Molecular Human Reproduction, Vol. 6, No. 12, 1119-1130,
December 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Calcium influx in human uterine epithelial RL95-2 cells triggers adhesiveness for trophoblast-like cells. Model studies on signalling events during embryo implantation
1 Max-Planck Institut für molekulare Physiologie, D-44227 Dortmund, and 2 Institut für Anatomie, Universitätsklinikum, D-45122 Essen, Germany
Abstract
RL95-2 is a human uterine epithelial cell line that exhibits adhesion competence on its apical surface for trophoblast-like JAR cells. Using confocal microscopy and an adhesion assay we have found that changes in intracellular free calcium ([Ca2+]i) in RL95-2 cells are involved in binding of JAR spheroids. Impact of spheroids upon, and movement of spheroids across, monolayers of RL95-2 cells produced a transient increase in [Ca2+]i. Pretreatment of RL95-2 cells with the Ca2+ channel inhibitor, diltiazem, reduced the [Ca2+]i increase. Interestingly, resting of JAR spheroids on RL95-2 cells caused no detectable alterations in [Ca2+]i although cell–cell bonds were formed during prolonged contact. However, separation of established bonds did produce an increase in [Ca2+]i which could be reduced by the Ca2+ channel blocker, SKF-96365, but not by diltiazem. SKF-96365 also reduced adhesion of JAR spheroids to RL95-2 cells. In all experiments, the increase in [Ca2+]i was due to influx from the external medium, as it could be blocked both by removing extracellular Ca2+ and by nickel. These results suggest that the plasma membrane of uterine RL95-2 cells contains two types of Ca2+ channels that are involved in trophoblast adhesion, i.e. diltiazem-sensitive channels contributing to initiation of JAR cell binding and SKF-96365-sensitive channels participating in a feedback loop that controls the balance of bonds.
calcium influx/cell adhesion/implantation/polarized phenotype/uterine epithelium
Introduction
Cell–cell adhesion between the blastocyst and the endometrial mucosa is critical for successful implantation of mammalian embryos. During the initial phase of embryo implantation, the trophoblast of the blastocyst has to establish contact with the free surface of the uterine epithelium and to adhere there. Adhesion to an apical plasma membrane is a very unusual phenomenon, and the mechanisms involved are poorly understood (Denker, 1993
).
With respect to the human uterine epithelium, apical contact formation with trophoblast cells seems to be possible only if the epithelium enters a specific functional state called receptivity which is thought to last from days 19–21 of a normal menstrual cycle (Finn and Martin, 1974; Navot et al., 1986; Lessey et al., 1995; Nikas and Psychoyos, 1997). Experimental studies in animals and data from human in-vitro implantation models have helped to shed light on features by which uterine cells may change their adhesive properties in this particular state. The starting point, i.e. the pre-receptive state, is a structurally and functionally polarized epithelial cell with distinct basal, lateral and apical plasma membrane domains. A reduction in the thickness of the glycocalyx of these cells and in cell surface charge has been observed (Murphy and Rogers, 1981; Morris and Potter, 1984). On the other hand, the biosynthesis and expression of new cell surface proteins has also been reported (Aplin, 1997; Lessey, 1998; Kirn-Safran and Carson, 1999). However, recent data suggest that, not only is the expression of apical membrane-associated molecules changed in the uterine epithelium at acquisition of receptivity, but changes in the lateral and basal sites of these cells can also be observed (Rogers and Murphy, 1992; Albers et al., 1995; Murphy, 1995; Nikas, 1999; Skinner et al., 1999). Putting all these data together, adhesion competence of the apical surface of uterine epithelial cells may be attained by changes in the structural and functional organization of these cells by partially down-regulating or destabilizing their apico–basal polarized phenotype (Denker, 1983, 1994b). Although still poorly understood, these alterations have consequences for the initiation of adhesion during embryo implantation.
