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5β1 IntegrinDepartments of Obstetrics and Gynecology (E.Y., K.Ino, S.S., A.I., H.K., K.S., A.N., F.K.) and Hematology (A.A.), Nagoya University Graduate School of Medicine, 466-8550 Nagoya, Japan; and Departments of Molecular Biochemistry and Clinical Investigation (E.M.) and Biochemistry (E.M., K.Ina.), Osaka University Graduate School of Medicine, 565-0871 Suita, Japan
Address all correspondence and requests for reprints to: Department of Obstetrics and Gynecology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: yamaeiko{at}med.nagoya-u.ac.jp.
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Abstract |
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5β1 integrin was significantly reduced in small interfering GnT-V-transfected Jar cells compared with mock transfectants, although the expression of 1, 5, 6, and β1 integrin on the cell surface was not changed. These results suggest that GnT-V is expressed in human EVT and is involved in regulating trophoblast invasion through modifications of the oligosaccharide chains of 5β1 integrin. |
Introduction |
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EVT differentiation and invasion is regulated by a variety of factors and multilevel processes, most of which are shared with invasive cancer cells. In addition to matrix metalloproteinases (MMPs), urokinase-type plasminogen activator and their inhibitors and adhesion molecules such as cadherin and integrin are involved in the regulation of both cancer and trophoblast cell invasion (3); however, trophoblast invasion is thought to have additional invasion check points because this cell type is largely confined to the endometrial-myometrial junction and continues until mid-gestation.
Extracellular matrix (ECM) associations of EVT are mediated through cell surface integrins. EVT differentiation is associated with changes in the cellular integrin profile and this combines with alterations in the ECM to form a microenvironment unique to placentation. Cytotrophoblast stem cells, which are located at the base of the anchoring villus, strongly express 6β4 integrin (laminin receptor). The first step in EVT differentiation involves the formation of large cell columns, which is accompanied by up-regulated 5β1 integrin (fibronectin receptor) expression within a fibronectin-rich microenvironment. When individual EVTs leave the cell columns and invade the uterine wall, they express 1β1 integrin (collagen/laminin receptor), in addition to the continued expression of 5β1 integrin, but 6β4 integrin is no longer detectable (1, 4). As an additional aid to invasion, migrant EVTs are also capable of secreting their own ECM, including fibronectin (5).
Oligosaccharides on glycoproteins are altered in tumorigenesis, and they often play a role in regulating the metastatic potential of tumor cells (6). A specific glycosyltransferase typically catalyzes the formation of a precise linkage between particular donor and acceptor molecules. To date, a number of genes encoding glycosyltransferases have been cloned worldwide (7). N-acetylglucosaminyltransferase V (GnT-V), a key glycosyltransferase involved in the formation of branching asparagine-linked oligosaccharides, is strongly linked to tumor metastasis (8, 9). Because integrins contain approximately 20–30 consensus N-glycosylation sites in both - and β-subunits, modifications of these N-glycans on integrins have the potential to alter their function (10). 5β1 integrin is a GnT-V target molecule and increased β1, 6-N-acetylglucosamine (β1-6GlcNAc) branching on β1 integrin by GnT-V has been shown to lead to inhibition of cisplatin-induced apoptosis by inhibiting 5β1 integrin clustering and can also induce migration of neck squamous cell carcinoma and fibrosarcoma (11, 12).
In this study, we show strong GnT-V expression in proximal, noninvasive EVT, which gradually reduced to low levels in distal, invasive EVT; In addition, we demonstrate that β1-6GlcNAc branching, the end product of GnT-V, is evident in EVT cells and localizes to the cell surface. Furthermore, to explore the underlying molecular mechanisms of this enzyme, we devised a knockdown approach using small interfering (si) RNA directed against GnT-V and show that GnT-V knockdown enhanced the migratory and invasive capacity of EVT cells through the decrease of β1-6GlcNAc branching on 5β1 integrin. Thus, GnT-V might be involved in regulating trophoblast invasion in first-trimester placenta.
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Materials and Methods |
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Immunohistochemistry
Placental samples from the first trimester were fixed in formalin and embedded in paraffin. Sections (thickness 4 µm) were immunostained as previously described (13), using anti-GnT-V antibody (Ab) (m21-b) at a dilution of 1:400 (14) and antihuman cytokeratin7 (CK7) Ab (Dako, Carpinteria, CA) at the dilution recommended by the manufacturer. For negative controls, the primary antibody was replaced with a nonspecific IgG (Santa Cruz Biotechnology, Santa Cruz, CA) at the same dilution.
