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Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst

Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the... Abstract Chemokines are implicated in the implantation process. The aim of this study was to investigate mRNA expression and protein levels of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle, during HRT and in the human blastocyst. The regulation of chemokine receptors in the endometrial epithelium was also studied using an in‐vitro model for the apposition phase of human implantation. We found up‐regulation of endometrial CXCR1 mRNA (419‐fold increase), CCR5 mRNA (612‐fold increase) and CCR2B mRNA (657 fold‐increase) during the luteal phase peaking in the pre‐menstrual endometrium. CXCR4 mRNA levels presented a specific although modest (18‐fold increase) up‐regulation during the implantation window. These findings were corroborated at the protein level in natural and HRT cycles. Immunoreactive CCR5 and CCR2B receptors were detected in human blastocysts whereas CXCR4 and CXCR1 were not present. Chemokine receptors in cultured endometrial epithelial cells showed an up‐regulation and polarization of CXCR1, CXCR4 and CCR5 receptors when a human blastocyst was present. The specific distribution and regulation of chemokine receptors in the endometrial epithelium and the human blastocyst suggest a possible implication of these receptors in the apposition and adhesion phases of human implantation. Key words: blastocyst/chemokine receptors/endometrial epithelial cells/human endometrium/implantation Submitted on July 30, 2002; resubmitted on September 5, 2002. accepted on December 13, 2002 Introduction Chemokines (short for chemoattractant cytokines), a family of small polypeptides with a molecular weight in the range of 8–12 kDa, are specialized in the attraction of specific leukocyte subsets through their binding to cell‐surface receptors. In reproductive biology, these molecules have been implicated in crucial processes such as ovulation, menstruation, embryo implantation, parturition and pathological processes such as preterm delivery, HIV infection, endometriosis and ovarian hyperstimulation syndrome (Cocchi et al., 1995; Simón et al., 1998). Chemokines and their receptors are divided into two families based on structural and genetic considerations. All chemokines are structurally similar, having at least three β‐pleated sheets and a C‐terminal α‐helix. Most chemokines also have at least four cysteines in conserved positions (Kayisli et al., 2002). In the CXC chemokine family (α‐chemokines), the two nearest cysteines (C) to the N‐termini of family members are separated by a single (and variable) amino acid (X). Interleukin‐8 (IL‐8) (α‐chemokine) is a potent chemoattractant and activator for neutrophils (Mukeida et al., 1989) and T lymphocytes (Larsen et al., 1989). IL‐8 is produced by a variety of cell types: monocytes, fibroblasts (Dudley et al., 1993), lymphocytes, epithelial and endothelial cells (Baggiolini and Dahinden, 1994). It has been detected in the human reproductive tract, at the cervix (Barclay et al., 1993), placenta (Saito et al., 1994) and endometrium (Arici et al., 1993). Monocyte chemotactic protein (MCP‐1; β‐chemokine) has been reported to attract NK cells (Loetscher et al., 1996) as well as T lymphocytes (Loetscher et al., 1994). MCP‐1 is secreted by a number of cell types such as endothelial cells (Sica et al., 1990), fibroblasts (Yoshimura and Leonard, 1990), monocytes and lymphocytes (Yoshimura and Leonard, 1989). It has been detected in normal endometrium (Arici et al., 1995) and endometriotic cells (Akoum et al., 1996). The β‐chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is a chemoattractant for monocytes, eosinophils and basophils, and is localized in eutopic endometrium and ectopic endometriotic implants (Hornung et al., 1997). Stromal‐derived factor‐1 (SDF‐1; α‐chemokine) is involved in regulating a wide array of leukocytes, lymphopoiesis and controls integrin‐dependent adhesion of T‐cells to the endothelium (Campbell et al., 1998). Most chemokines bind to the cell‐surface or connective‐tissue components such as glycosaminoglycans. Therefore, dimerization is favoured when chemokines associate with these molecules (Witt et al., 1994). These interactions provide strong evidence that chemokine dimerization is a critical process in vivo. Chemokine receptors belong to the superfamily of G‐protein‐coupled receptors (GPCR). The former receptors have seven sequences of 20–25 hydrophobic residues that form an α‐helix and span the plasma membrane, an extracellular N‐terminus, three extracellular loops, three intracellular domains and an intracellular C‐terminal tail. These receptors transmit information to the cell about the presence of chemokine gradients in the extracellular environment. They are named depending on the structure of their ligand (CXC or CC). CXCR4 is expressed on neutrophils, monocytes, B and T lymphocytes, and its primary ligand is SDF‐1 (Nagasawa et al., 1996). CCR5, receptor for RANTES and macrophage inflammatory protein‐1 (MIP‐1) α and β is expressed on monocytes, dendritic cells, activated T lymphocytes and natural killer (NK) cells (Chantakru et al., 2001). CCR2B, expressed in monocytes, basophils, dendritic cells, NK cells and activated T lymphocytes is the main receptor for MCP‐1, ‐2, ‐3 and ‐4 (Polentarutti et al., 1997). CXCR1, a receptor for interleukin 8 (IL‐8) and GCP‐2, is expressed mainly in neutrophils and dendritic cells (Wuyts et al., 1998). The binding of chemokines to their receptors is followed by the involvement of heterotrimeric G proteins (Reif and Cantrell, 1998; Ward et al., 1998) and the triggering of intracellular second messengers such as cAMP and calcium. One of the most impressive effects of the binding of chemokines to their receptors on leukocytes is the morphological change which this provokes; the cytoskeleton is rearranged, integrin‐mediated focal adhesions are formed and the cell binds and detaches from the substrate in a coordinated manner, with extension and retraction of pseudopods responsible for directional migration (Bokoch, 1995; Ward et al., 1998). A specific molecular crosstalk between embryo and endometrium has been reported during the human implantation process (Glasser et al., 1991; De los Santos et al., 1996). The endometrial epithelium is an important element where the molecular interactions between the embryo and the endometrium seem to be initiated (Simón et al., 1997; Galan et al., 2000; Meseguer et al., 2001). The endometrial epithelium produces and secretes chemokines (Arici et al., 1998; Caballero‐Campo et al., 2002). In the present study, we have analysed mRNA expression and immunolocalization of chemokine receptors (CXCR1, CCR5, CXCR4 and CCR2B) in the human endometrium throughout the natural menstrual and HRT cycles and in the human blastocyst. Furthermore, we have also studied the embryonic regulation of these endometrial chemokine receptors using an in‐vitro model for the apposition phase of human implantation. Materials and methods Institutional approval and informed consent This study was approved by the Institutional Review Board on the use of human subjects in research at the Instituto Valenciano de Infertilidad (IVI), and complies with Spain’s Law of Assisted Reproductive Technologies (35/88). All patients participating in this study signed a written statement of consent and were informed of the details of the study. Experimental design To investigate the mRNA expression of endometrial chemokine receptors (CXCR1, CXCR4, CCR2B and CCR5) throughout the menstrual cycle and their hormonal regulation in vivo, we used two different models: (i) endometrial samples from fertile patients undergoing natural cycles and (ii) HRT in mock cycles from participants in the ovum donation programme. To study the embryonic regulation of endometrial chemokine receptors, we have used an in‐vitro model of the apposition phase of human implantation, with endometrial epithelial cells co‐cultured with or without human blastocysts. Triploid embryos were analysed in order to localize the chemokine receptors on the human blastocyst. Endometrial samples Human endometrial tissues were obtained throughout the menstrual cycle from normal fertile women aged 23–39 years (n = 15). A small portion of each specimen was histologically examined and dated according to Noyes et al. (1950). Endometrial biopsies were distributed into five groups: group I, early–mid‐proliferative (days 1–8); group II, late proliferate phase (days 9–14); group III, early secretory phase (days 15–18); group IV, mid‐secretory phase (days 19–22); and group V, late secretory phase (days 23–28). Endometrial expression (real time PCR) and localization (immunohistochemistry) of the four receptors were analysed using these samples. Hormonal regulation of these receptors was also investigated at the protein level in endometrial biopsies from ovum donation recipients undergoing HRT (Meseguer et al., 2001; Martin et al., 2000). Serum and endometrial samples were obtained in mock cycles from five patients (23–29 years) receiving ovum donation and HRT, as previously described (Caballero‐Campo et al., 2002). Briefly, serum samples (S) and uterine biopsies (B) were taken from each patient at day 13 (S1, B1), 18 (S2, B2) and 21 (S3, B3). Therefore, at the time when serum and biopsies were collected, patients were treated for 3 days with 6 mg/day of estradiol valerate (EV) and for 6 days with 6 mg/day of EV plus 800 mg/day of progesterone. Biopsies were dated histologically according to Noyes et al. (1950). E2 was measured in the serum by immunoenzymatic assay (MEIA, Imx; Abbot Scientific, Madrid, Spain). Progesterone was measured by radioimmunoassay (biomerieux, Charbonnieres Les Bains, France). RNA isolation RNA extraction was performed according to Chomczynski and Sacchi (1987), with minor modifications using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Briefly, each tissue was weighed and 500 µl Trizol reagent was added for every 100 mg. Total RNA was separated from DNA and proteins by adding chloroform, and was precipitated with isopropanol (overnight, –20°C). The precipitate was washed twice in ethanol, air‐dried and resuspended in 75% diethylpyrocarbonate (DEPC)‐treated water. RNA was quantified by spectrophotometry on a SmartSpec 3000 spectrophotometer (Biorad, Barcelona, Spain). A260/A280 ratios for all samples used varied between 1.6 and 1.9. Reverse transcription RT was carried out using a Advantage RT‐for‐PCR kit (Clontech, Palo Alto, CA, USA). The mastermix per sample was prepared as follows: 5×reaction buffer, dNTP mix (10 mmol/l each), recombinant RNase inhibitor and MMLV (Moloney‐Murine Leukemia Virus) reverse transcriptase. One µg of each sample was diluted in DEPC‐treated water with oligo (dT)18. The mixture was then heated for 2 min at 70°C and kept on ice until the mastermix was added. For each RT, a blank was prepared using all the reagents except the RNA sample, for which an equivalent volume of DEPC‐treated water was substituted. The RT blank was used to prepare the PCR blank (below). Once all the components were mixed, the samples were incubated for 1 h at 42°C, and heated for 5 min at 94°C to prevent cDNA synthesis and destroy DNAse activity. The product was diluted with DEPC‐treated water to a final volume of 100 µl and stored at –20°C until PCR analysis was performed. Real time fluorescent PCR The LightCycler (Roche Diagnostics, GmbH Mannheim, Germany) Instrument was used to determine the relative quantification of gene expression of CXCR1, CXCR4, CCR2B and CCR5 receptors; GAPDH was chosen as the housekeeping gene control. The SYBR® Green I double‐stranded DNA binding dye (Roche Diagnostics, GmbH Mannheim, Germany) was chosen for these assays. Oligonucleotides were designed using Primer Express® software (AB, Foster City, CA, USA). Oligonucleotide sequences designed for the amplification of the different genes are shown in Table I. All real time PCR assays were run using SYBR® Green PCR Master Mix and the universal thermal cycling parameters indicated by the manufacturer (60°C annealing temperature for all primers). Relative quantification was carried out by employing the standard curve method using the SYBR® Green I dye. Data were presented as the relative average value for each gene investigated and then normalized with the average value of the housekeeping gene obtained on different days of each designated phase of the menstrual cycle. No direct comparison among different genes could be made as the standard was composed of different cDNA species, each at different concentrations. Quantification data were analysed at the beginning of the exponential phase (cycles 30–35) with the LightCycler analysis software 3.5 version. Background fluorescence was removed by setting a noise band. Duplicates showing >5% variation were discarded. To validate a real time PCR, standard curves with r > 0.95 and slope values between 3.1 and 3.4 were required. To explore whether other non‐expected products were also amplified, PCR products after 40 cycles were subjected to a subsequent agarose 2% gel electrophoresis with ethidium bromide to confirm amplification specificity (data not shown). Immunohistochemistry of human endometrium Formalin‐fixed and paraffin‐embedded endometrial biopsies were sectioned and mounted on glass slides coated with VectabondTM (Vector Laboratories, Burlingame, CA, USA). Twelve serial sections (6 µm) from each sample were prepared and the first and last sections stained with haematoxylin–eosin. After