中短链脂肪酸调节猪早期胚胎发育和胚胎附植的分子机制

High early pregnancy loss is an important factor in restriction of swine reproduction in China. How to enhance early embryonic survival through modulation of sows’ nutrition status during pregnancy is the critical scientific issue highly concerned in swine industry. Fatty acid metabolism plays important roles in preimplantation embryo development. Our comparative study of sows with high and low reproductive performance indicates that the abnormal lipid metabolism is highly associated with the reduce in reproductive performance. Our preliminary results in rats indicates that short and medium chain fatty acids such as sodium butyrate, Sodium hexanoate, and Sodium octanoate significantly improve embryo implantation and increase litter size. Based on our previous work, this project will investigate the following three main contents: (1) establish the high through-put screen system based on the Luciferase reporter gene combined with swine embryo transplantation to screen important short and medium fatty acids in modulation of swine embryo development and embryo implantation; (2) focus on mitochondrial energy metabolism and Steroid hormone synthesis to elucidate the molecular mechanism of short and medium chain fatty acids in modulation of embryo implantation; (3) in vivo verify and optimize the combination and ratio of short and medium chain fatty acids to exert sows’ maximal reproductive potential. Finishing the above systemic work will uncover the molecular mechanism of short chain and medium chain fatty acids in regulation of porcine early embryo development and embryo implantation. This has practical significance for better understanding the relationship between nutrition and reproduction and enhancing sows' reproductive performance in China.

早期胚胎死亡率高是限制母猪繁殖性能的关键因素。如何通过调整妊娠母猪的营养状况来提高早期胚胎成活率是领域内关注的关键科学问题。脂肪酸代谢对附植前胚胎发育具有重要作用，申请者对高低产母猪的比较研究发现，脂类代谢异常与繁殖性能降低高度相关，我们在模式动物上的研究表明，中短链脂肪酸丁酸钠、己酸钠和辛酸钠均可显著促进大鼠早期胚胎着床，增加窝产活仔数。本项目拟在前期工作基础上，建立基于荧光素酶报告基因的高通量筛选模型，结合猪早期胚胎移植试验，筛选调节猪早期胚胎发育和胚胎附植的重要中短链脂肪酸；以调节线粒体能量代谢和促进类固醇激素合成为研究重点，揭示中短链脂肪酸调节猪早期胚胎附植的分子机制；体内验证和优化中短链脂肪酸发挥母猪最大繁殖潜能的最佳中短链脂肪酸组合及配比。通过完成上述系统工作，揭示中短链脂肪酸在调节猪早期胚胎发育和胚胎附植的分子机制，对深入认识营养与繁殖的关系，提高我国母猪性能有重要实际意义。

母猪繁殖性能是制约养猪生产效率和经济效益的关键因素。本研究通过五个试验研究了中短链脂肪酸调节母猪早期胚胎发育和附植的分子机制。试验一以SD大鼠为模型，发现日粮短中链脂肪酸（0.1%丁酸钠、0.1%己酸钠或0.1%辛酸钠）能够显著增加大鼠窝产仔数和窝重，并且改善妊娠早期孕鼠胚胎着床。此外，还显著增加妊娠早期孕鼠血清花生四烯酸、孕酮（Progesterone，P4）的水平，促进血清磷脂代谢和鞘脂代谢，并且增强关键基因COX2、LPA3、StAR和CYP11A1的表达水平。试验二发现丁酸钠能够促进大鼠和人卵巢颗粒细胞雌二醇（Estrodiol，E2）和P4的合成。证明了丁酸的膜受体GPR41和GPR43以及脂类核受体PPARγ参与介导了丁酸钠对KGN细胞类固醇激素合成的促进作用。此外，丁酸钠显著降低了KGN细胞的组蛋白去乙酰化酶（Histone deacetylase，HDAC）的活性，并增强乙酰化组蛋白的表达水平；提高了一系列基因启动子区域的组蛋白H3K9乙酰化（Acetylated histone H3K9，H3K9ac）水平。并提高PGC1α的转录水平以增强线粒体合成以及降低细胞内氧化损伤，改善线粒体动态和提高细胞抗氧化能力进一步增强类固醇激素合成。试验三在妊娠第5天大鼠侧脑室注射丁酸钠之后4 h显著增加了血清中P4、E2、促黄体生成素（Luteinizing hormone，LH）和胆固醇的水平。其次，显著增加了卵巢类固醇激素合成相关基因的转录水平，同时改变了肝脏、卵巢和子宫脂质合成和代谢相关基因转录水平，并且显著增加了血清、肝脏和子宫花生四烯酸的水平。下丘脑和垂体组织体外培养试验发现丁酸钠能够显著增加下丘脑GnRH、Kiss1和GPR54的转录水平以及下丘脑组织培养液中GnRH的水平。试验四通过猪卵巢原代颗粒细胞和猪子宫内膜细胞发现了中短链脂肪酸组合（1mM丁酸钠，0.04mM辛酸钠，0.01mM月桂酸钠）能显著提高卵巢颗粒细胞激素合成能力和猪子宫内膜细胞的容受性，此外成功构建了基于荧光素酶报告基因的猪胚胎滋养层细胞高通量筛选模型。试验五验证了母猪妊娠早期日粮添加中短链脂肪酸组合（0.1%丁酸钠，0.05%辛酸钠，0.1%月桂酸钠）能显著提高窝产活仔数约1.43头，并提高母猪妊娠早期雌激素水平。