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Instituto Valenciano de Infertilidad (IVI-FIVIER) (F.D., A.C., J.M., A.P., C.S.) and Department of Pediatrics, Obstetrics, and Gynecology (F.D., J.M., A.P., C.S.), School of Medicine, University of Valencia, 46010 Valencia; and Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) (S.A., J.L.C.), Universidad Autónoma of Madrid, Cantoblanco, 28049 Madrid, Spain
Address all correspondence and requests for reprints to: Carlos Simón, M.D., Instituto Valenciano de Infertilidad (IVI-FIVIER), C/Policia Local, 3, 46015 Valencia, Spain. E-mail: csimon{at}interbook.net.
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Introduction |
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This period, termed the window of implantation, opens 4–5 d and closes 9–10 d after P production or administration. Therefore, the receptive window is limited to d 19–24 of the menstrual cycle in humans (3) and 8–10 d post ovulation in other primates (4). Indeed, the administration of P antagonist (5, 6) or E2 antiserum (7) during the preimplantation period disrupts endometrial receptivity in primates. Using this concept of E2 and P priming, a clinical endometrial receptivity window is routinely induced in ovum donation programs to synchronize the timing of embryo transfer (8).
Using different animal models, including the human, we have learned that to acquire the functional receptive phenotype, the endometria suffers structural and biochemical modifications that must be induced by specific gene regulation. These morphological changes include modifications in the plasma membrane (9) and cytoskeleton (10, 11). A number of biochemical markers for endometrial receptivity have been proposed (12), until now without clinical application. However, a hierarchical perspective of the genes modified during this process in humans is still lacking.
The human endometrial cell line RL95-2 is an epithelial cell line derived from a moderately differentiated endometrial adenocarcinoma (13) with specific morphological and biological characteristics (14). This cell line exhibits more pronounced adhesiveness for trophoblast-derived cells (JAR cells) (15) and mouse blastocysts (11) than any other human endometrial epithelial cell (EEC) line, including HEC-1-A and primary epithelium. The HEC-1-A cell line, in contrast, has poor adhesive properties and exhibits a polarized distribution of integrins, but the RL95-2 cell line shows atypical features in adherens junctions, with nonpolarized actin cytoskeleton and integrin distribution (16). Embryonic adhesion experiments using mouse blastocysts showed pronounced receptive and nonreceptive phenotypes in RL95-2 and HEC-1-A cells (81% vs. 46% of blastocyst adhesion, respectively), when compared with an intermediate adhesion rate in primary EEC cultured on extracellular matrix (67% of blastocyst adhesion) (11). Therefore, we used these cell lines as in vitro models for higher receptivity (RL95-2) and lower receptivity (HEC-1-A).
In the present study, we differentially analyzed the expression pattern of 375 human genes including cytokines, chemokines, adhesion molecules, and their receptors in receptive (LH+7) vs. prereceptive (LH+2) human endometrium and human endometrial cell lines with higher (RL95-2) and lower (HEC-1A) adhesiveness to JAR cells and mouse blastocysts. Using this combined approach, we found that IGF-binding protein-related protein 1 (IGFBP-rP1) was the second most up-regulated gene in the receptive status in both models. In addition, its quantitative mRNA expression, mRNA and the gene’s protein localization was assessed throughout the human menstrual cycle by quantitative fluorescent RT-PCR (QF-PCR), in situ hybridization, and immunohistochemistry.
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Materials and Methods |
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Human endometrial samples were obtained for research after written consent from patients. 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 Spanish Law of Assisted Reproductive Technologies (35/1988). To investigate the differential expression pattern between LH+2 and LH+7 phases, two endometrial samples from each of the two fertile patients (aged 23–39 yr) were obtained in the luteal phase at LH+2 and LH+7 d. A portion of each specimen was dated according to the criteria of Noyes et al. (17). We also obtained endometrial biopsies from 10 additional patients at different days of the menstrual cycle to analyze the expression pattern of the selected gene by QF-PCR and immunohistochemistry. Endometrial biopsies were distributed in five groups according to each phase: group I, early-mid-proliferative (d 1–8); group II, late proliferate phase (d 9–14); group III, early secretory (d 15–18); group IV, midsecretory (d 19–22); and group V, late secretory (d 23–28).