deparaffinization and rehydratation, sections were washed three times for 5 min with phosphate‐buffered saline (PBS). Non‐specific binding was blocked with non‐fat milk (50 mg/ml in PBS). Sections were then washed three times with PBS/0.05% Tween 20, pH 7.4 (PBS‐T) and incubated for 1.5 h at 37°C with the following specific antibodies: primary monoclonal antibody (Ab) against human CXCR4 and CCR5 from Pharmingen (San Diego, CA, USA), primary rabbit polyclonal Ab for human CXCR1 and CCR2B from Santa Cruz Biotechnology (Santa Cruz, CA, USA), each at 10 mg/ml. Negative controls were incubated with PBS with 1% bovine serum albumin (BSA) and 0.1% Tween 20. After being washed four times with PBS‐T, sections were incubated for 1.5 h at 37°C with the secondary antibody, fluorescein isothiocyanate (FITC)‐conjugated goat anti‐mouse IgG whole molecule (Sigma, St Louis, MO, USA). Afterwards, sections were washed four times with PBS‐T and gently washed with distilled water. Sections were mounted in aqueous mounting medium (Dako, Barcelona, Spain) and immunolocalization of HRT endometrial chemokines was visualized and photographed using an Olympus 35 mm camera attached to a fluorescence microscope (Nikon, Japan). Natural cycle endometrium photomicrographs were obtained using a Nikon digital camera coolpix 995. Immunostaining intensity was evaluated in at least three different specimens and interpreted as absent (0), weak (+), moderate (++) or intense (+++) by three independent observers. Positive controls were tonsil sections and negative controls were endometrial samples incubated in PBS with 1% BSA and 0.1% Tween 20 without primary antibody. Immunocytochemistry of human blastocysts For the immunostaining of human blastocysts, we employed an avidin–peroxidase staining method using primary monoclonal antibody (Ab) against human CXCR4 and CCR5 (Pharmingen), primary rabbit polyclonal Ab for human CXCR1 and CCR2B (Santa Cruz) each at 10 mg/ml. Embryos were previously fixed for 30 min at 4°C with freshly prepared 2% paraformaldehyde in PBS micro‐drops covered by oil (Simón et al., 1994). After fixation, blastocysts were treated with 0.2% Triton X‐100 (Sigma) in PBS for 10 min at 4°C to permeabilize the fixed cells and thereby facilitate the access of the antibody. Biotin‐labelled goat secondary antibodies (Sigma) were incubated for 30 min at 37°C. Incubation for 4–6 min with a working substrate solution of avidin–peroxidase (Sigma), diluted 1/40, was carried out. Blastocysts were photographed using an Olympus 35 mm camera attached to an inverted microscope (Nikon, Japan). Of a total of 24 blastocysts used, six human triploid blastocysts were analysed for each molecule and three were used as negative controls. In‐vitro model for apposition Based on our previous study, we have developed an in‐vitro model to observe interactions between the human embryo and endometrial epithelial cells (EEC). This model has led to a clinical programme in which embryos are co‐cultured with EEC until blastocyst stage and then transferred back to the mother (Simón et al., 1999). Embryos were obtained after ovulation induction and insemination, employing routine IVF or ICSI procedures. Endometrial biopsies from patients were minced into small pieces (<1 mm) and digested with a mild collagenase solution (0.1%) for 1 h at 37°C. The endometrial epithelium was isolated and purified as previously described (Simón et al., 1993). EEC were cultured to confluence in a steroid‐depleted medium containing a 3:1 mixture of DMEM (Sigma), MCDB‐105 (Sigma) and 5 mg insulin (Sigma) and supplemented with 10% charcoal–dextran‐treated bovine fetal serum (Hyclone, Logan, UT, USA). The homogeneity and purity of EEC cultures were assessed using immunohistochemical markers (Simón et al., 1994) and morphological characteristics (scanning electron microscopy) (Simón et al., 1999). After confluence, the culture media were replaced by a 1:1 mixture of IVF:S2 medium (Scandinavian IVF Science AB, Gothenburg, Sweden). Forty‐eight hours after insemination of oocytes, each 2–4‐cell human embryo was transferred to an EEC monolayer. When embryos reached the 8‐cell stage, the medium was replaced by S2 medium (Scandinavian IVF) until blastocyst stage. Embryonic development was checked daily and the medium changed every 24 h. On day 6 of co‐culture, blastocysts were transferred to the recipient using a Frydman catheter. EEC cultured alone under the same conditions were used as controls. Individual human embryos were co‐cultured with EEC for 5 days (from day 2 to day 6 of embryonic development). After embryo transfer, EEC wells were divided into two groups: EEC with embryos which had reached the blastocyst stage and EEC without embryos (controls). Confocal analysis Immunocytochemistry was performed following the same protocol as that previously described with the same antibodies but using EEC from our in‐vitro apposition model. Confocal analysis was performed with an NRC 1024 instrument (Bio‐Rad, Hempstead, UK). The excitation line used was 488 (FITC). The filter used was HQ515/10 (FITC). Transmitted light images were acquired for every field. Results mRNA expression and protein localization of CXCR1, CXCR4, CCR2B and CCR5 receptors throughout the menstrual cycle The mRNA expression of the four chemokine receptors was analysed throughout the menstrual cycle in three separate experiments each with five patients using real time PCR. Data is presented as a relative average value for each gene investigated and normalized with the average value of the housekeeping gene obtained on different days of each phase of the menstrual cycle in three different experiments. The lowest value for each receptor in each phase of the menstrual cycle was considered as basal expression and quantified as 1. The intensity of expression for a given receptor in a specific phase of the menstrual cycle is expressed as fold increase compared with the basal expression. CXCR1 (Figure 1A), CCR2B (Figure 1C) and CCR5 (Figure 1D) mRNA are highly regulated throughout the menstrual cycle with maximal expression in the luteal phase. The mRNA expression of these three receptors suggested a progesterone‐dependent pattern starting low in the early secretory phase and continuing through the mid‐secretory phase and peaking in the late secretory phase (612‐fold increase for CCR5, 419‐fold increase for CXCR1 and 657‐fold increase for CCR2b). Unlike the other receptors, CXCR4 receptor mRNA (Figure 1B) presented a specific increase in the mid‐luteal phase compared to the early and late luteal phases (17.7‐fold increase in mid‐luteal phase versus 1‐ and 6.8‐fold increase in early and late luteal phases respectively). This illustrates that this receptor is specifically up‐regulated during the implantation window and its lowest expression is noted in the early secretory phase. We have also studied the four receptors at the protein level, throughout the natural menstrual cycle (see Table III for semi‐quantitative analysis). CXCR1 receptor shows higher staining when compared to the other receptors. CXCR1 peaks in the early and mid‐secretory phases (groups III and IV) (Figure 2A and B). CXCR4 receptor also displays high staining across the whole cycle as in the case of CXCR1 receptor, it shows maximal expression in the mid‐secretory phase (group IV) (Figure 2C), confirming the real time RT–PCR experiments. CCR receptors in general show lower expression in the endometrium than CXC receptors. CCR2B staining appears in the late proliferate phase (group II) and reaches a moderate signal in the early secretory phase (group III), maintaining a low‐to‐moderate staining in the rest of the cycle (Figure 2D and E). CCR5 receptor signal starts in the late proliferate phase (group II) and remains low to moderate in different compartments (Figure 2F and G) (see Table II for localization and semi‐quantitative analysis). Hormonal regulation of immunoreactive CXCR1, CXCR4 CCR5 and CCR2B in human endometrium Peripheral E2 and progesterone levels were determined at the time of obtaining endometrial biopsies (non‐receptive, pre‐receptive and receptive phases) and were consistent with the physiological hormone levels expected. At day 13 (non‐receptive phase), E2 levels were 333.2 ± 92.9 pg/ml and progesterone was undetectable. At day 18 (pre‐receptive phase), E2 and progesterone levels were 331.6 ± 39.1 pg/ml and 9.5 ± 3.8 ng/ml respectively. At day 21 (receptive phase) E2 and progesterone levels were 362.6 ± 78.5 pg/ml and 10.5 ± 6.1 ng/ml respectively. These hormonal levels stimulated endometrial differentiation as assessed by classical histological criteria (Noyes et al., 1950). On day 13 (n = 3), when patients were treated solely with estradiol, a very weak staining for CCR2B, CCR5 and CXCR4 was localized in the luminal and glandular epithelium and endothelial cells (Figure 3A–C). During the pre‐receptive and receptive periods (days 18 and 21 respectively), an increase in staining intensity was noted for CXCR1 receptor at the glandular compartment (Figure 3D, G). A slight signal was observed in stromal cells. CCR5 receptor was also immunolocalized, mainly at the luminal epithelium but also at the stromal and perivascular cells (Figure 3E, H), showing a slight increase compared with the non‐receptive phase. CCR2B receptor shows a moderate increase in staining on day 18 and 21 in the luminal epithelium, while no staining was observed in endothelial cells or stroma (Figure 3F, L). CXCR4 receptor shows the same staining as CCR5, mainly expressed in epithelium on days 18 and 21 (data not shown). Endothelial and stromal cells were also positive. We used human tonsil sections (data not shown) as positive controls and negative controls were performed by deletion of the first antibody. Immunostaining intensity was evaluated in at least three different specimens and interpreted as absent (0), weak (+), moderate (++) or intense (+++) by three independent observers (Table II). Immunolocalization of CXCR1, CXCR4, CCR2B and CCR5 in human blastocysts We have detected immunoreactive CCR2B (Figure 4A, B) and CCR5 receptors (Figure 4E, F) in the human blastocyst. CCR2B staining is localized mainly at the inner cell mass, whereas CCR5 staining can be visualized across the trophectoderm. In all cases (n = 3) CCR5 staining was more intense than that of CCR2B receptor and the pellucide zonae was not stained in any case. Immunoreactive CXCR4 and CXCR1 were not detected in human blastocysts when the same technique was used (Figure 4C, D, G, H). Embryonic regulation of chemokine receptors in human EEC The embryonic impact on immunolocalization and polarization of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in cultured EEC was investigated using our apposition model for human implantation. Chemokine receptors CXCR1 (Figure 5A), CXCR4 (Figure 5D) and CCR5 (Figure 5G) produced a barely detectable staining in few cells at the EEC monolayer when the blastocyst was absent. However, when a human blastocyst was present there was an increase in the number of stained cells for CXCR1 (Figure 5B), CXCR4 (Figure 5E), and CCR5 (Figure 5H) and polarization of these receptors in one of the cell poles of the endometrial epithelium (Figure 5I). Immunolocalization and polarization changes in CCR2B receptor were not present in the EEC monolayer and this receptor was not up‐regulated by the presence of the human blastocyst. Discussion Chemokines were originally defined as host defence proteins and it is now clear that their repertoire of functions extends well beyond this role. Chemokines can mediate or inhibit angiogenesis and may be important targets for leukocyte adhesion (Payne and Cornelius, 2002). In this study, we present data that highlight the involvement of chemokine receptors at the human blastocyst–endometrial interface and which expand the biological relevance of chemokines in the embryonic implantation process. In this paper, we have detected that the protein staining for CXCR1, CCR2B and CCR5 receptors in epithelium and stroma decreases in the late secretory phase whereas mRNA expression for these receptors analysed by real time RT–PCR is maximal in this phase of the menstrual cycle. Therefore, a possible explanation to reconcile these findings is the high number of white blood cells which express chemokine receptors recruited during this period in the human endometrium. Remarkably, CXCR4 mRNA levels present a specific though modest up‐regulation (18‐fold) during the implantation window versus the early implantation phase. These findings echoed the semi‐quantitative immunohistochemical data and were corroborated using an in‐vivo model of hormonal regulation. Our in‐vivo model demonstrated that these receptors were present mainly in the endometrial epithelium but they were also present in the stromal and endothelial cells. The selective hormonal‐dependent up‐regulation of these receptors suggests that CXCR1, CCR2B and CCR5 display specific function(s) in the pre‐menstrual endometrium, whereas CXCR4 seems to be implicated in endometrial receptivity. A possible limitation of the in‐vivo model of hormonal regulation used in this study is that three consecutive endometrial biopsies (day 15, 18 and 21) were obtained in the same patient and therefore a non‐specific imflammatory reaction in the last biopsy may be present. Nevertheless, this possibility seems unlikely because the pattern of chemokine receptors expression reported in this model was reproduced when single biopsies were obtained in natural cycles from different patients. The chemotactic response is fundamental in leukocyte physiology and implies recognition of an external