Embryo implantation is initiated by the recognition and adhesion of the embryo to the endometrial epithelium, and many adhesive molecules are involved the initial molecular interactions at the maternal–fetal interface (1). The oligosaccharides carried by the adhesive molecules, for example, sialyl Lewis X (sLeX), Lewis Y (LeY), and Lewis X (LeX), are stage-specifically expressed by the endometrial epithelium. They reach relatively a high level on the cell surface during the implantation stage (2–5). Sialyl Lewis X, the ligand of L-selectin, plays an important role through L-selectin-ligand adhesion system.
L-selectin-ligand adhesion system are involved in some physiological and pathological processes, for example, inflammation, tumor metastasis and embryo implantation (6–8). In the implantation stage, the human endometrium upregulates sLeX oligosaccharide synthesis, and on the fetal side, the expression ofL-selectin increases in the trophoblasts (9, 10). It is considered that sLeX binds with L-selectin and mediates the initial recognition and adhesion between embryo and endometrium, which is crucial to establish human pregnancy (1, 10).
Sialyl Lewis X (NeuAcα2→3Galβ1 →4 [Fucα1-3] GlcNAcβ1→3Galβ1→R) is a fucosylated oligosaccharide. The last step of sLeX synthesis is catalyzed by the specific α1,3-fucosyltransferases (α1,3-FUTs). Currently, six kinds of α1,3-FUTs, FUT3, FUT4, FUT5, FUT6, FUT7 and FUT9, have been identified (11–13). Among these, the acceptor specificity of FUT7 is restricted to the distal N-acetylglucosamine on α2,3-sialylated lactosamines forming sLeX antigen, therefore, FUT7 is a key enzyme for the synthesis of sLeX (14, 15). The study shows that inhibition of FUT7 expression suppress the production of sLeX, thereby reducing carbohydrate-dependent cell adhesion in the endothelial cells (16), but the inhibitory effect of FUT7 on sLeX and embryo implantation remains unknown.
In this study, an in vitro implantation model composed of human uterine epithelial cell line (RL95-2) and human embryonic cell line (JAR) was utilized. RL95-2 cells and JAR cells can mimic the receptive uterine epithelium and adhesion-competent embryo, respectively (17–19). To inhibit the expression of FUT7 gene, an antisense oligodeoxynucleotide targeting specific FUT7 sequence was synthesized and transiently transfected to RL95-2 cells. We found that reduction of FUT7 expression by the antisense oligodeoxynucleotide was associated with the decreased synthesis of sLeX in RL95-2 cells, and the reduced adhesion of JAR embryonic cells to RL95-2 uterine epithelial cells.
MATERIALS AND METHODS
An antisense oligodeoxynucleotide (5′-CAGCATTATT CATCCACAGT-3′, 19-38) and a control sense oligodeoxynucleotide (5′-ATGAATAATGCTGGGCACGG-3′, 26–45) were designed against the initiation region of the published sequence for human FUT7 (GenBank accession number: BC074746) by RNA Structure Software. Oligodeoxynucleotides were synthesized, purified and modified by phosphorothioate at the Shanghai Company (China). The specificity of the oligodeoxynucleotides was confirmed by BLAST sequence analysis.
The human uterine epithelial RL95-2 cells and human embryonic JAR cells were obtained from the American Type Culture Collection (Manassas, VA). RL95-2 cells were grown in DMEM/F12(1:1) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 5 μg/mL insulin (Sigma–Aldrich, St. Louis, MO), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO2 in humidified air. The JAR cells were maintained in RPMI 1640 (Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C under 5% CO2 in humidified air according to standard procedures. The growth medium was renewed every 2–3 days.
RL95-2 cells (5 × 104/mL) were trypsinized and seeded onto six-well plates. When cells reached 90% confluence, the transfection was performed. The cells were transfected with 200 nM sense oligodeoxynucleotides, and either 50 nM, 100 nM, or 200 nM antisense oligodeoxynucleotides using 2 μL of Lipofectamine™ Reagent and Plus™ Reagent following the manufacturer's instructions (Invitrogen). An untransfected control was maintained. RNA and protein samples were obtained from RL95-2 cells after incubation for 48 h.
