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Regular Article |
b Instituto Valenciano de Infertilidad, Research Department (FIVIER), Valencia 46020, Spain
c Department of Pediatrics, Obstetrics and Gynecology, School of Medicine, Valencia University, Valencia 46010, Spain
ABSTRACT
Human endometrial epithelial cells (EECs) are nonadhesive for embryos throughout most of the menstrual cycle. During the so-called implantation window, the apical plasma membrane of EECs acquire adhesive properties by undergoing a series of morphological and biochemical changes. The human endometrial-derived epithelial cell line, RL95-2, serves as an in vitro model for receptive uterine epithelium because of its high adhesiveness for trophoblast-derived cells. In contrast, the HEC-1-A cell line, which displays poor adhesive properties for trophoblast cells, is considered to be less receptive. The ezrin, radixin, and moesin protein family members, which are present underneath the apical plasma membrane, potentially act to link the cytoskeleton and membrane proteins. In the present study, we have further investigated the adhesive features in these two unrelated endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also analyzed the protein pattern and mRNA expression of ezrin and moesin in RL95-2 cells versus HEC-1-A cells. The results demonstrate that RL95-2 cells were indeed more receptive (81% blastocyst adhesion) compared with HEC-1-A cells (46% blastocyst adhesion). An intermediate adhesion rate was found in primary EECs cultured on extracellular matrix gel, thus allowing a partial polarization of these cells (67% blastocyst adhesion). Furthermore, we found that moesin was absent from RL95-2 cells. In contrast, ezrin is expressed in both cell lines, yet it is reduced in adherent RL95-2 cells. Data are in agreement with the hypothesis that uterine receptivity requires down-regulation or absence of moesin, which is a less-polarized actin cytoskeleton.
embryo, growth factors, implantation, signal transducers, uterus
INTRODUCTION
It is well known that ovarian hormone priming of the endometrium is essential for implantation [1, 2]. In the mouse model, if endometrial epithelial cells (EECs) are removed from the uterus, the embryo will attach, independent of any regulatory control [3]. Thus receptivity is a property of EECs. Human EECs show a typical polarized phenotype of simple epithelia, which is nonadhesive for trophoblasts throughout most of the menstrual cycle. Therefore, EECs must modify its phenotype at the time of window implantation to become adhesive [4]. To date, the molecular details of how the surface of human EECs acquire receptive features are unclear. The evidence suggests that the apical plasma membrane of EECs may acquire adhesive properties by undergoing a series of morphological changes, which are collectively referred to as ‘the plasma membrane transformation’ [5], primarily as a result of the disruption of the cytoskeleton in response to external signals. In addition, it has been proposed that a remodeling of the epithelial organization, from a polarized to a nonpolarized phenotype, may prepare the apical pole for cell-to-cell adhesion [6, 7].
A role for the membrane/cytoskeleton interface in the development of the receptive status has been demonstrated, yet it is poorly understood. Because few integral membrane proteins have been found to directly interact with actin, linker proteins could serve to connect the actin cytoskeleton with the plasma membrane [8]. The closely related ezrin, radixin, and moesin (ERM) proteins are concentrated in specialized sites at which actin filaments are densely associated with the plasma membrane (i.e., microvilli, ruffling membranes, and cleavage furrows) [9–11]. Biochemical data support the proposed role of ERM family members as membrane-cytoskeletal linkers. Transient expression of a partial sequence of ezrin cDNA in cell cultures shows amino terminus-plasma membrane association [12], and the capability of the COOH-terminal domain to bind F-actin [13]. Moreover, with the expression of the partial sequence corresponding to the NH2-terminus, cellular membrane extensions were inhibited [14].
The human EEC line, RL95-2, was derived from a moderately differentiated adenosquamous carcinoma of the endometrium [15]. The RL95-2 cell line exhibits more pronounced adhesiveness for trophoblast-derived cells (JAR cell line) than other human EEC lines, including HEC-1-A [16] and, thus, serves as an in vitro model for receptive uterine epithelium. The HEC-1-A cell line that has poor adhesive properties exhibits the well-known pattern of cell-to-cell contacts of polarized epithelial cells with polarized distribution of integrins; meanwhile, RL95-2 cells show atypical features in adherens junctions, with nonpolarized actin cytoskeleton and integrin distribution [6, 17].
In the present study, we have corroborated the adhesive features of these endometrial-derived cell lines using an established in vitro model for embryonic adhesion. We have also investigated the cellular expression of ezrin and moesin proteins, and their respective mRNAs in these human endometrial cell lines with different adhesive properties for trophoblast-derived cells. Using this in vitro model, we have demonstrated decreased expression of membrane-cytoskeleton linkers in cells that displayed increased adhesiveness for blastocysts.