gradient of chemokines (Sanchez‐Madrid and Del Pozo, 1999). Leukocyte trafficking through the endothelium is, in some aspects, comparable to the implantation process, where the contact and invasion of the endometrium with and by the blastocyst is similar to the rolling, attachment and crossing process of the lymphocytes through the endothelium. It has been demonstrated that CCR2B and CCR5 receptors are polarized at the leading edge of migrating lymphocytes (Nieto et al., 1997) and that leukocyte adhesion through integrins is required to induce cell polarization (Del Pozo et al., 1997) and redistribution of chemokine receptors (Del Pozo et al., 1995). These data further suggest that chemokines may act in combination with adhesion molecules to steer leukocyte traffic to tissues (Butcher, 1991; Moser and Loetcher, 2001). Leukocyte migration is a complex phenomenon where chemokines and their receptors are important players. The patching of chemokine receptors to the leading edge of a cell implies two remarkable consequences: (i) the functional specialization of this cell’s domain in signal transduction; and (ii) the establishment of an endogenous polarity in the cell, which may be crucial for chemotaxis and other immune responses involving chemokines (Nieto et al., 1998). In our study, when a human blastocyst was present, there was an increase in the number of endometrial epithelial cells stained for CXCR1, CXCR4 and CCR5, and polarization of these receptors in one of the cell poles became evident. These chemokines, secreted locally by the endometrium in the implantation window or by the human blastocyst in the apposition phase (Caballero‐Campo et al., 2002), may act as a signal for receptor polarization/dimerization, thereby acting as a sensor mechanism for increasing local cell responsiveness in the activation of endometrial adhesion molecules. Recently, it has been shown that the binding of a chemokine to its receptors induces homo or heterodimerization of the latter (Rodriguez‐Frade et al., 2001). This effect has been noted on CCR2B and CCR5 receptors (Mellado et al., 2001). The simultaneous presence of RANTES and MCP‐1 induces the heterodimeric receptor complex CCR2B–CCR5, which has unique features, including the reduction of the threshold concentration of chemokine required to induce a response. This might have functional relevance in the cell. The formation of these complexes in lymphocytes activates cell adhesion in contrast to the cell migration triggered by homodimers. We have demonstrated that immunoreactive CCR2B and CCR5 receptors are localized at the human blastocyst. Could the blastocyst form these complexes in response to endometrial chemokines and therefore trigger a similar response? An array of different chemokines is expressed (Kao et al., 2002) and produced in the human endometrium at the time of implantation (Caballero‐Campo et al., 2002; Kayisli et al., 2002). These chemokines could be immobilized by low‐affinity binding to heparin‐bearing proteoglycans on the vascular endothelial or epithelial surface, thereby facilitating the oligomerization of chemokines (Hoogewerf et al., 1997). This effect permits effective presentation of chemokines to cells or groups of cells, which are then able to respond to the chemokines’ presence. In this way, variations in the availability of these chemokines would affect the ability of a ligand to trigger homo‐ or heterodimerization. At low concentrations of chemokines, receptor heterodimerization is favoured and cell adhesion is triggered (Mellado et al., 2001). Caballero‐Campo et al. (2002) have investigated the hormonal and embryonic regulation of IL‐8, RANTES and MCP‐1 in the endometrium. IL‐8 and MCP‐1 were present in the glandular and luminal epithelium, and RANTES was mainly localized on stromal cells. IL‐8 and MCP‐1 were up‐regulated in the presence of E2 and progesterone. IL‐8 mRNA expression and protein were up‐regulated in the presence of the human blastocyst using our co‐culture model. In this paper, we have detected that some chemokine receptors (CXCR1, CXCR4 and CCR5) are present and regulated hormonally in EEC. They display the typical polarization present in lymphocytes, creating a polarized cell capable of responding to different chemokines. We have also detected two of these receptors, CCR5 and CCR2B, in the human blastocyst. White blood cells are not an organized group of cells such as the cells which form the human blastocyst, but the polarization of the latter at the time of adhesion is a constant process that is intriguing and deserves special consideration. The blastocyst is guided to the implantation site in a polarized manner that is species specific, driven by unknown mechanisms and crosses the epithelial barrier penetrating into the stroma. Although the chemokine receptor expression in the blastocyst is homogeneous over the surface of the blastocyst, including the trophectoderm and the inner cell mass, these receptors may polarize if they encounter an adequate gradient of chemokines secreted by the endometrium, (epithelium, stroma or white blood cells) either free or bound to proteoglycans of the epithelial surface. The blastocyst could respond at this point with chemokine polarization and/or homo/heterodimerization. The possible heterodimerization of receptors, in our case CCR2B and CCR5 receptors, might develop in the human blastocyst an expression pattern of different integrins and adhesion molecules such as β1, β5, α6 or E‐cadherin. These have been discovered in the blastocyst at the time of implantation and are necessary for the first steps of embryo adhesion (Bloor et al., 2002). Further studies of chemokine polarization and blastocyst chemotaxis are needed to confirm our hypothesis. Acknowledgements We would like to thank all of the IVF group of IVI, especially Amparo Mercader, for the collection of samples. We also thank Marcos Meseguer, Nicolas Garrido and Pedro Caballero‐Campo for their support and Dr Alvarez Barrientos for his help with the confocal analysis. This study was financed by grants MT1999‐B24364784, FISS 00/0643 and SAF2001.2948 from the MCYT of the Spanish Government. View largeDownload slide Figure 1. Quantitative mRNA analysis of chemokine receptors in human endometrium throughout the menstrual cycle by real time fluorescent RT–PCR. (A) CXCR1 receptor expression. (B) CXCR4 receptor. (C) CCR2B receptor. (D) CCR5 receptor. Endometrial biopsies were distributed in five groups, each corresponding to a different phase: group I; early–mid‐proliferative (days 1–8), group II; late proliferative (days 9–14), group III; early secretory (days 15–18), group IV; mid‐secretory (days 19–22), and group V; late secretory (days 23–28). Data are presented as mRNA expression fold increase compared with the group of basal expression for each receptor. Three experiments were performed in a total of 15 endometrial samples to obtain the mean shown in the graphs. CXCR1, CCR2B and CCR5 show a typical pattern of decidualization increasing their expression in the late proliferative phase (group V) whereas CXCR4 shows an implantation expression pattern, increasing in the mid‐secretory phase (receptive phase). Error bars show SD. View largeDownload slide Figure 1. Quantitative mRNA analysis of chemokine receptors in human endometrium throughout the menstrual cycle by real time fluorescent RT–PCR. (A) CXCR1 receptor expression. (B) CXCR4 receptor. (C) CCR2B receptor. (D) CCR5 receptor. Endometrial biopsies were distributed in five groups, each corresponding to a different phase: group I; early–mid‐proliferative (days 1–8), group II; late proliferative (days 9–14), group III; early secretory (days 15–18), group IV; mid‐secretory (days 19–22), and group V; late secretory (days 23–28). Data are presented as mRNA expression fold increase compared with the group of basal expression for each receptor. Three experiments were performed in a total of 15 endometrial samples to obtain the mean shown in the graphs. CXCR1, CCR2B and CCR5 show a typical pattern of decidualization increasing their expression in the late proliferative phase (group V) whereas CXCR4 shows an implantation expression pattern, increasing in the mid‐secretory phase (receptive phase). Error bars show SD. View largeDownload slide Figure 2. Immunolocalization of CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle. (A) Group I, CXCR1 receptor shows moderate staining in glandular epithelium (arrow). (B) Group II, CXCR1 receptor. Arrows indicate strong staining in luminal epithelium and stromal cells. (C) Group II CXCR4 receptor. Moderate‐to‐strong staining is observed in luminal, glandular and stromal cells. (D) Group II, CCR2 receptor. Faint staining in luminal epithelium and some stromal cells. (E) Group III, CCR2 receptor. Arrows show moderate staining in luminal epithelium. Glandular epithelium also stained. (F) Group III CCR5 receptor. Faint‐to‐moderate staining in the three compartments. (G) Group V CCR5 receptor. Gland showing staining in the lumen. (H) negative control. (I) Positive control ThP1 cells. The semi‐quantitative analysis of the data is presented in Table III. View largeDownload slide Figure 2. Immunolocalization of CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle. (A) Group I, CXCR1 receptor shows moderate staining in glandular epithelium (arrow). (B) Group II, CXCR1 receptor. Arrows indicate strong staining in luminal epithelium and stromal cells. (C) Group II CXCR4 receptor. Moderate‐to‐strong staining is observed in luminal, glandular and stromal cells. (D) Group II, CCR2 receptor. Faint staining in luminal epithelium and some stromal cells. (E) Group III, CCR2 receptor. Arrows show moderate staining in luminal epithelium. Glandular epithelium also stained. (F) Group III CCR5 receptor. Faint‐to‐moderate staining in the three compartments. (G) Group V CCR5 receptor. Gland showing staining in the lumen. (H) negative control. (I) Positive control ThP1 cells. The semi‐quantitative analysis of the data is presented in Table III. View largeDownload slide Figure 3. Immunolocalization and hormonal regulation of CXCR1, CCR5 and CCR2B in human endometrium. (A, D, G) CXCR1 receptor. (B, E, H) CCR5 receptor. (C, F, L) CCR2B receptor. Endometrial samples from hormonal replacement therapy cycles in ovum donation recipients were obtained during non‐receptive (day 13; A, B, C), pre‐receptive (day 18; D, E, F) and receptive phases (day 21; G, H, I). The semi‐quantitative analysis of the data is presented in Table II. View largeDownload slide Figure 3. Immunolocalization and hormonal regulation of CXCR1, CCR5 and CCR2B in human endometrium. (A, D, G) CXCR1 receptor. (B, E, H) CCR5 receptor. (C, F, L) CCR2B receptor. Endometrial samples from hormonal replacement therapy cycles in ovum donation recipients were obtained during non‐receptive (day 13; A, B, C), pre‐receptive (day 18; D, E, F) and receptive phases (day 21; G, H, I). The semi‐quantitative analysis of the data is presented in Table II. View largeDownload slide Figure 4. Immunolocalization of chemokine receptors in human blastocysts. (A) Negative control for CCR2B receptor. (B) Arrow indicates the staining for CCR2B at the inner cell mass. (C) CXCR4 negative control. (D) CXCR4 staining in the human blastocyst (E). CCR5 negative control. (F) Arrows indicate the positive staining for CCR5 at the trophectoderm. (G) CXCR1 negative control. (H) A human blastocyst stained for CXCR1 receptor. The staining for CCR2B is localized mainly at the inner cell mass whereas CCR5 staining can be visualized across the trophectoderm. CXCR1 and CXCR4 staining was not detected in the human blastocyst. View largeDownload slide Figure 4. Immunolocalization of chemokine receptors in human blastocysts. (A) Negative control for CCR2B receptor. (B) Arrow indicates the staining for CCR2B at the inner cell mass. (C) CXCR4 negative control. (D) CXCR4 staining in the human blastocyst (E). CCR5 negative control. (F) Arrows indicate the positive staining for CCR5 at the trophectoderm. (G) CXCR1 negative control. (H) A human blastocyst stained for CXCR1 receptor. The staining for CCR2B is localized mainly at the inner cell mass whereas CCR5 staining can be visualized across the trophectoderm. CXCR1 and CXCR4 staining was not detected in the human blastocyst. View largeDownload slide Figure 5. Embryonic effect on immunolocalization and polarization of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in cultured endothelial endometrial epithelial cells (EEC). (A, B) CXCR1 receptor staining in non‐polarized EEC cultured without and with an individual blastocyst respectively. (C) Phase contrast of EEC monolayer. (D, E) CXCR4 receptor staining in EEC without or with blastocyst, respectively. (F) Negative control with deletion of the first antibody. CCR5 staining in EEC cultured without (G) or with an individual blastocyst (H). (I) Detail of a single non‐polarized epithelial cell expressing CXCR1 receptor (see arrows). Inner square: contrast phase of the EEC monolayer with the stained cell. CXCR1, CXCR4 and CCR5 staining were barely detected only in a few cells at the EEC monolayer without the blastocyst. Staining for CCR2B was not present in cultured EEC in the presence or absence of a human blastocyst. View largeDownload slide Figure 5. Embryonic effect on immunolocalization and polarization of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in cultured endothelial endometrial epithelial cells (EEC). (A, B) CXCR1 receptor staining in non‐polarized EEC cultured without and with an individual blastocyst respectively. (C) Phase contrast of EEC monolayer. (D, E) CXCR4 receptor staining in EEC without or with blastocyst, respectively. (F) Negative control with deletion of the first antibody. CCR5 staining in EEC cultured without (G) or with an individual blastocyst (H). (I) Detail of a single non‐polarized epithelial cell expressing CXCR1 receptor (see arrows). Inner square: contrast phase of the EEC monolayer with the stained cell. CXCR1, CXCR4 and CCR5 staining were barely detected only in a few cells at the EEC monolayer without the blastocyst. Staining for CCR2B was not present in cultured EEC in the presence or absence of a human blastocyst. Table I. Oligonucleotide primers with predicted respective PCR product sizes.     