Insight into 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced disruption of zebrafish spermatogenesis via single cell RNA-seq.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical. Our previous work demonstrated the latent reproductive maladies of early-life TCDD exposure in zebrafish. Zebrafish acutely exposed to low, environmentally relevant levels of TCDD (50 pg/mL) during two windows of sexual differentiation in development (1 hour of exposure at 3 and 7 weeks postfertilization) were later infertile, showed a reduction in sperm, and exhibited gene expression consistent with an altered microenvironment, even months after exposure. Due to the highly heterogeneous cell- type and -stage landscape of the testes, we hypothesized various cell types contribute markedly different profiles toward the pathology of TCDD exposure. To investigate the contributions of the diverse cell types in the adult zebrafish testes to TCDD-induced pathology, we utilized single-cell RNA-seq and the 10x Genomics platform. The method successfully captured every stage of testicular germ cell development. Testes of adult fish exposed during sexual differentiation to TCDD contained sharply decreased populations of late spermatocytes, spermatids, and spermatozoa. Spermatogonia and early spermatocyte populations were, in contrast, enriched following exposure. Pathway analysis of differentially expressed genes supported previous findings that TCDD exposure resulted in male infertility, and suggested this outcome is due to apoptosis of spermatids and spermatozoa, even years after exposure cessation. Increased germ cell apoptosis was confirmed histologically. These results provide support for an environmental exposure explanation of idiopathic male infertility.