Expression analysis of growth differentiation factor 9 (Gdf9/gdf9), anti-müllerian hormone (Amh/amh) and aromatase (Cyp19a1a/cyp19a1a) during gonadal differentiation of the zebrafish, Danio rerio.

In the zebrafish, no sex-determining gene has been identified, while some sex-related genes, such as cyp19a1a and amh, show sexually dimorphic expression. Interestingly, most of these genes are expressed in the somatic cells. With increasing evidence suggesting roles of germ cells in gonadal differentiation, there is an increasing interest in the factors released by the germ cells for the bidirectional communication between the two compartments. We have reported that Gdf9/gdf9 is an oocyte-specific factor in the zebrafish, similar to that of mammals. Whether and how Gdf9 is involved in gonadal differentiation is unknown. In this study, we compared the expression levels of gdf9, cyp19a1a, and amh among several other sex-related genes in the gonads before, during, and after sex differentiation. The expression of gdf9 started in the gonads before sex differentiation, and its level surged in the differentiated ovary. Its expression pattern was similar to that of cyp19a1a, but reciprocal to amh expression. Using recombinant zebrafish Gdf9 (rzfGdf9), we further showed that Gdf9 significantly suppressed the expression of amh while increased that of activin beta subunits (inhbaa and inhbb) in vitro. Although gdf9 and cyp19a1a showed co-expression during gonadal differentiation, we only observed a slight but not significant response of cyp19a1a to rzfGdf9. Knocking down the expression of gdf9 and cyp19a1a with vivo-morpholinos caused a male-skewed sex ratio. Our data suggested that Gdf9 is likely involved in promoting oocyte/ovary differentiation in the zebrafish and it may act by suppressing amh expression, at least partly, in the somatic cells.