View Large Table II. Semi‐quantitative analysis of immunohistochemistry in human endometrium     Summary of the immunohistochemical experiments identifying CXCR1, CXCR4, CCR5 and CCR2 in different compartments of the human endometrium during the non‐receptive (day 13), pre‐receptive (day 18) and receptive phases (day 21) in hormone replacement therapy cycles. Designations of 0 (negative), + (weakly positive) to +++ (intensely positive) indicate the relative intensities of the signals averaged for at least three different samples. Variability between readers is indicated with a slash mark. Epit. = epithelial cells; St. = stromal cells; End. = endothelial cells. View Large Table III. Semi‐quantitative analysis of immunohistochemistry results of chemokine receptors CXCR1, CXCR4, CCR2B and CCR5 in different compartments of the human endometrium throughout the menstrual cycle     Designations of – (negative), + (weakly positive), ++ (intensely positive) and +++ (strongly positive) indicate the relative intensities of the signals averaged for three different blind observers. LE = luminal epithelium; GE = glandular epithelium; SC = stromal cells. View Large References Akoum, A., Lemay, A., McColl, S., Turcot‐Lemay, L. and Maheux, R. ( 1996) Elevated concentration and biologic activity of monocyte chemotactic protein‐1 in the fluid of patients with endometriosis. Fertil. Steril. , 66, 17–23. Google Scholar Arici, A., Head, J.R., MacDonald, P.C. et al. ( 1993) Regulation of interleukin‐8 gene expression in human endometrial cells in culture. Mol. Cell. Endocrinol. , 94, 195–204. Google Scholar Arici, A., McDonal, P.C. and Casey, M.L. ( 1995) Regulation of monocyte chemotactic protein‐1 gene expression in human endometrial cells in cultures. Mol. Cell. Endocrinol. , 107, 189–197. Google Scholar Arici, A., Seli, E., Senturk, L.M., Gutierrez, L.S., Oral, E. and Taylor, H.S. ( 1998) Interleukin‐8 in the human endometrium. Clin. Endocrinol. Metab. , 83, 1783–1787. Google Scholar Baggiolini, M. and Dahinden, A. ( 1994) CC chemokines in allergic inflammation. Inmmunol. Today , 15, 127–133. Google Scholar Barclay, C.G., Brennard, J.E., Kelly, R.W. and Calder, A.A. ( 1993) Interleukin‐8 production by human cervix. Am. J. Obstet. Gynecol. , 169, 625–632. Google Scholar Bloor, D.J., Metcalfe, A.D., Rutherford, A., Brison, D.R. and Kimber, S.J. ( 2002) Expression of cell adhesion molecules during human preimplantation embryo development. Mol. Hum. Reprod. , 8, 237–245. Google Scholar Bokoch, G.M. ( 1995) Chemoattractant signalling and leukocyte activation. Blood , 86, 1649–1657. Google Scholar Butcher, E.C. ( 1991) Leukocyte–endothelial cell recognition: three (or more) steps to specificity and diversity. Cell , 67, 1033–1036. Google Scholar Caballero‐Campo, P., Dominguez, F., Coloma, J., Meseguer, M., Remohi, J., Pellicer, A. and Simon, C. ( 2002) Hormonal and embryonic regulation of chemokines IL‐8, MCP‐1 and RANTES in human endometrium during the window of implantation. Mol. Hum. Reprod. , 8, 375–384. Google Scholar Campbell, J.J., Hedrick, J. Zlotnik, A., Siani, M.A., Thompson, D.A. and Butcher, E.C. ( 1998) Chemokines and the arrest of lymphocytes rolling under flow conditions. Science , 279, 381–384. Google Scholar Chantakru, S., Kuziel, W.A., Maeda, N. and Croy, B.A. ( 2001) A study on the density and distribution of uterine natural killer cells at mid pregnancy in mice genetically‐ablated for CCR2B, CCR 5 and the CCR5 receptor ligand, MIP‐1 alpha. J. Reprod. Immunol. , 49, 33–47. Google Scholar Chomczynski, P. and Sacchi, N. ( 1987) Single step method of RNA isolation by acid guanidinium thiocyanate phenol‐chloroform extraction. Anal. Biochem. , 162, 156–159. Google Scholar Cocchi, F., DeVico, A.L., Garzino‐Demo, A., Arya, S.K., Gallo, R.C. and Lusso, P. ( 1995) Identification of RANTES, MIP‐1 alpha, and MIP‐1 beta as the major HIV‐suppressive factors produced by CD8+ T cells. Science , 270, 1811–1815. Google Scholar De los Santos, M.J., Mercader, A., Francés, A., Portoles, E., Remohi, J., Pellicer, A. and Simon, C. ( 1996) Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin‐1 system during embryonic development. Biol. Reprod. , 54, 563–574. Google Scholar Del Pozo, M.A., Sánchez‐Mateos, P., Nieto, M. and Sanchez‐Madrid, F. ( 1995) Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway. J. Cell Biol. , 131, 495–508 Google Scholar Del Pozo, M.A., Cabañas, C., Montoya, Ager, A., Sanchez‐Mateos, P. and Sanchez‐Madrid, F. ( 1997) ICAM’s redistributed by chemokines to cellular uropods as a mechanism for recruitment of T‐lymphocytes. J. Cell Biol. , 137, 1–16. Google Scholar Dudley, D.J., Trantman, M.S. and Mitchel, M.D. ( 1993) Inflammatory mediators regulate interleukin‐8 production by cultured gestational tissues: evidence for a cytokine network at the chorio‐decidual interface. Clin. Endocrinol. Metab. , 76, 404–410. Google Scholar Galan, A., O’Connor, J.E., Valbuena, D., Herrer, R., Remohi, J., Pampfer, S., Pellicer, A. and Simon, C. ( 2000) The human blastocyst regulates endommtrial epithelial apoptosis in embryonic adhesion. Biol. Reprod. , 63, 430–439. Google Scholar Glasser, S.R., Mulholland, J. and Mani, S.K. ( 1991) Blastocyst endometrial relationships: reciprocal interaccions between uterine epithelial and stromal cells and blastocysts. Trophoblast Res. , 5, 225–280. Google Scholar Hoogewerf, A.J., Kuschert, G.S.V., Proudfoot, A.E., Chung, C.W., Cooke, R.M., Hubbard, R.E., Wells, T.N. and Sanderson, P.N. ( 1997) Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry , 36, 13570–13578. Google Scholar Hornung, D., Ryan, I.P., Chao, V.A., Vigne, J.L., Schriock, E.D. and Taylor, R.N. ( 1997) Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. Clin. Endocrinol. , 82, 1621–1628. Google Scholar Kao, L.C., Tulac, S., Lobo, S., Imani, B., Yang, J.P., Germeyer, A., Osteen, K., Taylor, R.N., Lessey, B.A. and Giudice, L.C. ( 2002) Global gene profiling in human endometrium during the window of implantation. Endocrinology , 143, 2119–2138. Google Scholar Kayisli, U.A., Mahutte, N.G. and Arici, A. ( 2002) Uterine chemokines in reproductive physiology and pathology. Am. J. Reprod. Immunol. , 47, 213–221. Google Scholar Larsen, C., Anderson, A., Aella, E., Oppenheim, J.J. and Matsushima, K. ( 1989) The neutrophil‐activating protein (NAP‐1) is also chemotactic for T lymphocytes. Science , 243, 1469–1466. Google Scholar Loetscher, P., Seitz, M., Clark‐Lewis, I., Baggiolini, M. and Moser, B. ( 1994) Monocyte chemotactic proteins MCP‐1, MCP‐2, and MCP‐3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. , 8, 1055. Google Scholar Loetscher, P., Seitz, M., Clark‐Lewis, I., Baggiolini, M. and Moser, B. ( 1996) Activation of NK cells by CC chemokines. Chemotaxis, Ca2+ mobilization, and enzyme release. J. Immunol. , 156, 322. Google Scholar Martin, J.C., Jasper, M.J., Valbuena, D., Meseguer, M., Remohi, J., Pellicer, A. and Simon, C. ( 2000) Increased adhesiveness in cultured endometrial‐derived cells is related to the absence of moesin expression. Biol. Reprod. , 63, 1370–1376. Google Scholar Mellado, M., Rodriguez‐Frade, J.M., Vila‐Coro, A.J., Fernandez, S., Martin de Ana, A., Jones, D.R., Toran, J.L. and Martinez‐A.C. ( 2001) Chemokine receptor homo‐ or heterodimerization activates distinct signaling pathways. EMBO J. , 20, 2497–507. Google Scholar Meseguer, M., Aplin, D., Caballero‐Campo, P., O’Connor, J.E., Martin, J.C., Remohi, J., Pellicer, A. and Simon, C. ( 2001) Human endometrial mucin MUC1 is up‐regulated by progesterone and down regulated in vitro by the human blastocyst. Biol. Reprod. , 64, 590–601. Google Scholar Moser, B. and Loetscher, P. ( 2001) Lymphocyte traffic control by chemokines. Nat. Immunol. , 2, 123–128. Google Scholar Mukaida, N., Shiroo, M. and Matsushima, K. ( 1989) Genomic structure of the human monocyte derived neutrophil chemotactic factor IL‐8. J. Immunol. , 143, 1366–1371. Google Scholar Nagasawa, T., Nakajima, T., Tachibana, K., Iizasa, H., Bleul, C.C., Yoshie, O., Matsushima, K., Yoshida, N., Springer, T.A. and Kishimoto, T. ( 1996) Molecular cloning and characterization of a murine pre‐B‐cell growth‐stimulating factor/stromal cell‐derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin. Proc. Natl Acad. Sci. USA , 93, 14726–14729. Google Scholar Nieto, M., Frade, J.M.R., Sancho, D., Mellado, M., Martinez‐A, C. and Sanchez‐Madrid, F. ( 1997) Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis. J. Exp. Med. , 186, 153–158. Google Scholar Nieto, M., Navarro, F., Perez‐Villar, J.J., del Pozo, M.A., Gonzalez‐Amaro, R., Mellado, M., Frade, J.M., Martinez‐A, C., Lopez‐Botet, M. and Sanchez‐Madrid, F. ( 1998) Roles of chemokines and receptor polarization in NK‐target cell interactions. J. Immunol. , 161, 3330–3339. Google Scholar Noyes, R.W., Hertig, A.T. and Rock, J. ( 1950) Dating the endometrial biopsy. Fertil. Steril. , 1, 3–25. Google Scholar Payne, A.S. and Cornelius, L.A. ( 2002) The role of chemokines in melanoma tumor growth and metastasis. J. Invest. Dermatol. , 118, 915–922. Google Scholar Polentarutti, N., Allavena, P., Bianchi, G., Giardina, G., Basile, A., Sozzani, S., Mantovani, A. and Introna, M. ( 1997) IL‐2‐regulated expression of the monocyte chemotactic protein‐1 receptor (CCR2B) in human NK cells: characterization of a predominant 3.4‐kilobase transcript containing CCR2B and CCR2A sequences. J. Immunol. , 158, 2689–2694. Google Scholar Reif, K. and Cantrell, D.A. ( 1998) Networking Rho family GTPases in lymphocytes. Immunity , 8, 395–401. Google Scholar Rodriguez‐Frade, J.M., Mellado, M. and Martinez‐A, C. ( 2001) Chemokine receptor dimerization: two are better than one. Trends Immunol. , 22, 612–617. Google Scholar Saito, S., Kasahara, T., Sakakura, S., Umekage, H., Harada, N. and Ichijo, M. ( 1994) Detection and localisation of interleukin‐8 mRNA and protein in human placenta and decidual tissues. J. Reprod. Immuol. , 27, 161–172. Google Scholar Sanchez‐Madrid, F. and del Pozo, M.A. ( 1999) Leukocyte polarization in cell migration and immune interactions. EMBO J. , 18, 501–511. Google Scholar Sica, A., Wang, J.M., Colotta, F., Dejana, E., Mantovani, A., Oppenheim, J.J., Larsen, C.G., Zachariae, C.O. and Matsushima, K. ( 1990) Monocyte chemotactic and activating factor gene expression induced in endothelial cells by interleukin‐1 (IL‐1) and tumor necrosis factor (TNF‐a). J. Immunol. , 144, 3034–3038. Google Scholar Simón, C., Piquette, G., Francés, A. and Polan, M.L. ( 1993) Localization of interleukin‐1 type 1 receptor and interleukin‐1b in human endometrium throughout the menstrual cycle. Clin. Endocrinol. Metab. , 77, 549–555. Google Scholar Simón, C., Piquette, G.N., Francés, A., el‐Danasouri, I., Irwin, J.C. and Polan, M.L. ( 1994) The effect of interleukin‐1b on the regulation of IL‐1 receptor type 1 messenger ribonucleic acid and protein levels in cultured human endometrial stromal and glandular cells. Clin. Endocrinol. Metab. , 78, 675–682. Google Scholar Simón, C., Gimeno, M.J., Mercader, A., O’Connor, J.E., Remohi, J., Polan, M.L. and Pellicer, A. ( 1997) Embryonic regulation of integrins b3, a4 and a1 in human endometrial epithelial cells in vitro. Clin. Endocrinol. Metab. , 82, 2607–2616. Google Scholar Simón, C., Caballero‐Campo, P., García‐Velasco, J.A. and Pellicer, A. ( 1998) Potential implications of chemokines in reproductive function: an attractive idea. J. Reprod. Immunol. , 38, 169–193. Google Scholar Simón, C., Mercader, A., García‐Velasco, J.A., Nikas, G., Moreno, C., Remohi, J. and Pellicer, A. ( 1999) Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. Clin. Endocrinol. Metab. , 84, 2638–2646. Google Scholar Ward, S.G., Bacon, K. and Westwick, J. ( 1998) Chemokines and T lymphocytes: more than a attraction. Immunity , 9, 1–11. Google Scholar Witt, D.P. and Lander, A.D. ( 1994) Differential binding of chemokines to glycosaminoglycan subpopulations. Curr. Biol. , 4, 394. Google Scholar Wuyts, A., Proost, P., Lenaerts, J.P., Ben‐Baruch, A., Van Damme, J. and Wang, J.M. ( 1998) Differential usage of the CXC chemokine receptors 1 and 2 by interleukin‐8, granulocyte chemotactic protein‐2 and epithelial‐cell‐derived neutrophil attractant‐78. Eur. J. Biochem. , 255, 67–73. Google Scholar Yoshimura, T. and Leonard, E.J. ( 1990) Secretion by human fibroblasts of monocyte chemoattractant protein‐1, the product of the gene JE. J. Immunol. , 144, 2377–2383. Google Scholar Yoshimura, T., Yuhki, N. and Moore, S. ( 1989) Human monocyte chemoattractant protein 1 (MCP‐1): full‐length cDNA cloning, expression in mitogen‐stimulated blood mononuclear leukocytes and sequence similarity to mouse competence gene JE. FEBS Lett. , 244, 487–493. Google Scholar http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Human Reproduction Oxford University Press

Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst

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Oxford University Press
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1360-9947