MATERIALS AND METHODS
Institutional Approval and Informed Consent
This project was approved by the institutional review board on the use of human subjects in research at the Instituto Valenciano de Infertilidad, and complies with the Spanish Law of Assisted Reproductive Technologies (35/1988). Endometrial and placenta samples donated for research were obtained after written consent from patients.
Materials, Reagents, and Antibodies
Cell culture flasks and Petri dishes were obtained from FALCON (Becton Dickinson Labware Europe, Le Pont De Claix, France). Cell culture media and reagents were obtained from Sigma-Aldrich (Irvine, U.K.) and Gibco [smbrl]nm (Life Technologies, Paisley, Scotland). General chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany) or Sigma-Aldrich. The restriction enzyme, PvuII, was purchased from Roche Diagnostics GmbH (Mannheim, Germany).
Affinity-purified polyclonal antibodies 454 (antimoesin) and 464 (antiezrin) were a generous gift from Frank Solomon (Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA) [18]. Goat antimoesin and antiezrin (C-15) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit anti-goat peroxidase-conjugated antibody was from DAKO A/S (Glostrup, Denmark).
Cell Line Cultures
Human endometrial carcinoma HEC-1-A cells (HTB-112) and RL95-2 cells (CRL-1671) were purchased from American Type Culture Collection (ATCC; Rockville, MD). Cell lines were grown in plastic flasks in 5% CO2 in air at 37°C. HEC-1-A cells were seeded in McCoys 5A medium plus 10% fetal bovine serum (FBS), and RL95-2 cells in a 1:1 mixture of Dulbeccos modified Eagles medium (DMEM) and F-12K nutrient medium plus 10% FBS. This mixture was supplemented as indicated in the ATCC datasheet. All media were additionally supplemented with gentamicin (50 µg/ml) and fungizone (0.25 µg/ml). RL95-2 cells (3 x 105) and HEC-1-A cells (2 x 105) were seeded in 24-well culture plaques for 7 days, and the growth medium was renewed every 2–3 days.
Primary Endometrial Epithelial Cell Cultures
Primary cell cultures derived from human endometria were used in the same embryo adhesion assays. Samples were obtained in the luteal phase from fertile patients undergoing endometrial biopsy (ages 23–39 yr). The biopsies were processed as previously described [19]; EECs were cultured on 24-well plaques coated with a diluted solution (1:4) of extracellular matrix (ECM) gel (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany).
Embryo Recovery
B6C3F1 mice were purchased from Elevage Janvier (Le Genest, St. Isle, France) and housed in a conventional holding facility with controlled temperature and lighting (14L:10D) at the University of Valencia School of Medicine. Food and water were supplied ad libitum. Female mice 6–8 wk of age were primed to ovulate by i.p. administration of 10 IU eCG (Sigma-Aldrich) in 100 µl Dulbeccos PBS, free of Ca2+ and Mg2+ (pH 7.4) to induce synchronous follicle development, followed by i.p. administration of 10 IU hCG (Sigma-Aldrich) in 100 µl PBS 48 h later to initiate ovulation. Females were generally housed two per cage overnight with a stud male and examined the following morning for the presence of a vaginal plug (Day 1 of pregnancy). On Day 2 of pregnancy, mice were killed by cervical dislocation, and embryos were flushed from the oviduct with PBS using a blunt, 30-gauge needle attached to a 2-ml syringe.
Embryos were cultured for 3 days in S2 medium (Scandinavia IVF Science, Gothenburg, Sweden). Degenerated embryos were discarded, and only expanded blastocysts with a normal morphology were used. No zona pellucidae were artificially removed.
Embryo Adhesion Assay
The adhesion of mouse blastocysts to confluent EEC monolayers was measured using a mechanical assay [20]. After 48 h of incubation over confluent monolayers (5% CO2 in air at 37°C) of HEC-1-A cells, RL95-2 cells, and primary EECs from endometrium biopsies, the incubation plaques were moved along a 3-cm diameter circular path at a speed of one rotation per second for about 10 sec. Embryos floating in the medium were judged as not attached, and embryos not floating were considered to be attached. Embryos were examined under an inverted microscope (Nikon Diaphot 300, Nikon Corporation, Tokyo, Japan). As a control for slightly different medium composition, mouse blastocysts were cultured alone with the corresponding medium and, after 48 h, were examined for attachment to the plastic.