Total RNA was extracted from cells using Trizol reagent (Invitrogen). cDNA was synthesized using a RNA PCR Kit (AMV) Version 3.0 (Takara, Otsu, Shiga, Japan). For PCR, the FUT7 gene forward primer was 5′-CACCTCCGAGCATCTT CAACTG-3′ and reverse primer was 5′-CGTTGGTATCGG CTCTCATTCATG-3′ and produced a PCR product of 497 bp. The β-actin gene forward primer was 5′-ATCTGGCACCA CACCTTCTACAATGAGCTGCG-3′ and reverse primer was 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′ and produced a PCR product of 838 bp. PCR reactions to amplify the DNA fragment were carried out as follows: initial denaturation at 94°C for 5 min, 35 cycles of 94°C for 40 sec, 57°C for 45 sec, 72°C for 45 sec, and a final extension at 72°C for 5 min. The amplified products were analyzed by 1% agarose gel electrophoresis, and the bands were visualized by ethidium bromide staining, followed by analysis with LabWorks 4.6 gel imaging and analysis software (UVP. Inc). β-Actin expression was detected as the internal control. The experiments were repeated three times.
To prepare whole cell protein extracts, cells were washed in PBS and resuspended in cell lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, pH 8.0, 0.2 mM Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% Nonidet P-40) and incubated on ice for 30 min. The cell lysates were clarified by centrifugation at 9,000g for 10 min, and the supernatant was collected. Protein concentration was determined with the Coomassie Protein Assay Reagent (Bio-Rad, Hercules, CA) using bovine serum albumin as a standard. Total protein lysates (80 μg) were separated by 10% SDS-PAGE mini-gel. Samples were transferred electrophoretically to nitrocellulose membranes. After blocking with TTBS (50 mM Tris-HCl pH 7.5, 0.15 M NaCl, 0.1% Tween-20) containing 5% fat-free dry milk for 2 h, the membrane was incubated with goat anti-FUT7 (1:300) (Santa Cruz Biotech, Santa Cruz, CA) in TTBS containing 5% fat-free dry milk for 2 h at 37°C. After washing with TTBS three times, 5 min each, the membrane was incubated with biotinylated anti-goat IgG (1:500) (Santa Cruz Biotech) antibody. After washing in TTBS, the bound antibody was detected through the use of avidin-conjugated horseradish peroxidase (1:500) (Santa Cruz Biotech). Color reaction was obtained using NBT/BCIP. The same blot was reprobed with anti-β actin antibody (Santa Cruz Biotech) to confirm equal loading. The membranes were scanned and analyzed using LabWorks 4.6 software. The experiments were repeated three times.
Indirect Immunofluorescence Staining
Transfected cells were also grown on glass coverslips. Forty-eight hours after transfection, the cells were washed with PBS and then fixed in 4% paraformaldehyde for 15 min. Cells were permeabilized with 0.1% Triton-PBS for 10 min at room temperature for FUT7 staining. After blocking with 1% complete serum for 2 h at 37°C, goat anti-FUT7 antibody (1:100) and mouse anti-sLeX antibody (1:100) (Chemicon, Temecula, CA) were applied to the slide and incubated overnight at 4°C. The coverslips were washed three times with PBS, and then incubated with FITC-conjugated anti-goat IgG (1:200) (Sigma) or TRITC-conjugated rat anti-mouse IgM (1:200) (Sigma) for 30 min at 37°C. After rinsing, specimens were mounted in PBS containing 90% glycerol and 1.0% p-phenylenediamine and subsequently monitored under a BX51 Olympus microscope.
