Phenotypical Rescue of Bmp15 Deficiency by Mutation of Inhibinα(inha) Provides Novel Clues to How Bmp15 Controls Zebrafish Folliculogenesis

AbstractAs an oocyte-specific growth factor, bone morphogenetic protein 15 (BMP15) plays a critical role in controlling folliculogenesis. However, the mechanism of BMP15 action remains elusive. Using zebrafish as the model, we created abmp15mutant using CRISPR/Cas9 and demonstrated thatbmp15deficiency caused a significant delay in follicle activation and puberty onset followed by complete arrest of follicle development at previtellogenic stage without yolk accumulation. The mutant females eventually underwent female-to-male sex reversal to become functional males, which was accompanied by a series of changes in secondary sexual characteristics. Interestingly, the blockade of folliculogenesis and sex reversal inbmp15mutant could be rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic stage with yolk accumulation and the fish maintained their femaleness without sex reversal. Transcriptome analysis revealed up-regulation of pathways related to TGF-β signaling and endocytosis in the double mutant follicles. Intriguingly, the expression of inhibin/activin βAa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout ofinhbaain the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again in the oocytes although the follicles could continue to grow beyond the size range of previtellogenic stage. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly inbmp15single mutant females, returned to normal in the double mutant (bmp15-/-;inha-/-), but reduced again significantly in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-). E2 treatment could rescue the vitellogenic follicles inbmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation inbmp15-/-;inha-/-fish. In summary, the present study provided comprehensive genetic evidence for the interaction ofbmp15pathways and the activin-inhibin system in regulating folliculogenesis, in particular E2 production from the follicle, Vtg biosynthesis in the liver and its update by the developing oocytes.