Triploidy alters hormonal and paracrine signaling to promote male development in zebrafish

Sex differentiation in zebrafish is governed by a complex interplay of genetic and endocrine signals. Triploid zebrafish, which are largely sterile, consistently develop as males, but the underlying mechanisms remain elusive. Here, we combined histological and transcriptomic analyses to examine how triploidy and exposure to 17α-ethinylestradiol (EE2) modulate sex differentiation in zebrafish. Triploidy disrupted hormonal and paracrine signaling, with downregulation of fshr and amh, upregulation of igf3, potential activation of β-catenin pathway, and suppression of ptger2a and dio1, resulting in complete masculinization. In diploids, EE2 exposure resulted in a wide range of gonadal phenotypes, from testes and ovotestes to fully developed ovaries, reflecting the complexity and variable sensitivity of zebrafish sex differentiation to hormonal stimuli. Potential mechanistic insights underlying these outcomes are provided. By contrast, long exposure of triploid zebrafish to EE2 promoted the expansion of early germ cells, but failed to induce ovarian differentiation, suggesting a fixed male trajectory induced by triploidy. Triploids also showed a distinct endocrine state, lacking the EE2-induced suppression of cyp11c1 observed in diploids, suggesting altered corticosteroid homeostasis that may reinforce masculinization. Both triploidy and EE2 administration altered meiosis and spermiogenesis, consistent with the downregulation of klhl10 and constrained retinoic acid signaling through dhrs3a and/or cyp26b1. At the molecular level, both triploidy and EE2 converged on suppression of early steroidogenic genes, including star and cyp11a1, indicating limited androgen and estrogen biosynthesis. Together, these findings reveal how triploidy reshapes endocrine regulation and responsiveness and reveal shared and unique molecular pathways by which EE2 influences zebrafish gonadal fate.