A number of molecules have been proposed to contribute to the binding of trophoblast to uterine epithelium, specifically heparan sulphate proteoglycans and heparan sulphate-binding proteins (Carson et al., 1998), trophinin (Fukuda and Nozawa, 1999), and H-type-1 carbohydrate antigen (Illingworth and Kimber, 1999). However, little is known regarding signal transduction events that might form the reaction cascade leading to firm adhesion between trophoblast and uterine epithelium. To what extent these adhesion molecules might be involved in triggering such signalling events in the uterine epithelium is almost completely unknown. We have some preliminary evidence that rises in intracellular Ca2+ ([Ca2+]i) can be triggered mechanically via integrins that are apically located in RL95-2 cells (Thie et al., 1997). Furthermore, determination of binding forces using atomic force spectroscopy demonstrated that formation of strong cell–cell bonds between trophoblast and uterine epithelium might be a relatively slow process, possibly including sequential steps of bond formation (Thie et al., 1998). Therefore, it may be expected that a complex cascade of events is initiated at the free cell pole of uterine epithelial cells starting with signalling and culminating in the remodelling of this cell pole to allow firm adhesion of the trophoblast to take place. Some components involved in this process may contain elements regulated by Ca2+ ions, since Ca2+ is involved in many cellular signal transduction pathways, and regulation of cell adhesion by [Ca2+]i has been observed in many cell systems (Clark and Brugge, 1995; Evenas et al., 1998). Thus, changes in the intracellular concentration of this second messenger in uterine epithelial cells might play an important role in embryo implantation.
RL95-2 (Way et al., 1983) is a human uterine epithelial cell line which, in contrast to other endometrial cell lines, e.g. HEC-1-A (Kuramoto et al., 1972) and AN3-CA (Dawe et al., 1964), not only lacks epithelial polarity and shows loosening of lateral borders and changes in actin-based cytoskeleton, but its apical pole also exhibits adhesive properties for trophoblast-like cells (Thie et al., 1995, 1996, 1997). In this respect, RL95-2 cells have proven to be useful as a cell culture model system for the receptive human uterine epithelium (e.g. Raboudi et al., 1992; John et al., 1993; Rohde and Carson, 1993; Liu et al., 1998). Thus, this system may provide novel insights into the complex process of human embryo implantation regulation. Therefore, using confocal microscopy and an adhesion assay, we investigated whether the initial contact of trophoblast-like JAR cells with human uterine epithelial RL95-2 cells is associated with changes in [Ca2+]i in the uterine cells, and whether this signalling correlates with the adhesion competence of RL95-2 cells.
Materials and methods
Cell culture
Human endometrial carcinoma cell lines were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA), i.e. RL95-2 cells (CRL 1671; Way et al., 1983), HEC-1-A cells (HTB 112; Kuramoto et al., 1972), and AN3-CA cells (HTB 111; Dawe et al., 1964). For routine culture, cell lines were grown in plastic flasks at 37°C in a humidified atmosphere containing 5% CO2. In brief, RL95-2 cells were seeded out in a 1 + 1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 (Gibco-Life Technology, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS; Gibco), 10 mmol/l HEPES (Gibco), and 0.5 µg/ml insulin (Gibco), HEC-1-A cells in McCoy's 5A medium (Gibco) supplemented with 10% FCS, and AN3-CA cells in Eagle's minimum essential medium with Earle's salts and non-essential amino acids (Gibco) supplemented with 10% FCS. All media were additionally supplemented with penicillin (100 IU/ml; Gibco) and streptomycin (100 µg/ml; Gibco). The growth medium was changed every 2–3 days, and cells were subcultured by trypsinization (trypsin–EDTA solution; Gibco) when they became confluent.
For experiments, endometrial cell lines were cultured on coverslips. Cells were harvested by trypsinization from confluent routine cultures, counted (CASY1-Cell Counter; Schärfe System, Reutlingen, Germany), and adjusted to the desired concentration, i.e. RL95-2 700 000 cells, HEC-1-A 200 000 cells, and AN3-CA 300 000 cells each in 2.0 ml of the respective culture medium. Subsequently, the cell suspension was seeded out on poly-D-lysine-coated glass coverslips (12 mm in diameter) positioned in 4 cm2 Falcon multiwells. Cells were grown in medium to confluent monolayers and used for experiments within 3 days after initiation of culture.
Spheroids of human JAR choriocarcinoma cells (ATCC: HTB 144; Pattillo et al., 1971) were used to investigate whether the contact of trophoblast-like cells to endometrial monolayers elicits changes in cell behaviour of endometrial cells. For this purpose, JAR spheroids were prepared according to a previously described procedure (John et al., 1993). Briefly, a suspension of 100 000 JAR cells/ml in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco), supplemented with 10% FCS and penicillin–streptomycin, was incubated on a gyratory shaker at 110 rpm obtaining multicelluar spheroids 72 h after initiation of culture.
Cell–cell adhesion assay
The adhesiveness of the free surface of endometrial cell monolayers for human JAR spheroids was measured using a centrifugal force-based adhesion assay. The adhesion assay has been described previously (John et al., 1993). Briefly, JAR spheroids were harvested, counted, and gently delivered onto a confluent monolayer of human endometrial cell lines grown on coverslips. For the experiment, growth medium was changed to standard solution supplemented with drugs as described below. After 60 min, spheroid adhesion to the endometrial monolayers was quantified by centrifuging coverslips (with the cell spheroid surface facing down) at 12 g for 5 min. Attached spheroids were counted and expressed as a percentage of the number of spheroids seeded.