RL95-2 (CRL-1671) and HEC-1-A (HTB-112) human endometrial cell lines were obtained from American Type Culture Collection (Manassas, VA) and cultured and grown as described previously (11).
Separation of epithelial and stromal cells
The endometrial samples were minced into less than 1-mm pieces with two sterile blades, and we separated blood and mucus. Pieces were digested for 1 h in a 37 C shaker bath with 0.1% (wt/vol) collagenase type IA (Sigma, St. Louis, MO) in DMEM (Sigma).
Endometrial stromal cells were isolated by filtration through a 30-µm sieve, and the epithelial fraction was obtained by gravity sedimentation as previously described (18, 19, 20). Samples were collected in Trizol (Life Technologies, Inc./BRL, Madrid, Spain) and immediately frozen for RNA extraction. The purity of the fractions obtained has been assessed by immunohistochemistry using cytokeratin, vimentin, and CD68 antigens (19). Two samples of each phase of the cycle were separated in this manner.
RNA isolation and DNase I digestion
Total RNA was extracted from whole endometrial biopsies obtained at LH+2 and LH+7 and fresh RL95-2 and HEC-1 human endometrial cell lines. Samples were collected and processed in Trizol (Life Technologies, Inc./BRL) according to the manufacturer’s instructions. This was followed by two rounds of phenol/chloroform cleanup, precipitated overnight at -20 C with 0.5 volumes of isopropanol and washed with 70% (vol/vol) ethanol. Total RNA (20 µg) of endometrial biopsies and cell lines were treated for 30 min with 5 µl DNase I (CLONTECH Laboratories, Inc., Palo Alto, CA) at 37 C, followed by one round of phenol/chloroform and another solely with chloroform. The RNA was then precipitated overnight at -20 C, using 0.1 volume of 2 M sodium acetate (pH 5.2) and 2.5 volumes of 100% ethanol. The RNA was washed with 80% ethanol and the pellet dissolved in 20 µl RNase-free water. The integrity of the RNA was assessed using a 1% (wt/vol) guanidinium isothiocyanate agarose gel. The RNA concentrations were determined by OD with a GeneQuant II spectrophotometer (Pharmacia, Uppsala, Sweden). The 260/280-nm absorbance ratio for each sample was between 1.6 and 1.9.
cDNA array hybridization and statistical analysis
Two identical cDNA arrays membranes (human cytokine expresión array; R&D Systems, Minneapolis, MN) and their gene-specific primers were used. For the preparation of cDNA probes, the Atlas cDNA expression array kit (CLONTECH Laboratories, Inc.) was used, and the recommended protocols were followed. The cDNA probes were synthesized from purified total RNA using [
32-P] dATP, dCTP, dGTP and deoxythymidine 5'-triphosphate. Unincorporated 32P-labeled nucleotides were removed by CHROMA SPIN-200 column chromatography (CLONTECH Laboratories, Inc.). After prehybridization for 30 min at 68 C in Express Hyb solution (CLONTECH Laboratories, Inc.), the probes were hybridized overnight with the cDNA array filters. Membranes were washed at 68 C three times in 2x saline sodium citrate (SSC), 1% SDS (wt/vol) and once in 0.1x SSC, 0.5% SDS (wt/vol). Following this they were washed in 2x SSC at room temperature. After overnight exposure, membranes were analyzed using a bioimaging analyzer (BAS-MP 20240; Fuji Photo Film Co., Ltd., Tokyo, Japan). The image files were quantified using both the manufacturer and NIH Image 1.62 software. In addition, arrays were subjected for 3 d to x-ray film autoradiography (BioMax MS, Kodak, Rochester, NY), and the films were scanned using an Agfa StudioStar scanner (Agfa Corp., Mortsel, Belgium). The image files were quantified as before. The normalized signals on both arrays were used to determine fold induction or fold reduction in expression of gene-specific RNAs between samples.