Human chorionic villous explant culture
Villous explant cultures were established using placental tissues obtained from legal abortions (6–9 wk) as previously reported (13). After villous fragments were cultured for 6 d with or without 1 µg/ml swainsonine (SW; Wako Pure Chemical Industries, Osaka, Japan), the distance was measured from each villous tip to the furthest outgrowth. SW has been shown to cause the synthesis of hybrid-type oligosaccharides in vitro and in vivo, and it is a potent inhibitor of both lysosomal mannosidase and Golgi mannosidase II (15). As such, it inhibits the synthesis of complex-type oligosaccharides on N-glycans including β1-6 branching. Data were obtained from two separate experiments performed in six dishes for each group.
After culture, the explants were fixed with 4% paraformaldehyde for 30 min at room temperature and methanol for 10 min at –20 C. After blocking with 5% skim milk in PBS for 20 min at room temperature, the cultured tissue was immunostained for GnT-V and CK7, as described for immunohistochemistry. Identification of β1-6 branched, asparagine-linked oligosaccharides was done by lectin histochemistry, using horseradish peroxidase-conjugated leukoagglutinating phytohemagglutinin (L4-PHA; Seikagaku, Tokyo, Japan) at a dilution of 1:200, using a previously described method (16). This lectin preferentially recognizes β1-6 branches of tri- or tetraantennary sugar chains.
Cell lines and culture
Human choriocarcinoma cell lines (Jar, JEG-3, and BeWo) and HeLa cells (human cervical cancer cells) were purchased from the American Type Culture Collection (Manassas, VA). NaUCC-1, CC-3, and CC-4 are human choriocarcinoma cell lines that have been previously established in our laboratory (17). The human EVT cell line HTR-8/SVneo was kindly donated by Dr. Charles H. Graham (Queen’s University, Kingston, Ontario, Canada) (18). All cell lines were grown in RPMI 1640 (Sigma, St. Louis, MO), supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and 2 mM glutamine. 293 cells (American Type Culture Collection) were maintained in IMDM (Sigma) with 10% fetal calf serum. Cultures were incubated at 37 C in 5% CO2.
Western blot analysis and GnT-V activity assay
Western blot analysis for GnT-V protein was performed as previously described (16). The activity of GnT-V in whole-cell lysates was determined using a pyridylaminated, biantennary sugar chain as an acceptor substrate, as previously described (19). Briefly, 25 µl of a reaction buffer, which contained 250 mM Mes (pH 6.25), 80 mM UDP-GlcNAc, 20 mM EDTA, 400 mM N-acetylglucosamine and 1% Triton X-100, 10 µl of 400 mM the sugar chain substrate, and 15 µl of cellular proteins (10–50 µg) were incubated at 37 C for 4 h. After the enzyme reaction was stopped by boiling, the aliquot was applied to HPLC, using a TSK gel ODS-80TM column (4.6 x 150 mm; Tosoh, Tokyo, Japan). The specific activity of GnT-V was expressed as picomoles N-acetylglucosamine transferred per hour per milligram of proteins. Data were obtained from four individual experiments.
L4-PHA blot analysis
Protein-blotted nitrocellulose filters were prepared exactly as described for Western blotting. After blocking with 5% skim milk for 30 min at room temperature, the filter was incubated in PBS containing 1:1000 biotinylated L4-PHA (Seikagaku) for 1 h at room temperature. After washing, the filter was exposed to 1:1000 dilution of avidin-peroxidase conjugate (ABC kit; Vector Research, Burlingame, CA) in PBS for 30 min at room temperature. The membrane was washed and then developed using enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL).
In vitro cell proliferation assay
5 x 103 cells were plated in 100 µl of medium in 96-well plates and incubated for 96 h at 37 C. Cell viability was determined by modified tetrazolium salt (MTS) assay using the Cell Titer 96 Aqueous One solution proliferation assay kit (Promega, Madison, WI) according to the manufacturer’s instructions.