Protein Extraction and Immunoblot
HEC-1-A and RL95-2 cells were cultured as described earlier. Proteins were extracted in a cell lysis buffer (1% NP-40, 0.5% deoxycholate, 0.1% SDS, 25 mM Tris pH 8.0, 150 mM NaCl) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 20 min. Cell lysates were precleared by centrifugation at 13 000 x g for 20 min, at 4°C. Proteins were separated in 8% Tris-glycine pre-cast mini gels (NOVEX, Frankfurt, Germany) by SDS-PAGE [21] and then transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) using a PowerEase 500 Power Supply and XCell II Mini-Cell equipment (NOVEX).
Western blots were blocked overnight at 4°C with 3% nonfat dry milk in Tris-buffered saline (25 mM Tris pH 7.5, 0.17 M NaCl) plus 0.05% Tween (TBS-T). After a brief wash with TBS-T, the first antibody incubation (C-15, 5 µg/ml) was performed in 1% nonfat dry milk TBS-T for 90 min at room temperature. Following several washes with TBS-T, a mixture of the secondary antibody and ExtrAvidin peroxidase-conjugated antibody (1:2000 dilution, Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) in the same antibody buffer was incubated for 1 h at room temperature. Blots were then washed as before, except the final wash, which was performed with TBS. Proteins were visualized using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech Europe Gmbh, Freiburg, Germany).
Immunoprecipitation
In some experiments, the cell lysates were immunoprecipitated using specific 454 and 464 antibodies. Briefly, the extracts were preabsorbed to 25 µl protein A-Sepharose beads (pA-S; Sigma Chemical Company, St. Louis, MO). Thereafter, the supernatants were incubated with specific 454 or 464 antibodies, precipitated with pA-S, washed with the RIPA buffer, and eluted by boiling in Laemmli sample buffer.
Immunofluorescence Microscopy
For indirect immunofluorescence microscopy, cells cultured on cover-glass slides were fixed with 2% paraformaldehyde in PBS for 15 min at 4°C, then treated with 0.2% Triton X-100 in PBS for 10 min. Samples were subsequently treated with PBS containing 1% BSA for 15 min, and then incubated with the first affinity-purified antibody, antimoesin pAb 454 or antiezrin pAb 464, for 30 min at 4°C. Cells were washed with PBS and then incubated with a fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody (Sigma). Samples were then washed with PBS and then examined using an epifluorescence microscope (Labophot-2, Nikon, Tokyo, Japan). Negative controls were performed omitting primary antibodies.
Affinity-purified antibodies were prepared by slight modifications of established procedures [18, 22] from proteins transferred onto nitrocellulose. Antimoesin 454 was affinity-eluted from the moesin-immunoreactive band of microvascular lung endothelial cells (Clonetics Corporation, Walkersville, MD); antiezrin 464 was affinity-eluted from the ezrin-immunoreactive band of placenta protein extractions.
Oligonucleotides Synthesis
Oligonucleotides from reverse transcriptase-polymerase chain reaction (RT-PCR) were designed using Primer Designer software (Scientific and Educational Software, State Line, PA). Table 1 shows sequences and nucleotide positions (relative to the translation initiation site) that were chosen for the synthesis of oligonucleotides for the amplification of human ezrin cDNA [23] and human moesin cDNA [24].
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RNA Extraction and RT-PCR
To analyze the expression of ezrin and moesin genes, total RNA was prepared from cell cultures grown in 5-cm Petri dishes in the same conditions as for adhesion assays. RNA from the cells was extracted directly on the dishes using 1 ml of TRIZOL reagent (Gibco BRL) according to the method described by Chomczynski and Sacchi [25]. Total RNA was then DNase-treated (DNase I, Boehringer-Mannheim, Mannheim, Germany). To obtain the cDNA from cell lines and from placenta after cesarean section, the final ethanol-precipitated products were used for RT reactions using an Advantage RT-for-PCR kit (Clontech, Palo Alto, CA). Semiquantitative PCR was developed to obtain the specific moesin or ezrin products. The PCR cycle, repeated 30 times, consisted of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min. The PCR products were analyzed on 1.5% agarose gels (Pronadisa, Madrid, Spain). For ß-actin, the same PCR conditions were used except that the PCR cycle was repeated 24 times. Quantitative estimation of bands was performed by densitometric analysis with image software (1-D software, Gelprinter Plus, TDI, Madrid, Spain). PvuII restriction sites in the cDNA products were determined using Sequaid II software (Rhoads & Ronfa 1989; Molecular Genetics Laboratory, Kansas State University, Manhattan, KS).
Statistical Analysis
Data are presented as means of percentage ± SEM. The chi-square test was carried out between groups. Significance was defined as P < 0.05. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL).