RT-PCR and QF-PCR
Total RNA (1 µg) was reverse transcribed and PCR amplified by means of a single-buffer system (Access RT-PCR; Promega Corp., Madison, WI), using oligonucleotide primers specific to human IGFBP-rP1 transcript (forward primer 5'-CATCTGGAATGTCACTGGTGCCCAG-3' and reverse primer 5'-GAGGTTTATAGCTCGGCACCTTCACC-3') or human ß-actin transcript (forward primer 5'-GCATGGAGTCCTGTGGCATCCACG-3' and reverse primer 5'-GGTGTAACGCAACTAAGTCATAG-3'). Equivalent aliquots of each amplification reaction were separated in a 2% (wt/vol) agarose gel in 1x Tris-acetate/EDTA buffer and stained with ethidium bromide.
DNase I-treated RNA (50 ng) was reverse transcribed and PCR amplified using the one-step LightCycler-RNA amplification SYBR Green I kit with the LightCycler instrument (Roche, Mannheim Germany). ß-Actin was used as a housekeeping internal control and RT-PCR amplified in all the RNA samples. Oligonucleotide sequences designed for the amplification of both genes were those previously described. Relative quantification was carried out using the standard curve method and the SYBR Green I dye. Data are presented as a relative average value ± SEM for IGFBP-rP1 gene and then normalized with the average value of the ß-actin gene obtained at different days in each designated phase of the menstrual cycle.
In situ hybridization
Total RNA (1 µg) was reverse transcribed and PCR amplified using a single-buffer system (Access RT-PCR; Promega Corp.) and oligonucleotide-specific primers to human IGFBP-rP1/MAC25 mRNA (sense 5'-GCACCTGCGAGCAAGGTCC-3' and antisense 5'-GCACCTTCACCTTTTTTCACTGGC-3'). PCR products (382 bp) were inserted in the EcoRV site of pBluescript KSII (Stratagene, La Jolla, CA) via thymidine-adenine cloning and characterized by restriction analysis. DNA sequence was reconfirmed using T3 and T7 primers. Digoxigenin cRNA antisense and sense probes were created by either T3 (antisense) or T7 (sense) RNA polymerase-mediated transcription of linearized plasmids with EcoRI and HindIII. Paraffin-embedded sections were baked for 2 h at 60 C, dewaxed with two baths of xylene and hydrated in a series of alcohols. A further bath with 0.2 M HCl and diethylpyrocarbonate-treated water was performed. Digestion was implemented with proteinase K (10 mg/ml) (Life Technologies, Inc./BRL) for 30 min at 37 C. Following this, a further wash with 0.1 M triethanolamine with acetic anhydride (0.25% vol/vol) was performed. Sections were prehybridized for 2 h at 50 C with hybridization buffer containing 60% deionized formamide, 25 mM Tris (pH 7.4), 1 mM EDTA (pH 8), 0.4 M NaCl, dextran sulfate (12% wt/vol), and Denharts solution (1x). Then, 5 µg/ml tRNA and 5 µg/ml salmon sperm DNA were added to the buffer. Overnight hybridization was performed in hybridization buffer with 0.1 mM dithiothreitol, sodium trisulfate (0.1% vol/vol), and SDS (0.1%) at 50 C with 500 ng/ml sense and antisense probes. Sections were consecutively washed in 2x SSC at room temperature, 2x SSC at 50 C, 1x SCC, and finally 0.1x SCC. RNase A (20 µg/ml) digestion for 1 h at 37 C shaking was implemented. Afterward, 1x blocking solution (Roche) was added in buffer 1 (pH 7.5) containing 100 mM maleic acid and 150 mM NaCl. Sections were incubated for 1 h at room temperature with alkaline phosphatase anti-DIG antibody (casa commercial, diluted 1:500) in buffer 1 containing 1x blocking solution. Color development was performed for 2 h at room temperature in buffer 3 [0.1 M NaCl, 50 mM MgCl2, 0.1 M Tris (pH 9.5)] containing NBT/BCIP (Roche) (1% vol/vol) and 1 mM levamisole. Counterstaining was performed with 0.1% methyl green for 30 sec. Sections were mounted using Kaiser’s glycerol gelatin (Sigma). Photomicrographs were obtained using a digital camera (coolpix 995; Nikon, Tokyo, Japan).
