To confirm the masculinization of XX medaka by oxidative stress, H2O2 treatments were performed with 0.75 and 2 mM H2O2 (purity 30%, CAS RN: 7722-84-1; Wako Pure Chemical, Osaka, Japan) using wild-type larvae from 0 to 5 days post-hatching (dph) in 6-well culture plates (Corning, Glendale, AZ) with the water being changed daily. A rescue test to reduce oxidative stress through antioxidant supplementation was conducted with either 1 or 10 μM N-acetyl-L-cysteine (NAC; Wako) dissolved in Dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, MO) as previously described (20 (link)), using larvae from 0 to 5 dph in 6-well culture plates with the water being changed daily.
To investigate whether other XX medaka lines undergo masculinization from oxidative stress, pparaa KO, and gsdf KO medaka larvae were treated with 2 mM H2O2 under the conditions outlined above. Finally, treatment with 5 μM metyrapone (Sigma-Aldrich) dissolved in ethanol (Wako) as previously described (17 (link)), was used in conjunction with 2 mM H2O2 in FLFII medaka larvae to investigate the relationship between cortisol and oxidative stress-induced masculinization. The survival rates and the body sizes in adults are shown inSupplementary Tables S1 S2
To investigate whether other XX medaka lines undergo masculinization from oxidative stress, pparaa KO, and gsdf KO medaka larvae were treated with 2 mM H2O2 under the conditions outlined above. Finally, treatment with 5 μM metyrapone (Sigma-Aldrich) dissolved in ethanol (Wako) as previously described (17 (link)), was used in conjunction with 2 mM H2O2 in FLFII medaka larvae to investigate the relationship between cortisol and oxidative stress-induced masculinization. The survival rates and the body sizes in adults are shown in
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