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Regular Article |
a Department of Obstetrics, Gynecology and Women's Health, UMD-New Jersey Medical School, Newark, New Jersey 07103-2714
ABSTRACT
Episialin, which is found on the apical membrane of human endometrial epithelium, has been postulated to act as an antiadhesive factor through the steric hindrance generated by its extensively glycosylated structure. The present studies were designed to test this hypothesis in an in vitro model of endometrial-blastocyst attachment. Episialin was expressed in human endometrial carcinoma cells (HEC-1A > RL95-2), and attachment of JAr choriocarcinoma cells to the endometrial cell monolayers was inversely related to episialin expression. Treatment of endometrial monolayers with type III sialidase increased JAr binding, and this increase was suppressed by HMFG1, a monoclonal antibody specific for episialin. The effects of sialidase appear to have resulted from a contaminant protease rather than from a loss of sialic acid residues, because sialidase preparations other than type III were ineffective. After sialidase treatment, conditioned medium from cells treated with type III sialidase contained more episialin than medium from cells treated with other sialidase preparations. Similar attachment-assay results were obtained using O-sialoglycoprotein endopeptidase; after treatment, the increase in JAr binding (>50%) was suppressed by the antiepisialin antibody. These results demonstrate for the first time that episialin acts as an antiadhesive agent in a model of human endometrial-blastocyst attachment.
implantation/early development, uterus
INTRODUCTION
Attachment of the mammalian blastocyst to the endometrium is a highly regulated process. The human endometrium is unreceptive to blastocyst attachment except for a short period of the menstrual cycle known as the window of implantation [1]. During that period, which occurs between Days 20 and 24 of the menstrual cycle, uterine receptivity increases markedly, in turn increasing the probability of blastocyst attachment [2, 3]. It is widely accepted that the external surface of the trophectoderm and the luminal surface of the endometrial epithelium both present factors that promote or are involved in endometrial-blastocyst attachment. In addition to these proadhesive factors, the presence of inhibitory factors on these surfaces that prevent or reduce the probability of blastocyst attachment has also been suggested. One such putative factor is the mucin episialin, which is proposed as an antiadhesive agent because of its extended, glycosylated structure [4–7]. Episialin is a high-molecular-weight, integral membrane protein found on the apical membrane of the endometrial epithelium in humans and other mammalian species. Its extracellular domain consists of a variable number of repeat amino acid sequences, each containing serine and threonine residues that are sites for O-linked glycosylation. Human episialin has between 30 and 90 of these amino acid repeats depending on the specific tissue. Thus, carbohydrate makes up approximately 40% by weight of the mature glycoprotein. The molecule is predicted to have a rigid, secondary structure that is hypothesized to extend out from the cell surface to a height of 200–300 nm, some 10- to 20-fold higher than the normal glycocalyx [8]. Hilkens et al. [4] have suggested that episialin shields the apical domain of an epithelium, providing steric hindrance to other molecular interactions such as cell-cell adhesion. In addition to inhibition by steric hindrance, sialylation of the episialin side chains also may produce some degree of electrostatic repulsion, which is another potential antiadhesive mechanism. Supporting this antiadhesive role, Ligtenberg et al. [9] noted that human mammary epithelial cells and melanoma cells transfected with high levels of episialin had less aggregation capacity than the nontransfected parental cells. Moreover episialin also inhibits lymphocyte-target cell interaction and the integrin-mediated adhesion of cells to extracellular matrix, adding to the evidence supporting its antiadhesive nature [10, 11].
To be considered as a candidate adhesive or antiadhesive factor on the endometrium, a molecule must be present on the luminal surface of the endometrial epithelium and have some functional role in the attachment process. The existence of an implantation window implies changes in the endometrium that permit blastocyst attachment, changes which might include direct up- or down-regulation of adhesive or antiadhesive molecules. Evidence shows that episialin is present on the luminal surface of the human endometrial epithelium, and that its expression varies during the course of the menstrual cycle [12–15]. What has not been demonstrated is a functional role for episialin in the human blastocyst attachment process. The long-term goal of this research is to determine whether episialin acts as an antiadhesive factor in human endometrial-blastocyst attachment and how it is regulated. As an initial step, the role of episialin in cell-cell interaction has been explored using an in vitro attachment model, developed from those described by Rohde and Carson [16] and Thie et al. [17], and designed to represent some of the elements of endometrial-blastocyst adhesion in vivo. This model system measures the binding of JAr cells, a choriocarcinoma cell line derived from early human trophoblast and representing the trophectoderm, to carcinoma cell lines derived from the endometrial epithelium. The work reported here was designed to test the hypothesis that episialin hinders or reduces the ability of endometrial cells to bind JAr trophoblast cells.
