Low concentrations of 17β-trenbolone induce female-to-male reversal and mortality in the frog Pelophylax nigromaculatus.

Trenbolone, as a growth promoter in animal agriculture, has become an environmental androgen in surface water. Here, we aimed to reveal the effects of 17β-trenbolone on survival, growth, and gonadal differentiation in the frog Pelophylax nigromaculatus, which is widespread in East Asia and undergoing population decline. P. nigromaculatus tadpoles were exposed to 17β-trenbolone (0.1, 1, 10 μg/L) from Gosner stage 24/25 to complete metamorphosis. We found that 17β-trenbolone resulted in significantly high mortality in a concentration-dependent manner, with a decrease in body weight in the high concentration group compared with the solvent control. Based on gross gonadal morphology, no females were observed, instead of about 15% ambiguous sexes and 85% males, in all 17β-trenbolone treatment groups. Like normal testes, the gonads with sex-ambiguous morphology exhibited testicular histology, showing that the sex-ambiguous gonads were incomplete ovary-to-testis reversals (IOTTRs) with certain ovarian morphological features. In the IOTTRs, the transcriptional levels of ovary-biased genes decreased drastically relative to normal ovaries, and even declined to the levels in normal testes. These observations confirmed that all test concentrations of 17β-trenbolone resulted in 100% sex reversal, although some sex-reversed testes retained some ovarian characteristics at the morphological level. To our knowledge, this is the first report strongly demonstrating that trenbolone can cause female-to-male reversal in amphibians. Given that the lowest concentration tested is environmentally relevant, our study highlights the risks of trenbolone and other environmental androgens for P. nigromaculatus and other amphibians, in particular the species with high sensitivity of gonadal differentiation to androgenic chemicals.