Polystyrene nanoplastics disrupt epididymal initial segment by perturbing NK cell differentiation and epithelial homeostasis.

Spermatozoa exit the testis in an immotile and immature state, and obtain motility and maturity in the epididymis with the most abundant functions acquired in its initial segment (IS). The reshaping of polystyrene nanoplastics (PS-NPs) exposure on the epididymis, however, has never been studied in detail yet, let alone from a multi-omic perspective. Herein, we utilized single-cell transcriptomics, untargeted metabolomics and in vivo experiments to explore PS-NPs-induced cellular remodeling of rat IS. A dose-dependent decrease in the epithelial thickness was merely observed in the IS (Control vs. low vs. medium vs. high group, 38.33 ± 2.52 vs. 32.67 ± 3.15 vs. 21.67 ± 3.58 vs. 11.08 ± 1.67 μm, respectively; P < 0.001) instead of the other three epididymal segments. PS-NPs exposure inhibited the acidification of epididymal luminal fluid (Control vs. low vs. medium vs. high group, pH, 6.05 ± 0.19 vs. 6.35 ± 0.14 vs. 6.57 ± 0.23 vs. 6.70 ± 0.24, respectively; P < 0.001), downregulated sperm acrosome reaction (Control vs. low vs. medium vs. high group, 42.67 ± 4.62% vs. 36.38 ± 4.04% vs. 28.07 ± 3.46% vs. 17.69 ± 2.52%, respectively; P < 0.05), and reduced sperm motility (Control vs. low vs. medium vs. high group, 83.08 ± 4.02% vs. 76.33 ± 2.08% vs. 70.67 ± 3.79% vs. 65.33 ± 2.89%, respectively; P < 0.01). Cell apoptosis after exposure existed across the IS without cell type-specificity. Besides, we found altered metabolic landscape and dysregulatory amino acid metabolites after exposure. Interestingly, cell-cell communication together with pseudotime trajectory delineated that PS-NPs facilitated NK cell differentiation towards cytotoxic and proliferative subtypes, during which ICAM and TNF signalings were upregulated. Moreover, PS-NPs exposure upregulated AQP9 through activating ERK1/2 in principal cells. Furthermore, we observed dysfunctions and ultrastructural defects of blood-epididymal barrier with downregulated protein levels of tight junction (VIM, SOX9, KRT5 and β-catenin) and upregulated ROS levels in basal cells. In summary, we offer previously unidentified insights into the contribution of PS-NPs in epididymal IS and extend on existing knowledge of environmental contaminant-induced epididymis damage that merits future investigation.