Studies On The Upregulation Of Fucosyltransferase IV(FUT4) And Promotion Of Embryo Implantation Mediated By Baicalin

Backgrounds and Objective:1. Glycosylation plays a significant role in determining the receptivity of the uterine endometrium to embryo.2. Fucosyltransferase IV(FUT4) is stage-specifically expressed in the uterine endometrium of mammalians and considered as a marker of the endometrial receptivity.3. Baicalin, a monomer of flavonoids extracted from the herb Scutellaria Baicalensis,is known to have the functions in improving the reproduction in traditional Chinese medicine.4. However, whether the regulatory mechanism of baicalin is correlated with the expression of endometrial FUT4 in embryo-endometrium adhesion remains unclear.5. In this study, we are arming to explore the effect of baicalin on embryo adhesion/implantation and its underlying glycobiological mechanisms, which further reveal the important regulatory effect of baicalin on mammalian embryo implantation.Methods:1. RT-PCR, Western blot and immunofluorescence were used to observe the influence of baicalin on the expression levels of FUT4 mRNA and protein in human endometrialRL95-2 cells.2. By using Western blot, the levels of key molecules involved in Wnt/β-catenin signaling pathway, including phosphorylated glycogen synthase kinase 3β(p-GSK3β),GSK3β, β-catenin and specificity protein-5(SP5), were examined in 8 groups, which contain control, dimethylsulfoxide(DMSO), baicalin, dickkopf-1(DKK1), baicalin combined with DKK1, vector, β-catenin siRNA(si-β-catenin) and baicalin combined with si-β-catenin groups.3. To explore how SP5 regulates the expression of FUT4, we analyzed FUT4 expression levels in sh-SP5, SP1-cDNA and the co-transfection groups in RL95-2 cells by using RT-PCR and Western blot.4. Embryo adhesion rate in 15 groups, including control, DMSO, baicalin(3, 6μg/m L), sh-SP5 et al. were assayed in in vitro implantation model with the co-cultured endometrial RL95-2 cells and embryonic JAR cells.5. Baicalin and S. Baicalensis decoction(SBD) were also gavaged to Kunming mice to further detect SP5 and FUT4 mRNA and protein expression levels in mouse uterine endometrium at gestation day 4(GD 4) by using RT-PCR, Western blot and immunohistochemistry.6. Embryo implantation number in control, saline, baicalin and SBD groups were calculated at GD 8.Results:1. Baicalin elevates endometrial FUT4 m RNA and protein expression levels in human endometrial RL95-2 cells. RT-PCR and Western blot demonstrated that baicalin 3μg/m L(P<0.05) and 6 μg/mL(P<0.01) could significantly enhance FUT4 mRNA and protein expression levels in RL95-2 cells compared with the control and DMSO groups.Simultaneously, immunofluorescent staining obtained the same results as above.2. Baicalin activates Wnt/β-catenin signaling pathway in human endometrial RL95-2cells. Western blot revealed that baicalin(6 μg/mL) could obviously increase the expression levels of p-GSK3β, β-catenin and SP5(P<0.001) in RL95-2 cells compared with the control; also baicalin treatment recovered the expression levels of p-GSK3β,β-catenin and SP5 in RL95-2 cells that pre-treated with DKK1 or β-catenin si RNA,respectively.3. SP5 increases FUT4 mRNA and protein expression levels in human endometrial RL95-2 cells. RT-PCR and Western blot showed that the expression of FUT4 m RNA and protein in sh-SP5(P<0.001), SP1-cDNA(P<0.01) and the co-transfection(P<0.001) groups were strongly decreased compared with the controls.4. Baicalin enhances human endometrial RL95-2 cells adhere to human embryonic JAR cells by the upregulation of endometrial FUT4 mediated via Wnt/β-catenin signaling pathway. Adhesion experiment showed that there was no statistical significance among the control, DMSO, sh-control and mock groups; embryo adhesion rate of RL95-2 cells in baicalin groups were largely enhanced compared with the control and DMSO groups, while in sh-SP5 or SP1-cDNA group was significantly decreased in comparison with the respective control; also, baicalin recovered embryo adhesion rate of RL95-2 cells pre-treated with sh-SP5 or SP1-cDNA, but still lower than that in baicalin groups; baicalin could considerably increase the adhesion rate in the co-transfection group(P<0.01); further study demonstrated that baicalin significantly reversed the low embryo adhesion rate mediated by FUT4 siRNA(P<0.001).5. Baicalin increases endometrial FUT4 mRNA and protein expression levels in mouse endometrial tissues during implantation window. RT-PCR and Western blot revealed that baicalin and SBD both could significantly upregulate the gene and protein expression levels of FUT4 and SP5 in mouse uterine endometrium at GD 4 in comparison with the control and saline groups. Consistently, immunohistochemistry showed that SP5 and FUT4 stain in SBD group were stronger than that in control group,but weaker than that in baicalin group.6. Baicalin improves the rate of successful pregnancy in mouse model. The number of implanted embryo in baicalin(P<0.01) and SBD(P<0.05) groups were highly increased compared to the control and saline groups.1. Baicalin elevates endometrial FUT4 m RNA and protein expression levels in human endometrial RL95-2 cells and mouse endometrial tissues.2. Baicalin enhances human endometrial RL95-2 cells adhesion to human embryonic JAR cells.3. Baicalin facilitates embryo implantation in mouse model.4. Baicalin increases endometrial receptivity by upregulation of FUT4 mediated by Wnt/β-catenin signaling pathway.