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Original Article |
Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
Address all correspondence and requests for reprints to: Hiroshi Fujiwara, M.D., Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail: fuji{at}kuhp.kyoto-u.ac.jp.
Abstract
Eph receptor tyrosine kinases and their cell membrane-bound ligands, ephrins, are well known to function in cell-to-cell interaction and to play an important role in cell migration and adhesion during embryonic development in mammals. To investigate the involvement of the Eph-ephrin system in human embryo implantation, the expression of Eph receptors and ephrins was examined in human blastocysts and the endometrium.
Immunohistochemical examination showed that ephrin A1 was expressed on human endometrial luminal and glandular epithelial cells in both the proliferative (cycle d 8–13; n = 8) and secretory (cycle d 18–24; n = 7) phases. RT-PCR analysis of isolated endometrial epithelial cells and stromal cells showed that ephrin A1 mRNA was predominantly expressed in endometrial epithelial cells. Northern blot analysis also confirmed the expression of ephrin A1 mRNA in the endometrium. In addition, nested RT-PCR analysis revealed the mRNA expression of Eph A1, one of the representative receptors for ephrin A1, in human blastocysts obtained from patients undergoing in vitro fertilization treatment.
These findings indicate a possible interaction between human blastocysts and endometrial epithelial cells via the Eph-ephrin system. Because intracytoplasmic signals are induced by Eph receptors after ephrin stimulation, this system may be involved in the activation process of the human embryo during the implantation period.
EMBRYONIC IMPLANTATION IN the uterus is an essential process in mammalian pregnancy. Before invading the endometrium, the human embryo attaches to endometrial luminal epithelial cells. Although the precise mechanisms have not yet been clarified thoroughly, several adhesion-related molecules have been proposed to play roles in this initial phenomenon (1, 2). For example, integrins that mediate the cell-to-extracellular matrix interaction are expressed on the luminal sites of endometrial surface epithelial cells (3, 4). On the other hand, large molecules such as Muc-1 have also been suggested to interfere with the binding between the embryo and endometrial luminal epithelial cells (5, 6). During the implantation stage, the human embryo is activated, and the trophectoderm of the blastocysts differentiates to develop the invasive phenotype of trophoblasts. Although direct cell-to-cell interaction of the trophectoderm with the endometrium is considered to be a key process in implantation, the activating molecules that induce the transformation of trophoblasts to the invasive phenotype are still unknown.
Ephrins are membrane-bound ligands for the Eph family of protein tyrosine kinase receptors. The Eph-ephrin system functions in cell-to-cell communications. The interaction of ephrin with Eph receptors was reported to control cellular responses such as cell migration, boundary formation of structures, and control of cell shape (7, 8). Both Eph receptors and ephrins are dramatically expressed in a wide range of regions of the vertebrate embryo including the ectoderm, mesoderm, and endoderm (9). These molecules have been proposed to regulate angiogenesis and axonal guidance (10, 11). Because this system can induce cellular responses after cell-to-cell interaction, it is one of the candidate systems that might activate human embryos during the implantation process.
Despite the large number of studies describing important functions of the Eph-ephrin system during embryonic development, there are few reports showing the role of this system in the adult. Ephrins can be divided into two subclasses: ephrin A and ephrin B. Ephrin A ligands (ephrin A1-A5) are anchored to the cell surface via a glycosylphosphatidylinositol anchor and bind to Eph A receptors (Eph A1-A8), whereas ephrin B ligands (ephrin B1-B3) have transmembrane and cytoplasmic domains and interact with Eph B receptors (Eph B1-B6; Ref. 12). In the present study, the expression of Eph-ephrins was screened by RT-PCR. Ephrin A1 mRNA was detected thereby in the human endometrium. We further studied the localization of ephrin A1 by immunohistochemistry and confirmed its mRNA expression by RT-PCR and Northern blot analyses. In addition, using nested RT-PCR, we also investigated the expression of Eph A family members, which are receptors for ephrin A1, on human embryos that were obtained from patients undergoing in vitro fertilization (IVF) treatment.
Materials and Methods
Reagents
The rabbit antihuman ephrin A1 (SC911) polyclonal antibody (pAb) and its blocking peptide, which was used for immunization, and rhodamine-conjugated goat antimouse Ig were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The other rabbit pAb for antimouse ephrin A1, which also reacted with human ephrin A1, was obtained from Zymed Laboratories, Inc. Corp. (San Francisco, CA). Antihuman cytokeratin-7 mouse monoclonal antibody (mAb; clone OV-TL, IgG1) and fluorescein isothiocynate (FITC)-conjugated swine antirabbit Ig were purchased from DAKO Corp. (Glostrup, Denmark). Mouse IgG1 negative control mAb (X0931, DAKO Corp.) was used as a negative control.
