Li Qi, Tang Ying, Chen Yanru, Li Bo, Wang Hongzhan, Liu Shicheng, Adeniran Samson O, Zheng Peng
College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China.
Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Animals (Basel). 2024 Sep 25;14(19):2771. doi: 10.3390/ani14192771.
Melatonin plays a critical role in regulating embryo attachment in ruminants. While numerous studies have investigated its effects on early embryo development in vitro, the precise mechanisms by which melatonin influences the receptivity of endometrial epithelial cells in dairy cows remain unclear. The prerequisite for embryo implantation is the specific physiological condition of the endometrium that allows the embryo to implant, also known as endometrial receptivity. In addition to this, endometrial cells undergo processes such as proliferation, differentiation, and renewal, which makes the embryo more easily implanted. In this study, bovine endometrial epithelial cells were cultured and treated with melatonin, Silent Information Regulator 1 (SIRT1) inhibitor (EX527), and protein kinase B (AKT) phosphorylation inhibitor (periposine). RT-qPCR, Western blot, and immunofluorescence analysis were performed to investigate the effects of melatonin on the expression of target gene (SIRT1); cell proliferative genes, phosphatidylinositol-4,5-bisphosphate 3-Kinase (PI3K), AKT, cyclinD1, cyclinE1; and receptive genes (Leukemia Inhibitory Factor (LIF), Vascular Endothelial Growth Factor (VEGF), Homeobox Structure Gene 10 (HOXA10)). Additionally, microRNA (miRNA) mimics and inhibitors were used to transfect the cells to study the regulatory relationship between miRNA and receptive genes. Results indicated that melatonin activates the PI3K/AKT signaling pathway, upregulates cyclinD1 and cyclinE1, and promotes the proliferation of bovine endometrial epithelial cells. Melatonin also upregulated the expression of VEGF and HOXA10 and downregulated the expression of bta-miR-497 and bta-miR-27a-3p through SIRT1/PI3K/AKT signaling pathway. Further, bta-miR-497 and bta-miR-27a-3p were found to negatively regulate VEGF and HOXA10, respectively. Therefore, melatonin regulates the expression of VEGF and HOXA10 through the SIRT1/PI3K/AKT pathway and promotes the establishment of receptivity in bovine endometrial epithelial cells.
褪黑素在反刍动物胚胎着床调控中起关键作用。虽然众多研究已探究其对体外早期胚胎发育的影响，但褪黑素影响奶牛子宫内膜上皮细胞接受性的精确机制仍不清楚。胚胎着床的前提是子宫内膜具备允许胚胎着床的特定生理状态，即子宫内膜接受性。除此之外，子宫内膜细胞会经历增殖、分化和更新等过程，这使得胚胎更易于着床。在本研究中，培养牛子宫内膜上皮细胞，并分别用褪黑素、沉默信息调节因子1（SIRT1）抑制剂（EX527）和蛋白激酶B（AKT）磷酸化抑制剂（围脂素）进行处理。采用实时定量聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法（Western blot）和免疫荧光分析，研究褪黑素对靶基因（SIRT1）；细胞增殖相关基因，磷脂酰肌醇-4,5-二磷酸3-激酶（PI3K）、AKT、细胞周期蛋白D1（cyclinD1）、细胞周期蛋白E1（cyclinE1）；以及接受性相关基因（白血病抑制因子（LIF）、血管内皮生长因子（VEGF）、同源盒结构基因10（HOXA10））表达的影响。此外，使用微小RNA（miRNA）模拟物和抑制剂转染细胞，以研究miRNA与接受性相关基因之间的调控关系。结果表明，褪黑素激活PI3K/AKT信号通路，上调cyclinD1和cyclinE1的表达，并促进牛子宫内膜上皮细胞的增殖。褪黑素还通过SIRT1/PI3K/AKT信号通路上调VEGF和HOXA10的表达，下调bta-miR-497和bta-miR-27a-3p的表达。此外，发现bta-miR-497和bta-miR-27a-3p分别对VEGF和HOXA10起负调控作用。因此，褪黑素通过SIRT1/PI3K/AKT途径调节VEGF和HOXA10的表达，并促进牛子宫内膜上皮细胞接受性的建立。
