Developmental Trajectory and Regulation of Primary Males in Giant Grouper (epinephelus Lanceolatus)

The shortage of male broodstock is a major bottleneck that hinders the sustainable development of giant grouper (Epinephelus lanceolatus) industry, making effective male induction techniques a priority. However, developing effective male induction techniques remains elusive because the molecular basis of sex determination and gonadal development in this species is poorly understood. Here, we systematically characterize the molecular basis underlying gonadal differentiation of E. lanceolatus. We present a comprehensive atlas detailing the developmental trajectory of both primary males and females, showing that the species exhibits typical diandric protogynous hermaphroditism. Specifically, a critical window of primary sex reversal occurs at approximately two years of age, during which around 38.9% of individuals develop into primary males without reaching functional female maturation. RNA-seq analysis identified a key regulatory network composed of dmrt1, amh, gsdf, inha, and igf3, that is involved in male development. Additionally, the G protein-adenylyl cyclase signaling pathway was identified to play a crucial role in primary male differentiation. Using single-molecule fluorescence in situ hybridization, we revealed the co-localization of zbtb26 and dmrt1 mRNAs in both germline stem cells and gonadal somatic cells. Dual-luciferase reporter assay demonstrated that Zbtb26 directly activates the dmrt1 promoter in a dose-dependent manner. ChIP assay further supported that Zbtb26 can bind to the predicted motif-containing region of the dmrt1 promoter. These results indicate that Zbtb26 may directly promote dmrt1 transcription, thereby contributing to primary male sex determination. In summary, this study advances the understanding of diandric protogynous hermaphroditism in E. lanceolatus and provides a foundation for the development of male induction techniques for this species.