Role Of Sialyl Lewis X And Lewis Y Oligosaccharides In Embryo Implantation

I. Objective and purposeImplantation is a complex biological process. It requires the synchrony between the appropriately developed embryo and the receptive uterine endometrium. The apptoptiate developed of uterine and embryo is the basis for successful implantation. Oligosaccharides, as constituents of cell surface glycoconjugates, have been shown to have many biological activities during implantation process, which participate in the recongization of cell-cell and signal transduction. A variety of oligosaccharide structures have been identified at embryonic and uterine cell surfaces and change dynamically during progression of the implantation. Some reports show that strong L-selectin staining was present in the human embryo, whereas the expression of selectin oligosaccharide-based ligands was up-regulated during the implantation window, the binding of L-selectin and sialy Lewis X (sLeX) mediated the adhesion of embryo and uterine. Lewis Y (LeY) were highly expressed on both murine embryo and uterine endometrium during implantation period, and the functional blockage of LeY with specific anti-LeY antibody in either the embryo side or uterine endometrium side could reduce embryo implantation both in vitro and in vivo.Lewis oligosaccharide belongs to A, B, H Lewis blood group family. Fuco- syltransferases (FUTs) catalyze the synthesis of Lewis oligosaccharides. Until now, 11 fucosyltransferases have been identifided, which composed ofα1,2-,α1,3-,α1,6- fucosyltransferases. FUT7 belongs toα1,3 fucosyltransferases, which is the key enzyme of sLeX [NeuAcα2→3Galβ1→4 (Fuc1→3) GlcNAcβ1→R] synthesis. FUT4 also belongs to 1,3-fucosyltransferases. Based on H type 2 [Fucα→1Galβ1→4GlcNAcβ1→R] epitope, LeY is synthesized by the addition of a Fuc residue to N-acetyl- glucosamine (N-GlcNAc) with theα1, 3-linkage, FUT4 is the key enzyme for the synthesis of LeY. The regulation of FUT7 and FUT4 may control the expression of the sLeX and LeY.In this study, we utilized the FUT7 and FUT4 overexpression plasmid or FUT4- siRNA plasmid to interfere the expression of sLeX and LeY oligosaccharide on cell surface, and evaluate the roles of oligosaccharides on cell surface during implantation. The study will promte the further elucidation of the mechanism of oligosaccharides in embryo implantaion.II. Methods1.The recombinant vector was transfected into cells.2.The expression of FUT4 and FUT7 were detected by the methods of RT-PCR.3.The expression of FUT4 and FUT7, sLeX and LeY were detected by the methods of Western blot, indirect immunofluorescence staining and flow cytometry assay.4.Effect of FUT7 on cell apoptosis was analyzed by Annexin V/PI flow cytome- tric assay.5.Detect the percent adhesion of JAR cell adhesion to RL95-2 or HEC-1A cell monolayer.III. Results(I)Dual adhesion mediated by sLeX/L-selectin in vitro implantation model.1.The expression of sLeX and L-selectin in JAR and RL95-2 cells were detected by indirect immunofluorescence staining. The results showed that the staining of sLeX was present in JAR cellsand RL95-2 cells. L-selectin staining was also observed in JAR cells and RL95-2 cells. It suggests that sLeX and L-selectin were co-existed in either JAR cells or RL95-2 cells.2.To check if the expression of sLeX and L-selectin on both JAR cells and RL95- 2 cells was functionally involved in sLeX/L-selectin adhesion system, the effect of anti- body blocking was undergone using adhesion inhibition assay. The adhesion of JAR cells to RL95-2 cells were inhibited by the specific antibodies blockage. The percent adhesion was significantly decreased when JAR cells was pre-incubated with anti-sLeX antibody or anti-L-selectin antibody. The preincubation of anti-sLeX antibody or anti- L-selectin antibody in RL95-2 cells also caused the similar inhibitory effect.3.FUT7 expression plasmid was transfected into JAR or RL95-2 cells. The results showed that the gene and protein level of FUT7 were elevated by RT-PCR and flow cytometry assay in both JAR and RL95-2 cells transfected with FUT7 expression plasmid compared with that of the control and mock vector