MATERIALS AND METHODS
Cell Preparation
Cells were grown in Dulbecco's minimum essential medium (DMEM)/F12 supplemented with 25 mM Hepes and containing 10% fetal bovine serum (FBS). JAr cells (80% confluency) were trypsinized (0.25% w/v trypsin for 10 min at 37°C) on the night before assay and allowed to replate in the same flask. On the morning of the assay, the growth medium was replaced with Hanks' balanced salt solution (HBSS) containing 25 mM Hepes and 50 µM carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE), and the cells were incubated for 30 min at 37°C. After incubation, the CFDA-SE solution was removed, and the cells were rinsed with Enzyme-Free Cell Dissociation Solution (EFDS; Specialty Media, Lavallette, NJ) and then lightly trypsinized (0.04% w/v trypsin in EFDS for 2 min at 37°C). After trypsinization, DMEM/F12 containing 0.5% w/v BSA was added to the cells, which were washed and resuspended in the same medium to give 5.0 x 105 cells/ml. The trypsin pretreatment and light trypsinization procedures described were found to be the most effective method for producing a population of single JAr cells in suspension with a viability of more than 90% during the lifetime of the experiments.
For the attachment assay, HEC-1A, RL95-2, and AN3-CA endometrial carcinoma cell lines were plated in 12-well plates and grown to confluence in DMEM/F12/FBS. On the day of the assay, the growth medium was removed, and the endometrial cells were incubated in DMEM/F12/BSA for 60 min before the assay. For cell extraction and assessment of episialin expression by Western and slot-blotting analysis, cells were grown in 100-mm tissue culture dishes with DMEM/F12/FBS.
Attachment Assay
In the attachment assay, JAr cells (5 x 104 cells/0.1 ml), prepared as described earlier, were added to the wells containing confluent endometrial monolayers and incubated for 60 min at 37°C. As a negative control, JAr cells (5 x 104 cells/0.1 ml) were added to wells that had been preincubated with DMEM/F12/BSA but contained no endometrial cells. As a positive control, wells containing the endometrial cells under investigation were incubated with a double quantity of JAr cells (10 x 104 cells/well). To be included as a valid attachment experiment, the cells found attached in the positive control had to number 50% more than those in the control wells. After incubation with JAr cells, the medium in the wells was aspirated. and the wells were washed twice with 1 ml of DMEM/F12/BSA to remove nonadherent JAr cells. To achieve a consistent procedure, washing was performed using an electric pipettor that was programmed to repetitively eject an exact volume of wash buffer with an identical force each time (Electrapette; Matrix Technologies Corp., Hudson, NH). Wash volumes were directed onto the side of the well being washed to minimize direct shear forces on the endometrial monolayers. After washing, the wells were drained, and the cells were extracted by the addition of 0.5 ml of extraction buffer (0.5 N NaOH/1% SDS) and repeated passage through a 23-gauge needle. A sample of the extract was diluted in water, and the CDFA-SE fluorescence in the extract was measured using an SLM 8000C fluorimeter (SLM-Aminco, Urbana, IL) with an excitation wavelength of 490 nm and a long-pass emission filter of 530 nm.
In certain experiments, endometrial cells were pretreated with a capping antibody (214D4, 1:1, v:v) or an antiepisialin antibody (HMFG1, 1:20, v:v) for 30 min at 37°C. In other experiments, endometrial cells were pretreated with sialidase (100 mU/ml) or O-sialoglycoprotein endopeptidase (OSGEP, 60 µg/ml) for 60 min at 37°C. Cells were also treated with HMFG1 before treatment with sialidase, being incubated initially with HMFG1 alone for 30 min then for a further 60 min at 37°C in the presence of sialidase. For experiments in which cells were pretreated with sialidase, the pretreatment supernatants were removed, stored at -70°C, and replaced with DMEM/F12/BSA.
