不同品种猪妊娠早期子宫液差异分泌蛋白鉴定及其对附植的调控机制研究

Early embryonic loss that occurs during implantation is considered the major limitation for increasing litter size in swine. The development and implantation of the free-floating conceptuses is mediated by the uterine secretory proteins. The central hypothesis is that we can discover and characterize proteins and their genetic pathways controlling the economically important trait of embryo survival through secretome analysis of specific, relevant stages of the conceptus and endometrium during early gestation in animals that differ for this trait. Previous studies have shown the difference in embryonic loss and litter size between Meishan and Large White breeds during implantation. Thus, we will characterize the changes in the uterine protein profiles between Meishan and Large White breeds gilts on days 12 , 15 and 18 of gestation through Label-free LC MS/MS and Western blotting, in situ hybridization and immunocychemistry techniques. By integrating data of gene and miRNA profile of endometrium obtaind from our previous studies using stepwise regression and structural equation modelling, we will analyze the function and the signal pathways involved in silico and in vitro.In addition, we will use a newly developed successful technique for immobilizing osmotic pumps in the porcine reproductive tract to deliver substances into the luminal cavity of the uterus in order to investigate the function and regulation of the important secretory proteins in embryo implantation. This proposal addresses the lack of fundamental molecular knowledge in pig reproductive biology through a multidisciplinary approach involving molecular genetics, reproductive biology, statistics, and bioinformatics. Knowledge gained through this approach will be integrated with existing genetics to accelerate improvement in reproduction traits that have a substantial economical impact for pork production.

猪胚胎死亡是影响产仔数的重要因素。胚胎的死亡高峰在其附植期，此时游离于子宫腔中的胚胎其发育和附植直接受子宫液中分泌蛋白的调控, 因此子宫液中的分泌蛋白在胚胎附植中具有重要作用。本研究以在胚胎死亡和产仔数上有差异的梅山和大白猪为研究对象，在妊娠12天（附植初期）、15天（附植中期）和18天（胚胎完成定位结束游离状态的时期），采用差异分泌蛋白组学策略，利用基于液相色谱串联质谱的非标记定量技术，获得差异分泌蛋白谱，通过Western杂交、原位杂交和免疫组化等方法验证差异分泌蛋白的表达模式。联合申请人前期获得的子宫内膜基因及miRNA表达谱数据，分析分泌蛋白的功能和介导的信号通路，在细胞水平进行验证，借助猪子宫内渗透泵灌注封闭抗体法，通过测量胎盘和胚胎发育等指标，研究重要分泌蛋白对猪胚胎发育和附植的作用。研究结果可为发现影响猪胚胎附植的重要调控因子奠定基础，为猪产仔数的遗传改良提供新的基因素材。

本项目研究了分泌蛋白及其相关通路对猪胚胎附植的作用。（1）系统采集了大白和梅山猪4个妊娠时期（妊娠9天、12天、15天及18天）及发情期样品，进行了母胎界面表型组分析，确定了猪胚胎成功附植受宫腔闭合和定向附植两个因素影响。（2）获得了大白和梅山猪胚胎附植期分泌蛋白编码基因表达谱，利用免疫组化、原位杂交和Western 杂交等方法对差异表达的分泌蛋白进行了鉴定。（3）对重要分泌蛋白的功能进行了研究，(ⅰ)筛选了猪妊娠识别信号雌激素诱导的分泌蛋白基因，发现胚胎滋养层合成雌激素所需的胆固醇依赖于转运蛋白从母体子宫内膜外排至子宫腔液后再转运至胚胎，而该转运蛋白在子宫中胚胎附植侧（子宫系膜侧）的子宫内膜中显著高表达，说明存在相关的基因通路可引导胚胎定位至子宫系膜侧。（ⅱ）附植期猪孕体须经历快速的变形及增殖后在子宫中占位，本研究发现蛋白裂解酶matriptase以活化形式在猪滋养层上皮细胞高表达，并激活基质金属蛋白酶MMP1前体转化为活化的MMP1，降解滋养层上皮细胞外基质和基底膜。分泌蛋白乙酰肝素酶HPSE可释放与HSPG结合的生长因子，而HPSE也在附植期的滋养层上皮细胞中高表达。其次，这两类分泌蛋白在具有增殖能力的立方状滋养层上皮细胞中高表达，其中HPSE蛋白还可分泌到母体子宫内膜上皮细胞中，且HPSE基因与猪初生重显著关联。这些发现说明这两类分泌蛋白的共同作用可调控孕体的变形以占据足够的子宫空间，为后期的胎盘发育和胚胎正常发育奠定基础。（ⅲ）PLET1蛋白特异地在滋养层上皮细胞表达且其细胞定位在附植前后发生变化，该蛋白编码基因在滋养层上皮细胞中表达两个转录本且其表达可能受miR-365-3p的调控，结合敲除小鼠的研究结果推测PLET1在滋养层细胞的分化中有重要作用。发表标注基金资助的SCI文章7篇，其中有2篇发表在《Biology of Reproduction》 期刊，获得授权发明专利3项，培养博士生 2人，硕士生4人。