Abstract
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 achieved in vivo through social manipulation, inhibition of aromatase activity, or steroid treatment. However, the induction of sex change in vitro has been largely unexplored. In this study, we established an in vitro culture 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 change in vitro. The in vitro gonadal 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.
Highlights
In most vertebrates, sex is fixed after birth and remains the same throughout life [1]
Histological analysis of day 0 reference tissues (D0) reference tissues confirmed that all 5 Initial phase (IP) spotty wrasse captured were female
Atresia of previtellogenic oocytes (PVO) appeared to be a background effect of the in vitro culture [8]. This was reflected in reduced oocyte surface area in controls compared to D0 reference tissues, with the proportion of total tissue area filled with non-atretic PVO being significantly higher in D0 (73.6 ± 2.4 %) versus control ovaries (31.8 ± 3.7 %) (Figure 2A)
Summary
Sex is fixed after birth and remains the same throughout life [1]. Develop as one sex but remain able to change to the other during adulthood [2]. In vitro culture manipulations enable precise monitoring of the effects of biotic and abiotic factors (e.g. temperature, pH, microplastics, etc.) on gonadal development, but are underutilised in aquaculture and fisheries research. Ex vivo approaches show particular potential to study responses of gonadal tissue to sex steroids and other hormones (e.g. glucocorticoids). With the exception of a conference abstract on the effects of several hormones on the gonadal architecture of sex-changing three-spot wrasse (Halichoeres trimaculatus [8]), explant culture systems for the study of sex change in fish remain unreported
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