Immunohistochemistry
Immunohistochemistry was performed on endometrial sections using an LSAB peroxidase kit (DAKO Corp., Barcelona, Spain). Briefly, sections were incubated with 3% hydrogen peroxide for 5 min at room temperature (RT). After each step, the sections were washed with PBS incubated for 30 min at room temperature with antihuman IGFBP-rP1 polyclonal antiserum (obtained from rabbits immunized with a synthetic peptide of human IGFBP-rP1; IBT-Immunological and Biochemical Testsystems GmbH, Reutlingen, Germany) diluted 1:1000. No cross-reactivity was found with human IGFBPs 1–6. After 25-min incubation with the linker, streptavidin-peroxidase was added for 15 min and the substrate-chromogen solution used for 5 min to stain the slides. A counterstain with Mayer’s hematoxylin was performed. The slides were mounted with entellan (Merck, Darmstadt Germany). Negative control was provided by a commercial kit and performed by deletion of primary antibody.
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Results |
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The results of the differential gene expression pattern in LH+2 vs. LH+7 endometrium when 375 candidate genes were analyzed are shown in Table 1
. In the list of regulated genes, we identified genes that were already known to be expressed differently in the LH+2and LH+7 phases, such as placental protein 14 (PP14) (14.4 fold-up), osteopontin (3.7 fold-up), integrin alpha3 (2.3 fold-up), and IL-1RtI (1.8 fold-up). However, we also detected a number of genes that have not been previously identified in the human endometrium and whose difference of expression in the LH+2 and the LH+7 phases has not been described (Table 1). These genes can be classified in different groups: extracellular matrix proteins (decorin), heparin-binding molecules (pleiotrophin), genes related to tyrosine kinases (EFNA2), and growth factors (bone morphogenetic protein-7).
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Only four genes were minimally down-regulated in the receptive endometrium (between 1.32- and 1.89-fold change). These genes were two IL receptors (IL-15R and IL-9R); bone morphogenetic protein 7; a growth factor of the TGF-ß family; and ephrin-A2 (a tyrosine kinase ligand).
In Table 2 we present the comparative results obtained after the cDNA array hybridization of the endometrial cell lines, HEC-1-A vs. RL95-2. The two highly expressed genes in the RL95-2 cell line were neurite growth-promoting factor 2 (NEGF2/midkine) and IGFBP-rP1, with a 16- and 12-fold change, respectively. The rest of the up-regulated genes were chemokines (GRO oncogene 1 and 2), growth factor receptors (erbB1 and TNFRSF16), and growth factors (TGF). The group of adhesion molecules appears minimally up-regulated (between 1.30- and 3.69-fold change) and includes EpCAM, integrins ß 1, 4, and 1.
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, A and B). RT-PCR was performed using the same RNA of both cell lines and endometrial biopsies to confirm the RNA expression of the cDNA arrays (Fig. 1C). IGFBP-rP1 expression in both models exhibits a clear trend, reflecting the results obtained in the cDNA arrays (Fig. 1C).
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-3, were down-regulated in the high-adhesive cell line and down-regulated genes in the receptive endometrium, such as ephrin-A2, were up-regulated in the high-adhesive cell line. Expression and localization of IGFBP-rP1 mRNA in the human endometrium
To further corroborate these findings, we investigated the expression pattern and distribution of IGFBP-rP1 in the human endometrium. QF-PCR for this gene was investigated in different patients (n = 10) throughout the menstrual cycle.