Adhesion of JAR Cells to RL95-2 Cell Monolayer
The adhesion assay was performed as described (19). In brief, RL95-2 cells were grown on the coverslips to form confluent monolayers, and then differently treated before addition of JAR cells as follows: cells of untransfected (control); transfected with 200 nM sense oligodeoxynucleotides; 50 nM, 100 nM, or 200 nM antisenseoligodeoxynucleotides; preincubated with anti-sLeX antibody (5 μg/mL) for 30 min; and cells transfected with 200 nM antisense oligodeoxynucleotides followed by incubation with anti-sLeX antibody (5 μg/mL) for 30 min. Then, the cultured JAR cells (5 × 104) were harvested, counted, and gently delivered onto the confluent monolayer of RL95-2 cells in JAR growth medium. After 1 hr, JAR cells unadhered to the endometrial cell monolayer were removed by centrifugation of the coverslips with the cell surface facing down at 12g for 5 min. The unadhered cells are counted after resuspension of the pellet with PBS, and the attached JAR cells on the coverslip were also counted for further confirmation. The assay was repeated three times. Adhesion potential was calculated as the percentage of the JAR cells that had attached compared with the total number of JAR cells plated.
Results are expressed as mean ± standard error of mean (SEM) of three independent experiments. Statistical significance of difference between test groups was assessed by one-way ANOVA followed by Scheffe's test (post hoc), with P < 0.05 considered to be significant.
FUT7 Gene Expression Detected by RT-PCR
To determine the effect of antisense oligodeoxynucleotides on FUT7 gene expression, different doses of antisense oligodeoxynucleotides were transiently transfected into RL95-2 cells. A semi-quantitative RT-PCR was adopted to analyze the expression of FUT7 mRNA (Fig. 1). The relative density of a 497 bp product of the FUT7 gene and an 838 bp product of internal control β-actin was calculated after separation by 1% agarose gel electrophoresis. Results showed that the expression of FUT7 gene was dramatically decreased in antisense oligodeoxynucleotide transfected RL95-2 cells in comparison with the untransfected control or sense oligodeoxynucleotide transfected cells. The inhibition of gene expression in the cells treated with 50 nM, 100 nM, or 200 nM antisenseoligodeoxynucleotides was 21.8%, 43.5%, and 60.2%, respectively, and in a dose dependent manner.
FUT7 Protein Expression Detected by Western Blotting
To detect the protein expression level of FUT7 in RL95-2 cells after oligodeoxynucleotide transfection, Western blotting was employed (Fig. 2). Results revealed that the expression level of FUT7 was much lower in antisense oligodeoxynucleotide transfected RL95-2 cells in comparison with the untransfected control or sense oligodeoxynucleotide transfected cells. The level of reduction was 26.2%, 44.2% and 74.0%, respectively, with the strongest inhibition in 200 nM antisense oligodeoxynucleotide transfection. Results indicate that antisense oligodeoxynucleotide transfection effectively interferes with the expression of FUT7 and the effect is dose dependent.
Indirect Immunofluorescence Analysis of FUT7 and sLeX
Both FUT7 and sLeX synthesis in RL95-2 cells were analyzed after oligodeoxynucleotide transfection by indirect immunofluorescence. Results showed that FUT7 was mainly located in the cytoplasm (Figs. 3b and 3c) and sLeX on the cell membrane (Figs. 3e and 3f). The fluorescence intensity of both FUT7 and sLeX in the cells transfected with antisense oligodeoxynucleotide (200 nM) was weaker than that of the untransfected controls (Figs. 3c vs. 3b, 3f vs. 3e). This indicates that the synthesis of FUT7 and sLeX in RL95-2 cells is decreased after antisense oligodeoxynucleotide transfection.
Analysis of Adhesion
To observe the influence of antisense oligodeoxynucleotide on the adhesion of JAR cells to RL95-2 cell monolayer, the in vitro adhesion model of RL95-2 cells and JAR cells was used (Fig. 4A). Statistical analysis showed that the percent adhesion had no difference between the untransfected control (68.6% ± 1.5%) and sense oligodeoxynucleotide transfected cells (63.0% ± 2.1%). However, the percent adhesion in RL95-2 cells transfected with antisense oligodeoxynucleotides was significantly decreased compared with the untransfected control (P < 0.01), and the lowest percent adhesion of the cells transfected with 200 nM antisense oligodeoxynucleotide was 48.0% ± 2.0%. In addition, blocking the RL95-2 cells with anti-sLeX antibody caused a decreased percent adhesion (42.3% ± 2.4%) compared with the untransfected control. The percent adhesion (36.4% ± 2.5%) was the strongest inhibited in the cells with 200 nM antisense oligodeoxynucleotide transfection followed by incubation with anti-sLeX antibody. The results indicate that antisense oligodeoxynucleotides of FUT7 gene inhibits the adhesion of embryo cells to the endometrial cells through decreased expression of FUT7 enzyme and sLeX antigen.