Processing for electron microscopy
Endometrial cell monolayers were grown on poly-D-lysine-coated thermanox coverslips (Nunc, Napperville, IL, USA). For subsequent transmission electron microscopy, samples were rinsed twice in phosphate-buffered saline (PBS) and fixed in 2.5% glutardialdehyde in 0.1 mol/l cacodylate buffer, pH 7.4, for 30 min at room temperature, washed in cacodylate buffer, post-fixed with 1% OsO4 in cacodylate buffer, dehydrated with ethanol and propylene oxide and embedded in epoxy resin (Cross, 1989). The embedded cells were separated from the thermanox coverslip by snap-freezing in liquid nitrogen. Ultrathin sections were mounted on 200-mesh copper grids, double-stained with uranyl acetate and lead citrate and examined with a Zeiss TEM 902A.
For scanning electron microscopy, samples were fixed in 2.5% glutardialdehyde in 0.1 mol/l cacodylate buffer, pH 7.4, for 30 min at room temperature. After repeated washings in distilled water, samples were dehydrated with ethanol and, at a critical point, dried using methanol as intermedium and carbon dioxide as drying medium. Then samples were sputtered with a conductive layer of gold and imaged with a Leo SEM 420.
Measurement of [Ca2+]i
Confluent monolayers of endometrial cells grown on coverslips were loaded with fluorescent Ca2+-sensitive probes for 60 min at 37°C, i.e. 5 µmol/l Oregon green 488 BAPTA-1 AM and 10 µmol/l Fura-Red AM in standard solution containing 0.025% Pluronic F127. After loading, cell monolayers were rinsed and incubated in standard solution without Ca2+ probes for 10 min. Thereafter, monolayers were transferred into a Perspex chamber mounted on a stage of a confocal laser scanning microscope (Noran, Bruchsal, Germany). Cell fluorescence was excited by use of the 488-nm band of an argon ion laser. The ratio of emission <580 nm and >580 nm for Oregon green 488 BAPTA-1 and Fura-Red respectively, was used to represent [Ca2+]i.
To study the effect of impact of JAR spheroids upon endometrial cells, spheroids being held horizontally by a glass pipette using the holding mode of a microinjector (Eppendorf, Hamburg, Germany) were lowered to and placed on the free surface of endometrial monolayers. The glass pipettes were pulled from borosilicate filament glass capillaries (Hilgenberg, Malsfeld, Germany) on a horizontal puller (Zeitz, München, Germany). The exact position of the tip of the pipette was controlled using a micromanipulator (Luigs & Neumann, Rattingen, Germany). JAR spheroids resting on endometrial monolayers were subsequently moved over the endometrial cells by pushing the holding pipette horizontally. Glass beads (250–300 µm in diameter; Braun Melsung, Melsung, Germany) were used as control. All experiments were performed at room temperature.
Solutions
The standard solution contained 118 mmol/l NaCl, 10 mmol/l Na+-N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), 10 mmol/l H+-HEPES, 3.2 mmol/l KCl, 2.5 mmol/l CaCl2, 1.8 mmol/l MgSO4, 1.8 mmol/l KH2PO4, 5 mmol/l glucose, 10% FCS, pH 7.4. In some experiments, as specifically indicated, cells were incubated in standard solution without CaCl2 and FCS (Ca2+-free solution).
Inhibiting drugs (SKF-96365, Calbiochem, Bad Soden, Germany; diltiazem and nifedipin, Sigma, Deisenhofen, Germany) were dissolved first in dimethyl sulphoxide (final solvent concentration 0.1%). The cells were preincubated with these drugs in standard solution for 10 min at 37 °C. Medium containing 0.1% dimethyl sulphoxide only served as a control.
Pluronic F-127, Oregon green 488 BAPTA-1 AM, and Fura-Red AM were purchased from Molecular Probes (Eugene, OR, USA). All other chemicals were purchased from Serva (Heidelberg, Germany).
Statistical analysis
Values are presented as mean ± SE, with n denoting the number of experiments. Paired and unpaired t-tests were applied as appropriate. P < 0.05 was considered to be statistically significant.