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10.1093/molehr/gag024
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Abstract

Abstract Chemokines are implicated in the implantation process. The aim of this study was to investigate mRNA expression and protein levels of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle, during HRT and in the human blastocyst. The regulation of chemokine receptors in the endometrial epithelium was also studied using an in‐vitro model for the apposition phase of human implantation. We found up‐regulation of endometrial CXCR1 mRNA (419‐fold increase), CCR5 mRNA (612‐fold increase) and CCR2B mRNA (657 fold‐increase) during the luteal phase peaking in the pre‐menstrual endometrium. CXCR4 mRNA levels presented a specific although modest (18‐fold increase) up‐regulation during the implantation window. These findings were corroborated at the protein level in natural and HRT cycles. Immunoreactive CCR5 and CCR2B receptors were detected in human blastocysts whereas CXCR4 and CXCR1 were not present. Chemokine receptors in cultured endometrial epithelial cells showed an up‐regulation and polarization of CXCR1, CXCR4 and CCR5 receptors when a human blastocyst was present. The specific distribution and regulation of chemokine receptors in the endometrial epithelium and the human blastocyst suggest a possible implication of these receptors in the apposition and adhesion phases of human implantation. Key words: blastocyst/chemokine receptors/endometrial epithelial cells/human endometrium/implantation Submitted on July 30, 2002; resubmitted on September 5, 2002. accepted on December 13, 2002 Introduction Chemokines (short for chemoattractant cytokines), a family of small polypeptides with a molecular weight in the range of 8–12 kDa, are specialized in the attraction of specific leukocyte subsets through their binding to cell‐surface receptors. In reproductive biology, these molecules have been implicated in crucial processes such as ovulation, menstruation, embryo implantation, parturition and pathological processes such as preterm delivery, HIV infection, endometriosis and ovarian hyperstimulation syndrome (Cocchi et al., 1995; Simón et al., 1998). Chemokines and their receptors are divided into two families based on structural and genetic considerations. All chemokines are structurally similar, having at least three β‐pleated sheets and a C‐terminal α‐helix. Most chemokines also have at least four cysteines in conserved positions (Kayisli et al., 2002). In the CXC chemokine family (α‐chemokines), the two nearest cysteines (C) to the N‐termini of family members are separated by a single (and variable) amino acid (X). Interleukin‐8 (IL‐8) (α‐chemokine) is a potent chemoattractant and activator for neutrophils (Mukeida et al., 1989) and T lymphocytes (Larsen et al., 1989). IL‐8 is produced by a variety of cell types: monocytes, fibroblasts (Dudley et al., 1993), lymphocytes, epithelial and endothelial cells (Baggiolini and Dahinden, 1994). It has been detected in the human reproductive tract, at the cervix (Barclay et al., 1993), placenta (Saito et al., 1994) and endometrium (Arici et al., 1993). Monocyte chemotactic protein (MCP‐1; β‐chemokine) has been reported to attract NK cells (Loetscher et al., 1996) as well as T lymphocytes (Loetscher et al., 1994). MCP‐1 is secreted by a number of cell types such as endothelial cells (Sica et al., 1990), fibroblasts (Yoshimura and Leonard, 1990), monocytes and lymphocytes (Yoshimura and Leonard, 1989). It has been detected in normal endometrium (Arici et al., 1995) and endometriotic cells (Akoum et al., 1996). The β‐chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is a chemoattractant for monocytes, eosinophils and basophils, and is localized in eutopic endometrium and ectopic endometriotic implants (Hornung et al., 1997). Stromal‐derived factor‐1 (SDF‐1; α‐chemokine) is involved in regulating a wide array of leukocytes, lymphopoiesis and controls integrin‐dependent adhesion of T‐cells to the endothelium (Campbell et al., 1998). Most chemokines bind to the cell‐surface or connective‐tissue components such as glycosaminoglycans. Therefore, dimerization is favoured when chemokines associate with these molecules (Witt et al., 1994). These interactions provide strong evidence that chemokine dimerization is a critical process in vivo. Chemokine receptors belong to the superfamily of G‐protein‐coupled receptors (GPCR). The former receptors have seven sequences of 20–25 hydrophobic residues that form an α‐helix and span the plasma membrane, an extracellular N‐terminus, three extracellular loops, three intracellular domains and an intracellular C‐terminal tail. These receptors transmit information to the cell about the presence of chemokine gradients in the extracellular environment. They are named depending on the structure of their ligand (CXC or CC). CXCR4 is expressed on neutrophils, monocytes, B and T lymphocytes, and its primary ligand is SDF‐1 (Nagasawa et al., 1996). CCR5, receptor for RANTES and macrophage inflammatory protein‐1 (MIP‐1) α and β is expressed on monocytes, dendritic cells, activated T lymphocytes and natural killer (NK) cells (Chantakru et al., 2001). CCR2B, expressed in monocytes, basophils, dendritic cells, NK cells and activated T lymphocytes is the main receptor for MCP‐1, ‐2, ‐3 and ‐4 (Polentarutti et al., 1997). CXCR1, a receptor for interleukin 8 (IL‐8) and GCP‐2, is expressed mainly in neutrophils and dendritic cells (Wuyts et al., 1998). The binding of chemokines to their receptors is followed by the involvement of heterotrimeric G proteins (Reif and Cantrell, 1998; Ward et al., 1998) and the triggering of intracellular second messengers such as cAMP and calcium. One of the most impressive effects of the binding of chemokines to their receptors on leukocytes is the morphological change which this provokes; the cytoskeleton is rearranged, integrin‐mediated focal adhesions are formed and the cell binds and detaches from the substrate in a coordinated manner, with extension and retraction of pseudopods responsible for directional migration (Bokoch, 1995; Ward et al., 1998). A specific molecular crosstalk between embryo and endometrium has been reported during the human implantation process (Glasser et al., 1991; De los Santos et al., 1996). The endometrial epithelium is an important element where the molecular interactions between the embryo and the endometrium seem to be initiated (Simón et al., 1997; Galan et al., 2000; Meseguer et al., 2001). The endometrial epithelium produces and secretes chemokines (Arici et al., 1998; Caballero‐Campo et al., 2002). In the present study, we have analysed mRNA expression and immunolocalization of chemokine receptors (CXCR1, CCR5, CXCR4 and CCR2B) in the human endometrium throughout the natural menstrual and HRT cycles and in the human blastocyst. Furthermore, we have also studied the embryonic regulation of these endometrial chemokine receptors using an in‐vitro model for the apposition phase of human implantation. Materials and methods Institutional approval and informed consent This study was approved by the Institutional Review Board on the use of human subjects in research at the Instituto Valenciano de Infertilidad (IVI), and complies with Spain’s Law of Assisted Reproductive Technologies (35/88). All patients participating in this study signed a written statement of consent and were informed of the details of the study. Experimental design To investigate the mRNA expression of endometrial chemokine receptors (CXCR1, CXCR4, CCR2B and CCR5) throughout the menstrual cycle and their hormonal regulation in vivo, we used two different models: (i) endometrial samples from fertile patients undergoing natural cycles and (ii) HRT in mock cycles from participants in the ovum donation programme. To study the embryonic regulation of endometrial chemokine receptors, we have used an in‐vitro model of the apposition phase of human implantation, with endometrial epithelial cells co‐cultured with or without human blastocysts. Triploid embryos were analysed in order to localize the chemokine receptors on the human blastocyst. Endometrial samples Human endometrial tissues were obtained throughout the menstrual cycle from normal fertile women aged 23–39 years (n = 15). A small portion of each specimen was histologically examined and dated according to Noyes et al. (1950). Endometrial biopsies were distributed into five groups: group I, early–mid‐proliferative (days 1–8); group II, late proliferate phase (days 9–14); group III, early secretory phase (days 15–18); group IV, mid‐secretory phase (days 19–22); and group V, late secretory phase (days 23–28). Endometrial expression (real time PCR) and localization (immunohistochemistry) of the four receptors were analysed using these samples. Hormonal regulation of these receptors was also investigated at the protein level in endometrial biopsies from ovum donation recipients undergoing HRT (Meseguer et al., 2001; Martin et al., 2000). Serum and endometrial samples were obtained in mock cycles from five patients (23–29 years) receiving ovum donation and HRT, as previously described (Caballero‐Campo et al., 2002). Briefly, serum samples (S) and uterine biopsies (B) were taken from each patient at day 13 (S1, B1), 18 (S2, B2) and 21 (S3, B3). Therefore, at the time when serum and biopsies were collected, patients were treated for 3 days with 6 mg/day of estradiol valerate (EV) and for 6 days with 6 mg/day of EV plus 800 mg/day of progesterone. Biopsies were dated histologically according to Noyes et al. (1950). E2 was measured in the serum by immunoenzymatic assay (MEIA, Imx; Abbot Scientific, Madrid, Spain). Progesterone was measured by radioimmunoassay (biomerieux, Charbonnieres Les Bains, France). RNA isolation RNA extraction was performed according to Chomczynski and Sacchi (1987), with minor modifications using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Briefly, each tissue was weighed and 500 µl Trizol reagent was added for every 100 mg. Total RNA was separated from DNA and proteins by adding chloroform, and was precipitated with isopropanol (overnight, –20°C). The precipitate was washed twice in ethanol, air‐dried and resuspended in 75% diethylpyrocarbonate (DEPC)‐treated water. RNA was quantified by spectrophotometry on a SmartSpec 3000 spectrophotometer (Biorad, Barcelona, Spain). A260/A280 ratios for all samples used varied between 1.6 and 1.9. Reverse transcription RT was carried out using a Advantage RT‐for‐PCR kit (Clontech, Palo Alto, CA, USA). The mastermix per sample was prepared as follows: 5×reaction buffer, dNTP mix (10 mmol/l each), recombinant RNase inhibitor and MMLV (Moloney‐Murine Leukemia Virus) reverse transcriptase. One µg of each sample was diluted in DEPC‐treated water with oligo (dT)18. The mixture was then heated for 2 min at 70°C and kept on ice until the mastermix was added. For each RT, a blank was prepared using all the reagents except the RNA sample, for which an equivalent volume of DEPC‐treated water was substituted. The RT blank was used to prepare the PCR blank (below). Once all the components were mixed, the samples were incubated for 1 h at 42°C, and heated for 5 min at 94°C to prevent cDNA synthesis and destroy DNAse activity. The product was diluted with DEPC‐treated water to a final volume of 100 µl and stored at –20°C until PCR analysis was performed. Real time fluorescent PCR The LightCycler (Roche Diagnostics, GmbH Mannheim, Germany) Instrument was used to determine the relative quantification of gene expression of CXCR1, CXCR4, CCR2B and CCR5 receptors; GAPDH was chosen as the housekeeping gene control. The SYBR® Green I double‐stranded DNA binding dye (Roche Diagnostics, GmbH Mannheim, Germany) was chosen for these assays. Oligonucleotides were designed using Primer Express® software (AB, Foster City, CA, USA). Oligonucleotide sequences designed for the amplification of the different genes are shown in Table I. All real time PCR assays were run using SYBR® Green PCR Master Mix and the universal thermal cycling parameters indicated by the manufacturer (60°C annealing temperature for all primers). Relative quantification was carried out by employing the standard curve method using the SYBR® Green I dye. Data were presented as the relative average value for each gene investigated and then normalized with the average value of the housekeeping gene obtained on different days of each designated phase of the menstrual cycle. No direct comparison among different genes could be made as the standard was composed of different cDNA species, each at different concentrations. Quantification data were analysed at the beginning of the exponential phase (cycles 30–35) with the LightCycler analysis software 3.5 version. Background fluorescence was removed by setting a noise band. Duplicates showing >5% variation were discarded. To validate a real time PCR, standard curves with r > 0.95 and slope values between 3.1 and 3.4 were required. To explore whether other non‐expected products were also amplified, PCR products after 40 cycles were subjected to a subsequent agarose 2% gel electrophoresis with ethidium bromide to confirm amplification specificity (data not shown). Immunohistochemistry of human endometrium Formalin‐fixed and paraffin‐embedded endometrial biopsies were sectioned and mounted on glass slides coated with VectabondTM (Vector Laboratories, Burlingame, CA, USA). Twelve serial sections (6 µm) from each sample were prepared and the first and last sections stained with haematoxylin–eosin. After deparaffinization and rehydratation, sections were