/L for 72 h, and then the cell cycle, oxidative stress and DNA damage effect were detected. Western blotting was used to determine the protein levels of the key genes as well as the core molecules of related pathways.

The key genes CYP1A1 and CYP17A1 associated with BkF-related reproductive system diseases were screened out from the CTD database. Gene Ontology (GO) enrichment analysis found that they were involved in male reproductive system and cell cycle arrest, obviously enriched in biological processes such as oxidative stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these genes were significantly enriched in cytochrome P450 pathway (P<0.01). After GC-2 cells were exposed to BkF for 72 h, the proliferation was significantly decreased in a dose-dependent manner, as compared with the control group. When the exposure dose was higher than 40 μmol/L, the cell cycle arrest was obvious (P<0.05); Both the intracellular reactive oxygen species (ROS) level (P<0.05) and the protein level of DNA damage marker γ-H2AX (P<0.01) were remarkably increased in a dose-dependent manner. In addition, the protein levels of CYP1A1 and CYP17A1 were greatly higher in the exposure group than the control group (P<0.05), while treatment of AhR inhibitor CH223191 resulted in notably down-regulated the expression of the 2 proteins (P<0.05).

BkF induces the activation of AhR receptors, then causes elevated expression of downstream genes CYP1A1 and CYP17A1 as well as increased intracellular ROS, and finally leads to oxidative stress and genetic damage in GC-2 cells.