猪妊娠早期子宫腔液外泌体差异表达miRNA的鉴定及其对胚胎附植的机制研究

About 30% embryonic mortality during implantation is the most important factors that restrict the litter size of western pigs, however, there is no embryonic loss during implantation in Chinese Meishan pigs. The successful-implantation of the free-floating embryos is mediated by the signalling molecules in uterine luminal fluid (ULF). Recent studies demonstrated that exosome-associated miRNA in ULF is an important factor in regulating embryo implantation, but there was no related studies in pig. Our previous work found that pig embryos can take in exosomes in ULF which secreted by endometrium. Therefore, in this study we will investigate the changes in the exosome-associated miRNA profiles between Meishan and Yorkshire gilts on days 9, 12 and 15 of pregnancy through RNA-Seq and Northern blot and in situ hybridization techniques. The target genes of differential expression miRNAs will be predicted and the signaling pathways will be analyzed by a series of bioinformatics software. Then, the important miRNAs and their signaling pathways related to implantation will be analyzed and identified in trophoblast cell culture and in mouse embryo implantation model. The study could contribute to clarify the important factors of regulation the embryo implantation and could provide novel idea to improve the success rate of implantation.

外种猪附植期约30%的胚胎死亡是制约母猪产仔数的重要因素，而我国梅山猪附植期胚胎死亡率极低。游离于子宫腔中的胚胎能否成功附植受子宫腔液中信号分子所调控，近年来发现子宫腔液外泌体能转运子宫内膜来源的miRNA到胚胎细胞从而调控胚胎附植。申请人前期研究已鉴定猪子宫腔液富含外泌体并能被胚胎所摄取。因此本项目以附植期胚胎死亡有显著差异的梅山猪和大白猪为研究对象，在胚胎附植三个关键时期利用RNA-Seq技术筛选子宫腔液外泌体中差异表达miRNA，通过Northern blot、原位杂交等方法进行验证。分析差异表达miRNA靶基因及其信号通路并在滋养层细胞中验证，利用小鼠附植模型鉴定影响胚胎附植的重要miRNA，在繁殖母猪群体中检测重要miRNA 和靶基因的多态位点，并进行产仔数性状的关联分析。本研究将可为发现调控猪胚胎附植的重要因子奠定基础，为提高胚胎附植成功率提供新的研究思路。

外种猪附植期约30%的胚胎死亡是制约母猪产仔数的重要因素，游离于子宫腔中的胚胎能否成功附植受子宫腔液中信号分子所调控，近年来发现子宫腔液外泌体能转运子宫内膜来源的miRNA到胚胎细胞从而调控胚胎附植。本项目以附植期胚胎死亡有显著差异的梅山猪和大白猪为研究对象，利用RNA-Seq技术筛选子宫腔液外泌体中差异表达miRNAs并研究其对胚胎附植的调控机制。研究结论如下：（1）猪胚胎附植期子宫腔液富含外泌体，外泌体粒径大小在30-150nm之间，成椭圆形或杯状，表达CD63,CD9,TSG101等标记蛋白；（2）附植过程中从妊娠12天开始子宫腔液外泌体浓度显著增加，子宫腔液外泌体主要由子宫内膜腔上皮和子宫腺上皮细胞所分泌，体外细胞及胚胎共孵育实验表明子宫腔液外泌体能靶向运输至胚胎滋养层细胞并调控细胞的增殖与迁移能力；（3）外泌体中富含miRNAs分子，从妊娠12天开始外泌体miRNAs的表达模式发生显著变化，与大白猪相比，梅山猪妊娠12天外泌体miRNAs中有29个miRNAs下调表达，44个miRNAs上调表达；（4）子宫腔液外泌体miRNAs能靶向运输至胚胎滋养层细胞发挥调控作用，实验表明子宫腔液来源的外泌体miR-378和miR-21-5p能靶向进入胚胎滋养层细胞抑制细胞的增殖；（5）通过体外细胞实验证实miR-378/21-5p通过抑制靶基因RDX和StAR的表达来抑制胚胎滋养层细胞增殖，进一步通过小鼠胚胎附植模型证实miR-378/21-5p能够促进胚胎附植。本项目初步解析了猪胚胎附植期子宫腔液外泌体miRNAs对胚胎附植的调控作用，为提高胚胎附植成功率提供新的研究资料。