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

Abstract

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.

Identification of fish spermatogenic cells through high-throughput immunofluorescence against testis with an antibody set

“…In mice, the interconnected syncytia of two to 16 spermatogonia are considered as SSCs in a context-dependent manner, and they share similar nuclear morphology and molecular markers ( 59 ). In our study, the nuclear morphology of SPG-A(2,4,8-cell) and the expression pattern of the four proteins in it are also similar to those of SPG-A(1-cell), suggesting that SPG-A(2,4,8-cell) are either SSCs or can achieve stemness under certain condition ( Figure 6K ), which is consistent with a recent single cell sequencing study ( 60 ). Nevertheless, more solid conclusion should be concluded by SSC transplantation approach in the future.…”

Germ cells do not progress through spermatogenesis in the infertile zebrafish testis

“…Notably, lymphocyte and macrophage populations form the majority of cells in infertile testes. In a previous study of endocrine disrupted testes, spermatogenesis failure in post-meiotic cells showed arrest or apoptosis of developing germ cells but does not characterize the immune cells of such tissues (Haimbaugh et al 2022). In our data, it is unclear whether defects in meiosis or a post-meiotic event is responsible for the absence of these more differentiated cell types in naturally infertile testes.…”

“…Together, our imaging and single-cell transcriptomic data suggest that infertile testes have a large population of spermatogonia and some spermatocytes but a dearth of post-meiotic cell types. We hypothesized that the expansion of immune cells we observe in infertile testes may help facilitate the apoptosis and removal of dying cells in the testis, perhaps explaining the absence of post-meiotic germ cells in infertile testes by a mechanism similar to what is observed in endocrine-disrupted testes (Haimbaugh et al 2022). However, we did not detect differences in the expression of markers for apoptosis between fertile and infertile testes (Fig.…”

“…However, this stage of life marks just the sunrise of a dynamic and continually maintained process throughout the adult life of a zebrafish. Another study compared 18-month testes from control zebrafish and infertile fish that were exposed to an endocrine-disruptive chemical early in life (Haimbaugh et al 2022). Notably, endocrine disruption caused arrest or apoptosis of post-meiotic germ cells after typical development through spermatogonial and meiotic phases.…”