washed three times for 5 min with phosphate‐buffered saline (PBS). Non‐specific binding was blocked with non‐fat milk (50 mg/ml in PBS). Sections were then washed three times with PBS/0.05% Tween 20, pH 7.4 (PBS‐T) and incubated for 1.5 h at 37°C with the following specific antibodies: primary monoclonal antibody (Ab) against human CXCR4 and CCR5 from Pharmingen (San Diego, CA, USA), primary rabbit polyclonal Ab for human CXCR1 and CCR2B from Santa Cruz Biotechnology (Santa Cruz, CA, USA), each at 10 mg/ml. Negative controls were incubated with PBS with 1% bovine serum albumin (BSA) and 0.1% Tween 20. After being washed four times with PBS‐T, sections were incubated for 1.5 h at 37°C with the secondary antibody, fluorescein isothiocyanate (FITC)‐conjugated goat anti‐mouse IgG whole molecule (Sigma, St Louis, MO, USA). Afterwards, sections were washed four times with PBS‐T and gently washed with distilled water. Sections were mounted in aqueous mounting medium (Dako, Barcelona, Spain) and immunolocalization of HRT endometrial chemokines was visualized and photographed using an Olympus 35 mm camera attached to a fluorescence microscope (Nikon, Japan). Natural cycle endometrium photomicrographs were obtained using a Nikon digital camera coolpix 995. Immunostaining intensity was evaluated in at least three different specimens and interpreted as absent (0), weak (+), moderate (++) or intense (+++) by three independent observers. Positive controls were tonsil sections and negative controls were endometrial samples incubated in PBS with 1% BSA and 0.1% Tween 20 without primary antibody. Immunocytochemistry of human blastocysts For the immunostaining of human blastocysts, we employed an avidin–peroxidase staining method using primary monoclonal antibody (Ab) against human CXCR4 and CCR5 (Pharmingen), primary rabbit polyclonal Ab for human CXCR1 and CCR2B (Santa Cruz) each at 10 mg/ml. Embryos were previously fixed for 30 min at 4°C with freshly prepared 2% paraformaldehyde in PBS micro‐drops covered by oil (Simón et al., 1994). After fixation, blastocysts were treated with 0.2% Triton X‐100 (Sigma) in PBS for 10 min at 4°C to permeabilize the fixed cells and thereby facilitate the access of the antibody. Biotin‐labelled goat secondary antibodies (Sigma) were incubated for 30 min at 37°C. Incubation for 4–6 min with a working substrate solution of avidin–peroxidase (Sigma), diluted 1/40, was carried out. Blastocysts were photographed using an Olympus 35 mm camera attached to an inverted microscope (Nikon, Japan). Of a total of 24 blastocysts used, six human triploid blastocysts were analysed for each molecule and three were used as negative controls. In‐vitro model for apposition Based on our previous study, we have developed an in‐vitro model to observe interactions between the human embryo and endometrial epithelial cells (EEC). This model has led to a clinical programme in which embryos are co‐cultured with EEC until blastocyst stage and then transferred back to the mother (Simón et al., 1999). Embryos were obtained after ovulation induction and insemination, employing routine IVF or ICSI procedures. Endometrial biopsies from patients were minced into small pieces (<1 mm) and digested with a mild collagenase solution (0.1%) for 1 h at 37°C. The endometrial epithelium was isolated and purified as previously described (Simón et al., 1993). EEC were cultured to confluence in a steroid‐depleted medium containing a 3:1 mixture of DMEM (Sigma), MCDB‐105 (Sigma) and 5 mg insulin (Sigma) and supplemented with 10% charcoal–dextran‐treated bovine fetal serum (Hyclone, Logan, UT, USA). The homogeneity and purity of EEC cultures were assessed using immunohistochemical markers (Simón et al., 1994) and morphological characteristics (scanning electron microscopy) (Simón et al., 1999). After confluence, the culture media were replaced by a 1:1 mixture of IVF:S2 medium (Scandinavian IVF Science AB, Gothenburg, Sweden). Forty‐eight hours after insemination of oocytes, each 2–4‐cell human embryo was transferred to an EEC monolayer. When embryos reached the 8‐cell stage, the medium was replaced by S2 medium (Scandinavian IVF) until blastocyst stage. Embryonic development was checked daily and the medium changed every 24 h. On day 6 of co‐culture, blastocysts were transferred to the recipient using a Frydman catheter. EEC cultured alone under the same conditions were used as controls. Individual human embryos were co‐cultured with EEC for 5 days (from day 2 to day 6 of embryonic development). After embryo transfer, EEC wells were divided into two groups: EEC with embryos which had reached the blastocyst stage and EEC without embryos (controls). Confocal analysis Immunocytochemistry was performed following the same protocol as that previously described with the same antibodies but using EEC from our in‐vitro apposition model. Confocal analysis was performed with an NRC 1024 instrument (Bio‐Rad, Hempstead, UK). The excitation line used was 488 (FITC). The filter used was HQ515/10 (FITC). Transmitted light images were acquired for every field. Results mRNA expression and protein localization of CXCR1, CXCR4, CCR2B and CCR5 receptors throughout the menstrual cycle The mRNA expression of the four chemokine receptors was analysed throughout the menstrual cycle in three separate experiments each with five patients using real time PCR. Data is presented as a relative average value for each gene investigated and normalized with the average value of the housekeeping gene obtained on different days of each phase of the menstrual cycle in three different experiments. The lowest value for each receptor in each phase of the menstrual cycle was considered as basal expression and quantified as 1. The intensity of expression for a given receptor in a specific phase of the menstrual cycle is expressed as fold increase compared with the basal expression. CXCR1 (Figure 1A), CCR2B (Figure 1C) and CCR5 (Figure 1D) mRNA are highly regulated throughout the menstrual cycle with maximal expression in the luteal phase. The mRNA expression of these three receptors suggested a progesterone‐dependent pattern starting low in the early secretory phase and continuing through the mid‐secretory phase and peaking in the late secretory phase (612‐fold increase for CCR5, 419‐fold increase for CXCR1 and 657‐fold increase for CCR2b). Unlike the other receptors, CXCR4 receptor mRNA (Figure 1B) presented a specific increase in the mid‐luteal phase compared to the early and late luteal phases (17.7‐fold increase in mid‐luteal phase versus 1‐ and 6.8‐fold increase in early and late luteal phases respectively). This illustrates that this receptor is specifically up‐regulated during the implantation window and its lowest expression is noted in the early secretory phase. We have also studied the four receptors at the protein level, throughout the natural menstrual cycle (see Table III for semi‐quantitative analysis). CXCR1 receptor shows higher staining when compared to the other receptors. CXCR1 peaks in the early and mid‐secretory phases (groups III and IV) (Figure 2A and B). CXCR4 receptor also displays high staining across the whole cycle as in the case of CXCR1 receptor, it shows maximal expression in the mid‐secretory phase (group IV) (Figure 2C), confirming the real time RT–PCR experiments. CCR receptors in general show lower expression in the endometrium than CXC receptors. CCR2B staining appears in the late proliferate phase (group II) and reaches a moderate signal in the early secretory phase (group III), maintaining a low‐to‐moderate staining in the rest of the cycle (Figure 2D and E). CCR5 receptor signal starts in the late proliferate phase (group II) and remains low to moderate in different compartments (Figure 2F and G) (see Table II for localization and semi‐quantitative analysis). Hormonal regulation of immunoreactive CXCR1, CXCR4 CCR5 and CCR2B in human endometrium Peripheral E2 and progesterone levels were determined at the time of obtaining endometrial biopsies (non‐receptive, pre‐receptive and receptive phases) and were consistent with the physiological hormone levels expected. At day 13 (non‐receptive phase), E2 levels were 333.2 ± 92.9 pg/ml and progesterone was undetectable. At day 18 (pre‐receptive phase), E2 and progesterone levels were 331.6 ± 39.1 pg/ml and 9.5 ± 3.8 ng/ml respectively. At day 21 (receptive phase) E2 and progesterone levels were 362.6 ± 78.5 pg/ml and 10.5 ± 6.1 ng/ml respectively. These hormonal levels stimulated endometrial differentiation as assessed by classical histological criteria (Noyes et al., 1950). On day 13 (n = 3), when patients were treated solely with estradiol, a very weak staining for CCR2B, CCR5 and CXCR4 was localized in the luminal and glandular epithelium and endothelial cells (Figure 3A–C). During the pre‐receptive and receptive periods (days 18 and 21 respectively), an increase in staining intensity was noted for CXCR1 receptor at the glandular compartment (Figure 3D, G). A slight signal was observed in stromal cells. CCR5 receptor was also immunolocalized, mainly at the luminal epithelium but also at the stromal and perivascular cells (Figure 3E, H), showing a slight increase compared with the non‐receptive phase. CCR2B receptor shows a moderate increase in staining on day 18 and 21 in the luminal epithelium, while no staining was observed in endothelial cells or stroma (Figure 3F, L). CXCR4 receptor shows the same staining as CCR5, mainly expressed in epithelium on days 18 and 21 (data not shown). Endothelial and stromal cells were also positive. We used human tonsil sections (data not shown) as positive controls and negative controls were performed by deletion of the first antibody. Immunostaining intensity was evaluated in at least three different specimens and interpreted as absent (0), weak (+), moderate (++) or intense (+++) by three independent observers (Table II). Immunolocalization of CXCR1, CXCR4, CCR2B and CCR5 in human blastocysts We have detected immunoreactive CCR2B (Figure 4A, B) and CCR5 receptors (Figure 4E, F) in the human blastocyst. CCR2B staining is localized mainly at the inner cell mass, whereas CCR5 staining can be visualized across the trophectoderm. In all cases (n = 3) CCR5 staining was more intense than that of CCR2B receptor and the pellucide zonae was not stained in any case. Immunoreactive CXCR4 and CXCR1 were not detected in human blastocysts when the same technique was used (Figure 4C, D, G, H). Embryonic regulation of chemokine receptors in human EEC The embryonic impact on immunolocalization and polarization of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in cultured EEC was investigated using our apposition model for human implantation. Chemokine receptors CXCR1 (Figure 5A), CXCR4 (Figure 5D) and CCR5 (Figure 5G) produced a barely detectable staining in few cells at the EEC monolayer when the blastocyst was absent. However, when a human blastocyst was present there was an increase in the number of stained cells for CXCR1 (Figure 5B), CXCR4 (Figure 5E), and CCR5 (Figure 5H) and polarization of these receptors in one of the cell poles of the endometrial epithelium (Figure 5I). Immunolocalization and polarization changes in CCR2B receptor were not present in the EEC monolayer and this receptor was not up‐regulated by the presence of the human blastocyst. Discussion Chemokines were originally defined as host defence proteins and it is now clear that their repertoire of functions extends well beyond this role. Chemokines can mediate or inhibit angiogenesis and may be important targets for leukocyte adhesion (Payne and Cornelius, 2002). In this study, we present data that highlight the involvement of chemokine receptors at the human blastocyst–endometrial interface and which expand the biological relevance of chemokines in the embryonic implantation process. In this paper, we have detected that the protein staining for CXCR1, CCR2B and CCR5 receptors in epithelium and stroma decreases in the late secretory phase whereas mRNA expression for these receptors analysed by real time RT–PCR is maximal in this phase of the menstrual cycle. Therefore, a possible explanation to reconcile these findings is the high number of white blood cells which express chemokine receptors recruited during this period in the human endometrium. Remarkably, CXCR4 mRNA levels present a specific though modest up‐regulation (18‐fold) during the implantation window versus the early implantation phase. These findings echoed the semi‐quantitative immunohistochemical data and were corroborated using an in‐vivo model of hormonal regulation. Our in‐vivo model demonstrated that these receptors were present mainly in the endometrial epithelium but they were also present in the stromal and endothelial cells. The selective hormonal‐dependent up‐regulation of these receptors suggests that CXCR1, CCR2B and CCR5 display specific function(s) in the pre‐menstrual endometrium, whereas CXCR4 seems to be implicated in endometrial receptivity. A possible limitation of the in‐vivo model of hormonal regulation used in this study is that three consecutive endometrial biopsies (day 15, 18 and 21) were obtained in the same patient and therefore a non‐specific imflammatory reaction in the last biopsy may be present. Nevertheless, this possibility seems unlikely because the pattern of chemokine receptors expression reported in this model was reproduced when single biopsies were obtained in natural cycles from different patients. The chemotactic response is fundamental in leukocyte physiology and implies recognition of an external gradient of chemokines (Sanchez‐Madrid