Results
Effect of impact, resting and movement of JAR spheroids on the [Ca2+]i of endometrial cells
Prior to adhesion and final implantation, the blastocyst initially contacts the uterine epithelium. We developed a model of blastocyst–uterus interaction in order to investigate changes in [Ca2+]i of uterine epithelial cells during this phase of initial contact. We measured [Ca2+]i in either RL95-2, HEC-1-A, or AN3-CA monolayer-cultured cells under three sets of conditions: (i) during impact of JAR spheroids upon monolayer-cultured cells; (ii) during rest of unmoved spheroids on the monolayers; and (iii) while spheroids were moved across the free surface of monolayers.
Impact of JAR spheroids upon monolayers of RL95-2 cells evoked a marked increase of [Ca2+]i. A representative reaction is shown in Figure 1A. The fluorescence ratio increased within 15 s after initial contact, reached a maximum and decreased towards the pre-contact level within 120 s. In HEC-1-A cells, this Ca2+ response was significantly lower (Figure 1B). The impact of spheroids on AN3-CA cells had no significant effect on the [Ca2+]i (Figure 1B).
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After recovery from the initial impact, subsequent resting of JAR spheroids on monolayers of RL95-2, HEC-1-A and AN3-CA cells for up to 60 min had no effect on the [Ca2+]i in the endometrial cells (data not shown).
Movement of JAR spheroids across monolayers of RL95-2 cells, but not across monolayers of HEC-1-A and AN3-CA, evoked changes in [Ca2+]i. As shown in Figure 2A, the [Ca2+]i of RL95-2 cells increased transiently throughout spheroid movement. The latency of the rise of [Ca2+]i along the route of the spheroid movement (trace) was related to the speed of spheroid movement (Figure 2B). Spreading of the Ca2+ wave away from the area of spheroid contact (trace) was not observed during the experiment. As a control, we checked whether the increase of [Ca2+]i in RL95-2 cells was based on specific cell–cell interactions or was evoked by mechanical stimulation. As an alternative to JAR spheroids, glass beads were used. Figure 3 shows the changes in [Ca2+]i due to the movement of a glass bead across a RL95-2 monolayer. The pattern of the transient [Ca2+]i increase caused by glass beads was similar to that of JAR spheroids. The trace of the [Ca2+]i increase, however, was narrower in experiments with glass beads than in experiments with spheroids due to the more regular shape of beads.
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Adhesion competence of endometrial cells for JAR spheroids
To explore whether the differences in Ca2+ signalling were associated with differences in adhesive properties of endometrial cell lines, we measured the adhesion competence of RL95-2, HEC-1-A, and AN3-CA monolayers for JAR spheroids using the centrifugal force-based adhesion assay. At the chosen time point of 60 min after confrontation, a high percentage of JAR spheroids (86.4 ± 0.44%; number of spheroids tested: n = 1294) were attached to confluent monolayers of RL95-2 cells. In comparison, JAR spheroids attached with significantly lower efficiency to HEC-1-A cells (33.8 ± 0.38%; number of spheroids tested: n = 1089; P < 0.05) and AN3-CA cells (2.4 ± 0.19%; number of spheroids tested: n = 1174; P < 0.05). In control, glass beads used instead of JAR spheroids did not attach to RL95-2, HEC-1-A or AN3-CA cells.
Attachment morphology of spheroids adhering to RL95-2 cells
We subsequently analysed RL95-2 cells in greater detail, since, in contrast to HEC-1-A and AN3-CA cells, they showed both a strong Ca2+ response to an impact or movement of JAR spheroids and a high rate of adhesiveness for JAR spheroids.
As shown in Figure 4, stable cell–cell bonds developed between JAR spheroids and monolayer-cultured RL95-2 cells. Figure 4 A,B show the specific attachment morphology of a JAR spheroid adhering to the RL95-2 monolayer after 60 min of contact. Morphologically, stable cell–cell adhesion is characterized by plasma membranes coming close together and running parallel for long stretches, as well as by microvillous projections interacting with each other across an irregularly formed space at a distance of 
100–200 nm. Formation of organized intercellular junctions is not observed in this system.
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Effect of separation of cell–cell bonds between spheroids and RL95-2 cells on the [Ca2+]i of RL95-2 cells
Next, we investigated whether separation of cell–cell bonds after prolonged contact between JAR spheroids and RL95-2 monolayer-cultured cells leads to Ca2+ signals in RL95-2 cells and whether this signal depends on the duration of cell–cell contact.
When spheroids were separated from the monolayer 10 min after contact, a transient increase of [Ca2+]i was observed in the underlying RL95-2 cells. The magnitude of the Ca2+ rise increased continuously when the duration of contact was increased, as shown in Figure 5A. For example, the value of the maximal fluorescence ratio after 60 min of contact was 2.4-fold higher than the value after 10 min of contact. In experiments using glass beads instead of JAR spheroids, however, the magnitude of the ratio signal in RL95-2 cells was independent of the duration of contact (Figure 5B), i.e. values after 10 min of contact with a glass bead were comparable with those after 60 min of contact.
