17β-Trenbolone binds to androgen receptor, decreases number of primordial germ cells, modulates expression of genes related to sexual differentiation, and affects sexual differentiation in zebrafish (Danio rerio)

Abstract

Exposure to 17β-trenbolone caused a skewed sex ratio in fish. However, the molecular initiating event and key molecular event(s) remain unknown. In this study, zebrafish were exposed to 17β-trenbolone at nominal concentrations of 2 ng/L, 20 ng/L, 200 ng/L, and 2000 ng/L from fertilization to 60 days post fertilization (dpf). First, the sex ratio at 60 dpf was calculated to evaluate adverse outcomes on sexual differentiation. 17β-Trenbolone caused a skewed sex ratio toward males, with intersex individuals observed in the 20 ng/L group and all-male populations found in the 200 ng/L and 2000 ng/L groups. Then, the distribution and number of primordial germ cells, the expression of sex differentiation-related genes, and plasma vitellogenin concentrations were detected in wild-type zebrafish and the EGFP-nanos-3′UTR transgenic line using whole-mount in situ hybridization, real-time PCR, EGFP fluorescence quantification, and enzyme-linked immunosorbent assay. The results indicated that 17β-trenbolone exposure decreased the number of primordial germ cells at 1 dpf and 3 dpf, decreased expression of ovarian differentiation-related genes foxl2 and cyp19a1a at 60 dpf, increased expression of testis differentiation-related genes dmrt1, sox9a, and amh at 60 dpf, and decreased plasma vitellogenin levels at 60 dpf, revealing the key molecular events at different time points involved in affected sexual differentiation by 17β-trenbolone. Finally, molecular docking showed that 17β-trenbolone docked into ligand-binding domain of zebrafish androgen receptor with high binding energy (−3.72 kcal/mol), suggesting that binding to androgen receptor is the molecular initiating event affecting sexual differentiation by 17β-trenbolone. We found that 17β-trenbolone can bind to the zebrafish androgen receptor, decrease the number of primordial germ cells during the early embryonic stage, modulate the expression of genes related to sexual differentiation during gonadal differentiation, and eventually cause a skewed sex ratio toward males in adults.