Gonadectomy, which was shown to drastically reduce circulating sex steroids for at least 4 months (25 ), was performed in male and female medaka as previously described (26 (link)). Briefly, after anesthesia using 0.02% tricaine methanesulfonate (MS222; Sigma), fertile males and females (indicated by spawning and/or the existence of oviposited eggs in the females) were incised to access the intraperitoneal cavity. We entirely removed the gonads, and the incision was sutured using a nylon thread (10-0 USP; Crownjun). The fish were kept in recovery medium (0.9% NaCl) for 3 days before experiment.
Sex steroids were supplemented via feeding as demonstrated previously (16 ), with some adjustments. The sex steroid–containing feed was prepared by mixing 5 mg of dry feed with 10 to 100 ng of 11-KT or E2 (Sigma) using 96% ethanol as the vehicle and then evaporated at 40 °C overnight. Due to the short half-life of sex steroids in prepared feeds (16 ), the fish were fed 5 hours before and sampled 2 hours after the onset of the dark phase (night). We performed the tests in tanks with or without water recirculation. Supplementation with 30 ng of sex steroids/day was found to closely mimic the circulating sex steroid level in medaka in a circulating system (Figure S1 (27 (link))) and thus was used for the rest of the study. After postsurgery recovery, each fish was fed with sex steroid–supplemented or control feed (with vehicle alone) once a day for 3 (fluorescence in situ hybridization [FISH] and immunofluorescence) or 5 (quantitative polymerase chain reaction [qPCR] and enzyme-linked immunosorbent assay [ELISA]) days.
Sex steroids were supplemented via feeding as demonstrated previously (16 ), with some adjustments. The sex steroid–containing feed was prepared by mixing 5 mg of dry feed with 10 to 100 ng of 11-KT or E2 (Sigma) using 96% ethanol as the vehicle and then evaporated at 40 °C overnight. Due to the short half-life of sex steroids in prepared feeds (16 ), the fish were fed 5 hours before and sampled 2 hours after the onset of the dark phase (night). We performed the tests in tanks with or without water recirculation. Supplementation with 30 ng of sex steroids/day was found to closely mimic the circulating sex steroid level in medaka in a circulating system (Figure S1 (27 (link))) and thus was used for the rest of the study. After postsurgery recovery, each fish was fed with sex steroid–supplemented or control feed (with vehicle alone) once a day for 3 (fluorescence in situ hybridization [FISH] and immunofluorescence) or 5 (quantitative polymerase chain reaction [qPCR] and enzyme-linked immunosorbent assay [ELISA]) days.