Tissues
Human endometrial tissues were obtained from normally cycling premenopausal women who had undergone hysterectomy for the treatment of uterine myoma or uterine cervical carcinoma in situ. Each endometrial specimen was examined histologically. Eight endometria were in the proliferative phase (cycle d 8–13), and seven were in the secretory phase (cycle d 18–24). Endometria in the secretory phase were further staged according to the criteria of Noyes et al. (13). These endometrial specimens were used for immunohistochemistry, isolation of endometrial epithelial and stromal cells, and Northern blot analysis. Informed consent for the use of these tissues in this study was obtained from all donors.
Human blastocysts
Twenty-three spare embryos from 15 patients were examined in this study. Ovarian stimulation, oocyte collection, and embryo culture were performed as described previously, with minor modifications (14). In brief, administration of a GnRH agonist (buserelin acetate; Aventis Pharma Co., Tokyo, Japan) was initiated in the midluteal phase or the early follicular phase. All patients subsequently received pure FSH (Serono Japan Co., Tokyo, Japan) or human menopausal gonadotropin (Organon Japan Co., Tokyo, Japan) from cycle d 3 for ovarian stimulation until the dominant follicle reached a diameter of more than 18 mm, followed by an injection of human chorionic gonadotropin (Mochida Pharmaceutical Co., Osaka, Japan) 36 h before oocyte retrieval. After the oocytes were picked up, they were cultured in Quinn’s Advantage Fertilization Medium (Sage BioPharma, Inc., Bedminster, NJ) with 10% serum protein substitute (Sage BioPharma, Inc.) in 5% CO2, 5% O2, and 90% N2. Conventional insemination was performed for IVF. After fertilization was confirmed on the next day (d 1), the zygotes were cultured for another 1 or 2 d. The quality of all embryos was evaluated as one of five grades according to Veeck’s classification (15). The three embryos of the highest grade were transferred into the uterine cavity on d 2 or 3. When the patients desired cryopreservation, all of the grade 1 embryos and some of the grade 2 embryos were frozen and preserved. The other residual embryos of grades 2 and 3 were used for this study as spare embryos and further cultured for another 2 or 3 d. Three days after fertilization, embryos were cultured in Blastocyst Medium (Irvine Scientific Sales Co., Inc., Santa Ana, CA) with 10% serum protein substitute. Eph A receptor expression was examined as described below using 13 spare embryos that developed to various stages such as 8-cell stage (n = 4), morula stage (n = 3), and blastocyst stage (n = 6).
This study was performed according to the ethical guidelines regarding studies in which human gametes or embryos are used as materials; these guidelines are issued by the Ethics Committee of the Japan Society of Obstetrics and Gynecology. The use of spare embryos was approved by the Ethics Committee of Kyoto University Hospital, and informed consent for use of the spare embryos in this study was obtained from all donor couples attending the IVF unit of the Kyoto University Hospital.
Indirect double immunofluorescence staining of the human endometrium
Human endometrial frozen sections were prepared as previously described (16). Eight endometrial specimens in the proliferative phase, consisting of cycle d 8 (n = 1), d 9 (n = 3), d 10 (n = 1), d 11 (n = 1), d 12 (n = 1), and d 13 (n = 1), and seven specimens in the secretory phase, consisting of d 18 (n = 1), d 20 (n = 2), d 21 (n = 2), d 23 (n = 1), and d 24 (n = 1), were subjected to immunohistochemical analysis. Each specimen was embedded in OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL), snap-frozen in liquid nitrogen, and stored at -80 C. Frozen tissues were sliced at 6-µm thickness using a cryostat microtome (Cryocut 1800, Reichert-Jung, Heidelberg, Germany), immediately air-dried on neoprene (Nisshin EM, Tokyo, Japan)-coated glass slides, and fixed in acetone at -20 C for 5 min. The slides were immediately examined or stored at -20 C until use.