Transwell migration and invasion assay
The migration and invasion assay of HTR-8/SVneo cells was performed as previously reported (13). Briefly, cells were incubated with or without 1 µg/ml SW for 48 h before seeding and during migration and invasion periods. Invasion and migration assay of Jar was performed after 40 and 20 h of incubation, respectively. The number of cells was counted under a microscope at x200 magnification. Data were obtained from three individual experiments performed in triplicate.
Construction of siRNA vector and retroviral infection
We constructed a retroviral siRNA vector of human GnT-V using a modified, previously established method (20). Briefly, small interfering oligonucleotides specific for GnT-V were designed using the siRNA software on the Takara Bio web site (Ohtsu, Japan). The oligonucleotides were annealed and then ligated into BamHI/ClaI sites of the pSINsi-hU6 vector (Takara Bio). The pCGCGP construct, which expresses vesicular stomatitis virus glycoprotein, was used to produce high-titer viral supernatants rapidly (22). To generate pseudotype retrovirus, we cotransfected 10 µg of the siRNA vector and 10 µg of pCGCGP (21) into 293 cells using calcium phosphate coprecipitation. The culture medium was replaced with 8 ml of fresh medium 8 h after transfection and the retroviral supernatant was collected 48 h after transfection.
Jar cells were infected with the viral supernatant, and the cells were then selected with 0.5 mg/ml G418 for 2–3 wk. Stable GnT-V-knockdown was confirmed by GnT-V expression of protein and mRNA levels. Total RNA (1 µg) isolated from cells using an RNeasy kit (QIAGEN, Tokyo, Japan) was reverse transcribed using 2.5 µM random hexamers (Applied Biosystems, Foster City, CA) in 20-µl reactions. GnT-V and glyceraldehyde-3-phosphate dehydrogenase were amplified by PCR in 50-µl mixtures, as previously described (22, 23). The PCR products were resolved by electrophoresis on 1.0% agarose gels.
Zymography
2 x 105 cells were plated in a 12-well chamber and incubated with 400 µl serum-free medium for 24 h. The conditioned medium was mixed with the same volume of 2x sample buffer [0.125 M Tris-HCl (pH 6.8), 20% glycerol, 4% sodium dodecyl sulfate, 0.005% bromophenol blue] and loaded on nonreducing, 10% polyacrylamide gels containing 0.1% gelatin. The proteins were separated by electrophoresis at a constant voltage of 100 V for 150 min. Gels were washed to remove the sodium dodecyl sulfate and incubated overnight at 37 C in a solution of 50 mM Tris and 5 mM CaCl2 to allow for the enzymes to digest the gelatin. Gels were stained with 0.5% Coomassie brilliant blue and destained in 10% acetic acid/30% methanol.
Cell adhesion assay
Cells (4 x 104) were plated in 96-well plates coated with fibronectin, collagen type I and type IV (Becton, Dickinson and Co., Franklin Lakes, NJ) and allowed to attach to each matrix at 37 C for 30 min after centrifugation at 1500 rpm for 15 sec. After washing with PBS, absorbance readings at 492 nm (A492nm) were performed. The rate of cell adherence was calculated as follows: [A492nm (matrix) – A492nm (no matrix)]/A492nm (no matrix). Data were obtained from three individual experiments performed in eight wells.
Lectin blot analysis on immunoprecipitated 5β1 integrin
Immunoprecipitation was performed using 1 mg of proteins extracted from cells with antihuman 5β1 integrin monoclonal Ab (mAb) (MAB2247; Chemicon International, Temecula, CA), as previously described (12). Anti-β1 integrin mAb (Chemicon International) and anti-5 integrin Ab (H-104) (Santa Cruz Biotechnology) were used at a dilution of 1:500 and Western blotting was performed using the above protocol.
Flow cytometry
Cells were incubated with anti-β1 integrin mAb, anti-1 integrin mAb, anti-5 integrin mAb, and anti-6 integrin mAb for 1 h at 4 C. All mAbs were purchased from Chemicon International and diluted 1:40 in PBS. After washing with PBS, the cells were incubated with phycoerythrin (PE)-conjugated goat antimouse immunoglobulin Ab (Beckman Coulter Co., Fullerton, CA) at a dilution of 1:40 at 4 C for 30 min in the dark. The cells were washed twice and resuspended in 500 µl PBS. Data were acquired using a FACS Calibur (Becton, Dickinson and Co.), and analyzed using CELL Quest software (Becton, Dickinson and Co.).