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In addition, we analysed whether the duration of preceding contact of a JAR spheroid to RL95-2 cells influences the Ca2+ signal in RL95-2 cells along the route of subsequent spheroid movement. Figure 6
shows the values of the maximal fluorescence ratio in RL95-2 cells underlying a spheroid and in cells situated 150 µm along the route of the spheroid. While values obtained in cells of the resting area significantly increased with prolonged contact (see also above), values in cells along the trace did not. In the control, glass beads evoked increases in Ca2+ at the beginning of the trace similar to those observed at a distance of 150 µm. Moreover, values of maximal fluorescence ratio evoked by glass beads on their route were similar to those evoked by moving spheroids.
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These experiments indicate that a rise in the Ca2+ signal of RL95-2 cells following detachment of JAR spheroids may be associated with separation of specific cell–cell bonds formed after prolonged spheroid contact.
Characterization of the Ca2+ signal in RL95-2 cells evoked by separation of cell–cell bonds between JAR spheroids and RL95-2 cells
In order to explore the source of increased [Ca2+]i in the RL95-2 cells during separation of an attached JAR spheroid, we first performed experiments in Ca2+-free solution (Figure 7). In the absence of Ca2+ in the extracellular medium, the increase of the [Ca2+]i in RL95-2 cells was completely abolished. Next, we supplemented the standard solution with the Ca2+ channel blocker Ni2+ (5 mmol/l), which reduced the increase of [Ca2+]i by 81.7% (Figure 7). Collectively, these data indicate that the increase of the [Ca2+]i in RL95-2 cells was due to Ca2+ influx from the extracellular medium.
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In further experiments, we characterized the nature of Ca2+-channels associated with the Ca2+ influx in RL95-2 cells upon separation of stable cell–cell bonds. As shown above, the magnitude of the Ca2+ response was dependent upon the duration of contact. These data suggested that an accessory mechanism may lead to an augmentation of Ca2+ signalling over time. We therefore tested SKF-96365, an inhibitor of receptor-mediated Ca2+ channel. After 10 min of contact, the increase of [Ca2+]i in the presence of SKF-96365 (100 µmol/l) did not differ significantly from the value of control. With prolonged contact, however, SKF-96365 inhibited the increase of [Ca2+]i in RL95-2 cells (Figure 5A
), i.e. after 20, 30, 40, 50, and 60 min of contact the increase of the value of maximal fluorescence ratio was significantly reduced by 51.1, 35.0, 40.0, 40.5, and 45.2% respectively. These results indicate that the Ca2+ influx induced by separation of cell–cell bonds between spheroids and RL95-2 cells consist of two components. One part of the signal is a basic Ca2+ influx (which is time-independent). The second part increases with the duration of contact and is probably based on receptor–ligand interactions activating a receptor-mediated Ca2+ channel.
As mechano-sensitive Ca2+ channels could be responsible for the basic part of the Ca2+ signal, we supplemented the incubation medium with gadolinium that is known to inhibit stretch-activated channels. However, gadolinium (500 µmol/l) showed no significant effect on the [Ca2+]i increase (Figure 7). Furthermore, we tested inhibitors of voltage-gated Ca2+ channels, which have been reported in many epithelial cells (Zhang and O'Neil, 1999). Neither nifedipin (10 µmol/l) nor diltiazem (10 µmol/l), both well-known blockers of voltage-activated Ca2+-channels, showed any effect on the Ca2+ response in RL95-2 cells upon separation of cell–cell bonds (Figure 7).
Characterization of the Ca2+ signals evoked by movement of spheroids
We also analysed the effect of Ca2+ channel blockers on the Ca2+ influx observed when spheroids were moving across the surface of monolayer-cultured RL95-2 cells. While SKF-96365 (100 µmol/l) showed no significant effect on the increase of [Ca2+]i, diltiazem (10 µmol/l) significantly reduced the increase of [Ca2+]i by 58.3% (Figure 8). Moreover, the characteristics of the Ca2+ channels involved in the influx in experiments with spheroid movement were similar to those observed in experiments with glass beads, i.e. gadolinium (500 µmol/l), nifedipin (10 µmol/l), and SKF-96365 (100 µmol/l) showed no effect on the increase of [Ca2+]i (Figure 9), while diltiazem (10 µmol/l) again significantly reduced the Ca2+ response by 40.1% (Figure 5B). These results indicate that the mechanisms of Ca2+ influx in RL95-2 cells evoked by movement of spheroids are similar to those evoked by movement of glass beads. These mechanisms seem to be based on an activation of Ca2+ channels by short contact and to differ from those activated by separation of cell–cell bonds between spheroids and RL95-2 cells.