RESULTS
Adhesion Assays in Primary EECs, and RL95-2 and HEC-1-A Cells
Adhesion experiments showed significant differences in blastocyst adhesiveness in RL95-2 cells versus HEC-1-A cells (81.0% ± 11.5% and 46.5% ± 24.5%, respectively; Fig. 1). Data were collected from five different experiments in which a total of 286 mouse blastocysts were examined. Mouse blastocysts (n = 33) incubated on EECs cultured on 24-well plaques coated with ECM gel showed an intermediate adhesion rate (67.4% ± 9.4%; Fig. 1). Differences in distinct culture media (the media for both cell lines were slightly different as indicated by ATCC) were ruled out by appropriate control wells. A total of 45 blastocysts were used to rule out media-induced changes in embryo adhesion. Embryos attached to plastic regardless of media (100% adhesion rate with HEC-1-A and RL95-2 media; 93% with EEC medium).
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Moesin and Ezrin Protein Expression in RL95-2 and HEC-1-A Cells
To identify whether moesin and ezrin were expressed equally in both cell lines, we examined cell lysates from monolayers cultured in the same conditions that were used for blastocyst adhesion assays. After SDS-PAGE, immunoblots were performed using C-15 antibodies, which recognized both ezrin and moesin proteins from lysates before (Fig. 2A) or after immunoprecipitation with specific antibodies for ezrin and moesin (Fig. 2B). Unexpectedly, both commercial C-15 antibodies showed cross-reactivity between ezrin and moesin proteins (Fig. 2A, lanes H and P); bearing in mind the purpose of the study, the different expression patterns of moesin in the two cell lines became clear. We determined that moesin protein was absent in lysates from RL95-2 cells (Fig. 2, lane R). In contrast, HEC-1-A cells were found to express moesin (Fig. 2, lane H). Immunoblots also revealed that both cell lines expressed the ezrin protein (Fig. 2, lanes H and R). Endothelial cell lysates, which express moesin but not ezrin [14], and placenta lysates that have been reported to express both ezrin and moesin [26], were used as positive controls (Fig. 2, lanes Ed and P, respectively).
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To further examine these results, immunofluorescence experiments were performed using affinity-purified antibodies 454 and 464 (Fig. 3). Intense ezrin signals were detected in both cell lines, whereas moesin was present in HEC-1-A cells, but not in RL95-2 cells. The staining for ezrin and moesin proteins was mainly localized to cellular extensions at the upper surface level and was concentrated along the lateral surface at cell-to-cell boundaries (Fig. 3, B and C). Negative controls for each cell line were performed but the primary antibody was omitted (see representative, inset in Fig. 3A). A faint signal can be seen in Figure 3D; whereas affinity-purified antibodies were used for immunofluorescence experiments, these purified antibodies also displayed a minor cross-reactivity (Fig. 2B, moesin, lane R).
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Moesin and Ezrin mRNA Expression in RL95-2 and HEC-1-A Cells
Semiquantitative RT-PCR was developed to verify protein data and to exclude the possibility that the epitope recognized by the antibody was masked in an inactive conformation of the protein, which diminished moesin detection. After 30 cycles, the moesin-amplified PCR product was not observed in RL95-2 cells (Fig. 4, MOESIN, lane R). A decreased expression of ezrin mRNA in RL95-2 cells was also demonstrated (Fig. 4, EZRIN, lane R compared to lane H; see also Fig. 5). cDNA from placenta was used as a positive control (Fig. 4, lanes E and Mo). These PCR products were used for enzymatic digestion to assure the specificity of the synthesized oligonucleotides. Fragments digested with PvuII confirmed that the PCR-amplified products were ezrin and moesin (data not shown). Beta actin-amplified PCR product (ß) after 24 cycles was used as the housekeeping gene (Fig. 4, ß-actin).
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DISCUSSION
The plasma membrane of EECs is the first site of contact between embryonic and maternal cells in a wide variety of species [27]. Whereas the type of implantation differs widely among species, the initial process seems to start with trophoblast attachment to the altered plasma membrane of uterine epithelial cells, and this appears to be a general phenomenon found not only in invasive types of implantation, but also in noninvasive epitheliochorial types [4, 28]. This is particularly relevant when the alterations that occur in the plasma membrane at the time of adhesion are considered [29–31]; these are referred to as the ‘plasma membrane transformation’ [5].
In a preliminary study we demonstrated the presence of ezrin in human endometrium, which was specifically localized to the apical membrane of epithelial gland cells [32].