and Del Pozo, 1999). Leukocyte trafficking through the endothelium is, in some aspects, comparable to the implantation process, where the contact and invasion of the endometrium with and by the blastocyst is similar to the rolling, attachment and crossing process of the lymphocytes through the endothelium. It has been demonstrated that CCR2B and CCR5 receptors are polarized at the leading edge of migrating lymphocytes (Nieto et al., 1997) and that leukocyte adhesion through integrins is required to induce cell polarization (Del Pozo et al., 1997) and redistribution of chemokine receptors (Del Pozo et al., 1995). These data further suggest that chemokines may act in combination with adhesion molecules to steer leukocyte traffic to tissues (Butcher, 1991; Moser and Loetcher, 2001). Leukocyte migration is a complex phenomenon where chemokines and their receptors are important players. The patching of chemokine receptors to the leading edge of a cell implies two remarkable consequences: (i) the functional specialization of this cell’s domain in signal transduction; and (ii) the establishment of an endogenous polarity in the cell, which may be crucial for chemotaxis and other immune responses involving chemokines (Nieto et al., 1998). In our study, when a human blastocyst was present, there was an increase in the number of endometrial epithelial cells stained for CXCR1, CXCR4 and CCR5, and polarization of these receptors in one of the cell poles became evident. These chemokines, secreted locally by the endometrium in the implantation window or by the human blastocyst in the apposition phase (Caballero‐Campo et al., 2002), may act as a signal for receptor polarization/dimerization, thereby acting as a sensor mechanism for increasing local cell responsiveness in the activation of endometrial adhesion molecules. Recently, it has been shown that the binding of a chemokine to its receptors induces homo or heterodimerization of the latter (Rodriguez‐Frade et al., 2001). This effect has been noted on CCR2B and CCR5 receptors (Mellado et al., 2001). The simultaneous presence of RANTES and MCP‐1 induces the heterodimeric receptor complex CCR2B–CCR5, which has unique features, including the reduction of the threshold concentration of chemokine required to induce a response. This might have functional relevance in the cell. The formation of these complexes in lymphocytes activates cell adhesion in contrast to the cell migration triggered by homodimers. We have demonstrated that immunoreactive CCR2B and CCR5 receptors are localized at the human blastocyst. Could the blastocyst form these complexes in response to endometrial chemokines and therefore trigger a similar response? An array of different chemokines is expressed (Kao et al., 2002) and produced in the human endometrium at the time of implantation (Caballero‐Campo et al., 2002; Kayisli et al., 2002). These chemokines could be immobilized by low‐affinity binding to heparin‐bearing proteoglycans on the vascular endothelial or epithelial surface, thereby facilitating the oligomerization of chemokines (Hoogewerf et al., 1997). This effect permits effective presentation of chemokines to cells or groups of cells, which are then able to respond to the chemokines’ presence. In this way, variations in the availability of these chemokines would affect the ability of a ligand to trigger homo‐ or heterodimerization. At low concentrations of chemokines, receptor heterodimerization is favoured and cell adhesion is triggered (Mellado et al., 2001). Caballero‐Campo et al. (2002) have investigated the hormonal and embryonic regulation of IL‐8, RANTES and MCP‐1 in the endometrium. IL‐8 and MCP‐1 were present in the glandular and luminal epithelium, and RANTES was mainly localized on stromal cells. IL‐8 and MCP‐1 were up‐regulated in the presence of E2 and progesterone. IL‐8 mRNA expression and protein were up‐regulated in the presence of the human blastocyst using our co‐culture model. In this paper, we have detected that some chemokine receptors (CXCR1, CXCR4 and CCR5) are present and regulated hormonally in EEC. They display the typical polarization present in lymphocytes, creating a polarized cell capable of responding to different chemokines. We have also detected two of these receptors, CCR5 and CCR2B, in the human blastocyst. White blood cells are not an organized group of cells such as the cells which form the human blastocyst, but the polarization of the latter at the time of adhesion is a constant process that is intriguing and deserves special consideration. The blastocyst is guided to the implantation site in a polarized manner that is species specific, driven by unknown mechanisms and crosses the epithelial barrier penetrating into the stroma. Although the chemokine receptor expression in the blastocyst is homogeneous over the surface of the blastocyst, including the trophectoderm and the inner cell mass, these receptors may polarize if they encounter an adequate gradient of chemokines secreted by the endometrium, (epithelium, stroma or white blood cells) either free or bound to proteoglycans of the epithelial surface. The blastocyst could respond at this point with chemokine polarization and/or homo/heterodimerization. The possible heterodimerization of receptors, in our case CCR2B and CCR5 receptors, might develop in the human blastocyst an expression pattern of different integrins and adhesion molecules such as β1, β5, α6 or E‐cadherin. These have been discovered in the blastocyst at the time of implantation and are necessary for the first steps of embryo adhesion (Bloor et al., 2002). Further studies of chemokine polarization and blastocyst chemotaxis are needed to confirm our hypothesis. Acknowledgements We would like to thank all of the IVF group of IVI, especially Amparo Mercader, for the collection of samples. We also thank Marcos Meseguer, Nicolas Garrido and Pedro Caballero‐Campo for their support and Dr Alvarez Barrientos for his help with the confocal analysis. This study was financed by grants MT1999‐B24364784, FISS 00/0643 and SAF2001.2948 from the MCYT of the Spanish Government. View largeDownload slide Figure 1. Quantitative mRNA analysis of chemokine receptors in human endometrium throughout the menstrual cycle by real time fluorescent RT–PCR. (A) CXCR1 receptor expression. (B) CXCR4 receptor. (C) CCR2B receptor. (D) CCR5 receptor. Endometrial biopsies were distributed in five groups, each corresponding to a different phase: group I; early–mid‐proliferative (days 1–8), group II; late proliferative (days 9–14), group III; early secretory (days 15–18), group IV; mid‐secretory (days 19–22), and group V; late secretory (days 23–28). Data are presented as mRNA expression fold increase compared with the group of basal expression for each receptor. Three experiments were performed in a total of 15 endometrial samples to obtain the mean shown in the graphs. CXCR1, CCR2B and CCR5 show a typical pattern of decidualization increasing their expression in the late proliferative phase (group V) whereas CXCR4 shows an implantation expression pattern, increasing in the mid‐secretory phase (receptive phase). Error bars show SD. View largeDownload slide Figure 1. Quantitative mRNA analysis of chemokine receptors in human endometrium throughout the menstrual cycle by real time fluorescent RT–PCR. (A) CXCR1 receptor expression. (B) CXCR4 receptor. (C) CCR2B receptor. (D) CCR5 receptor. Endometrial biopsies were distributed in five groups, each corresponding to a different phase: group I; early–mid‐proliferative (days 1–8), group II; late proliferative (days 9–14), group III; early secretory (days 15–18), group IV; mid‐secretory (days 19–22), and group V; late secretory (days 23–28). Data are presented as mRNA expression fold increase compared with the group of basal expression for each receptor. Three experiments were performed in a total of 15 endometrial samples to obtain the mean shown in the graphs. CXCR1, CCR2B and CCR5 show a typical pattern of decidualization increasing their expression in the late proliferative phase (group V) whereas CXCR4 shows an implantation expression pattern, increasing in the mid‐secretory phase (receptive phase). Error bars show SD. View largeDownload slide Figure 2. Immunolocalization of CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle. (A) Group I, CXCR1 receptor shows moderate staining in glandular epithelium (arrow). (B) Group II, CXCR1 receptor. Arrows indicate strong staining in luminal epithelium and stromal cells. (C) Group II CXCR4 receptor. Moderate‐to‐strong staining is observed in luminal, glandular and stromal cells. (D) Group II, CCR2 receptor. Faint staining in luminal epithelium and some stromal cells. (E) Group III, CCR2 receptor. Arrows show moderate staining in luminal epithelium. Glandular epithelium also stained. (F) Group III CCR5 receptor. Faint‐to‐moderate staining in the three compartments. (G) Group V CCR5 receptor. Gland showing staining in the lumen. (H) negative control. (I) Positive control ThP1 cells. The semi‐quantitative analysis of the data is presented in Table III. View largeDownload slide Figure 2. Immunolocalization of CXCR1, CXCR4, CCR5 and CCR2B in human endometrium throughout the menstrual cycle. (A) Group I, CXCR1 receptor shows moderate staining in glandular epithelium (arrow). (B) Group II, CXCR1 receptor. Arrows indicate strong staining in luminal epithelium and stromal cells. (C) Group II CXCR4 receptor. Moderate‐to‐strong staining is observed in luminal, glandular and stromal cells. (D) Group II, CCR2 receptor. Faint staining in luminal epithelium and some stromal cells. (E) Group III, CCR2 receptor. Arrows show moderate staining in luminal epithelium. Glandular epithelium also stained. (F) Group III CCR5 receptor. Faint‐to‐moderate staining in the three compartments. (G) Group V CCR5 receptor. Gland showing staining in the lumen. (H) negative control. (I) Positive control ThP1 cells. The semi‐quantitative analysis of the data is presented in Table III. View largeDownload slide Figure 3. Immunolocalization and hormonal regulation of CXCR1, CCR5 and CCR2B in human endometrium. (A, D, G) CXCR1 receptor. (B, E, H) CCR5 receptor. (C, F, L) CCR2B receptor. Endometrial samples from hormonal replacement therapy cycles in ovum donation recipients were obtained during non‐receptive (day 13; A, B, C), pre‐receptive (day 18; D, E, F) and receptive phases (day 21; G, H, I). The semi‐quantitative analysis of the data is presented in Table II. View largeDownload slide Figure 3. Immunolocalization and hormonal regulation of CXCR1, CCR5 and CCR2B in human endometrium. (A, D, G) CXCR1 receptor. (B, E, H) CCR5 receptor. (C, F, L) CCR2B receptor. Endometrial samples from hormonal replacement therapy cycles in ovum donation recipients were obtained during non‐receptive (day 13; A, B, C), pre‐receptive (day 18; D, E, F) and receptive phases (day 21; G, H, I). The semi‐quantitative analysis of the data is presented in Table II. View largeDownload slide Figure 4. Immunolocalization of chemokine receptors in human blastocysts. (A) Negative control for CCR2B receptor. (B) Arrow indicates the staining for CCR2B at the inner cell mass. (C) CXCR4 negative control. (D) CXCR4 staining in the human blastocyst (E). CCR5 negative control. (F) Arrows indicate the positive staining for CCR5 at the trophectoderm. (G) CXCR1 negative control. (H) A human blastocyst stained for CXCR1 receptor. The staining for CCR2B is localized mainly at the inner cell mass whereas CCR5 staining can be visualized across the trophectoderm. CXCR1 and CXCR4 staining was not detected in the human blastocyst. View largeDownload slide Figure 4. Immunolocalization of chemokine receptors in human blastocysts. (A) Negative control for CCR2B receptor. (B) Arrow indicates the staining for CCR2B at the inner cell mass. (C) CXCR4 negative control. (D) CXCR4 staining in the human blastocyst (E). CCR5 negative control. (F) Arrows indicate the positive staining for CCR5 at the trophectoderm. (G) CXCR1 negative control. (H) A human blastocyst stained for CXCR1 receptor. The staining for CCR2B is localized mainly at the inner cell mass whereas CCR5 staining can be visualized across the trophectoderm. CXCR1 and CXCR4 staining was not detected in the human blastocyst. View largeDownload slide Figure 5. Embryonic effect on immunolocalization and polarization of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in cultured endothelial endometrial epithelial cells (EEC). (A, B) CXCR1 receptor staining in non‐polarized EEC cultured without and with an individual blastocyst respectively. (C) Phase contrast of EEC monolayer. (D, E) CXCR4 receptor staining in EEC without or with blastocyst, respectively. (F) Negative control with deletion of the first antibody. CCR5 staining in EEC cultured without (G) or with an individual blastocyst (H). (I) Detail of a single non‐polarized epithelial cell expressing CXCR1 receptor (see arrows). Inner square: contrast phase of the EEC monolayer with the stained cell. CXCR1, CXCR4 and CCR5 staining were barely detected only in a few cells at the EEC monolayer without the blastocyst. Staining for CCR2B was not present in cultured EEC in the presence or absence of a human blastocyst. View largeDownload slide Figure 5. Embryonic effect on immunolocalization and polarization of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in cultured endothelial endometrial epithelial cells (EEC). (A, B) CXCR1 receptor staining in non‐polarized EEC cultured without and with an individual blastocyst respectively. (C) Phase contrast of EEC monolayer. (D, E) CXCR4 receptor staining in EEC without or with blastocyst, respectively. (F) Negative control with deletion of the first antibody. CCR5 staining in EEC cultured without (G) or with an individual blastocyst (H). (I) Detail of a single non‐polarized epithelial cell expressing CXCR1 receptor (see arrows). Inner square: contrast phase of the EEC monolayer with the stained cell. CXCR1, CXCR4 and CCR5 staining were barely detected only in a few cells at the EEC monolayer without the blastocyst. Staining for CCR2B was not present in cultured EEC in the presence or absence of a human blastocyst. Table I. Oligonucleotide primers with predicted respective PCR product sizes.     