QF-PCR experiments in total endometrium throughout the menstrual cycle revealed that expression increases 35-fold during the LH+7 phase (d 19–22), compared with the LH+2 phase (d 15–18), followed by a sharp increase in the late luteal phase. This conforms to a profile consistent with a marker of endometrial decidualization (Fig. 2A).
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). A similar experiment was performed using glyceraldehyde-3-phosphate dehydrogenase as an internal housekeeping gene, with identical results.
To confirm these results, in situ hybridization was performed using endometrial tissue throughout the menstrual cycle. Our results indicated that IGFBP-rP1 mRNA is localized to the luminal and glandular epithelium, stromal cells, and blood vessels (Fig. 3). In the luminal epithelium, higher expression is noted in the early secretory phase (group III), with a slight decrease in mid and late secretory phases (groups IV and V) (Table 3). Glandular epithelium has a different expression pattern, showing minimal expression in early secretory (group III) and increasing in mid and late luteal phases. Finally, stromal cells show very slight expression in proliferative and early secretory phases, which increases sharply in the luteal phase (groups IV and V).
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). The intensity of staining increases in the midsecretory phase (group IV) (Fig. 4F) but remains throughout the cycle with more or less intensity. Endothelial cells were also positive for IGBBP-rP1 (Fig. 4I).
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Discussion |
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The IGF axis is a network of ligands (IGF-I and -II), receptors (IGF-RI and IGF-RII), and binding proteins (IGFBPs) (26). The actions of IGFs are regulated in part by IGFBPs, which are responsible for the former’s bioavailability for activating the receptors. IGFBPs are subdivided into two groups: high-affinity binding proteins (IGFBP-1 to -6) and low-affinity IGFBPs (IGFBP-7 to -10) (27). Their low affinity for IGF and the conserved structural homology with the IGFBP family suggests that these molecules may have unique biological properties independent of their capacity to bind IGF.
Mac-25 (also known as IGFBP-7 or IGFBP-rP1) was initially cloned as a gene whose expression was decreased in meningioma cells and tumor-related leptomeningeal cells (28) and subsequently reisolated, through differential display as a sequence preferentially expressed in senescent human mammary epithelial cells (29). The deduced amino acid sequence of the human mac25 polypeptide shares a 20–25% identity with human IGFBPs, and recombinant mac25 was found to function as an IGFBP (30). Mac25 was, therefore, renamed as IGFBP-7 and, more recently, as IGFBP-rP1 (31).
This molecule was the second most expressed gene of the 375 analyzed in the receptive endometrium and adhesive cell line (RL95-2). Because a role for IGFBP-rP1 in human endometrial receptivity has not been previously described, we have investigated its gene expression and localization, as well as its protein localization, throughout the menstrual cycle. QF-PCR experiments identified a 35-fold increase during the receptive phase when compared with the prereceptive phase, followed by a sharp increase in the late luteal phase. In addition, QF-PCR experiments using the epithelial and stromal endometrial fraction separately confirmed that IGFBP-rP1 expression was present in both compartments but mainly up-regulated in the stromal fraction. In general, in situ hybridization experiments corroborate the quantitative results obtained in the QF-PCR experiments. However, there were some discrepancies; QF-PCR showed minimal expression in the epithelial compartment in the early secretory phase (group III), but we detected, through in situ hybridization, higher expression of IGFBP-rP1 in the luminal epithelium when compared with the glandular epithelium. Considering that glandular epithelium represents almost 80% of the epithelial compartment, probably this may be the reason mRNA expression for this molecule is not picked up when the complete compartment is analyzed by QF-PCR. Immunohistochemical experiments corroborated the epithelial and stromal localization for this molecule and showed to be also IGFBP-rP1 localized at the endothelial cells. The localization of this protein reflects findings of a previous study that explored the distribution of IGFBP-rP1 in normal human tissues (32).
