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To examine the effect and mechanism of thyroid hormone on gonadal sex differentiation,  Takifugu rubripes  larvae were treated with goitrogen (methimazole, MET, 1000 g/g), and thyroxine (T4, 2nM) from 25 to 80 days after hatching (dah). Gonadal histology and sex ratios of fish were then determined at 80 dah. MET treatment induced masculinization, but T4 treatment did not induce feminization in  T. rubripes  larvae. Transcriptomic analysis of gonads at 80 dah was then conducted. Among the large number of differentially expressed genes between the groups, the expression of  foxl2 ,  cyp19a1a , and  dmrt1  was altered. The expression of  foxl2 ,  cyp19a1a ,  dmrt1  and  gsdf  at 25, 40, 55 days after treatment (dat) was further analyzed by qPCR. MET treatment suppressed the expression of  foxl2  and  cyp19a1a , and induced the expression of  dmrt1  in genetic females ( p  < 0.05). Additionally, T4 treatment induced an increase in the expression of  cyp19a1a  in genetic XY gonads only at 25 dat. However, the increase in  cyp19a1a  expression did not continue to 40 and 55 dat. This may explain why feminization of larvae was not found in the T4-treated group. Thus, the present study provides the first evidence that MET treatment causes masculinization in teleost fish. The effects of MET-induced masculinization in  T. rubripes  may act primarily  via  suppression of the expression of  foxl2  and  cyp19a1a , and stimulation of the expression of  dmrt1 . Moreover, the effects of higher concentrations of T4 or different concentrations of T3, on sex differentiation require further testing.

Effects of the Thyroid Endocrine System on Gonadal Sex Ratios and Sex-Related Gene Expression in the Pufferfish Takifugu rubripes