Immunoblotting
Extraction of cells for Western and slot-blotting analysis was performed by washing the cells with HBSS, scraping them in extraction buffer (50 mM Hepes/Tris [pH 7.4] containing 1 % SDS, 0.5 mM EDTA, and 0.5 µg/ml aprotinin and leupeptin), and repeatedly passing the extract through a 23-gauge needle. Protein concentrations were measured using the Bradford protein assay [18].
For Western blotting analysis, extracted cell samples were diluted into Western sample buffer to give a final concentration of 2.7 M urea, 1.7% SDS, 20 mM Tris-HCl, and 150 mM dithiothreitol. The samples were vortexed and then boiled for 5 min before loading. Samples (30–60 µg) were electrophoresed either on 6% SDS gels (4% stacking gel) or 4%–15% SDS gradient gels according to the method of Laemmli [19] and then blotted on nitrocellulose by electrophoretic transfer (100 mA) for 18 h. After transfer, the nitrocellulose membranes were air-dried and then blocked with 3% BSA in Tris-buffered saline (TBS; 150 mM NaCl, 50 mM Tris-HCl [pH 7.5]) for 60 min at room temperature. To increase the sensitivity of the assay, the membrane-bound episialin was desialylated, thus enabling improved antibody access to the core protein of episialin, as described elsewhere [20, 21]. Membranes were incubated for 18 h at 37°C with 30 mU/ml of sialidase (Vibrio cholerae; Boehringer Mannheim, Indianapolis, IN) in 50 mM sodium acetate, 4 mM CaCl2, and 10% BSA. After rinsing with TBS, membranes were blocked for 60 min at room temperature in 5% casein (in TBS). Membranes were then incubated for 18 h at 4°C with HMFG1, a monoclonal antibody specific for the PDTR repeat epitope in the repeat region of episialin (diluted 1:100 v:v in TBS containing 0.5% BSA). After washing with TBS containing 0.05% Tween (TBST; 1 x 15 min, 2 x 5 min), the membranes were incubated for 60 min at room temperature with the secondary antibody, rabbit antimouse immunoglobulin (Ig) G labeled with horseradish peroxidase (1:10,000 in TBS/BSA). The secondary antibody was removed by washing with TBST (1 x 15 min, 2 x 5 min), and immunoreactive bands were visualized on film by chemiluminescence (Renaissance; NEN Life Science Products, Boston, MA).
Preliminary studies by Western blotting analysis using HMFG1 revealed a single immunoreactive band with minimal background signal (see Results), enabling the use of slot blots for quantification of episialin. Samples were diluted in TBS to reduce the SDS concentration to 0.02%, and then 30 µg of membrane protein in 0.1–0.2 ml was blotted onto a nitrocellulose membranes using a Minifold II slot blot apparatus (Schleicher & Schuell, Keene, NH). Blocking, antibody blotting, and detection were performed as described earlier. Blots were visualized on x-ray film and quantified by two-dimensional scanning densitometry (Silverscanner; La Cie, Beaverton, OR). Blot density was measured using Image software (NIH, v1.61). A series of control samples containing a range of protein concentrations was included on each membrane to construct a standard curve, which was subsequently used to correct experimental values for nonlinearity in the blotting, film, and scanner responses.
Data Analysis
Data are shown as mean ± SEM. Comparison of JAr binding to endometrial cells was performed by ANOVA using Student-Newman-Keuls test. Other comparisons were performed using Student's t-test or a paired t-test.
