Oestrogen synthesis mediated by Hsd17b12 isoforms and dynamic regulation by Cyp19a in the olive flounder...

17β-oestradiol (E2) is essential for ovarian development. In teleosts, the current understanding of oestrogen synthesis primarily focuses on Cyp19a, which catalyses the synthesis of E2 from testosterone (T). In contrast, the conversion of oestrone (E1)-to-E2, mediated by Hsd17b12, and the role of this process in ovarian development remain understudied. This study investigated two Hsd17b12 isoforms in the commercially cultured fish, olive flounder (Paralichthys olivaceus). In vitro ovarian assays revealed isoform-specific functions. Hsd17b12a preferentially mediated E2 biosynthesis, whereas Hsd17b12b regulated T metabolism. Subsequent detection in HEK293T cells indicated that Hsd17b12a catalyses E1-to-E2 conversion, whereas Hsd17b12b mediates T-to-androstenedione (A) conversion. Site-directed mutagenesis targeting the conserved YxxxK catalytic motif showed that an alanine-to-serine substitution in Hsd17b12a and a serine-to-threonine substitution in Hsd17b12b significantly reduced enzymatic activity. In vivo overexpression of Hsd17b12a and -12b in the flounder ovaries revealed distinct phenotypes. Hsd17b12a overexpression elevated A and E1 levels without inducing histological changes. In contrast, Hsd17b12b overexpression induced proliferation of oogonium-like cells, significantly upregulated the expression of cyp26b1 and vasa, and increased A and E2 levels. Co-immunoprecipitation assays showed an interaction between Cyp19a and Hsd17b12a, and in vitro experiments showed co-expression of Hsd17b12b and cyp19a. These findings clarify the roles of the flounder Hsd17b12a and -12b in steroidogenesis, and, for the first time, their interaction with Cyp19a in fish.