Scientists studied how fish gonads develop and found that many genes and pathways, including oxytocin signaling, are involved in early sex differentiation. The work is basic research on fish, not a human clinical trial or a protocol you can apply.

The molecular mechanisms underlying gonadal differentiation in vertebrates have long intrigued researchers. However, studies in this field have predominantly focused on mammals, with limited attention given to non-mammalian vertebrates, particularly at the cellular level. To address this knowledge gap, we selected the Chinese tongue sole as our research model. We collected samples from six developmental stages of ovaries and testes and performed in-depth transcriptomic analyses of 123,344 single-cell nuclei. This study identified 34 major cell types, including five gonadal cell types, enabling us to outline the early sex differentiation roadmap in a non-model teleost species. Both somatic and germ cells in the gonads were systematically studied using bioinformatics methods. Numerous sex-biased genes and markers were identified and experimentally validated, contributing to our understanding of the dynamic development and differentiation processes of gonadal supporting cells and germ cells in teleosts. Weighted Gene Co-expression Network Analysis (WGCNA) networks emphasized the critical roles of foxl2a and dmrt1 in sex differentiation. Pseudotime analysis revealed the early differentiation processes between male and female germ cells. We discovered a new germ cell marker gene, gpat2, and elucidated the differentiation pathway of gonadal stem cells in teleost fish. Our results highlight the significance of pathways such as oocyte meiosis, Target of Rapamycin (TOR) complex, oxytocin signaling, cGMP-PKG signaling, and arginine signaling in the differentiation of oocytes and spermatogonial stem cells. Cell-cell communication analyses further revealed interactions among different gonadal cell types, identifying the WNT and Notch pathways as crucial for the development of female and male gonads. Furthermore, we verified a feedback loop between dmrt1 and zfpm2 for the first time in teleosts, suggesting potential roles for zfpm2 in the formation and development of the teleost testis. Our findings provide a comprehensive transcriptomic resource for investigating the early sex differentiation processes of teleosts at the single-cell level and bridge the knowledge gap in research on non-mammalian vertebrates.