Materials
JAr, HEC-1A, RL95-2 and AN3-CA cell lines were obtained from the American Type Culture Collection (Rockville, MD). The antiepisialin antibody, HMFG1 (clone 1.10.F3), was obtained from Biodesign International (Kennebunk, ME), and horseradish peroxidase-coupled antimouse IgG was obtained from Sigma Chemical Co. (St. Louis, MO). Capping antibody (214D4) was a kind gift of Dr. John Hilkens (The Netherlands Cancer Institute, Amsterdam). The OSGEP was obtained from Cedarlane Laboratories (Hornby, Ontario, Canada). Sialidase (i.e., neuraminidase) from V. cholerae was obtained from Boehringer Mannheim, and types II and III sialidase from V. cholerae were obtained from Sigma. Hyperbond ECL nitrocellulose and Hyperfilm ECL x-ray film were obtained from Amersham Life Science (Arlington Heights, IL). Renaissance chemiluminescence detection kits were obtained from NEN Life Science Products. Other chemicals, including electrophoresis materials, were obtained from Sigma and Bio-Rad Laboratories (Hercules, CA).
RESULTS
Episialin Expression on Endometrial Cells
To develop a model for binding to the endometrial epithelium that could be used to investigate the role of episialin, it was necessary to determine the relative expression of episialin in the endometrial carcinoma lines AN3-CA, RL95-2, and HEC-1A. Western blotting analysis of endometrial cell extracts was performed as described in Materials and Methods. During the initial electrophoresis on 6% gels, a strong band at approximately 250 kDa was noted for the HEC-1A samples, whereas a band of similar size but much weaker intensity was observed for the RL95-2 samples. No immunoreactivity was apparent for the AN3-CA samples (data not shown). Because the bands were close to the stacking gel-separating gel interface and, therefore, difficult to visualize clearly, electrophoresis was repeated using a 4–15% gradient gel (Fig. 1). The two left lanes in Figure 1 are separate HEC-1A samples that displayed a very broad band in the region of 240–320 kDa and faint bands in the region of 190–210 kDa. Because of this broad band structure, determining whether the antibody was reacting with one or more proteins was difficult. Episialin is a highly glycosylated protein, and because varying degrees of glycosylation might produce such a broad band structure, HEC-1A cells were pretreated with sialidase (100 mU/ml; V. cholerae) for 60 min at 37°C before extraction, similar to the procedure described previously by Ligtenberg et al. [9]. After electrophoresis, a single, discrete band was observed at an apparently much higher molecular weight (Fig. 1, two right lanes). This suggests that the broad major band and the possible minor bands are products of differential glycosylation, and that the HMFG1 antiepisialin antibody reacts with a single protein moiety in these samples. The apparent increase in molecular weight after sialidase treatment probably resulted from the complex interactions observed for highly glycosylated proteins (e.g., mucins) in SDS gels [22].
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The demonstration, by Western blotting, that HMFG1 identified a single immunoreactive species and also that, under the conditions used here, there was a minimal background signal, enabled use of slot blotting to quantify episialin expression. This procedure avoided the problems associated with electrophoresis and transfer of high-molecular-weight components and, in particular, the anomalies associated with SDS-mucin interactions. In addition to bypassing the complexities of electrophoresis and transfer, use of slot-blotting permitted simultaneous analysis of many samples, thus avoiding inter-gel comparisons and allowing parallel analysis of a standard curve, which was used to correct densitometric measurements for nonlinearity in film and densitometer responses. Extracts of AN3-CA, HEC-1A, and RL95-2 monolayers were slot-blotted as described earlier, employing sialidase treatment after transfer to nitrocellulose and before treatment with the primary antibody. Figure 2 shows representative blots for two sets of each cell type. After quantification by scanning densitometry and background subtraction, AN3-CA episialin content was not significantly different from zero (i.e., buffer blank). Comparison of HEC-1A and RL95-2 expression of episialin was performed by paired t-testing of the raw densitometric values for eight pairs of the two cell types. Expression of episialin by RL95-2 cells was significantly lower than that in HEC-1A cells (P < 0.05, n = 8). Setting the RL95-2 expression arbitrarily to a value of unity, the relative expression in RL95-2 and HEC-1A cells was 1.00 ± 0.25 and 4.16 ± 0.83, respectively (n = 8).
