Anin vitroovarian explant culture system to examine sex change in a hermaphroditic fish

Many teleost fishes undergo natural sex change, and elucidating the physiological and molecular controls of this process offers unique opportunities not only to develop methods of controlling sex in aquaculture settings, but to better understand vertebrate sexual development more broadly. Induction of sex change in some sequentially hermaphroditic or gonochoristic fish can be achievedin vivothrough social manipulation, inhibition of aromatase activity, and steroid treatment. However, the induction of sex changein vitrohas been largely unexplored. In this study, we established anin vitroculture system for ovarian explants in serum-free medium for a model sequential hermaphrodite, the New Zealand spotty wrasse (Notolabrus celidotus). This culture technique enabled evaluating the effect of various treatments with 17β-estradiol (E₂), 11-ketotestosterone (11KT) or cortisol (CORT) on spotty wrasse ovarian architecture for 21 days. A quantitative approach to measuring the degree of ovarian atresia within histological images was also developed, using pixel-based machine learning software. Ovarian atresia likely due to culture was observed across all treatments including no-hormone controls, but was minimised with treatment of at least 10 ng/mL E₂. Neither 11KT nor CORT administration induced proliferation of spermatogonia (i.e. sex change) in the cultured ovaries indicating culture beyond 21 days may be needed to induce sex changein vitro. Thein vitrogonadal culture and analysis systems established here enable future studies investigating the paracrine role of sex steroids, glucocorticoids and a variety of other factors during gonadal sex change in fish.