Statistical analysis
Data are expressed as the mean ± SD. For data of in vitro and in vivo experiments, statistical comparisons among groups were performed using the Student’s t test or ANOVA with Bonferroni corrections. Differences were considered significant when P < 0.05.
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Results |
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). GnT-V was expressed strongly in the cytoplasm of proliferative EVTs, and this staining became weak in the distal, invasive EVTs within the maternal decidua (Fig. 1B, arrows).
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); these cells also exhibited positive immunoreactivity against the trophoblast marker, CK7 (Fig. 1C). GnT-V staining was stronger in the proximal cells than the distal cells, consistent with the immunohistochemical results observed in placental sections. We also examined the expression of β1-6 branching asparagine-linked oligosaccharides in cultured EVTs by immunohistochemistry using L4-PHA, which preferentially binds to GlcNAc residues on β1-6 branches of tri- or tetraantennary sugar chains. The cells exhibited positive L4-PHA staining on the cell membrane (Fig. 1E), suggesting that a target glycoprotein of GnT-V is expressed on the cell surface of EVT.
GnT-V expression and β1-6 branching in trophoblast cell lines and human placenta
GnT-V protein expression was examined in six choriocarcinoma cell lines, one EVT cell line, and three samples of human placental lysates by Western blotting. In all samples, GnT-V protein was detected as an approximately 100-kDa band (Fig. 2A). We also measured the level of GnT-V activity in cell extracts using HPLC to assess the functional capacity of this enzyme in the various lysates. Although the protein level of GnT-V expression varied little in all cell lines, its activity was higher in Jar and HTR-8/SVneo cells than the other cell lines (Fig. 2B); hence, we decided to continue experimentation with these two cell lines.
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). This analysis revealed the existence of GnT-V target proteins in human placenta and trophoblastic cell lines.
Effect of swainsonine on the migration of cultured primary EVT cells
To investigate GnT-V function and β1-6GlcNAc branching in EVT, we incubated cultured EVTs with SW, which inhibits Golgi -mannosidase II and ultimately inhibits N-linked β1-6 oligosaccharide formation upstream of the action of GnT-V. First-trimester villi were cultured with or without 1 µg/ml SW for 6 d and examined for the extent of β1-6GlcNAc branching and the distance of cell migration. L4-PHA lectin staining was significantly decreased in cultured EVTs treated with SW compared with control (Figs. 1E and 3A). After measuring the distance between the villous tip to the furthest EVT (Fig. 3B), we observed significantly increased cell migration (169 ± 71%, P < 0.01) with SW treatment (Fig. 3C). These results suggest that GnT-V might play a role in inhibiting EVT migration via β1-6GlcNAc catalysis.
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); however, SW had no effect on cell proliferation, as assessed by MTS assay (Fig. 4B). Additionally, we sought to determine whether SW-mediated suppression of β1-6GlcNAc branching affected the migratory and invasive capacities of this EVT cell line. Indeed, the percentage of migratory HTR-8/SVneo cells treated with SW significantly increased to 172 ± 11% (P < 0.01) of control (Fig. 4C). Similarly, the percentage of SW-treated cells penetrating the matrigel layer was significantly higher (184 ± 17%, P < 0.01) to that observed in control cultures.
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). RT-PCR analysis also indicated RNA interference-directed down-regulation of GnT-V mRNA expression in these cells (Fig. 5B). Lastly, we confirmed the efficacy of siRNA transfection by examining the extent of β1-6GlcNAc branching, which was significantly reduced in KD cells, as determined by lectin blotting (Fig. 5C).
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). In contrast, migration and invasion analyses revealed that KD cells have a significantly greater potential to migrate and invade (213 ± 21 and 334 ± 43%, respectively) than WT and mock-transfected cells (Fig. 5E). These results were consistent with those seen in primary EVTs and HTR-8/SVneo cells treated with SW and showed that suppression of GnT-V function promoted the invasive and migratory capabilities of EVTs.