Although the function of this molecule remains to be demonstrated, this study strongly suggests that IGFBP-7/mac25/IGFBP-rP1 is implicated in human endometrial receptivity. It is unlikely that the role of this molecule in endometrial receptivity is that of IGFBP because of the low affinity of the former for IGF. However, several independent functions of vascular biology that may be involved in endometrial receptivity have been attributed to this molecule. IGFBP-rP1 has been reported as a prostacyclin-stimulating factor in vascular endothelial cells (33) and has been shown to contribute to the organization of new capillary vessels in tumor tissues by modulating the interaction of endothelial cells with type IV collagen (34). IGFBP-rPI contains an amino-terminal domain with homology with IGFBPs that is responsible for low-affinity binding to IGFs. This sequence is followed by a follistatin-like module that has a low but significant homology with the cysteine-rich, follistatin-like module of hevin/SC1, which is known to mediate cytokine binding in other proteins. These two modules in IGFBP-rP1 are likely to be involved in growth factor binding and may facilitate the retention of growth factors or chemokines (35). This molecule has also been defined as a tumor-derived adhesion factor (recently renamed angiomodulin) (36). At the ultrastructural level, a noteworthy feature associates IGFBP-rP1 with microvillous structures that constitute the initial point of contact between the endothelium and the adherent lymphocytes. It has been demonstrated that this molecule may harbor chemokines (37) and adhesion molecules such as the L-selectin countereceptor CD3453 and L-selectin ligands (38), suggesting that this molecule may contribute to the lymphocyte migration through high endothelial venules.
Therefore, in addition to its vascular functions, these properties indicate that IGFBP-rP1 is a good candidate for the presentation of adhesion-triggering cytokines to the lymphocytes rolling on, or migrating across, in and through the human endothelial epithelium, this process being remarkably similar to the implantation process. We hypothesized that this molecule acts in this way in the luminal endometrium. As we have seen with the immunohistochemistry, IGFBP-rP1 behaves as a secreted protein, perhaps secreted by stromal cells and transported to the luminal and glandular epithelium in which it accumulates, mainly in the receptive phase. In this localization, it is possible that IGFBP-rP1 binds cytokines, chemokines, growth factors, or key adhesion molecules implicated in the implantation process.
In summary, using this combined approach, we have discovered that IGFBP-rP1 is the second most up-regulated gene in both models of endometrial and epithelial receptivity. These results have been validated by QF-PCR and immunohistochemistry and suggest a novel function for this molecule in human endometrial receptivity. Further experiments need to be undertaken to unravel the implication of this molecule in the human endometrium at the time of implantation.
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Acknowledgments |
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Footnotes |
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F.D. and S.A. made equal contributions to this work.
Abbreviations: E2, 17ß-Estradiol; EEC, endometrial epithelial cell; IGFBP, IGF-binding protein; IGFBP-rP1, IGF-binding protein-related protein 1; LH+2, prereceptive phase; LH+7, receptive phase; P, progesterone; QF-PCR, quantitative fluorescent RT-PCR; SSC, saline sodium citrate; TIMP, tissue inhibitor of metalloproteinase.
Received May 8, 2002.
Accepted December 4, 2002.
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4 and 1 in human endometrial epithelial cells in vitro. J Clin Endocrinol Metab 82:2607–2616vß3 integrin are coexpressed in the human endometrium during the menstrual cycle but regulated differentially. J Clin Endocrinol Metab 86:4991–5000This article has been cited by other articles:
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) and stromal (
) fraction separated from the above-mentioned endometrial samples. Three independent experiments were performed with each sample, and data are presented as a relative average value ± SEM for IGFBP-rP1 gene and then normalized with the average value of the ß-actin gene obtained at different days in each designated phase of the menstrual cycle. EEC, Endometrial epithelial cells; SC, stromal cells.