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Endometrial Cell Attachment
The attachment assay described here is similar to that described initially by Rohde and Carson [16]. The major modification in the present assay was in preparation of the JAr cell suspension. Rohde and Carson trypsinized JAr cells to produce a suspension of single cells and then allowed them to recover for 4–6 h in the presence of 16.5 mM EDTA before use in the attachment assay. In the present study, despite use of EDTA, aggregation and loss of viability was observed, as measured by lack of trypan blue exclusion. Instead, an alternate procedure was used in which adhesion between JAr cells was eliminated through an initial trypsinization 18 h before the attachment assay using 0.25% trypsin. The single cells produced by this process were allowed to replate and recover overnight at a dilution sufficient to disperse them and preclude re-establishment of cell-cell interactions. As a result, the cell-substrate adhesion was susceptible to a minimal trypsinization immediately before the assay, which yielded a single cell suspension with more than 90% viability. A fluorescent marker, described by Rohde and Carson for use in assessing aggregates, was used here routinely as the marker for measuring JAr binding, thus avoiding the longer labeling required with [32P]O4. Finally, to provide reproducible forces when washing the cells, an automated dispenser was employed.
In view of the distribution of episialin between the three endometrial cell types as determined from the slot-blotting experiments, studies were performed to examine the degree of JAr binding in an attachment assay. Cells expressing episialin might be expected to show a lower degree of JAr adhesion because of the steric hindrance produced by episialin. Experiments were performed in which CFDA-labeled JAr cells were incubated for 60 min with confluent monolayers of AN3-CA, RL-95-2, or HEC-1A cells. After washing off the nonadherent cells, the extent of JAr adhesion was quantified by measuring CFDA fluorescence in the cells remaining adherent to the monolayers. In each experiment, JAr binding to the endometrial cells was expressed as a percentage of the cells added (Fig. 3). JAr binding to blank (i.e., DMEM/F12/BSA-treated) wells was 1.1% ± 0.2%, whereas binding to AN3-CA, RL95-2, and HEC-1A cells was 6.7% ± 1.2%, 26.6% ± 2.2% and 12.3% ± 2.1%, respectively (n = 9). All cell types showed significant JAr binding compared with blank (P < 0.05, ANOVA, Student-Newman-Keuls test). RL95-2 showed a significantly greater degree of JAr binding than HEC-1A, which, in turn, showed a greater level of binding than AN3-CA.
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Episialin "Capping"
In human A375 melanoma cells transfected with episialin, Wesseling et al. [11] demonstrated that incubation with monoclonal antibody 214D4 led to clustering of episialin in a "cap" on the cell surface, thus enabling an increased degree of cell adhesion to extracellular proteins. To determine if manipulation of episialin by use of this antibody might alter JAr binding to endometrial cells, its effect was tested in the attachment assay. Confluent monolayers of HEC-1A or RL95-2 cells were incubated both with or without this antibody (diluted 1:1, v:v, in DMEM/F12/BSA) for 30 min at 37°C, and JAr cells were then added to measure adhesion. In three experiments using the capping antibody, no significant difference was found between control and treated cells for either HEC-1A (1.00 ± 0.05 vs. 1.06 ± 0.07) or RL95-2 (1.00 ± 0.03 vs. 0.96 ± 0.12).
Effects of Sialidase on JAr Binding
The high number of sialic acid residues on episialin likely produces a substantial negative charge on the molecule. The antiadhesive effect of episialin might, therefore, result from electrostatic repulsion of negatively charged cells. This idea was tested by pretreating endometrial cells used for the attachment assay with sialidase before the attachment assay to remove sialic acid residues. HEC-1A cells were treated with sialidase (V. cholerae, type III from Sigma) before the attachment assay (200 mU/ml for 60 min at 37°C). In the same assays, a separate group of cells was pretreated with a specific monoclonal antiepisialin antibody (HMFG1, 1:20, v:v, for 60 min) both before and during treatment with sialidase to determine if the antiepisialin antibody could protect episialin from the effects of sialidase. The results of these experiments (Table 1) show that sialidase treatment increased JAr binding by more than 40%, whereas antiepisialin antibody treatment suppressed the effects of sialidase on JAr binding in six of seven experiments. The antiepisialin antibody alone had no effect on attachment. The experiments involving sialidase treatment of HEC-1A cells were then repeated with RL95-2 cells, which is the other endometrial cell type expressing episialin. After treatment with sialidase, binding of JAr cells to RL95-2 cells increased by 25% ± 4% (P < 0.05, Student's t-test, n = 3).
