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

Zebrafish are increasingly used as experimental models in studies of human disease, environmental toxicology, and reproductive biology. However, sex differences in hormone production are rarely examined, despite evidence from gene mutation studies indicating differential effects in females and males. The emerging reproductive peptide secretoneurin (SN) has not been quantitatively compared between sexes in any species. Here, we employed a newly developed extraction and LC-MS method to simultaneously quantify and compare levels of five steroids and thirteen peptides in brain, pituitary, and gonads. As expected, testosterone (T) and 11-ketotestosterone (11-KT) were higher in male tissues, while estrone (E1) and estradiol (E2) were elevated in the female pituitary/ovary and brain, respectively. Estriol (E3) 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 also showed higher levels in testes. Similarly, 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 future studies on sexually differentiated neuropeptide production and function across tissues.