View Large Table II. Semi‐quantitative analysis of immunohistochemistry in human endometrium     Summary of the immunohistochemical experiments identifying CXCR1, CXCR4, CCR5 and CCR2 in different compartments of the human endometrium during the non‐receptive (day 13), pre‐receptive (day 18) and receptive phases (day 21) in hormone replacement therapy cycles. Designations of 0 (negative), + (weakly positive) to +++ (intensely positive) indicate the relative intensities of the signals averaged for at least three different samples. Variability between readers is indicated with a slash mark. Epit. = epithelial cells; St. = stromal cells; End. = endothelial cells. View Large Table III. Semi‐quantitative analysis of immunohistochemistry results of chemokine receptors CXCR1, CXCR4, CCR2B and CCR5 in different compartments of the human endometrium throughout the menstrual cycle     Designations of – (negative), + (weakly positive), ++ (intensely positive) and +++ (strongly positive) indicate the relative intensities of the signals averaged for three different blind observers. LE = luminal epithelium; GE = glandular epithelium; SC = stromal cells. View Large References Akoum, A., Lemay, A., McColl, S., Turcot‐Lemay, L. and Maheux, R. ( 1996) Elevated concentration and biologic activity of monocyte chemotactic protein‐1 in the fluid of patients with endometriosis. Fertil. Steril. , 66, 17–23. Google Scholar Arici, A., Head, J.R., MacDonald, P.C. et al. ( 1993) Regulation of interleukin‐8 gene expression in human endometrial cells in culture. Mol. Cell. Endocrinol. , 94, 195–204. Google Scholar Arici, A., McDonal, P.C. and Casey, M.L. ( 1995) Regulation of monocyte chemotactic protein‐1 gene expression in human endometrial cells in cultures. Mol. Cell. Endocrinol. , 107, 189–197. Google Scholar Arici, A., Seli, E., Senturk, L.M., Gutierrez, L.S., Oral, E. and Taylor, H.S. ( 1998) Interleukin‐8 in the human endometrium. Clin. Endocrinol. Metab. , 83, 1783–1787. Google Scholar Baggiolini, M. and Dahinden, A. ( 1994) CC chemokines in allergic inflammation. Inmmunol. Today , 15, 127–133. Google Scholar Barclay, C.G., Brennard, J.E., Kelly, R.W. and Calder, A.A. ( 1993) Interleukin‐8 production by human cervix. Am. J. Obstet. Gynecol. , 169, 625–632. Google Scholar Bloor, D.J., Metcalfe, A.D., Rutherford, A., Brison, D.R. and Kimber, S.J. ( 2002) Expression of cell adhesion molecules during human preimplantation embryo development. Mol. Hum. Reprod. , 8, 237–245. Google Scholar Bokoch, G.M. ( 1995) Chemoattractant signalling and leukocyte activation. Blood , 86, 1649–1657. Google Scholar Butcher, E.C. ( 1991) Leukocyte–endothelial cell recognition: three (or more) steps to specificity and diversity. Cell , 67, 1033–1036. Google Scholar Caballero‐Campo, P., Dominguez, F., Coloma, J., Meseguer, M., Remohi, J., Pellicer, A. and Simon, C. ( 2002) Hormonal and embryonic regulation of chemokines IL‐8, MCP‐1 and RANTES in human endometrium during the window of implantation. Mol. Hum. Reprod. , 8, 375–384. Google Scholar Campbell, J.J., Hedrick, J. Zlotnik, A., Siani, M.A., Thompson, D.A. and Butcher, E.C. ( 1998) Chemokines and the arrest of lymphocytes rolling under flow conditions. Science , 279, 381–384. Google Scholar Chantakru, S., Kuziel, W.A., Maeda, N. and Croy, B.A. ( 2001) A study on the density and distribution of uterine natural killer cells at mid pregnancy in mice genetically‐ablated for CCR2B, CCR 5 and the CCR5 receptor ligand, MIP‐1 alpha. J. Reprod. Immunol. , 49, 33–47. Google Scholar Chomczynski, P. and Sacchi, N. ( 1987) Single step method of RNA isolation by acid guanidinium thiocyanate phenol‐chloroform extraction. Anal. Biochem. , 162, 156–159. Google Scholar Cocchi, F., DeVico, A.L., Garzino‐Demo, A., Arya, S.K., Gallo, R.C. and Lusso, P. ( 1995) Identification of RANTES, MIP‐1 alpha, and MIP‐1 beta as the major HIV‐suppressive factors produced by CD8+ T cells. Science , 270, 1811–1815. Google Scholar De los Santos, M.J., Mercader, A., Francés, A., Portoles, E., Remohi, J., Pellicer, A. and Simon, C. ( 1996) Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin‐1 system during embryonic development. Biol. Reprod. , 54, 563–574. Google Scholar Del Pozo, M.A., Sánchez‐Mateos, P., Nieto, M. and Sanchez‐Madrid, F. ( 1995) Chemokines regulate cellular polarization and adhesion receptor redistribution during lymphocyte interaction with endothelium and extracellular matrix. Involvement of cAMP signaling pathway. J. Cell Biol. , 131, 495–508 Google Scholar Del Pozo, M.A., Cabañas, C., Montoya, Ager, A., Sanchez‐Mateos, P. and Sanchez‐Madrid, F. ( 1997) ICAM’s redistributed by chemokines to cellular uropods as a mechanism for recruitment of T‐lymphocytes. J. Cell Biol. , 137, 1–16. Google Scholar Dudley, D.J., Trantman, M.S. and Mitchel, M.D. ( 1993) Inflammatory mediators regulate interleukin‐8 production by cultured gestational tissues: evidence for a cytokine network at the chorio‐decidual interface. Clin. Endocrinol. Metab. , 76, 404–410. Google Scholar Galan, A., O’Connor, J.E., Valbuena, D., Herrer, R., Remohi, J., Pampfer, S., Pellicer, A. and Simon, C. ( 2000) The human blastocyst regulates endommtrial epithelial apoptosis in embryonic adhesion. Biol. Reprod. , 63, 430–439. Google Scholar Glasser, S.R., Mulholland, J. and Mani, S.K. ( 1991) Blastocyst endometrial relationships: reciprocal interaccions between uterine epithelial and stromal cells and blastocysts. Trophoblast Res. , 5, 225–280. Google Scholar Hoogewerf, A.J., Kuschert, G.S.V., Proudfoot, A.E., Chung, C.W., Cooke, R.M., Hubbard, R.E., Wells, T.N. and Sanderson, P.N. ( 1997) Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry , 36, 13570–13578. Google Scholar Hornung, D., Ryan, I.P., Chao, V.A., Vigne, J.L., Schriock, E.D. and Taylor, R.N. ( 1997) Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells. Clin. Endocrinol. , 82, 1621–1628. Google Scholar Kao, L.C., Tulac, S., Lobo, S., Imani, B., Yang, J.P., Germeyer, A., Osteen, K., Taylor, R.N., Lessey, B.A. and Giudice, L.C. ( 2002) Global gene profiling in human endometrium during the window of implantation. Endocrinology , 143, 2119–2138. Google Scholar Kayisli, U.A., Mahutte, N.G. and Arici, A. ( 2002) Uterine chemokines in reproductive physiology and pathology. Am. J. Reprod. Immunol. , 47, 213–221. Google Scholar Larsen, C., Anderson, A., Aella, E., Oppenheim, J.J. and Matsushima, K. ( 1989) The neutrophil‐activating protein (NAP‐1) is also chemotactic for T lymphocytes. Science , 243, 1469–1466. Google Scholar Loetscher, P., Seitz, M., Clark‐Lewis, I., Baggiolini, M. and Moser, B. ( 1994) Monocyte chemotactic proteins MCP‐1, MCP‐2, and MCP‐3 are major attractants for human CD4+ and CD8+ T lymphocytes. FASEB J. , 8, 1055. Google Scholar Loetscher, P., Seitz, M., Clark‐Lewis, I., Baggiolini, M. and Moser, B. ( 1996) Activation of NK cells by CC chemokines. Chemotaxis, Ca2+ mobilization, and enzyme release. J. Immunol. , 156, 322. Google Scholar Martin, J.C., Jasper, M.J., Valbuena, D., Meseguer, M., Remohi, J., Pellicer, A. and Simon, C. ( 2000) Increased adhesiveness in cultured endometrial‐derived cells is related to the absence of moesin expression. Biol. Reprod. , 63, 1370–1376. Google Scholar Mellado, M., Rodriguez‐Frade, J.M., Vila‐Coro, A.J., Fernandez, S., Martin de Ana, A., Jones, D.R., Toran, J.L. and Martinez‐A.C. ( 2001) Chemokine receptor homo‐ or heterodimerization activates distinct signaling pathways. EMBO J. , 20, 2497–507. Google Scholar Meseguer, M., Aplin, D., Caballero‐Campo, P., O’Connor, J.E., Martin, J.C., Remohi, J., Pellicer, A. and Simon, C. ( 2001) Human endometrial mucin MUC1 is up‐regulated by progesterone and down regulated in vitro by the human blastocyst. Biol. Reprod. , 64, 590–601. Google Scholar Moser, B. and Loetscher, P. ( 2001) Lymphocyte traffic control by chemokines. Nat. Immunol. , 2, 123–128. Google Scholar Mukaida, N., Shiroo, M. and Matsushima, K. ( 1989) Genomic structure of the human monocyte derived neutrophil chemotactic factor IL‐8. J. Immunol. , 143, 1366–1371. Google Scholar Nagasawa, T., Nakajima, T., Tachibana, K., Iizasa, H., Bleul, C.C., Yoshie, O., Matsushima, K., Yoshida, N., Springer, T.A. and Kishimoto, T. ( 1996) Molecular cloning and characterization of a murine pre‐B‐cell growth‐stimulating factor/stromal cell‐derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin. Proc. Natl Acad. Sci. USA , 93, 14726–14729. Google Scholar Nieto, M., Frade, J.M.R., Sancho, D., Mellado, M., Martinez‐A, C. and Sanchez‐Madrid, F. ( 1997) Polarization of chemokine receptors to the leading edge during lymphocyte chemotaxis. J. Exp. Med. , 186, 153–158. Google Scholar Nieto, M., Navarro, F., Perez‐Villar, J.J., del Pozo, M.A., Gonzalez‐Amaro, R., Mellado, M., Frade, J.M., Martinez‐A, C., Lopez‐Botet, M. and Sanchez‐Madrid, F. ( 1998) Roles of chemokines and receptor polarization in NK‐target cell interactions. J. Immunol. , 161, 3330–3339. Google Scholar Noyes, R.W., Hertig, A.T. and Rock, J. ( 1950) Dating the endometrial biopsy. Fertil. Steril. , 1, 3–25. Google Scholar Payne, A.S. and Cornelius, L.A. ( 2002) The role of chemokines in melanoma tumor growth and metastasis. J. Invest. Dermatol. , 118, 915–922. Google Scholar Polentarutti, N., Allavena, P., Bianchi, G., Giardina, G., Basile, A., Sozzani, S., Mantovani, A. and Introna, M. ( 1997) IL‐2‐regulated expression of the monocyte chemotactic protein‐1 receptor (CCR2B) in human NK cells: characterization of a predominant 3.4‐kilobase transcript containing CCR2B and CCR2A sequences. J. Immunol. , 158, 2689–2694. Google Scholar Reif, K. and Cantrell, D.A. ( 1998) Networking Rho family GTPases in lymphocytes. Immunity , 8, 395–401. Google Scholar Rodriguez‐Frade, J.M., Mellado, M. and Martinez‐A, C. ( 2001) Chemokine receptor dimerization: two are better than one. Trends Immunol. , 22, 612–617. Google Scholar Saito, S., Kasahara, T., Sakakura, S., Umekage, H., Harada, N. and Ichijo, M. ( 1994) Detection and localisation of interleukin‐8 mRNA and protein in human placenta and decidual tissues. J. Reprod. Immuol. , 27, 161–172. Google Scholar Sanchez‐Madrid, F. and del Pozo, M.A. ( 1999) Leukocyte polarization in cell migration and immune interactions. EMBO J. , 18, 501–511. Google Scholar Sica, A., Wang, J.M., Colotta, F., Dejana, E., Mantovani, A., Oppenheim, J.J., Larsen, C.G., Zachariae, C.O. and Matsushima, K. ( 1990) Monocyte chemotactic and activating factor gene expression induced in endothelial cells by interleukin‐1 (IL‐1) and tumor necrosis factor (TNF‐a). J. Immunol. , 144, 3034–3038. Google Scholar Simón, C., Piquette, G., Francés, A. and Polan, M.L. ( 1993) Localization of interleukin‐1 type 1 receptor and interleukin‐1b in human endometrium throughout the menstrual cycle. Clin. Endocrinol. Metab. , 77, 549–555. Google Scholar Simón, C., Piquette, G.N., Francés, A., el‐Danasouri, I., Irwin, J.C. and Polan, M.L. ( 1994) The effect of interleukin‐1b on the regulation of IL‐1 receptor type 1 messenger ribonucleic acid and protein levels in cultured human endometrial stromal and glandular cells. Clin. Endocrinol. Metab. , 78, 675–682. Google Scholar Simón, C., Gimeno, M.J., Mercader, A., O’Connor, J.E., Remohi, J., Polan, M.L. and Pellicer, A. ( 1997) Embryonic regulation of integrins b3, a4 and a1 in human endometrial epithelial cells in vitro. Clin. Endocrinol. Metab. , 82, 2607–2616. Google Scholar Simón, C., Caballero‐Campo, P., García‐Velasco, J.A. and Pellicer, A. ( 1998) Potential implications of chemokines in reproductive function: an attractive idea. J. Reprod. Immunol. , 38, 169–193. Google Scholar Simón, C., Mercader, A., García‐Velasco, J.A., Nikas, G., Moreno, C., Remohi, J. and Pellicer, A. ( 1999) Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure. Clin. Endocrinol. Metab. , 84, 2638–2646. Google Scholar Ward, S.G., Bacon, K. and Westwick, J. ( 1998) Chemokines and T lymphocytes: more than a attraction. Immunity , 9, 1–11. Google Scholar Witt, D.P. and Lander, A.D. ( 1994) Differential binding of chemokines to glycosaminoglycan subpopulations. Curr. Biol. , 4, 394. Google Scholar Wuyts, A., Proost, P., Lenaerts, J.P., Ben‐Baruch, A., Van Damme, J. and Wang, J.M. ( 1998) Differential usage of the CXC chemokine receptors 1 and 2 by interleukin‐8, granulocyte chemotactic protein‐2 and epithelial‐cell‐derived neutrophil attractant‐78. Eur. J. Biochem. , 255, 67–73. Google Scholar Yoshimura, T. and Leonard, E.J. ( 1990) Secretion by human fibroblasts of monocyte chemoattractant protein‐1, the product of the gene JE. J. Immunol. , 144, 2377–2383. Google Scholar Yoshimura, T., Yuhki, N. and Moore, S. ( 1989) Human monocyte chemoattractant protein 1 (MCP‐1): full‐length cDNA cloning, expression in mitogen‐stimulated blood mononuclear leukocytes and sequence similarity to mouse competence gene JE. FEBS Lett. , 244, 487–493. Google Scholar

Journal

Molecular Human Reproduction – Oxford University Press

Published: Apr 1, 2003

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