17β-Trenbolone activates androgen receptor, upregulates transforming growth factor beta/bone morphogenetic protein and Wnt signaling pathways, and induces masculinization of caudal and anal fins in female guppies (Poecilia reticulata)

Sexually mature female guppies (Poecilia reticulata) were exposed to environmentally relevant concentrations (20, 200, and 2000 ng/L) of 17 beta-trenbolone for four weeks. As evidenced by the increased caudal fin index and anal fins developing into gonopodium-like structures, exposed females displayed masculinized secondary sexual characteristics. Differential gene expression and subsequent pathway analysis of mRNA sequencing data revealed that the transcription of transforming growth factor beta/bone morphogenetic protein signaling pathway and Wnt signaling pathway were upregulated following 17 beta-trenbolone exposure. Enzyme-linked immunosorbent assays showed that the bone morphogenetic protein 7 protein content was elevated after 17 beta-trenbolone exposure. Finally, real-time PCR revealed that 17 beta-trenbolone treatment significantly increased androgen receptor mRNA levels, and molecular docking showed potent interaction between 17 beta-trenbolone and guppy androgen receptor. Furthermore, 17 beta-trenbolone-induced masculinization of caudal and anal fins in female guppies, concomitant to the upregulated expression of differentially expressed genes involved in the above-mentioned two signaling pathways, was significantly inhibited by flutamide (androgen receptor antagonist). These findings demonstrated that 17 beta-trenbolone masculinized fins of female guppies by activating the androgen receptor. This study revealed that 17 beta-trenbolone could upregulate signaling pathways related to fin growth and differentiation, and eventually cause caudal and anal fin masculinization in female guppies.