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.

Triploid turbot (Scophthalmus maximus) exhibit superior growth and survival, yet the molecular basis of their sterility—a key trait for aquaculture—remains largely unexplored. This study investigated ovarian development and transcriptomic profiles in diploid and triploid S. maximus at three key stages (6, 10, and 20 months post-hatch, mph) to elucidate the stage-specific molecular mechanisms underlying triploid sterility. Histological analysis revealed that diploid ovaries progressed through normal oogenesis to the early vitellogenic stage by 20 mph, whereas triploid ovaries were arrested at the oogonial stage, with only occasional primary oocytes and extensive connective tissue infiltration. Comparative transcriptomic analysis identified 13,305, 14,599, and 13,331 differentially expressed genes (DEGs) between triploid and diploid ovaries at 6, 10, and 20 mph, respectively. Functional enrichment analysis showed that DEGs were significantly associated with meiotic processes, cell cycle regulation, energy metabolism, and apoptosis. Key meiotic genes (spo11, dmc1, sycp3) were consistently upregulated in triploids across all stages, while the DNA repair gene rad51 was paradoxically downregulated, indicating attempted but aberrant meiotic initiation. Oogenesis regulators (gdf9, bmp15, pou5f3) and energy metabolism genes (ndufa11, sdha, cox5a) were significantly downregulated, whereas apoptosis-related genes (eif2ak3, apaf1) were upregulated. Notably, KEGG pathway analysis revealed stage-specific shifts from stress-induced apoptosis and p53 signaling at early stages to proteasome activation at later stages, suggesting a transition from active germ cell elimination to maintenance of cellular homeostasis in developmentally arrested ovaries. Collectively, these findings demonstrate that triploid sterility is associated with coordinated dysregulation of meiotic progression, metabolic, and apoptotic pathways, providing a high-resolution molecular framework for understanding reproductive failure in triploid fish and informing strategies for optimizing triploid production in aquaculture.