FHL2促进牛卵泡颗粒细胞对FSH反应敏感性的作用及机制研究

Follicle stimulating hormone (FSH) drives follicle development by binding to FSHR located on granulosa cells (GCs) to activate the downstream signaling pathways. Therefore, the expression abundance of FSHR in GCs determines the sensitivity of cell response to FSH stimulation and significantly influences the bovine reproductive performance and reproductive technologies outcome. Our previous study showed that, FHL2, a transcriptional cofactor, upregulated FSHR mRNA expression and activated the downstream PI3K/AKT signaling pathway in bovine GCs. We also found that FHL2 promoted FSH-induced secretion of estradiol in bovine GCs. These observations demonstrate that FHL2 may improve bovine FSH responsive sensitivity via promoting FSHR transcription and activating its downstream signaling pathway. In the proposed studies, we will use bovine GC as cellular model to uncover the molecular mechanisms underlying FHL2 regulation of FSHR expression by exploring FHL2 interaction proteins, validating FSHR potential transcriptional factors, and determining binding activity of FHL2 to specific transcriptional factor(s). Moreover, we will examine the role of FHL2 in the regulation FSHR downstream gene expression and signaling interaction network. In addition, we have designed experiments to figure out how FHL2 interacts with FSHR signaling pathway to regulate FSH-stimulated bovine GC proliferation and steroidogenesis. Finally, we will use transgenic mouse model (FHL2 knockout mouse) to illuminate the role of FHL2 in animal reproductive performance and FSH responsive sensitivity. In conclusion, accomplishment of the proposed studies will unveil the regulatory mechanism of FHL2 on bovine FSH responsive sensitivity, and will improve the follicle development mechanism, and facility the application of reproductive techniques and improve the efficiency of in vitro embryo production.

促卵泡素结合位于卵泡颗粒细胞的受体FSHR激活下游信号促进卵泡发育，FSHR表达丰度不同导致的促卵泡素反应敏感性差异影响牛繁殖性能与繁殖技术应用效果。前期研究表明，转录因子共结合子FHL2上调牛颗粒细胞FSHR表达，促进促卵泡素诱导的雌激素分泌，影响其下游PI3K/AKT信号转导，提示FHL2可能通过促进FSHR转录，影响其下游信号通路，调控牛颗粒细胞对促卵泡素的反应敏感性。本项目先以牛颗粒细胞为模型，筛选FHL2互作蛋白和FSHR转录因子，揭示FHL2结合转录因子调控FSHR表达的机制，研究FHL2对FSHR下游基因表达及信号通路网络的调控作用，探明FHL2通过FSHR对促卵泡素诱导的颗粒细胞增殖、类固醇激素分泌的影响；再利用已构建的FHL2敲除小鼠模型揭示其对动物繁殖性能及促卵泡素反应敏感性的影响。项目研究成果对完善卵泡发育调控网络、促进繁殖技术应用及体外胚生产具有重要理论与现实意义。

促卵泡素结合位于卵泡颗粒细胞的受体FSHR激活下游信号促进卵泡发育，FSHR表达丰度及下游信号通路传导影响母畜对促卵泡素反应敏感性，从而影响牛繁殖性能与繁殖技术应用效果。本项目研究表明，FHL2正向调控颗粒细胞对FSH的反应敏感性，FHL2缺失抑制卵泡发育，FHL2不仅调控FSHR表达，而且调控了FSHR共激活的RTKs及其下游的多条信号通路，此外，FHL2还调控了FSHR下游的多个转录因子。主要研究结果包括：⑴FHL2在牛卵泡颗粒细胞中高表达，对卵泡颗粒细胞的增殖、凋亡和激素分泌具有重要调控作用；⑵FHL2可在细胞和个体水平调控颗粒细胞对FSH的反应敏感性，FHL2敲低不仅影响FSH诱导的颗粒细胞类固醇激素分泌水平、类固醇合成酶等的表达，还显著降低促性腺激素诱导的超数排卵效果；⑶FSH/FSHR除可激活经典的cAMP/PKA/CREB等通路外，还可高效共激活多个RTKs通路，其中较为重要的有EGFR、FGFR等；FSH还可通过PKA或RTKs通路，激活下游PI3K/AKT、MAPK/ERK等通路，及AP-1、EGR家族、NR4A家族转录因子等，促进卵泡发育；⑷FHL2可作为转录因子共激活子，对卵泡颗粒细胞FSHR受体的表达具有重要的正向调控作用；进一步研究证实，FHL2不仅作为转录因子共结合子调控了FSHR的表达，还调控了FSHR下游多个信号通路，包括cAMP/PKA、雌激素信号通路、类固醇合成等与激素分泌等相关的信号通路，及FSH共激活的Ras、EGFR、FGFR等RTKs通路，以及再下游的MAPK、mTOR、Hippo等信号通路，并能够调控FSH下游早期响应因子，如AP-1、EGR、NR4A等重要转录因子；⑸FHL2敲除小鼠表现为初情期延迟、发情周期紊乱，卵泡发育受阻，生长卵泡数量低；繁殖力测试结果表明，成年敲除雌鼠窝产仔数、产仔总数等繁殖力指标显著低于野生型；机制研究表明，敲除小鼠FSHR、EGFR、YAP1等关键基因下调，FSHR下游多条通路受抑制；⑹基于理论成果，建立了母牛发情排卵调控新技术。本项目累计发表论文10篇，其中SCI论文8篇，优秀会议论文1篇，获授权专利3项。项目研究成果进一步丰富了卵泡发育调控网络，并建立了母牛卵泡发育调控新技术在全国进行了推广应用，提高了母牛繁殖率。