Effect of GnT-V knockdown on gelatinase activity and cell adhesion to extracellular matrix
Invasion of tissues by trophoblast and tumor cells is a multistep process involving attachment to a basement membrane or ECM components, followed by degradation and subsequent migration through the degraded components. Type IV collagenases such as MMP-2 and MMP-9, are believed to be the principal mediators of trophoblast invasion. Hence, we performed gelatin zymography, revealing that Jar cells predominantly secreted MMP-2 and GnT-V knockdown did not significantly affect the expression of either the pro (72 kDa) or active form (62 kDa) of this enzyme (Fig. 6A). Next, we examined the adherence potential of Jar cell attachment to ECM proteins after GnT-V knockdown. The rate of cell attachment to fibronectin, collagen type I and collagen type IV was increased approximately 3-fold by knockdown of GnT-V expression (Fig. 6B).
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5β1 integrin but not 5β1 integrin expression6β4 and a gain of 5β1 integrins, implicating 5β1 in EVT cell function. Irving and Lala (24) reported that access to cell surface 5β1 is essential for EVT cell migration. We investigated whether integrin subtypes expressed on the cell surface were changed by GnT-V knockdown. Flow cytometric analysis demonstrated that expression levels of 1, 5, 6, and β1 integrin in KD cells were not significantly altered compared with WT and mock-transfected cells (Fig. 6C).
It has been reported that 5β1 integrin is one of the target molecules of GnT-V (11, 12): thus, modification of the sugar chain is an important step in determining glycoprotein function. Using 5β1 integrin immunoprecipitated followed by L4-PHA blotting, we examined whether GnT-V knockdown exerted any effects on the glycosylation state of 5β1 integrin. As shown in Fig. 6D, β1-6GlcNAc branching on both 5 and β1 integrins (immunoprecipitated from KD cells) was significantly decreased compared with WT and mock-transfected controls (Fig. 6D, upper panel); however, 5 and β1 integrin expression levels were not affected by siGnT-V (Fig. 6D, lower panels). These results suggest that diminished β1-6GlcNAc branching on 5β1 integrin by GnT-V knockdown is associated with the induction of EVT cell invasion.
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Discussion |
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Numerous studies have investigated integrin expression within different populations of trophoblasts in human placenta through immunohistochemical analyses (1, 25, 26), demonstrating that first-trimester cytotrophoblasts express 6β4 integrin (laminin receptor). As trophoblast cells migrate from the anchoring villus, they down-regulate 6β4 integrin and up-regulate both 5β1 (fibronectin receptor) and 1β1 integrin (laminin/collagen receptor). Intriguingly, the interactions between fibronectin and its receptor, 5β1 integrin, in EVT invasion has been reported to be both suppressive and permissive (4, 24, 27); however, these conflicting results may be due to ECM protein concentrations in each assay system (28). The present studies focuses on 5β1 integrin glycosylation in human EVT and, for the first time, reveals a novel regulatory mechanism of 5β1 integrin function by GnT-V in human trophoblast invasion.
Several GnT-V substrates with diverse roles in cell adhesion, migration, and proliferation are known, including β1integrin, matriptase, lamp-1, N-cadherin, and epidermal growth factor receptor (29, 30, 31, 32). Our lectin blotting experiments demonstrated that numerous proteins exhibit β1-6GlcNAc glycosylation in human trophoblasts. Additionally, these assays revealed that critical targets of GnT-V were relatively large, with extensive β1-6GlcNAc branching evident on proteins approximately 80–150 kDa. Lastly, L4-PHA histochemistry of cultured EVTs indicated that the target glycoprotein of GnT-V expressed was on the plasma membrane. These results support that the concept that one such GnT-V target could include 5β1 integrin, which is protein heterodimer of 150 and 130 kDa, expressed on the EVT cell surface; however, there remains a distinct possibility that other target glycoproteins may play a role in migration and invasion of human trophoblast cells.
Our results suggest that decreased GnT-V expression and subsequent reduction of β1-6GlcNAc branching during EVT differentiation from proximal (noninvasive) to distal (invasive) cell types may play a role in trophoblast invasion into the maternal compartment of the placenta; however, the factors regulating GnT-V expression during this process remains unclear. Previous reports have shown that the promoter of GnT-V has E2b transformation-specific sequence-1 (Ets-1) transcription factor binding sites in human bile duct carcinoma cells (33) and mRNA levels of GnT-V closely correlate to Ets-1 expression in various cancer cell lines (34). Moreover, Ets-1 protein expression exhibits strong immunoreactivity in EVT of the anchoring villus cell column, with a clear gradient of expression from the proximal to the distal end (35). These studies suggest that gradient expression of Ets-1 may be a factor in down-regulating GnT-V expression in the distal EVT cells of the first-trimester human placenta. Another possible regulatory mechanism may be mediated through the TGF-β signaling pathway because TGF-β has been reported to regulate GnT-V expression (36). TGF-β is produced primarily by the decidua and is a key negative regulator of EVT cell migration and invasion (24, 37); however, further studies are needed to identify those factors regulating GnT-V expression in trophoblast.