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Effect of Ca2+-channel blockers on adhesive properties of RL95-2 cells for JAR spheroids
We have attempted to characterize the contribution of Ca2+ influx to adhesive properties of RL95-2 cells for JAR spheroids by blocking the influx using Ca2+ channel inhibitors or by reducing the concentration of extracellular Ca2+ (Figure 10
). In the absence of Ca2+, attachment of JAR spheroids to RL95-2 cells was reduced by 97.5% when compared with standard solutions containing Ca2+ (adhesiveness: 2.2 ± 0.3% versus 86.4 ± 0.4%). Treatment of monolayers with Ni2+ (5 mmol/l) resulted in a 20.3% reduction of JAR cell attachment (adhesiveness: 68.9 ± 0.8% versus 86.4 ± 0.4%), whilst treatment with SKF-96365 (100 µmol/l) in a 7.9% reduction of attachment (adhesiveness: 79.6 ± 0.7% versus 86.4 ± 0.4%). However, treatment with diltiazem (10 µmol/l) showed no significant reduction of attachment (adhesiveness: 86.1 ± 0.5% versus 86.4 ± 0.4%) (Figure 10). No effect was observed in vehicle-treated monolayers.
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Discussion
The cellular basis of interactions between trophoblast and endometrial epithelium at the beginning of embryo implantation remains poorly understood, not only with respect to the human, but even for various animal species (reviewed in Denker, 1993, 1994a; Edwards, 1995; Rogers, 1995; Tabibzadeh and Babaknia, 1995; Lopata, 1996; Klentzeris, 1997). In the human, ethical and practical restraints are important obstacles in experimental studies of this aspect of reproduction. Apart from a few studies that have involved supernumerary human blastocysts obtained from IVF clinics (Lindenberg et al., 1986; Lindenberg, 1991; Bentin-Ley et al., 1999), experimental investigation has relied on model experiments using human endometrial cell lines. Among them, human uterine epithelial RL95-2 cells appear to be a useful cell culture system to investigate mechanisms driving the apical adhesiveness of uterine epithelium (Raboudi et al., 1992; John et al., 1993; Rohde and Carson, 1993; Liu et al., 1998). As previously shown, RL95-2 cells allow trophoblast-like JAR cells to attach to their apical pole forming strong cell–cell bonds (Thie et al., 1998). Additionally, we have some preliminary evidence that rises in [Ca2+]i can be triggered mechanically via integrins that are apically located in RL95-2 cells (Thie et al., 1997). In the present study, we demonstrate that Ca2+ signalling is elicited in RL95-2 cells upon contact with human trophoblast-like JAR cells and that this plays a role in their subsequent binding. Although it remains unclear how this mechanism works, it seems to require the opening of at least two different types of Ca2+ channels in the plasma membrane of RL95-2 cells and an influx of Ca2+ from the extracellular fluid, transiently increasing the cytosolic Ca2+ concentration. Notably, Ca2+ release from intracellular stores in RL95-2 cells was not observed during the process of JAR cell binding.
Endometrial RL95-2 cells, but not HEC-1-A and AN3-CA cells, showed an increase of [Ca2+]i upon distinct stimulation (e.g. impact or movement of JAR spheroids as well as separation of contact). This correlates with adhesion competence, features of cell architecture and expression of certain component molecules of membranes and cytoskeleton (Thie et al., 1995, 1997). According to these data, it appears that a particular cellular architecture as well as a molecular hierarchy of signalling pathways in RL95-2 cells enables these cells to respond to trophoblast-like cells. There is strong evidence that modulation of their epithelial phenotype, i.e. loss of apical–basal polarity, might prepare the apical cell pole for cell–cell interaction with the trophoblast. Previous studies have shown that expression of a polar phenotype, like in the case of HEC-1-A cells, prevents adhesion of trophoblast-like cells to the apical plasma membrane. However, a simple lack of cell polarity (= AN3-CA cells) of uterine cells is not sufficient to allow adhesion. Expression of adhesion molecules (e.g. 
6, ß1, ß4 integrin subunits) as well as proper organization of the cortical actin cytoskeleton, lacking in AN3-CA cells, seem to be prerequisites for binding of JAR spheroids (Thie et al., 1995). In this complicated network, changes in the spatial and temporal characteristics of Ca2+ signalling appear to be an important element in embryo–endometrium interaction.
