Zinc Oxide Nanoparticles-induced Testis Damage at Single-cell Resolution: Depletion of Spermatogonia Reservoir and Disorder of Sertoli Cell Homeostasis

The widespread application of zinc oxide nanoparticles (ZnO NPs) in our daily life has initiated an enhanced awareness of their biosafety concern. An incredible boom of evidence of organismal disorder has accumulated for ZnO NPs, yet there has been no relevant study at the single-cell level. Here, we profiled > 28,000 single-cell transcriptomes and assayed > 25,000 genes in testicular tissues from two healthy Sprague Dawley (SD) rats and two SD rats orally exposed to ZnO NPs. We identified 10 cell types in the rat testis. ZnO NPs had more deleterious effects on spermatogonia, Sertoli cells, and macrophages than on the other cell types. Cell-cell communication analysis indicated a sharp decrease of interaction intensity for all cell types except macrophages in the ZnO NPs group than in the control group. Interestingly, two distinct maturation states of spermatogonia were detected during pseudotime analysis, and ZnO NPs induced reservoir exhaustion of undifferentiated spermatogonia. Mechanically, ZnO NPs triggered fatty acid accumulation in GC-1 cells through protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling and peroxisome proliferator-activated receptor alpha (PPARα)/acyl-CoA oxidase 1 (Acox1) axis, contributing to cell apoptosis. In terms of Sertoli cells, downregulated genes were highly enriched for tight junction. In vitro and in vivo experiments verified that ZnO NPs disrupted blood-testis barrier formation and growth factors synthesis, which subsequently inhibited the proliferation and induced the apoptosis of spermatogonia. As for the macrophages, ZnO NPs activated oxidative stress of Raw264.7 cells through nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway and promoted cell apoptosis through extracellular signal-regulated kinase (ERK) 1/2 pathway. Collectively, our work reveals the cell type-specific and cellularly heterogenetic mechanism of ZnO NPs-induced testis damage and paves the path for identifying putative biomarkers and therapeutics against this disorder.