小分子化合物在水牛原始卵泡体外激活中的作用及其分子机制的研究

As one of the top-ten scientific breakthroughs of 2016, artificial oocytes stand as one of the most attractive life-science research areas in the field of animal reproductive physiology. Activation of primordial follicles is the basis of follicular development where mTOR and PI3K/Akt pathways exist as two critical pathways for the activation of primordial follicles. These two pathways have parallel and synergistic characteristics,with no relationship between the upstream and downstream cascades. However, it remains unclear whether there is a primary and secondary point. This study follows nearly 30 years of buffalo reproductive physiology research as performed by our research group. The development of buffalo primordial follicles through mTOR and PI3K/Akt activation or inhibition were of interest in this study. This was accomplished through testing the concentration capability of agonists and antagonists of the aforementioned pathways.Various techniques including immunohistochemistry, RNA-Seq, QRT-PCR, and protein imprinting were employed to elucidate the molecular mechanisms of small molecular compounds regulating the development of buffalo primordial follicles. This study will enrich the molecular mechanism theory of follicular development of a single foetus of large livestock, such as buffalo, and establish a theoretical foundation for the full development and application of excellence in breeding livestock genetic resources of buffalo.

人造卵母细胞被《科学》评为2016年度十大科学突破之一，动物生殖生理研究也是生命科学研究中最具吸引力的领域之一。原始卵泡激活是动物卵泡发生的基础，mTOR通路与PI3K/Akt通路是原始卵泡激活的两条关键通路，具有平行和协同效应的特点，不存在上下游的级联关系，但是否存在主次之分仍不清楚。本研究拟在课题组从事水牛生殖生理研究近30 年的基础上，通过单独或联合添加mTOR和PI3K/Akt通路激活剂与抑制剂，比较不同添加浓度和配伍方式对水牛卵泡发育的影响，建立小分子化合物促进水牛原始卵泡体外激活的最佳方案；在此基础上，应用免疫组化、RNA-Seq、QRT-PCR和蛋白印迹技术，阐明小分子化合物调节水牛原始卵泡发育的分子机制。本研究将丰富水牛等单胎大家畜卵泡发育的分子机制理论，并为水牛优良种畜遗传资源的充分开发应用奠定理论基础。

水牛繁殖力低是水牛产业面临的突出问题之一。通过获取卵母细胞，结合繁殖技术可提高水牛繁殖率。但通过超排仅可获得2-5枚水牛卵母细胞。原始卵泡激活是卵泡发生的基础，PI3K/Akt与mTOR是原始卵泡激活的关键通路，具平行或协同效应，但是否存在主次效应仍不清楚。本研究首先建立水牛卵巢皮质原始卵泡体外激活培养体系。确定采用mTOR激活剂200 μM phosphatidic acid、50μM propranolol，PI3K/AKT激活剂150 μM bpV、500 μg/mL 740Y-P可将水牛卵巢皮质在体外培养7天。其次通过激活或抑制mTOR 和 PI3K/AKT 通路研究其在水牛原始卵泡体外激活中的主次地位。发现单独添加两种通路激活剂均显著提高了体外培养卵巢皮质中的发育卵泡占比，而联合添加后其发育卵泡占比降低，说明两种通路同时激活可能存在拮抗作用。rpS6是mTOR通路的下游标志基因，其在PI3K/AKT通路激活后表达最高，也说明了PI3K/AKT通路能活化mTOR促进原始卵泡激活，提示PI3K/AKT的主导作用。从原始卵泡激活及后续卵泡发育中，99%以上的卵泡发生闭锁退化。颗粒细胞（GC）凋亡是卵泡闭锁的主要原因之一，通过RNA-seq构建健康和闭锁卵泡GC的差异表达谱和非编码调控网络。结果表明，在两种卵泡之间共有1,861和1,075个mRNAs，159和24个miRNAs，123和100个lncRNAs分别上调和下调，并注释为PI3K-AKT、mTOR和雌激素等通路，验证了lnc4040通过ceRNA机制作为miR-709海绵调控HIF1a的表达。最后，明确了FSH浓度与水牛卵泡发育过程中GC糖酵解呈正相关，AMPK/SIRT1通路的激活在FSH调控GC糖酵解中发挥重要作用。以上研究对于提高水牛可用卵母细胞数和繁殖效率，及阐明动物卵泡发育的分子机理具有重要意义。