猪胚胎附植定位类型差异机制解析及关键基因挖掘

Failure in embryo implantation is the cause of embryo loss and has impact on litter size in pigs.The mesometrial allocation of the porcine implanting embryo along the uterine vertical axis is critical for subsequent establishment of implantaion. Previously, by using digital slide scanning system, we found that the implanting embryos have two types of orientation within the uterine cavity: positioning at mesometrial or antimesometrial region along the vertical axis, which result in successful or abnormal implantation, respectively. The mechanisms of orientation of implanting embryo in pigs remain unclear. In this project, we plan to prepare the cross sections of porcine uteri derived from the implantation stage and collect the embryos and endometrium tissue using laser capture microdissection system. The orientation type of implanting embryo is considered as a discrete quality trait and the genome-wide association “embryo-maternal” interaction analysis using SNP chip will be performed to dissect effects of the embryo, maternal and embryo-maternal interaction, respectively. Target-region capture sequencing will be applied for detecting the significant genome regions. The transcriptomes of embryos and endometrium from the two types of embryo orientation will be investigated to identify the candidate genes. Then the characterization of key genes and mutation site involved in orientation of implanting embryo will be carried out using approaches from different levels including bioinformatics cytology, molecule and population. The results are of importance for elucidation of the mechanisms that determine embryo orientation during the initial time of implantation and will be useful for identification of genes responsible for litter size in pigs.

猪胚胎附植失败导致胚胎死亡，从而影响母猪产仔数。猪胚胎在子宫腔系膜侧的定位是成功附植的前提，申请人在前期研究中利用数字切片扫描系统，发现猪附植期胚胎存在两种定位类型：系膜侧定位和系膜对侧定位，分别导致附植成功和失败两种结果，但猪胚胎附植定位的机制尚不清楚。本项目拟制备猪胚胎附植期的子宫组织切片，用激光捕获显微切割系统分离附植点胚胎和子宫内膜。创造性地将胚胎附植定位类型作为离散型质量性状，利用SNP芯片开展“胚胎-母体”基因互作全基因组关联分析，剖分胚胎、母体及胚胎与母体互作效应，结合目标区域捕获测序，筛选显著关联的基因组区域。检测两种定位类型的胚胎及子宫内膜转录组，并整合全基因组关联分析数据，获得重要候选基因。综合应用生物信息学、分子细胞生物学、群体关联分析等手段，鉴定影响猪胚胎附植定位的重要基因及其关键突变位点，解析两种胚胎附植定位类型差异的机制，为阐明猪产仔数的分子机制提供依据。

猪胚胎附植失败导致胚胎死亡多、母猪产仔数少，进而降低养殖效益。猪胚胎在子宫腔系膜侧的定位是胚胎成功附植的前提，项目研究目标是鉴定调控猪胚胎附植定位的重要基因和通路、筛选重要基因及调控元件中关键突变、挖掘具有育种价值的分子标记。本研究围绕上述目标展开研究，取得的重要结果如下：（1）采集了纯种大白猪胚胎附植期的母胎组织样品，鉴定到尚未见诸报道的与猪胚胎附植定位重要事件密切相关的母胎界面组织学特征（包括子宫腔闭合，胚胎在子宫腔的定位及子宫腔中胚胎横向排位密度），并进行了表型分型和统计，为猪的胚胎附植相关基因的功能研究提供了组织学水平表型数据。（2）获得了猪子宫内膜中调控胚胎附植定位的lncRNA-miRNA-gene通路，利用激光显微切割及单细胞测序技术检测到猪胚胎附植期在子宫腔呈现空间特异性表达模式的基因和细胞，筛选到胚胎附植侧子宫腔上皮-胚胎-胚胎非附植侧子宫腔上皮细胞的互作通路，进而鉴定到与猪胚胎附植定位相关的重要候选基因。（3）发现ST6GAL1可通过介导唾液酸结构的形成调控子宫腔闭合，完成胚胎的定位附植。转录因子C/EBP-β通过调控猪胚胎附植期子宫内膜上皮细胞中MMP8基因的表达，降解细胞外基质II型胶原，在促进子宫内膜转变为容受性状态以接受胚胎的附植中发挥作用。仅在邻近胚胎附植位置子宫腔上皮表达的B4GALT1基因可通过催化形成Lewis x-carrying O-glycans结构，与胚胎粘附分子SPP1-integrin receptor 复合物互作，调控猪胚胎定位附植。（4）建立了具自主知识产权的猪重要性状关键基因和突变位点效应分析工具和数据库，利用调控组学等多组学技术鉴定到具有调控猪胚胎附植基因表达的功能调控元件及突变位点，在此基础上研发了首款60K母猪繁殖性状功能位点基因芯片。小结：本项目鉴定到表征猪胚胎定位附植的组织学特征，筛选到猪胚胎附植定位相关基因，发现了重要基因通过介导猪子宫腔闭合、子宫容受性转化及胚胎粘附调控胚胎附植定位的机制，挖掘到具有育种价值的分子标记，研发的基于功能突变位点的母猪繁殖性状基因芯片是培育高产母猪的新工具，研究结果为阐明猪产仔数的调控机制提供了重要依据。