R-spondin3在猪妊娠早期中的作用及其调控机制研究

The embryo or fetal death is one of the important factors affecting the number of per litter in sows. During the early pregnancy, especially the peri-implantation, the loss of embryo reaches a peak in sows. Rapid elongation of the blastocyst and its adhesion to uterine luminal epithelium to initiate the placentation, and the maternal endometrial changes to receive the embryo during the peri-implantation, these key processes are strictly regulated by many molecules from both embryo and maternal. The R-Spondin 3 (Rspo3), a member of the secreted protein family R-Spondin, is implicated in involvement of the development of mouse embryo and the placentation. Our preliminary study also showed that Rspo3 had a high expression in sow endometrium and placenta on day 40 of pregnancy, and showed a lower level in term placenta. These data suggested the role of Rspo3 in porcine early pregnancy. In this study, we will use gilts as the animal model. The expression and localization of the Rspo3 will be investigated in the porcine tissues including uteri and/or placentas collected on estrus day, day 12, 18 and 30 of the gestation, and association between Rspo3 expression and porcine embryo development will also be explored. To study its molecular regulatory mechanism at the maternal-fetal interface, primary cell culture system including porcine endometrial cells (stromal cells and luminal epithelial cells), trophoblast cells and in vitro embryo culture will be developed. Lentiviral vector systems for over-expression of Rspo3 and its specific siRNA will be constructed to mediate the up- and down-regulation of Rspo3. Effects of Rspo3 on the target cell functions such as proliferation, migration and their secretion will be determined. All these results will provide new scientific theoretical basis to decrease the embyo or fetal loss and improve the sow reproduction.

母猪胚胎或胎儿死亡是影响母猪产仔数的重要因素，其中附植期是胚胎死亡的一个高峰期，胚胎的迅速扩张启动胎盘发生、母体子宫内膜发生变化建立容受性，这些关键过程受许多分子的严格调控。研究发现R-spondin家族成员Rspo3对小鼠胚胎和胎盘发育有重要作用，前期研究发现其在猪妊娠40天的子宫内膜和胎盘中有高表达，而在足月材料中较低，这提示Rspo3可能在猪早期妊娠过程中起着重要作用。本项目以猪为动物模型，在体研究Rspo3在发情期、妊娠12天、18天和30天的母猪子宫内膜胚胎附植点和非附植点以及胎盘组织中的表达定位，并探讨Rspo3与猪胚胎发育的关联性；同时离体原代培养子宫内膜细胞、胚胎滋养层细胞及体外胚胎培养等，通过构建过表达和特异siRNA慢病毒载体分别介导Rspo3的上调和下调，研究其对母胎界面相关细胞功能的分子调控机制。相关结果可为减少母猪胚胎或胎儿死亡、提高母猪年生产力提供科学理论依据。

母猪胚胎或胎儿死亡是影响母猪产仔数的重要因素，其中胎盘的正常发育是维持正常妊娠的关键。分泌蛋白R-Spondin 3(RSPO3)缺失会造成小鼠胎盘发育异常而导致胚胎死亡。为研究RSPO3在猪早期妊娠过程中的作用，本研究首先克隆了猪胎盘RSPO3的编码序列，同源性与人的高度相似，其蛋白结构富含半胱氨酸；RSPO3在妊娠不同时期母猪母胎界面主要定位在滋养层柱状上皮细胞和子宫内膜腺体上皮细胞中，而在血管中几乎检测不到；RSPO3在妊娠40天，即胎盘生长处于较活跃的阶段时，在母胎界面的表达量较高。以猪滋养层细胞系为模型，通过慢病毒载体系统获得稳定的过表达或下调表达的细胞系，对细胞功能研究结果显示上调RSPO3后可显著抑制猪滋养层细胞的增殖和毛细小管样形成，而下调时则显著促进，对细胞的凋亡无显著影响发现；另外，猪滋养层细胞分泌激素（包括hCG、P4和E2）极低或不分泌，相关激素合成酶及受体的表达量也极低，上调或下调RSPO3后对相关激素无影响。由于猪胎盘RSPO3蛋白序列与人一致，而人胎盘绒毛膜癌细胞（JEG-3）被广泛用于胎盘相关功能研究，因此，本项目中探讨了RSPO3对JEG-3功能的影响，结果显示RSPO3通过AKT、ERK、PCNA、CD31以及β-catenin的表达，从而促进JEG-3异种移植瘤的生长。进一步通过小鼠模型验证，RSPO3主要定位于小鼠子宫内膜上皮、胎盘蜕膜、连接区和迷回路层，在妊娠早期的表达较高；通过用小鼠RSPO3过表达慢病毒系统感染囊胚使其在胎盘中特异性上调，结果发现过表达RSPO3后在妊娠后期可以增大胎儿重、胎盘面积增大，结果说明RSPO3可促进小鼠胎儿的发育。综合以上结果，RSPO3在猪妊娠早期母胎界面的高表达，提示RSPO3对胎盘发育起着主要的调控作用；对滋养层细胞功能的调节可能通过AKT/ERK/PCNA/CD31/β-catenin信号途径发挥，同时也提示RSPO3的异常表达可能与一些妊娠疾病相关。