Triploidy disrupts hormonal and paracrine signaling and enforces male development in zebrafish

Sex differentiation in zebrafish is governed by a complex interplay of genetic and endocrine cues. Triploid zebrafish, which are largely sterile, consistently develop as males, but the mechanisms remain elusive. Here, we combined histological and transcriptomic analyses to examine how triploidy and exposure to 17a-ethinyl estradiol (EE2) influence sex differentiation in zebrafish. Triploidy disrupted hormonal and paracrine signaling, with downregulation of fshr and amh, upregulation of igf3, suppression of ptger2a and dio1, collectively driving complete masculinization. In diploids, EE2 produced diverse gonadal outcomes, ranging from testes and ovotestes to fully differentiated ovaries. By contrast, EE2 promoted early germ cell proliferation in triploids but failed to induce ovarian differentiation, reinforcing a fixed male trajectory. Triplody and EE2 administration altered meiosis and spermatogenesis, consistent with 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, limiting androgen and estrogen biosynthesis. Together, these findings reveal how ploidy reshapes endocrine regulation and responsiveness and reveal shared and unique molecular pathways by which EE2 influences zebrafish gonadal fate. Overall design: exposure to 17a-ethinyl estradiol (EE2) in 2n and 3n zebrafish