Because of the current ethical dilemma of using human embryos for research, we performed our adhesion assays with mouse embryos. We showed that the human EEC line, RL95-2, has increased receptivity for mouse blastocysts (81% embryo adhesion) compared with other completely polarized human EEC lines (46% embryo adhesion for HEC-1-A), as reported previously for trophoblast-derived cells (JAR cells) [9]. RL95-2 cells are not morphologically polarized [6, 10] and lack a clear apical-basal axis. However, another study shows that an intact cortical actin cytoskeleton may be essential for achieving the receptive status [7]. In this sense, it is remarkable that an intermediate adhesion rate was observed in EECs cultured with an underlying ECM, which favors the achievement of a partially polarized cortical actin cytoskeleton (67% adhesion rate). It would be worthwhile to evaluate blastocyst adhesion to EECs cultured on inserts plus stromal cells, which would display a complete epithelial polarization.
We further investigated the expression pattern of the membrane-cytoskeleton linkers, ezrin and moesin, and the results showed that more adhesive properties for blastocysts were associated with decreased expression of ezrin and total absence of moesin protein. Thus, we consider that for increased endometrial receptivity, cytoskeletal-associated proteins such as moesin and ezrin must be decreased. The presence of ERM proteins could stabilize the actin-microvilli-associated cytoskeleton and, hence, directly (via steric hindrance) or indirectly (by providing an unsuitable membrane proteins pattern [33]) impairs embryo attachment. However, we have to keep in mind that the relationship between the high embryo adhesiveness to RL95-2 cells and the decreased pattern of moesin could be coincidental and not functionally related.
It is known that ovarian hormone exposure is essential for the development of endometrial receptivity. Although there are differences between species, it is clear that these hormones produce dramatic changes in the length and frequency of occurrence of microvilli [34–38]. Specifically, in human uterus, an increasing flattening of microvilli has been described around the time of implantation [39–41].
ERM proteins accumulate underneath the plasma membrane in various cell surface structures such as microvilli, membrane ruffles, and cell-cell contact sites [14, 42]. Moreover, ERM proteins are involved in microvilli formation and breakdown [43–45], and are also necessary for cell-cell and cell-substrate adhesion [17, 33, 43]. Those proteins that showed significant identity with the erythrocyte membrane protein band, 4.1 [46], function as cross-linkers between cell surface molecules and the actin cytoskeleton [47], and they have been suggested to have an inactive, closed conformation in the cytoplasm [15]. It is remarkable that the binding of ERM proteins to actin and the translocation of these proteins from cytoplasm to plasma membrane appear to be regulated by the small guanosine triphosphatase Rho members [48]; furthermore, phosphorylation induced by several growth factors such as epidermal growth factor and hepatocyte growth factor can control the function of these molecules [26, 43].
Despite the high homology between members of the ERM family [23, 24], studies that have analyzed their tissue-expression patterns show different cellular distribution [14] and regulation of ERM proteins, which suggest they have unique functions; however, in vitro experiments have suggested that their function is redundant [12, 13]. In this respect, it is remarkable that the differential expression of ezrin and moesin in cell lines with different receptive status could be demonstrated.
To date, apart from structural studies, the biochemical functions of these proteins are unclear, but it has been reported that ezrin, the prototype of the family, is a signal transducer, which through the phosphorylation of Tyr-353, conveys an antiapoptotic signal for cells growing in a three-dimensional matrix [49]. In addition, it has been shown that ezrin dephosphorylation correlates with microvilli breakdown when apoptosis is induced [50]. Our group recently described the embryonic regulation of apoptosis in EECs [51], and suggested that for an embryo to implant, it is necessary for it to induce apoptosis in underlying EECs. Thus, ERM proteins would be good candidates for studying EEC targets that are affected by the presence or adhesion of an embryo to their apical pole.
In conclusion, our findings demonstrate a decreased expression pattern of membrane-cytoskeleton linkers in cells that display increased blastocyst adhesiveness. These results suggest that there is a correlation between the absence of microvilli-stabilizing elements, such as moesin, and increased endometrial receptivity; but, nevertheless, other cytoskeleton-membrane linkers such as ezrin may be present.
ACKNOWLEDGMENTS
We thank Dr. Solomon of the Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, for gifts of antibodies against ERM proteins.
FOOTNOTES
First decision: 31 January 2000.
1 Supported by the Spanish Government, grant FISs 98/0855. 
2 Correspondence: Carlos Simón, Instituto Valenciano de Infertilidad, c/ Guardia Civil, 23, Valencia 46020, Spain. FAX: 34 96 3694735; csimon{at}interbook.net
Accepted: May 31, 2000.
Received: December 27, 1999.
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