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During the work described earlier, identical experiments were performed using type II sialidase from Sigma or sialidase supplied by Boehringer Mannheim (both isolated from V. cholerae). In these experiments, sialidase showed no effect on JAr binding to the HEC-1A monolayers. Repetition of the protocol using Sigma type III sialidase produced results similar to the initial data (i.e., an increase in JAr binding to HEC-1A). The manufacturer's description of Sigma type III sialidase notes the possible presence of protease activities, so the effects observed with this preparation might have resulted from the action of a protease rather than from sialic acid cleavage. Sigma type II sialidase was described as a further purification of the type III sialidase, whereas the Boehringer-Mannheim enzyme was described as having no detectable protease activities. Thus, it is possible that these two enzyme preparations were free of the component in the Sigma type III sialidase that produced increased JAr binding. If increased JAr binding resulted from removal of episialin from the surface of HEC-1A by a protease contaminant of type III sialidase rather than from removal of sialic acid residues, then increased quantities of episialin should be detectable in the conditioned medium of cells that had been pretreated with this specific sialidase preparation. This idea was tested by slot-blotting analysis of the conditioned medium obtained after sialidase treatment from a series of attachment assays. Analysis of the medium from type III sialidase-treated cells showed 42% ± 19% more episialin than in medium from cells treated with the Boehringer-Mannheim sialidase (P < 0.05, paired t-test, n = 4).
O-Sialoglycoprotein Endopeptidase
O-sialoglycoprotein endopeptidase (OSGEP) is a protease whose target is O-linked sialic acid-containing glycoproteins, such as episialin [23–26]. It does not affect N-linked glycoproteins or unglycosylated proteins. In a series of experiments similar to those performed with sialidase, OSGEP was incubated with HEC-1A cells. It was predicted that OSGEP might also cleave episialin from the endometrial cell surface, leading to increased JAr binding. Thus, a parallel set of cells were preincubated with HMFG1 before and during OSGEP treatment to test for the potential suppression of OSGEP effects. In three of four experiments, OSGEP treatment increased JAr binding by more than 50% (Table 2), and this increase in binding was inhibited by the presence of HMFG1.
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DISCUSSION
In this model of endometrial-blastocyst attachment, the data strongly support the hypothesis that episialin acts as an antiadhesive factor to prevent cell-cell interaction, most probably by steric hindrance. Removal of episialin from the cell surface enhanced JAr binding, and this increase in binding was prevented by an episialin-specific monoclonal antibody.
The interaction between JAr and the endometrial cells should not be construed as an exact model for endometrial-blastocyst binding. Given the origins of the cells types, some of the interactions involved probably are representative of those observed in endometrial-blastocyst binding, but the data reported here concern a functional assessment of the role of episialin, independent of the adhesive factors involved in cell-cell attachment. The focus of these studies was the modulation of cell-cell attachment by episialin, not the mechanisms of adhesion. An important element of this model was the ability to examine the role of episialin not simply on interactions at the molecular level but also at the more complex, more physiologic level of cell-cell interaction. Thus, these experiments are significant independent of the nature of the adhesive interactions involved.
The inverse relationship observed between the slot-blotting measurement of episialin expression and the extent of JAr binding to RL95-2 and HEC-1A cells is consistent with the hypothesis of an antiadhesive role for episialin. The minimal JAr binding to AN3-CA cells suggests that these cells, which have a less differentiated phenotype than the other two endometrial lines, may lack factors necessary for attachment. Of the two lines that bound JAr, the HEC-1A cells, which express episialin to a much greater extent than RL95-2 cells, showed a lower level of JAr binding than the RL95-2 cells.
