The miR-200 Family Targeting <i>amh</i> Affects the Gonadal Development of Japanese Flounder

Four members of the miR-200 family in Japanese flounder (Paralichthys olivaceus) have sex-biased expression patterns, but their target genes and how they work in the development of the gonads are rarely known. Anti-Müllerian hormone (AMH) can inhibit the development of Muller’s duct in female mammals and regulate the formation of gametes after sexual maturity. There is no Muller’s duct in teleosts, but the amh gene still exists. Knockout of amh results in sex reversal from male to female. Therefore, it is essential to explore the relationship between the miR-200 family and amh to clarify what role miR-200 plays in the development of the gonads. In Japanese flounder, the two binding sites for the miR-200 family in the 3′UTR of amh were found through bioinformatic prediction. Double luciferase and green fluorescent protein reporter experiments demonstrated amh to be directly targeted by miR-200a and miR-200b. Moreover, miR-200a and miR-200b reduced the expression of amh through site 1 rather than site 2. To explore the regulatory role of miR-200a in gonadal development, we further overexpressed miR-200a in the primary Sertoli cells of the testis. With the overexpression of miR-200a, the expression of amh decreased, while the expression of the other two male sex-related genes, dmrt1 (doublesex and mab-3 related transcription factor 1) and gsdf (diagonal soma driven factor), increased significantly. This result indicates that the miR-200 family regulates the gonadal differentiation and development by targeting amh in Japanese flounder