The Expression Pattern Of TLR4 In Endometrium During Sheep Embryo Implantation Period

The sheep raising industry is a part of the economic development of animal husbandry in China.For the development of animal husbandry,more and more nuclear transfer,embryo transfer and other technologies have long been applied to production practice,but the development of these technologies has the problem of low embryo implantation rate,a complex physiological process,and a key link in pregnancy.TLR4 is the most frequently studied and widely distributed gene in recent years.Binding to LPS ligands can activate the TLR4 signaling pathway,causing the release of various inflammatory factors and hormones downstream,and can interact to regulate many functions of cells.Involving mammalian embryo attachment,research on gene expression in embryonic attachment has also become a hot topic.Objective:To study the expression patterns of TLR4 on the endometrium of sheep during the embryo implantation stages,and to verify the effect of TLR4 on the implantation at the cell level.The aim of research is that provide reference for improving the embryo implantation rate.Methods:The experiment was applied by semi quantitative RT-PCR and real-time fluorescence quantitative RT-PCR were used to detect the expression of TLR4 gene in the endometrium of 9 d,13 d,17d,21 d and 25 d sheep of gestation sheep respectively.Secondly,by immunohistochemistry and immunological antibody coloring detect the uterine tissues of early pregnancy sheep(9 d,13 d,17 d,21 d,25 d)were selected to detect the distribution and expression of TLR4 protein in various stages of uterus,and identifying the integrity of the stromal cells by the specificity of VIM protein.Finally,the eukaryotic expression vector of pcDNA3.1(+)-TLR4 was constructed.Then the vector was identified by bacterial liquid PCR and enzyme digestion,and the company was sequenced to verify whether the carrier was constructed successfully.Experiment four:The endometrial stromal cells were stimulated by 1μg·L~-11 LPS,and an inflammatory model of endometrial stromal cells was established.The expression of TLR4 mRNA,the expression of TLR4 protein and the expression level of IL-1 beta and IL-6 of the immune factors were detected by the fluorescence quantitative RT-PCR technique and the protein immunoblotting Western Blot method.Results:Not only found the expression of TLR4 gene in the oviduct,ovary,uterus,bladder,spleen and pineal body of the whole body of sheep is high,and the expression is weak in the liver,small intestine and muscle,but it is not expressed in the tissues of the heart,the pituitary,and the fat,but the expression level of TLR4 gene in the endometrium decreased from 9 d to 17 d,and then increased to 25 d.In addition,the expression level of TLR4 gene was significantly higher in the luteal phase than in the follicular phase(P<0.05)in both the Chinese merino sheep or the endometrium tissues of Hu sheep.Then TLR4 protein is expressed in all parts of the endometrium,showing a dynamic spatiotemporal pattern,especially in the matrix cells of the 17 d.In the end,the eukaryotic expression vector of pcDNA3.1(+)-TLR4 was constructed.And LPS decreased the expression of IL-6 in cells,increased the expression of IL-1β,caused inflammatory reaction,and made Th1/Th2 bias against the Th1 direction of pregnancy.The expression of TLR4 mRNA in 12 h,24 h,48 h and 72 h was significantly higher than that in the control group(P<0.05),in which 24 h,48 h and 72 h were significantly higher than those in the control group(P<0.01).The expression of TLR4 protein was always higher than that of the control group,which was consistent with the quantitative results.Conclusion:The expression of TLR4 is tissue specific and has some regularity in the endometrium of sheep embryo implantation and estrus cycle,which suggests that TLR4 plays a certain role in the breeding period of sheep.We found the expression pattern of TLR4 receptor protein in the early endometrium of embryo implantation.LPS effectively activates the TLR4 signaling pathway in the endometrium cells,and the TLR4 receptor protein of the uterine decidua plays an important role in maintaining the balance of the early pregnancy microenvironment.