Transcriptome Analysis of 17α-Methyltestosterone-Induced Sex Reversal in Pseudopleuronectes yokohamae

Marbled flounder (Pseudopleuronectes yokohamae) exhibits a distinct female growth advantage and an XX/XY sex determination system. To exploit these traits, we investigated 17α-methyltestosterone (MT)-induced transcriptomic changes in gonadal tissue with the goal of generating pseudomale XX broodstock for all-female fry production. Full-sibling diploid juveniles (60 days post-hatching, dph) were fed diets containing 0 (control), 0.5, or 2 mg/kg MT for 120 days, followed by a 60-day recovery period on a commercial diet prior to sampling. Testicular transcriptomes were profiled via high-throughput sequencing, and key differentially expressed genes were validated using qPCR. Both MT treatments resulted in 100% masculinization. Testicular transcriptome analysis revealed 972 differentially expressed genes (DEGs) (180 up, 792 down) in the 0.5 mg/kg MT-treated males (MT05M) compared to the control males, and 1245 DEGs (842 up, 403 down) in the 2 mg/kg MT group (MT20M). Gene Ontology terms were enriched for extracellular space and signaling receptor regulator activity. KEGG pathway analysis indicated significant enrichment in neuroactive ligand–receptor interaction, ovarian steroidogenesis, and TGF-β signaling. qPCR confirmed significant downregulation (p < 0.05) of sox17, bmp4, and smad6, while dmrt1 was downregulated only in the MT20M group. These findings demonstrate that MT effectively masculinizes P. yokohamae by modulating key sex-related genes and signaling pathways, providing a transcriptomic foundation and potential mechanistic insights for optimizing pseudomale induction to enable all-female aquaculture production.