As 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 a bmp15 mutant using CRISPR/Cas9 and demonstrated that bmp15 deficiency caused a significant delay in follicle activation and puberty onset followed by a complete arrest of follicle development at previtellogenic (PV) 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 in bmp15 mutant could be partially rescued by the loss of inhibin (inha-/-). The follicles of double mutant (bmp15-/-;inha-/-) could progress to mid-vitellogenic (MV) 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. Interestingly, the expression of inhibin/activin βAa subunit (inhbaa) increased significantly in the double mutant ovary. Further knockout of inhbaa in the triple mutant (bmp15-/-;inha-/-;inhbaa-/-) resulted in the loss of yolk granules again. The serum levels of estradiol (E2) and vitellogenin (Vtg) both decreased significantly in bmp15 single mutant females (bmp15-/-), 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 arrested follicles in bmp15-/-, and fadrozole (a nonsteroidal aromatase inhibitor) treatment blocked yolk accumulation in bmp15-/-;inha-/- fish. The loss of inhbaa also caused a reduction of Vtg receptor-like molecules (e.g., lrp1ab and lrp2a). In summary, the present study provided comprehensive genetic evidence that Bmp15 acts together with the activin-inhibin system in the follicle to control E2 production from the follicle, Vtg biosynthesis in the liver and its uptake by the developing oocytes.

中文翻译：

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通过 inha 突变挽救斑马鱼的 bmp15 缺陷揭示了 BMP15 调节卵泡发生的机制。

作为卵母细胞特异性生长因子，骨形态发生蛋白 15 (BMP15) 在控制卵泡发生中发挥着关键作用。然而，BMP15 的作用机制仍不清楚。以斑马鱼为模型，我们使用 CRISPR/Cas9 创建了 bmp15 突变体，并证明 bmp15 缺陷导致卵泡激活和青春期开始显着延迟，随后卵泡发育完全停滞在卵黄发生前 (PV) 阶段，而没有卵黄积累。突变的雌性最终经历了雌雄逆转，成为功能性雄性，同时伴随着第二性征的一系列变化。有趣的是，bmp15 突变体中卵泡发生和性逆转的阻断可以通过抑制素 (inha-/-) 的损失来部分挽救。双突变体(bmp15-/-;inha-/-)的卵泡可以进展到卵黄发生中期(MV)阶段，并有卵黄积累，并且鱼保持其雌性而没有性逆转。转录组分析显示双突变卵泡中与 TGF-β 信号传导和内吞作用相关的途径上调。有趣的是，双突变体卵巢中抑制素/激活素βAa亚基（inhbaa）的表达显着增加。在三重突变体（bmp15-/-；inha-/-；inhbaa-/-）中进一步敲除inhbaa会导致卵黄颗粒再次丢失。bmp15单突变雌性(bmp15-/-)中雌二醇(E2)和卵黄蛋白原(Vtg)的血清水平均显着下降，双突变体(bmp15-/-;inha-/-)中恢复正常，但再次降低在三重突变体（bmp15-/-；inha-/-；inhbaa-/-）中显着。E2 处理可以挽救 bmp15-/- 中停滞的卵泡，法屈唑（一种非甾体芳香酶抑制剂）处理可以阻止 bmp15-/-;inha-/- 鱼中的卵黄积累。inhbaa 的缺失还导致 Vtg 受体样分子（例如 lrp1ab 和 lrp2a）的减少。总之，本研究提供了全面的遗传证据，表明 Bmp15 与卵泡中的激活素抑制素系统共同作用，控制卵泡中 E2 的产生、肝脏中 Vtg 的生物合成以及发育中卵母细胞的摄取。