Implantation is a complex developmental process, and the cross-talking based on the molecular interactions between the mature embryo and receptive endometrium is crucial (20, 21). The elevated expression of the oligosaccharides carried by glycoconjugates are not only the receptive markers but also the functional adhesive ligands of the endometrial epithelium during implantation period. For example, LeY is highly expressed in the endometrial epithelium of human and mouse at implantation window, and preincubation of the cultured murine epithelium with anti-LeY antibody significantly blocks the adhesion and implantation of the embryo to the epithelium (22). Sialyl Lewis X expressed by the endometrium is also closely related to the functional state of implantation. Sialyl Lewis X of human endometrial epithelium increases in abundance after ovulation and reaches a maximum (23). It is also correlated with the further trophoblast emigration from the placenta (24).
The synthesis of stage-specifically expressed fucosylated oligosaccharides are controlled by their respective fucosyltransferases, the key synthesis enzymes. FUTs are grouped as α1, 2-, α1, 3- and α1, 6- FUTs according to the types of fucosylation linkage (11, 12). FUT1 and FUT4 take part in the synthesis of LeY oligosaccharide antigen (25), and FUT9 is the key enzyme for LeX production (26, 27). FUT7 is mainly expressed in leukocytes, endothelial cells (15, 28) and tumor cells (29, 30). FUT7 synthesizes only sLeX epitope (14). Transfection of FUT7 cDNA can increase the synthesis of sLeX antigen (31). Recently, we introduced the FUT7 gene into RL95-2 cells by transient transfection, and found that FUT7 up-regulation in the transfected cells was strongly associated with the augmentation of sLeX compared with the control (unpublished data). The introduction of antisense cDNA of FUT7 gene into the human lymphoid cell lines selectively down-regulated FUT7, and suppressed sLeX expression and E-selectin mediated binding (32). Transfection of FUT7 antisense sequence impairs the tumor proliferation (33). The tumor metastasis was also attenuated because of the reduction of L-selectin ligands in FUT7-deficient mice (6). The suppression of FUT7 expression and the determination of the effect of this inhibition on embryo implantation has not been performed.
Antisense oligodeoxynucleotides are small segments of single-strand DNA complementary to the mRNA regions of a target gene. They can interfere with translation by binding to the target mRNA, inducing degradation of the mRNA, and thereby inhibiting translation (34, 35). Oligodeoxynucleotides are relatively easier access to specific cellular targets and have fewer side effects. Therefore, oligodeoxynucleotides are useful in the research and practical application. In this work, we observed the effect of FUT7 suppression by introducing an antisense oligodeoxynucleotide of FUT7 gene to the cultured RL95-2 cells. We found that the expression of both FUT7 enzyme and sLeX antigen were decreased in a dose dependent manner, and the adhesion of JAR embryonic cells to RL95-2 epithelial cells was significantly reduced, compared with the untransfected control and sense oligodeoxynucleotide transfected cells. The decrease in the percent adhesion was also studied in the RL95-2 cells after single anti-sLeX antibody incubation or combined antisense oligodeoxynucleotide transfection and anti-sLeX antibody incubation. Although the combined treatment of antisense oligodeoxynucleotide transfection and anti-sLeX antibody incubation caused the strongest inhibition of adhesion, it had only a moderate effect, indicating that other ligands of L-selectin except sLeX, for example, MECA-79 epitope or heparin-like molecules, can also interact with L-selectin (36, 37). Further studies are needed.
In conclusion, the antisense oligodeoxynucleotide of FUT7 can significantly suppress the gene and protein expression of FUT7, and decrease sLeX synthesis in uterine epithelial cells, and thereby interrupt sLeX antigen mediated adhesion of embryonic cells to uterine epithelial cells. FUT7 may be a potential target to control embryo adhesion and implantation.
This work was supported by National Natural Science Foundation of China Research grants (30270329, 30670465) and Liaoning Provincial Core Laboratory of Glycobiology and Glycoengineering grant.