Previous studies have reported that overexpression of GnT-V resulted in increased migration and reduced 5β1 integrin-mediated adhesion to fibronectin, likely due to the inhibition of integrin clustering on the cell surface (38, 39, 40); however, our data indicate that EVT migration and invasion were enhanced by reduced GnT-V expression and β1-6GlcNAc branching. Such a discrepancy can be attributed to the type of cells or cell lines used. The majority of studies investigating the role of GnT-V on migration and invasion have used cancer cell lines; however, EVTs are not a malignant cell type. Whereas EVT invasion and tumor progression use similar biochemical mediators for invasion, trophoblast invasion is limited in both time and space. Invasion of the EVT occurs during the first trimester of pregnancy and does not progress beyond the proximal third of the myometrium.
The mechanisms by which GnT-V knockdown and subsequent reduction of β1-6GlcNAc branching stimulated EVT migration and invasion remain unclear; however, we speculate that this process may involve phosphorylation of focal adhesion kinase (FAK). Previous studies using cancer cells revealed that β1-6GlcNAc branching on 5β1 integrin or epidermal growth factor receptor by GnT-V resulted in altered cell function through changes in the phosphorylation status of FAK and/or ERK (12, 30). Furthermore, FAK has been reported to be expressed by trophoblasts at all stages of differentiation, but activated FAK was detected only at sites of EVT invasion (41). Under conditions that compromise cytotrophoblast differentiation (i.e. hypoxia in vitro, preeclampsia in vivo), FAK phosphorylation was substantially decreased, although total FAK levels did not change. Nonetheless, further studies are required to determine whether these signaling pathways induce trophoblast migration and invasion via suppression of GnT-V expression and/or function.
In summary, we provide the first evidence of a functional role of GnT-V in trophoblast migration and invasion in the human placenta. Our results show that GnT-V was strongly expressed in proximal EVT, which decreased in distal invasive types. This gradient decrease of GnT-V resulted in decreased β1-6 N-linked glycan branching of 5β1 integrin in invasive EVT. Consequently, cell attachment to ECM proteins in maternal decidua was stimulated, leading to a cellular phenotype with an increased invasive capacity. These findings suggest that GnT-V is involved in the regulation of trophoblast invasion.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online October 9, 2008
Abbreviations: Ab, Antibody; CK7, cytokeratin 7; ECM, extracellular matrix; EVT, extravillous trophoblast; FAK, focal adhesion kinase; β1-6GlcNAc, β1, 6-N-acetylglucosamine; GnT-V, N-acetylglucosaminyltransferase V; L4-PHA, leukoagglutinating phytohemagglutinin; mAb, monoclonal Ab; MMP, matrix metalloproteinase; MTS, modified tetrazolium salt; si, small interfering; SW, swainsonine.
Received July 8, 2008.
Accepted for publication September 26, 2008.
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References |
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-D-mannoside β1,6 N-acetylglucosaminyltransferase. J Biol Chem 257:13421–134275β1 integrin clustering and stimulates cell migration. Cancer Res 62:6837–68455β1 integrin in a cisplatin-resistant human squamous cell carcinoma cell line. Mol Cancer Ther 2:1207–12145β1. Biol Reprod 60:828–8385β1 integrin expression mediated by the protein kinase C signaling pathway. J Biol Chem 280:8332–8342This article has been cited by other articles:
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  K. K.W. Lam, P. C.N. Chiu, M.-K. Chung, C.-L. Lee, K.-F. Lee, R. Koistinen, H. Koistinen, M. Seppala, P.-C. Ho, and W. S.B. Yeung Glycodelin-A as a modulator of trophoblast invasion Hum. Reprod., September 1, 2009; 24(9): 2093 - 2103. [Abstract] [Full Text] [PDF]
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