transfection. Moreover, the synthesis of sLeX was also increased in JAR or RL95-2 cells after FUT7 expression plasmid transfection by flow cytometry assay. The percent adhesion was analyzed. When FUT7 expression plasmid was co-transfected into both of the cells, the percent adhesion was higher.4.Heparin, the inhibitor of L-selectin, was utilized. Statistical analysis showed that when JAR cells, RL95-2 cells, or both of the cells were incubated with heparin, the percent adhesions were also significantly decreased compared with that of the control. The inhibition was the most obvious when both cells were treated with heparin, comp- ared with the separate addition of heparin to either cell culture.5.The apoptosis was determined by flow cytometry assay after RL95-2 cells were transfected with FUT7 expression plasmid. The data indicated that after co-cultured with JAR cells, the extent of apoptosis in transfected RL95-2 cells was increased, compared with that of the control and mock vector transfected RL95-2 cells.(II)The relationship between potension of uterine receptivity and LeY oligo- saccharide.1.The percent adhesion of JAR to RL95-2 cell monolayer was higher than HEC- 1A cell monolayer; and co-culture the JAR cells with RL95-2 cell monolayer or HEC- 1A cell monolayer, respectively, the extension of JAR cells on RL95-2 cell monolayer was more obviously than that on HEC-1A cell monolayer.2.For LeY assay, the expression of LeY on RL95-2 cells was also higher than on HEC-1A cells by Western blot and indirect immunofluorescence staining.3.For FUT4 detect assay, RT-PCR, Western blot and indirect immunofluores- cence staining were utilized. Results revealed that the expression of FUT4 in RL95-2 cells was higher than in HEC-1A cells on gene level and protein level4.To detect the effect of FUT4-siRNA on the RL95-2 cells, different assays were employed. RT-PCR found that the expression of FUT4 gene was dramatically decreased by FUT4-siRNA transfection in comparison to untransfected controls. Western blot, indirect immunofluorescence staining and flow cytometry revealed all that the protein expression level of FUT4 was much lower in FUT4-siRNA transfected RL95-2 cells in comparison to untransfected controls. The percent adhesion of JAR cell to RL95-2 cell monolayer was been inhibited.5.To explore the effect of FUT4 on LeY synthesis, FUT4 expression plasmid was transfected into HEC-1A cells. The results showed that the gene and protein level of FUT4 were elevated in FUT4 expression plasmid transfected cell compare to untrans- fected control by RT-PCR, Western blot, indirect immunofluorescence staining and flow cytometry assay. The synthesis level of LeY is also augment parallel with the increased of FUT4. The percent adhesion of JAR cell to HEC-1A cell monolayer was been evaluated.6.To study the effect of FUT4 expression on EGFR/MAPK signaling pathway, tyrosine phosphorylation of EGFR and ERK1/2 was assessed by Western blotting. After stimulating with EGF (100 ng/ml) for 10 min, sample was harvested. In RL95-2 cells, FUT4 knocking down cells expressed less pEGFR, pTYr and pERK1/2. Meanwhile, overexpression of FUT4 in HEC-1A cells could increase the synthesis of pEGFR, pTYr and pERK1/2.IV. Conclusions(I) Dual adhesion mediated by sLeX/L-selectin in vitro implantation model.1.The binding of L-selectin on JAR cells and sLeX on RL95-2 cells mediates the embryo implantation.2.The binding of sLeX on JAR cells and L-selectin on RL95-2 cells mediates the embryo implantation. Dual adhesion mediated by sLeX/L-selectin participate the embryo implantation.3.Adhesion mediated by sLeX increases apoptosis of RL95-2 cells.(II) The relationship between potension of uterine receptivity and LeY oligo- saccharide.1.The expression FUT4 and LeY on RL95-2 cells is higher than which on HEC- 1A cells. The results indicate that FUT4 and LeY may been seemed as endometrial receptivity markers.2.Regulation of FUT4 can control the expression of LeY oligosaccharide, further increase the percent adhesion of embryo to uterine.3.FUT4 can regulate the LeY epression which further mediates the EGFR/MAPK signaling pathway and participates embryo implantation.