The slides were incubated for 30 min at room temperature with mouse antihuman cytokeratin-7 mAb (5 µg/ml) or mouse IgG1 negative control mAb (5 µg/ml), which was diluted with RPMI medium containing 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY) and 0.1% NaN3. After being washed in PBS, the slides were incubated with rhodamine-conjugated goat antimouse Ig (diluted 1:40) for 30 min at room temperature in the dark. The washed slides were then reacted with rabbit antihuman ephrin A1 (5 µg/ml) or rabbit antimouse ephrin A1 (5 µg/ml) antibody for 30 min, followed by incubation with FITC-conjugated swine antirabbit Ig (diluted 1:20) for 30 min. For negative controls of second staining, antihuman ephrin A1 pAb that was preincubated with a 5-fold (by weight) excess of blocking peptide for 2 h at room temperature was used. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA), which reduces fluorescence fading, and then examined under a confocal laser scanning microscope (Carl Zeiss Inc., Jena, Germany).
Isolation of endometrial epithelial cells and stromal cells
Endometrial tissues were minced into small pieces of less than 1 mm3 and incubated in RPMI supplemented with 10% fetal bovine serum, 0.5% collagenase I (Wako Pure Chemical Industries Ltd., Osaka, Japan), and 0.005% deoxyribonuclease I (DNAase I, Sigma, St. Louis, MO) at 37 C for 1 h. After this enzymatic digestion, most of the endometrial stromal cells, including immune cells, were present as single cells or small aggregates, whereas most of the epithelial cells remained in larger clumps. The single cell fraction containing endometrial stromal cells was isolated by three cycles of differential sedimentation at unit gravity (17) and was washed twice in RPMI. The separated single cells were used as an endometrial stromal cell fraction. On the other hand, the fraction of the large clumps containing epithelial cells was incubated in a 10-cm dish (Corning, Inc., Corning, NY) for 1 h at 37 C to remove adherent stromal cells. The nonadherent cell clumps were collected and used as an endometrial epithelial cell fraction.
RNA isolation
The endometrial tissues, isolated endometrial cells, and fresh blastocysts were immediately frozen in liquid nitrogen and stored at -80 C until RNA extraction. Total RNA was extracted using a commercial kit (TRIzol, Life Technologies, Inc., Gaithersburg, MD).
RT-PCR analysis of ephrin A1 mRNA expression in human endometrial epithelial and stromal cells
Five micrograms of total RNA from the endometrial epithelial cell fractions or endometrial stromal cell fractions was reverse-transcribed with random primers using a commercial kit (First Strand cDNA Synthesis Kit, Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotide primers designed on the basis of the sequence of human ephrin A1 cDNA (sense primer, 5'-TCT ACA TAG CAT CGG TCA CA-3', positions 598–617; antisense primer, 5'-TTT CCT TCA GTC AGT TCA TC-3', positions 1087–1106; Ref. 18) or with human S26 primers (sense primer, 5'-GGTCCGTGCCTCCAAGATGA-3', positions 8–27; antisense primer, 5'-TAAATCGGGGTGGGGGTGTT-3', positions 308–327; Ref. 19). After PCR amplification, 10 µl of each PCR product was electrophoresed on a 1.5% agarose gel, and amplified bands were detected by ethidium-bromide staining. After cloning the PCR product and verifying its sequence, the enzymatically digested cDNA insert was purified and used as a probe for subsequent Northern blot analysis.
Northern blot analysis of ephrin A1 mRNA expression in the human endometrium
Ten micrograms of total RNA from endometrial tissues (secretory phase; n = 2) were separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech, Arlington Heights, IL). The membrane was incubated with prehybridization solution (Rapid Hybridization Buffer; Amersham Pharmacia Biotech) for 30 min at 65 C and then hybridized with the 32P-labeled ephrin A1 cDNA probe for 2 h at 65 C in the same solution. After hybridization, the membrane was washed in 2x SSC [2 mM sodium citrate and 20 mM sodium chloride in distilled water (pH 7.0)] with 0.1% SDS at room temperature for 15 min and in 0.2x SSC with 0.1% SDS at 65 C for 30 min, and then subjected to autoradiography. The membrane was washed and rehybridized with the S26 probe to correct for the amount of loaded RNA.