Results from the manipulation of JAr binding by agents that affect presentation of episialin provide strong support for the antiadhesive nature of episialin. Because the increases in JAr binding after sialidase or OSGEP treatment might have resulted from the action of these agents on one or more cell surface components other than episialin, a third treatment group, beyond the control and sialidase- or OSGEP-treated groups, was added. This third group consisted of cells to which a specific antiepisialin antibody was added before treatment with sialidase or OSGEP. This antibody was present both before and during treatment. When this antibody was present, JAr binding was not increased by sialidase or OSGEP treatment, in contrast to the effect observed when the antibody was not present. The effect of a specific antiepisialin antibody clearly shows that whereas sialidase or OSGEP may degrade other cell surface glycoproteins, their effect on episialin causes the alterations in JAr binding. One can speculate that sialidase or OSGEP also might alter the function of adhesive molecules on the endometrial cells, but stimulation of JAr binding by degradation (and in an identical manner for both agents) seems improbable. It also does not explain the effects observed with the antiepisialin antibody. The simplest explanation consistent with all the data is that episialin acts as an antiadhesive factor in this model.
The experimental data suggest that the effect of the Sigma type III sialidase may actually be mediated by a contaminant protease, because this effect was not observed with other, more highly purified preparations of sialidase. The increase in immunoreactive episialin found in conditioned medium from HEC-1A cells treated with type III sialidase supports this suggestion. However, in these circumstances, the question of the effective agent is not relevant. The key element in these experiments is the demonstration that the action of sialidase or OSGEP can be suppressed by a monoclonal antibody specific for episialin.
Considering the apparent effects of episialin in limiting JAr binding to HEC-1A cells, a capping antibody, which has been shown previously to cluster episialin on the cell surface [11], might act to aggregate episialin and permit increased JAr binding after treatment. The capping antibody was ineffective, however, in changing the extent of JAr binding to either RL95-2 or HEC-1A cells. Even so, substantial differences exist between the circumstances in the experiments reported here and those in which the capping antibody was shown to be effective [11]. In the latter experiments, the capping antibody was used on cells in suspension, and episialin, which initially was distributed over the cell surface, eventually was clustered in one location, the "cap," by the action of the antibody. In the experiments described here, the cells on which the capping antibody was tested were confluent, adherent epithelial cells expressing episialin on an apical surface. This topology probably severely restricts the ability of the capping antibody to clear episialin from a sufficient area of the apical surface to permit increased JAr cell binding.
The results of these experiments confirm the hypothesis that episialin acts as an antiadhesive factor in this model of endometrial-blastocyst binding, and they raise the question of episialin's role in vivo. Research performed using a mouse model has shown that the high level of Muc1 (i.e., murine episialin) expression on the surface of the endometrial epithelium during the proestrous and estrous stages is down-regulated during diestrus, which is coincident with increased blastocyst receptivity [27]. This profile is consistent with a role for episialin as an antiadhesive factor, because its expression decreases during the implantation window. In the rabbit, a very different profile is apparent. Hoffman et al [28] demonstrated that Muc1 expression was up-regulated in the pre- and peri-implantation periods. At the implantation site itself, however, Muc1 expression was dramatically reduced on the luminal epithelium apposed to blastocysts, suggesting that some local effect, possibly mediated through blastocyst signaling, may be important in rabbit implantation.
In humans, episialin has been observed on the apical surface of the endometrial epithelium. Contradictory reports, however have appeared concerning its expression over the course of the menstrual cycle. Hey et al. [14, 15] have described up-regulation of episialin expression during the implantation phase, whereas others [12, 13] have demonstrated down-regulation during the same period. Further reports suggest that in addition to, or rather than, changes in expression of the protein core of episialin, alterations occur in the glycan chains carried by episialin over the menstrual cycle [29] such that moieties including the lactosaminoglycans, which are present during the proliferative phase, disappear during the secretory phase [30]. Thus, the true profile may comprise a very complex pattern of core protein and glycan chain expression.
