Distinct sex differences in the production of steroids and neuropeptides in the adult zebrafish brain–pituitary–gonadal axis

Zebrafish are valuable models for studies of human disease, environmental toxicology, and reproductive biology. However, sex differences in hormone production are rarely examined, despite gene mutation studies showing differential effects in females and males. The emerging reproductive peptide secretoneurin (SN) has not been compared between sexes in any species. Using a novel extraction and liquid chromatography–mass spectrometry method, we simultaneously quantified five steroids and thirteen peptides in brain, pituitary, and gonads. Testosterone and 11-ketotestosterone were higher in male tissues; estrone and estradiol were elevated in the female pituitary, ovary, and brain, respectively. Estriol was more abundant in testes. Gonadotropin-releasing hormones Gnrh2 and Gnrh3 were notably higher in testes. Oxytocin (isotocin) and vasopressin (vasotocin) were elevated in the female brain and in testes. Kisspeptins 1 and 2 were higher in testes. SNa and SNb were more abundant in the female brain and pituitary, and markedly higher in testes than ovaries. Several smaller SN fragments were detected at low levels with patterns suggesting sex-specific enzymatic processing. These findings reveal pronounced sex differences in both classical and emerging reproductive hormones and identify the SN peptide family as a new component of the brain–pituitary–gonadal axis. This dataset provides a foundation for studies on sexually differentiated neuropeptide function across tissues.