The impact of JAR spheroids upon, and movement of spheroids across, monolayers of RL95-2 cells evoked a significant increase of [Ca2+]i in RL95-2 cells. Evidence that increased [Ca2+]i is due to an activation of Ca2+ channels came from experiments using Ca2+-free solution as well as using the divalent cation Ni2+ that inhibits the passage of Ca2+ across the channels by blocking the channel pore (Hagiwara and Byerly, 1981). In addition, increases in [Ca2+]i as a result of cell damage during impact and movement of JAR spheroids across monolayers could be excluded as control experiments using monolayers of AN3-CA produced no change of [Ca2+]i. To characterize the types of Ca2+ channels involved in increases in [Ca2+]i, we tested the effects of various Ca2+ entry blockers on RL95-2 cells. The effects of diltiazem on the Ca2+ influx indicated that voltage-dependent Ca2+ channels are activated. The family of voltage-dependent Ca2+ channels has been described in excitable and non-excitable cells (Snutch and Reiner, 1992; Zhang and O'Neil, 1999) where they have an important role in signal transduction and Ca2+ transport. Diltiazem is a well-known blocker of voltage-gated L-type channels (Triggle, 1999) indicating that RL95-2 cells could posses L-type channels. However, nifedipin which is also a specific inhibitor of L-type channels (Triggle, 1999) showed no effect on the [Ca2+]i in RL95-2 cells. Thus, the Ca2+ influx seems to occur via Ca2+ channels which possesses pharmacological characteristics that differ from those of the classical L-type channel. This can be correlated with the large diversity of Ca2+ channels due to homologous genes for different types of subunits and several types of auxiliary subunits (Mori et al., 1993; Isom et al., 1994).
As diltiazem blocked only a part of the Ca2+ signal, other types of Ca2+ channels appear also to be involved in gating extracellular Ca2+ to the interior of RL95-2 cells during short contact with JAR spheroids. Stretch-activated channels could be responsible for this effect. Mechano-sensitive Ca2+ channels, which are mainly involved in cell volume regulation, have been found in various types of epithelial cells (Sackin, 1995). Gadolinium is reported to block stretch-activated Ca2+ channels (Yang and Sachs, 1989). Although application of gadolinium showed no effect on the increase of [Ca2+]i in RL95-2 cells, we cannot exclude the possibility that stretching of the plasma membrane might activate mechano-sensitive Ca2+ channels in RL95-2 cells, as gadolinium is not efficient in each case as has been shown in opossum kidney cells (Ubl et al., 1989; Sackin, 1995). A role for receptor-mediated Ca2+ channels in RL95-2 cells was clearly excluded as SKF-96365, which is known to block receptor-mediated channels with high efficiency (Merritt et al., 1990), did not reduce this Ca2+ signal in our experiments. The functional role of Ca2+ influx evoked by short contact with JAR spheroids requires further detailed investigation. Although diltiazem inhibited the increase of [Ca2+]i, it did not block the adhesiveness of JAR spheroids to RL95-2 cells. Nevertheless, complete blockade of adhesion may not necessarily be expected in such experiments. Considering that diltiazem reduced the increase of [Ca2+]i in RL95-2 cells by 58.3%, the remaining level of [Ca2+]i may be sufficient for JAR cell binding. Thus, having overcome repulsive interactions due to peripheral and structural features of the uterine epithelium (Thie et al., 1998), activation of this Ca2+ signalling pathway may help to initiate attachment and adhesion of the blastocyst in vivo.
While short contact, e.g. impact and movement of JAR spheroids, evoked a significant increase of [Ca2+]i in RL95-2 cells, further resting of spheroids on RL95-2 cells did not elicit a subsequent series of Ca2+ transients. This behaviour of uterine epithelial RL95-2 cells is in contrast to other cell types, e.g. vascular endothelial cells, where lymphocyte adhesion results in an oscillatory pattern of endothelial cell [Ca2+]i (e.g. Pfau et al., 1995; Ricard et al., 1997). The reason for this difference remains to be clarified in further investigations.