There are reports in the literature that describe episialin as an adhesive factor. It is worth considering therefore whether the anti-adhesive function described here for episialin is relevant to the human endometrial-blastocyst interaction or whether an adhesive role might be more probable. In one report describing an adhesive interaction, episialin bound intracellular adhesion molecule-1 (ICAM-1), an endothelial adhesive factor [31]. Although the embryo, blastocyst, and trophoblast cells all express ICAM-1 [32, 33], endometrial-blastocyst interactions between episialin and ICAM-1 are very unlikely; JAr choriocarcinoma cells (which also express ICAM-1) do not exhibit enhanced binding to cells expressing higher quantities of episialin, as might be predicted if their interaction was adhesive. In fact, the opposite is true: as demonstrated by the data in Figures 2 and 3, HEC-1A cells expressing the greater quantity of episialin bind JAr cells less efficiently than RL95-2 cells that express only approximately 25% of the episialin found on HEC-1A. Another report describes an adhesive interaction between E-selectin and the carbohydrate sialyl-Lewis a and x epitopes carried by episialin [34]. No data are available regarding E-selectin in the embryo or blastocyst, however, and E-selectin does not appear to be present on the human trophoblast [35–37], suggesting that an interaction of episialin with E-selectin also is unlikely. Combined with data demonstrating the antiadhesive function of episialin, this suggests that adhesive interactions are extremely unlikely in this model.
The results reported here demonstrate that episialin expressed on endometrial cells can inhibit cell-cell interactions with trophoblastic cells. The role played by this antiadhesive molecule in the complex process of blastocyst implantation, however, remains to be described.
ACKNOWLEDGMENTS
The authors gratefully acknowledge both the advice and the provision of antibodies by Dr. John Hilkens (The Netherlands Cancer Institute, Amsterdam).
FOOTNOTES
First decision: 7 September 1999.
2 Current address: Department of Obstetrics, Gynecology and Women's Health, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. 
1 Correspondence: Nicholas P. Illsley, Department of Obstetrics, Gynecology and Women's Health, Medical Sciences Building, E506, UMD-New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103-2714. FAX: 973 972 4256; illsleni{at}umdnj.edu
Accepted: March 6, 2000.
Received: August 18, 1999.
REFERENCES
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 M. Beck and M. R. Strand Glc1.8 from Microplitis demolitor Bracovirus Induces a Loss of Adhesion and Phagocytosis in Insect High Five and S2 Cells J. Virol., February 1, 2005; 79(3): 1861 - 1870. [Abstract] [Full Text] [PDF]
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 K. Mardberg, K. Nystrom, M. A. Tarp, E. Trybala, H. Clausen, T. Bergstrom, and S. Olofsson Basic amino acids as modulators of an O-linked glycosylation signal of the herpes simplex virus type 1 glycoprotein gC: functional roles in viral infectivity Glycobiology, July 1, 2004; 14(7): 571 - 581. [Abstract] [Full Text] [PDF]
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 H.-Y. Li, S.-P. Chang, C.-C. Yuan, H.-T. Chao, H.-T. Ng, and Y.-J. Sung Induction of p38 Mitogen-Activated Protein Kinase-Mediated Apoptosis Is Involved in Outgrowth of Trophoblast Cells on Endometrial Epithelial Cells in a Model of Human Trophoblast-Endometrial Interactions Biol Reprod, November 1, 2003; 69(5): 1515 - 1524. [Abstract] [Full Text] [PDF]
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J. Carver, K. Martin, I. Spyropoulou, D. Barlow, I. Sargent, and H. Mardon An in-vitro model for stromal invasion during implantation of the human blastocyst Hum. Reprod., February 1, 2003; 18(2): 283 - 290. [Abstract] [Full Text] [PDF]
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 C. Heneweer, L. H. Kruse, F. Kindhauser, M. Schmidt, K. H. Jakobs, H.-W. Denker, and M. Thie Adhesiveness of human uterine epithelial RL95-2 cells to trophoblast: Rho protein regulation Mol. Hum. Reprod., November 1, 2002; 8(11): 1014 - 1022. [Abstract] [Full Text] [PDF]
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G. Simmons, R. J. Wool-Lewis, F. Baribaud, R. C. Netter, and P. Bates Ebola Virus Glycoproteins Induce Global Surface Protein Down-Modulation and Loss of Cell Adherence J. Virol., March 1, 2002; 76(5): 2518 - 2528. [Abstract] [Full Text] [PDF]
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