Nested RT-PCR analysis of Eph A families in human blastocysts
Whole RNAs from each blastocyst were reverse-transcribed with random primers using a commercial kit (First Strand cDNA Synthesis Kit, Amersham Pharmacia Biotech). The resulting cDNA mixtures were subjected to 35 cycles of first-round PCR amplification with oligonucleotides from the cDNA of Eph A family members as primers (Table 1
). Aliquots (0.1 µl) of cDNAs obtained from the first amplification served as templates for a second DNA amplification reaction that used inner nested or semi-nested primers (Table 1). This second-round PCR was performed for 35 cycles. After PCR amplification, 10 µl of each PCR product was electrophoresed on a 1.5% agarose gel, and amplified bands were detected by ethidium-bromide staining. S26 was used as a positive control, and for all amplifications, negative controls (water only) were included. Primer sequences and sizes of PCR products are given in Table 1. The nested PCR product corresponding to Eph A1 was sequenced to confirm its identity.
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Immunohistochemical localization of ephrin A1 expression in human endometrium
In the normal endometrium, both in the proliferative and secretory phases, high levels of ephrin A1 were detected on the luminal surface and on glandular epithelial cells that were also stained by anticytokeratin 7 mAb (Fig. 1). The positive staining of endometrial epithelial cells for ephrin A1 was completely abolished by treatment with immunizing peptide (Fig. 1L). Almost all stromal cells were negative for the expression of ephrin A1 throughout the menstrual cycle. There were no significant differences in the intensity of expression of ephrin A1 on epithelial cells between the proliferative and secretory phases (Figs. 1 and 2).
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, G–I). RT-PCR analysis of ephrin A1 mRNA expression in human endometrial epithelial and stromal cells
Three pairs of endometrial cell fractions from one specimen in the proliferative phase (cycle d 12) and two specimens (cycle d 21 and 24) in the secretory phase were subjected to RT-PCR analyses. PCR products of the expected size of 509 bp for ephrin A1 were amplified from the cDNAs derived from the endometrial epithelial cell fraction, whereas they were not amplified from the cDNAs derived from the endometrial stromal cell fraction (Fig. 3A).
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Northern blot analysis showed ephrin A1 mRNA at 1.5 kbp in the RNAs derived from the endometrium in the secretory phase (cycle d 18 and 21; n = 2; Fig. 3B).
Nested RT-PCR analysis of Eph A family members in human blastocysts
By nested RT-PCR analysis using Eph A1 primers, a PCR product of 399 bp was detected by ethidium-bromide staining when the cDNA derived from four of six blastocysts was used as a template (Fig. 4). The size of this product was clearly different from that derived from PCR amplification of genomic DNA (1063 bp). The sequence of this product was identical to that encoding Eph A1. Using similar methods, we could not detect the RT-PCR products corresponding to Eph A2, A3, A4, A5, and A7 in this study.
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). Discussion
By immunohistochemistry using antihuman ephrin A1 antibody, we demonstrated here that immunoreactive ephrin A1 was expressed on endometrial luminal and glandular epithelial cells. No significant difference of the level of ephrin A1 expression was observed between endometrial specimens derived from proliferative and secretory phases. When the antibody was preincubated with the synthetic peptide that had been used for immunization, the positive immunohistochemical staining disappeared, supporting the specific expression of ephrin A1 protein on human endometrial epithelial cells. On the other hand, almost no expression of ephrin A1 protein was observed on the endometrial stromal cells. The specific localization of immunoreactive ephrin A1 on human endometrial epithelial cells was also demonstrated by antimouse ephrin A1 pAb. The expression of ephrin A1 mRNA was detected by RT-PCR, and this mRNA expression was predominantly observed in endometrial epithelial cells, in agreement with the immunohistochemical results. Northern blot analysis confirmed the expression of ephrin A1 mRNA in the human endometrium and demonstrated that the size of ephrin A1 mRNA isolated from endometrial tissues was 1.5 kbp, which is compatible with the reported length. From these findings, we concluded that ephrin A1 is expressed on human endometrial luminal and glandular epithelial cells.
