TGF-β信号通路是调控家畜卵巢颗粒细胞状态、卵泡发育命运和繁殖潜能的关键通路。前期研究发现在猪卵巢颗粒细胞中，SMAD4（TGF-β通路最后一个成员）可正反馈调控TGF-β通路，并与USP9X形成环路，且两者均可调控TGFBR2，因此我们推测SMAD4-USP9X环路可能通过TGFBR2参与SMAD4对TGF-β通路的反馈调控。基于以上假设，本项目拟采用ChIP和Co-IP等技术分析猪卵巢颗粒细胞中SMAD4-USP9X环路形成机制；采用过表达和Co-IP等技术分析SMAD4-USP9X轴通过影响TGFBR2去泛素化反馈调控TGF-β通路的表观遗传机制；采用EMSA和ChIP等方法分析USP9X-SMAD4轴通过miRNA-TGFBR2反馈调控TGF-β通路的表观遗传机制。研究结果对完善TGF-β通路的反馈调控，深入解析其调控猪卵泡发育机制，和最终阐明猪繁殖潜能的遗传机理等均有一定意义。

TGF-β signaling pathway plays an important role in ovarian granulosa cell status, follicular developmental fate and reproductive potential of domestic animals. In our previous studies, SMAD4 (the downstream molecule in the TGF-β pathway) was found as a positive feedback controller for the TGF-β pathway in the porcine ovarian granulosa cells, which could also form a loop with USP9X. Both SMAD4 and USP9X could modulate TGFBR2. Therefore, we speculate that SMAD4-USP9X loop may positively feedback to regulate TGF-β pathway mediated by TGFBR2. Based on the above hypothesis, the mechanism of SMAD4-USP9X loop formation will be analysed by ChIP and Co-IP in porcine ovarian granulosa cells. The overexpression and Co-IP techniques will be used to detect the effect of SMAD4-USP9X loop on the epigenetic mechanism of positive-feedback regulation for TGF-β pathway through the deubiquitination of TGFBR2. The epigenetic mechanism for positive-feedback regulation of TGF-β pathway will be also investigated via the USP9X-SMAD4 axis mediated by miRNA-TGFBR2 using EMSA and ChIP. This study will benefit the improvement for understanding the feedback regulation of TGF-β pathway, and further help to reveal the regulatory mechanism for follicular development and reproductive potential of pig.