Function Study Of Le～Y Glycan,LIF And β1,4-galactosyltransferase Ⅰ In Embryo Development And Implantation

During the process of embryo implantation of mammalian animal, The fine co-operation between invasive blastocyst and receptive endometrium is the criticality of successful implantation. Estrogen and progestogen act on uterine endometrium by in-cell receptor to stroma edema and vascular engorgement, which provide material grounding for embryo implantation. At the same time endometrium cells secrete many cytokines, adhesive molecules and proteinase and so on. Various factors changing with pregnancy progress promote the mutual recognition and adhesion of uterus-embryo.Glycans of glycoconjugate such as glycoprotein and glycolipid of cell surface participate in specific biology information transmission. Le~Y glycan of cell surface sustaining highly expressed on uterine endometrium and blastocyst during implantation. Specific Le~Y glycoprotein appeared in implantation window. Le~Y participated in the recognition and adhesion of uterus-embryo as a mediator molecule and regulated implantation by hormone as an information molecule. Embryos of high expression Le~Y before implantation had quick development and high implantation ability. Therefore, Le~Y may act as a functional marker of embryo development maturation and implantation. Regulating FUT1 gene expression, the key enzyme gene of Le~Y synthesis(α1,2-fucosyltransferase, FUT1) may further regulate embryo implantation, which provides new method and idea for interferecing embryo implantation.LIF is the most important cytokines of embryo implantation. It promoted embryo development and increased successful rate of transplantation as embryo nutrition factor. LIF expressed on uterine endometrium cells of human and mouse and ensured blastocyst successful imbedding. It has foundβ1,4-galactosyltransferaseⅠ(β1,4-GalTⅠ) participated in the adhesion and spreading of cell, recognition of sperm-ovum, growth of nerve neurite, the formation of tissue and tumor metabasis as an adhesive molecule. Embryo implantation is similar to tumor metabasis and invasion. The paper studied the effects of Le~Y,LIF andβ1,4-GalTⅠon embryo development and implantation. The correlation of LIF andβ1,4-GalTⅠon Le~Y expression was studied to investigate mutual relation of implantation factors and deepen the mechanism of embryo implantation.The major methods and results are summarized as follows:1. Uterine endometrium cells of pregnancy D3 were prepared and grouped. The effects of FUT1siRNA transfected into cells and LIF(10ng/ml)treating cells on Le~Y expression and embryo implantation were studied. The results indicated: After FUT1siRNA was transfected into cells, FUT1 gene and Le~Y expression declined by RT-PCR and Dot-blot analyses, compared to normal culture group and interference control group. LIF promoted FUT1 gene and Le~Y expression. The model of embryo implantation in vitro showed the rate of embryo attachment and outgrowth(32.7%) of interference group was lower than that of normal culture group(40.7%)and interference control group(40.0%). LIF promoted FUT1 gene and further increased the rate of attachment and outgrowth of embryo(52.0%), compared to control group(37.3%). These results hinted Le~Y expression level of cell surface affected the recognition and adhesion of embryo and uterine endometrium cells. Regulating FUT1 gene expression may further regulate embryo implantation. In addition, LIF may regulate the recognition and adhesion of uterus-embryo by up-regulating Le~Y expression.2. Culture medium of individual embryo was obtained from reproduction center lab after fertilization in vitro. Using Dot-blot to detect Le~Y secretion of human individual embryo. The correlation of embryo development,embryo quality, selection of transplantation embryo and Le~Y secretion level was studied. The results showed Le~Y secretion of human embryo started from 4-cell stage and increased with embryo development. Le~Y secretion levels of different patients'embryos with same development stage were different. Le~Y secretion levels of same patients'embryos with same development stage were also different. >6-cell embryo number(37/45)of Le~Y high secretion on D3 was significantly higher than that of low one(18/30). The number of embryo transplanted into uterine(71.1%)with high Le~Y secretion level was also higher than that of low one(40.0%).The results indicated Le~Y can promote embryo development and raise embryo quality and may act as a diagnosis index of selectinggood embryos.3.β1,4-GalTⅠgene interference and overexpression plasmids were transfected into human uterine endometrium cells (RL95-2). The effects ofβ1,4-GalTⅠon FUT1gene expression, Le~Y expression and embryo implantation were studied. After plasmids had been transfected into cells for 60 hours, fluorescence microscope detected the expression of green fluorescent proteins(GFP) and cytochemistry stain. The results showed GFP was light expression and the synthesis of Galβ1-4GlcNAc declined in interference group, but GFP expressed stongly and synthesis of Galβ1-4GlcNAc increased in overexpression group, compared to normal culture group, empty vector group and interference control group. These results indicated plasmids transfection were successful. FUT1 and Le~Y expression declined in interference group, and the rate of attachment and outgrowth(26.7%) was also lower than that of normal culture group(56.0%), empty vector group (56.0%)and interference control group(53.3%). In overexpression group, FUT1 and Le~Y expression increased and the rate of attachment and outgrowth (66.7%)was also higher. These results indicatedβ1,4-GalTⅠmay participate in mutual adhesion of uterus-embryo and embryo implantation course by regulating Le~Y expression. The function ofβ1,4-GalTⅠin embryo implantation will be investigated further.