dmrt1 Is Responsible for Androgen-Induced Masculinization in Nile Tilapia

17α-Methyltestosterone (MT) is a widely used androgen for all-male fish production in aquaculture. However, the molecular mechanism underlying MT-induced masculinization remains unclear. In this study, we aim to identify the key gene responsible for MT-induced masculinization using the Nile tilapia (<i>Oreochromis niloticus</i>) <i>amhy</i>, <i>dmrt1</i>, and <i>gsdf</i> mutants, which exhibit male-to-female sex reversal. Nile tilapia fry from these three mutant lines were treated with 50 μg/g MT from 5 to 30 days after hatching (dah). The results showed that <i>amhy</i> and <i>gsdf</i> mutants, but not <i>dmrt1</i> mutants, were masculinized by the MT treatment. Gonadal transcriptome analysis revealed that genes involved in steroidogenesis and germ cell development in MT-treated <i>dmrt1</i> mutants exhibited a similar expression pattern to that of the wild type (WT) XX. In addition, the <i>dmrt1</i> mutants cannot be masculinized by co-treatment with MT and the aromatase inhibitor fadrozole. The MT treatment completely blocked early steroidogenic enzyme (Star2, Cyp17a2, and Cyp19a1a) expression independent of <i>amhy</i>, <i>gsdf</i>, and <i>dmrt1</i>. A luciferase analysis showed that MT directly suppressed basal and Sf-1-activated <i>cyp19a1a</i> promoter activity through <i>ara</i> and <i>arb</i> in cultured HEK293 cells. Furthermore, MT treatment inhibited germ cell proliferation in <i>amhy</i> and <i>gsdf</i> mutants but not in <i>dmrt1</i> mutants. Consistently, <i>dmrt1</i> expression was induced in MT-treated WT XX, -<i>amhy</i>, and -<i>gsdf</i> mutants. Taken together, these results suggest that <i>dmrt1</i> is indispensable for MT-induced masculinization in Nile tilapia and that MT functions by inhibiting early steroid synthesis and activating <i>dmrt1</i> to promote testis development.