Synergistic role of β-catenin1 and 2 in ovarian differentiation and maintenance of female pathway in Nile tilapia

• Two β-cats were identified from Nile tilapia, their cellular localization was confirmed in teleosts ovary.
• Fish β-cat1 activates the TOPFlash reporter, whereas β-cat2 does not.
• Inhibition of the canonical Wnt/β-cat signal pathway cause the female gonad masculinization.
• Two β-cats probably play synergistic roles in ovarian differentiation and repression male pathway gene expression in tilapia.

Two β-catenin (β-cat) genes exist in teleosts but little is known about their expression and function in ovarian development. We identified β-cat1 and β-cat2 from the Nile tilapia. β-cat1 and β-cat2 displayed a similar expression pattern in the ovary during development, and were mainly expressed in the oogonia and oocytes. In luciferase assays, β-cat1 activated the TOPFlash reporter dose-dependently, whereas β-cat2 failed to do so. Cotransfection of β-cat1 and β-cat2 synergistically enhanced the expression of the reporter. A specific interaction between β-cat1 and β-cat2 was also observed in a mammalian two-hybrid assay. Furthermore, tilapia recombinant Dkk1, an inhibitor of the β-cat pathway, decreased β-cat1 and β-cat2, while increased sox9, dmrt1, cyp11b2 and foxl2 expression in the in vitro cultured tilapia ovary, which could be abolished by simultaneous treatment with Bio, an agonist of β-cat. Consistently, β-cat1 or β-cat2 knockdown in XX fish by TALENs caused the retardation of ovarian differentiation and masculinization, as reflected by the upregulation of dmrt1, cyp11b2, sox9, and serum 11-KT level. On the contrary, serum E2 level was unchanged even though foxl2 transcription was upregulated. These data suggestes that both β-cat1 and β-cat2 are important members and play synergistic roles in the canonical Wnt signal pathway in fish. Independent of Foxl2-leading estrogen pathway, they might be involved in ovarian differentiation and repression of the male pathway gene expression in tilapia.