褪黑素通过LIF调控小鼠胚胎附植的研究

Mammalian embryo implantation is precisely regulated by hormones, cytokines, growth factors and other molecules. Studies have shown that progesterone (P) and estradiol (E2) are the main hormone regulation of endometrial development. LIF is one of the the most important cytokines mediating embryo implantation. The embryo implantation sites in the pinealectomy rat are significantly reduced, and serum E2 increases, P drops. Melatonin can regulate the expression of p53. It have been reported that p53 can affect embryo implantation by regulating the expression of the LIF. In addition, LIF is also E2 sensitive genes. Study of our group shows that melatonin receptors MT1 and MT2 exist in pregnant mice uterus and oral supplemention melatonin can improve the implantation sites, litter size, and promote the expression of the uterus p53 upregulation. Therefore, in this program, mice were used to explore the mechanism of melatonin and its receptor promotes embryo implantation. Main experiments: Firstly, the pathway of melatonin and receptors in the regulation of LIF expression and embryo implantation will be determined. Secondly, it will be assessed whether melatonin can effect E2, P and those receptors ER and PR, so that regulate LIF expression and embryo implantation . Finally,melatonin's antioxidant role in improving the intrauterine environment will be assesed. The expected results will clarify the embryo implantation mechanisms and provide important theoretical basis.

哺乳动物的胚胎附植受到激素、细胞因子等精确调控。已有研究表明，孕酮（P）及雌二醇（E2）是调控子宫内膜发育的主要激素，而LIF是胚胎附植最为重要的细胞因子之一。切除大鼠松果体后胚胎附植点显著减少，E2上升，P下降；同时，褪黑素能调节p53的表达，p53又能调控LIF的表达而影响胚胎附植；此外，LIF也是E2敏感基因。但褪黑素促进胚胎附植的机理尚不清楚。本课题组研究表明，妊娠小鼠子宫存在褪黑素受体MT1和MT2；口服褪黑素能够提高附植数和产仔数，并促进子宫p53的上调表达。因此，本项目以小鼠为研究对象，首先，探索褪黑素是否通过MT1或MT2促进p53的表达和磷酸化来调控LIF转录而促进胚胎附植。其次，研究褪黑素是否调控E2、P分泌及其受体表达，进而影响LIF等相关基因的表达以促进附植。最后，研究褪黑素是否通过调节子宫抗氧化水平，改善子宫环境而促进附植。预期结果将阐明褪黑素促进胚胎附植的机理。

本项目通过褪黑素处理，并采用体内外结合、传统与前沿技术手段结合，从多角度精确研究褪黑素调控胚胎附植的机理，取得一系列研究成果。（1）妊娠早期小鼠子宫中褪黑素相关基因（SNAT和MT2）表达促进胚胎附植。在早期妊娠阶段，子宫腺与子宫腔上皮中褪黑素合成酶SNAT逐渐升高；褪黑素作用受体MT2促进子宫内膜发育，子宫内膜面积、子宫腺密度与指数均增加，在附植窗口期褪黑素通过上调附植关键基因PRA、P53以及囊胚激活信号HB-EGF/ErbB1信号促进胚胎附植；（2）褪黑素及其受体MT1参与调控颗粒细胞黄体化。首次证实了褪黑素及其受体MT1是LH的下游分子，在MT1峰前注射褪黑素可提高孕酮水平和黄体关键基因的表达，进而提高小鼠胚胎附植点数；通过RNA-seq证实了ECM-receptor、Focal adhesion和PI3K-Akt是褪黑素在颗粒细胞的下游通路，参与黄体形成的调控；（3） 褪黑素通过其受体调节p53促进胚胎附植并减缓延长光照（光污染）引起的妊娠效率下降。褪黑素上调了附植第4.5天的E2水平，并通过MT1/2受体上调了P53蛋白，同时期母鼠子宫中LIF蛋白持续表达；妊娠小鼠饮水中添加褪黑素能提高小鼠的窝产仔数1.3只，减少光污染引起的妊娠小鼠胚胎附植点下降；（4）褪黑素还通过调节Star、Cyp11a1和Ihh促进对小鼠早期妊娠卵巢中孕酮合成。小鼠妊娠后口服褪黑素，第0.5天和第7.5孕酮分泌量显著提高，附植点和产仔数量均增加；褪黑素可以调节卵巢中孕酮合成相关基因提高孕酮的合成，促进排卵后黄体发育；褪黑素促进妊娠后7.5d子宫内LIF 表达改善小鼠的子宫接受态从而促进胚胎附植。以通讯作者发表论文SCI收录论文5篇，累计影响因28.641；获得授权国家发明专利5项，申请国际发明专利1项，申请国家发明专利4项；培养研究生13名，已毕业研究生4名；通过第三方评价成果1项。