Male-biased zebrafish sex differentiation and metabolomics profile changes caused by dydrogesterone

Some progestins, including the widely used dydrogesterone (DDG), have been shown to cause male-biased sex ratio in teleost. However, there is a gap to fully understand the mechanisms of the sex differentiation disturbance by progestins, particularly from the metabolic aspect. We thus aimed to examine the sex changes by exposing zebrafish embryos to 4.4 (L), 44 (M) and 440 (H) ng/L DDG for up to 140 days, and investigated metabolomic profile changes during the critical period of sex differentiation at fry stage (35 dpf). DDG increased the percentage of male zebrafish in a dose-dependent manner, with 98% male fish in the high concentration group. In zebrafish fry, DDG increased the levels of some free fatty acids, monoglycerides, acylcarnitines, organic acids, free amino acids, while decreased lysophospholipids, uric acid and bile acids. DDG exposure also decreased the nucleoside monophosphates and UDP-sugars while increased nucleosides and their bases. These metabolite changes, namely increase in n-3 PUFAs (polyunsaturated fatty acids), myo-inositol, taurine, palmitoleic acid, oleic acid, lactic acid, fumaric acid, and uracil, and decrease in uric acid and bile acids, might account for the male-biased sex ratio in zebrafish. It appears that many of these metabolites could inhibit several pathways that regulate zebrafish gonad differentiation, including NF-κB/COX-2 and Wnt/β-catenin pathways, and activate p53 pathway. Thus we proposed a hypothesis that DDG might induce oocytes apoptosis through the above pathways and finally lead to female-to-male sex reversal. The results from this study suggest that DDG at environmentally relevant concentrations could affect zebrafish metabolomic profiles and finally disturb fish sex differentiation.