Because ephrin A1 is well known to be one of the representative ligands for Eph A family members, the expression of mRNAs encoding Eph A receptors on human blastocysts was examined to gain insight into the interaction between the embryo and endometrial luminal epithelial cells. RT-PCR products of 399 bp, corresponding to Eph A1 mRNA, were detected when cDNAs derived from human blastocysts were used as the templates in nested RT-PCR analysis. Because RT-PCR analysis was applied to whole-blastocyst homogenates, the primers for Eph A1 were designed to span an intron so that the cDNA-derived products could be distinguished by size from possible amplified false-positive signals due to contaminating genomic DNA. Because the size of the PCR product differed from that of genomic DNA and the nucleotide sequence of the PCR product was identical to that of Eph A1 mRNA, we concluded that Eph A1 mRNA is expressed in human blastocysts. As far as we know, this is the first report that demonstrates the Eph expression on the human blastocysts along with the ephrin expression on the endometrium, which is the first adult organ that a developing embryo interacts with. This study also showed sequential alteration of Eph A1 expression on developing embryos. Because the expression of Eph A1 mRNA was not detected in the cDNAs derived from embryos in the eight-cell or morula stages, it is suggested that Eph A1 appears on the developing embryos during blastocyst formation. On the basis of these results, we speculate that the Eph-ephrin A system plays some role(s) in the physiological interaction between the human embryo and endometrial luminal epithelial cells at the implantation site.
Ephrins are considered to regulate cellular responses such as cell migration, boundary formation of structures, and control of cell shape via Eph receptors (7, 8). For example, ephrin A1 was demonstrated to have chemoattractant effects on endothelial cells during inflammatory angiogenesis induced by TNF 
(20). Ephrins were reported to dimerize Eph receptors when they interact with Eph receptors. This further leads to the formation of clusters of Eph receptors, which in turn induce several cellular responses (9). Soluble forms of ephrins do not trigger receptor activation unless they are artificially clustered, whereas membrane-bound ephrins trigger Eph receptor phosphorylation, indicating that these molecules mediate contact-dependent cell interactions (21). Mechanisms of intracytoplasmic signaling pathways are now being extensively investigated (22). Recently, the activation of Eph A receptors by ephrin A 1 was shown to induce ephexin-dependent activation of 
A, one of the family GTPases, which regulates cell motility (23). In addition, the activation of Eph A2 receptor by ephrin A 1 was reported to regulate integrin function via dephosphorylation of FAK and paxillin (24). This molecule was also demonstrated to inhibit the Ras/MARK pathway and to attenuate cell proliferation by activating Eph A receptor tyrosine kinases (25). Furthermore, Eph receptors and ephrins were reported to play a role in not only up-regulation, but also down-regulation of adhesion during reorganization of tissues (26, 27). These mechanisms may be involved in cell activation or inactivation cascades in the early events of embryo implantation, when individual tissues initially contact each other.
Although ephrin A1 is a glycosylphosphatidylinositolanchored molecule, it is thought to receive signals from Eph receptors and transport these signals in reverse into its own intracytoplasmic sites (26). Thus, the interaction between Eph A1 and ephrin A1 can evoke a bidirectional signaling to both Eph A1- and ephrin A1-bearing cells, suggesting that this interaction may activate not only the embryo but also endometrial luminal epithelial cells during the implantation process. The regulation of integrin function in the luminal surface epithelial cells is considered to be important for the adhesion of blastocysts. Recently, we showed that the CD9 molecule is constitutively expressed on the endometrial luminal surface and glandular epithelial cells throughout the menstrual cycle. CD9 was shown to be associated with integrin ß1 that was expressed on endometrial epithelial cells (16). Because CD9 was also demonstrated to change the function of endometrial carcinoma cell lines via an integrin ß1-related pathway, this molecule was proposed to regulate endometrial receptivity for embryos by modulating integrin function (28). A recent study showed that activation of ephrin-A2 or ephrin-A5 by one of their receptors, EphA3, resulted in an integrin ß1-dependent increased adhesion of ephrin-A-expressing cells (29). It is possible that after cell-to-cell contact with embryos, ephrin A1, like CD9, regulates the functions of integrins that are expressed on the endometrial luminal epithelial cells, in addition to those expressed on the embryo.
In conclusion, this study demonstrated that ephrin A1 is expressed on human endometrial epithelial cells. On the other hand, mRNA expression of Eph A1 was detected in human embryos in the blastocyst stage, suggesting a physiological role of the Eph-ephrin system in embryo implantation. Further investigation of the expression and functions of these molecules will provide new insights into the mechanisms of the initial embryo-maternal interaction.
Acknowledgments
We are grateful to M. Oshima for technical assistance.
Footnotes
This work was supported in part by Grants-in-Aid for Scientific Research (no. 12470342, 13557140, 13671709, and 13671710).
Abbreviations: FITC, Fluorescein isothiocynate; IVF, in vitro fertilization; mAb, monoclonal antibody; pAb, polyclonal antibody.
Received March 31, 2002.
Accepted August 18, 2002.
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