The cell adhesion molecules that actually mediate trophoblast adhesion to the apical surface of uterine epithelial cells are still unknown. Various types of molecules have been proposed to play a role in this process, e.g. heparan sulphate proteoglycans and heparan sulphate-binding proteins (Carson et al., 1998), trophinin (Fukuda and Nozawa, 1999), and H-type-1 carbohydrate antigen (Illingworth and Kimber, 1999). Among them, integrins have received special interest as candidate molecules potentially mediating cell–cell binding between uterine epithelium and trophoblast (Albers et al., 1995; Aplin et al., 1996; Bronson and Fusi, 1996; Lessey, 1998). Indeed, in RL95-2 cells, integrins, e.g. 6, ß1, ß4 integrin subunits, are expressed along the entire plasma membrane including the free cell surface, in contrast to the non-receptive model cell line HEC-1-A, where they are missing in the apical plasma membrane (Thie et al., 1995). As previously shown, mechanical stimulation of RL95-2 cells via apical membrane-bound integrins (using paramagnetic beads and a magnetic force drag device) can elicit a Ca2+ response in RL95-2 cells (Thie et al., 1997).
When JAR spheroids were separated from the monolayers, a transient increase in [Ca2+]i was noted in the underlying RL95-2 cells. The magnitude of the Ca2+ rise increased continuously when the duration of contact was increased. Our data clearly show that stressing and/or separating of cell–cell bonds between uterine RL95-2 cells and trophoblast-like JAR cells activate different Ca2+ channels in the plasma membrane of RL95-2 cells. Our data demonstrate that the influx of Ca2+ may be co-regulated via receptor-mediated Ca2+ channels, as SKF-96365 reduced the Ca2+ increase up to the value observed after short contact time when bonds between the spheroid and the monolayer have not yet been established. The involvement of voltage-dependent or mechano-sensitive Ca2+ channels could be excluded, as neither diltiazem nor gadolinium had any effect on the increase of Ca2+. The nature of receptor molecules that activate the receptor-mediated Ca2+ channels of uterine epithelium in vivo remain unknown, as the cell adhesion molecules involved in human embryo implantation have not yet been identified. As shown in other cell types, [Ca2+]i can indeed play a key role in the regulation of cell–cell contacts. In the hepatocyte cell line mHepR1, [Ca2+]i is necessary for the physical connection between integrins and the cytoskeleton and can link integrin signal transduction with other pathways (Nebe et al., 1995, 1996). In Madin-Darby Canine Kidney (MDCK) cells, integrin-mediated Ca2+ signalling is important in rapid feedback regulation of cell–substrate adhesion (Sjaastad et al., 1996). In these cells, an inositol trisphosphate (IP3)-independent Ca2+ influx mechanism is responsible for integrin-mediated adhesion. The authors postulate that this Ca2+ influx generates Ca2+ gradients in the vicinity of the plasma membrane, which then modulates the adhesion processes (Sjaastad and Nelson, 1997). Similar mechanisms might regulate the adhesion of JAR spheroids. It is known that cells are able to distinguish between Ca2+ signals of different properties. In neurons it has been shown that a Ca2+-dependent gene expression can be strengthened with Ca2+ concentration over a certain range (Hardingham et al., 1997). Thus, the amplitude of Ca2+ increase contains information about the magnitude of effect.
At least two different types of Ca2+ channels in the plasma membrane of RL95-2 cells seem to play a role in Ca2+ influx and activation of yet unknown Ca2+ sensitive response mechanisms involved in adhesiveness of uterine cells to trophoblast. One type of channel that is sensitive for diltiazem contributes to the initiation of JAR cell binding. The second channel is sensitive to SKF-96365 and participates in a feedback loop that controls the balance of bonds between uterine epithelial cells and trophoblast-like cells. Similarly, the presence of different Ca2+ influx mechanisms that fulfil different functions have been described in other cells. For example, neurons express both receptor-mediated and voltage-gated Ca2+ channels. Interestingly, the effect of Ca2+ entry in those cells is dependent upon the channels through which it flows. It has been shown (Bading et al., 1993) that the influx of Ca2+ through voltage-dependent L-type channels, but not through receptor-mediated channels, activated CRE (cAMP response element)-dependent gene expression. Both types of Ca2+ influx could, however, activate SRE (serum response element)-dependent gene expression. With regard to uterine epithelial RL95-2 cells, cellular responses to the elevation of [Ca2+]i remain a matter for further investigations.
Acknowledgments
The authors wish to thank Prof. R.K.H. Kinne (Head of the Department of Epithelial Physiology of the Max-Planck-Institute for Molecular Physiology, Dortmund) for valuable discussions and continuous support of the project. The skilful technical assistance of Alexander Giffey, Bärbel Gobs, Birgit Maranca and Dorothea Schünke is gratefully acknowledged.
Notes
3 To whom correspondence should be addressed. E-mail: michael.thie{at}uni-essen.de 
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Submitted on June 23, 2000; accepted on September 14, 2000.
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