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Department of Gynecology and Obstetrics (H.F., K.T., K.K., Y.S., T.H., S.Y., S.F.), Faculty of Medicine, Institute for Frontier Medical Science (M.M.), Institute for Virus Research (M.U.), Kyoto University, Sakyo-ku, Kyoto 606-8397, 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 606-8397, Japan. E-mail: fuji{at}kuhp.kyoto-u.ac.jp.
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Abstract |
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Immunohistochemical study showed that ALCAM was expressed on endometrial luminal and glandular epithelial cells but not on the endometrial stromal cells in either the proliferative or secretory phase. Northern blot analysis of isolated endometrial epithelial cells and stromal cells showed that ALCAM mRNA was expressed in endometrial epithelial cells. Flow cytometry confirmed cell surface expression of ALCAM on endometrial epithelial cells. On the other hand, nested RT-PCR analysis demonstrated that ALCAM mRNA was expressed in human blastocysts but not in the embryos in the 8-cell or morula stages, which were obtained from patients undergoing in vitro fertilization treatment.
These findings indicate that ALCAM is expressed on human endometrial epithelial cells and blastocysts. The developing stage-specific expression on the embryo suggests that the ALCAM-ALCAM cell adhesion system is involved in an initial interaction of the embryo with maternal endometrium.
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Introduction |
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Recently, we raised a new monoclonal antibody (mAb, clone 37D) that specifically reacted with activated leukocyte cell adhesion molecule (ALCAM)/CD166 by immunizing mice with human luteal cells (in preparation). Preliminary immunohistochemical study showed that ALCAM is expressed on endometrial epithelium. ALCAM is initially reported and cloned as a cell surface ligand for CD6 molecule, which is a member of the scavenger receptor cysteine-rich family expressed on T lymphocytes (9). It has over 90% homology with the chicken adhesion molecule BEN/SC1/DM-GRASP (10, 11, 12); it also has 30% identity and 50% similarity with human melanoma cell adhesion molecule Mel-CAM/MUC18/CD146 (13). The ALCAM-CD6-mediated interaction is reported to regulate T cell development in the thymus, where thymic epithelial cells express ALCAM (14). Recent studies showed that ALCAM functions as not only a heterophilic (CD6-ALCAM) but also a homophilic (ALCAM-ALCAM) cell adhesion molecule (15). This interaction was shown to be involved in human melanoma invasion (16, 17) and hematopoietic cell differentiation (18). It was also reported that ALCAM-mediated homophilic cell-to-cell interaction is associated with lateral homooligomerization of ALCAM on the cell surface. Such oligomerization and ligand binding can form a tight ALCAM network between the cells (15).
Because these reactions can induce tight cell-to-cell interaction and stem cell differentiation, they can be candidates that anchor and stimulate human embryos on the maternal endometrial luminal surface during the implantation process. Therefore, in the present study, the precise expression profiles of ALCAM in the human endometrium were examined by immunohistochemical study and Northern blot analysis. In addition, using nested RT-PCR, we also investigated the mRNA expression of ALCAM and CD6 on human blastocysts obtained from patients undergoing in vitro fertilization (IVF) treatment.
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Materials and Methods |
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Fluorescein isothiocyanate (FITC)-conjugated and nonconjugated antihuman ALCAM (clone 3A6, IgG1 class) mAbs were obtained from Ancell Corporation, Bayport, MN. The mouse antihuman CD9 mAb (ALB-6, IgG1 class) and antihuman CD13 mAb (MCS-2, IgG1 class) were purchased from Cosmo Bio Co. Ltd. (Tokyo, Japan) and Nichirei Co. (Japan) (Tokyo, Japan), respectively. The mouse antihuman CD45 (leukocyte common antigen) mAb (T29/33, IgG1 class), mouse antihuman cytokeratin-7 mAb (clone OV-TL, IgG1), and FITC-conjugated rabbit antimouse Igs were purchased from DAKO Corp. (Kyoto, Japan). FITC-conjugated and nonconjugated mouse IgG1 negative controls (clone X0931) were also obtained from DAKO Corp.. Rhodamine-conjugated goat antimouse Igs were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The murine antitrinitrophenol mAbs (IgG1 class) were used as blocking mAb for double staining in immunohistochemistry (19).
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. Ten endometria were in the proliferative phase (cycle d 8–13) and 11 in the secretory phase (cycle d 17–24). Endometria in the secretory phase were further staged according to the criteria proposed by Noyes et al. (20). 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 (21). In brief, administration of a GnRH agonist (buserelin acetate; Aventis Pharma Co., Tokyo, Japan) was initiated in the midluteal phase or early follicular phase. All patients subsequently received pure FSH (Serono Laboratories, Inc., Tokyo, Japan) or human menopausal gonadotropin (Organon, 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 the next day (d 1), the zygotes were cultured for another 1 or 2 d . The quality of each embryo was evaluated as one of five grades according to Veeck’s classification (22). Three embryos showing the highest grade were transferred to 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.
The mRNA expression of ALCAM and/or CD6 was examined by nested RT-PCR, as described below, using 13 spare embryos that had developed to various stages: 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 immunofluorescence staining of the human endometrium
Human endometrial frozen sections were prepared as previously described (23). Ten endometrial specimens in the proliferative phase and 11 specimens in the secretory phase, consisting of d 17 (n-1), d 18 (n = 2), d 20 (n = 3), d 21 (n = 3), 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 to 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), antihuman CD13 mAb (5 µg/ml), or mouse IgG1 negative control mAb (5 µg/ml), which were diluted with RPMI 1640 medium containing 10% fetal bovine serum (Life Technologies, Inc., Grand Island, NY) and 0.1% NaN3. After washing in PBS, the slides were incubated with Rhodamine-conjugated goat antimouse Igs (diluted 1:100), for 30 min, at room temperature, in the dark. The washed slides were blocked with antitrinitrophenol mAb (40 µg/ml) for 30 min, then washed again and reacted with FITC-conjugated mouse antihuman ALCAM mAb (10 µg/ml) or FITC-conjugated mouse negative control mAb (10 µg/ml) for 30 min. 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, Jena, Germany).
Isolation of endometrial epithelial cells and stromal cells
Endometrial tissues were minced to 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 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 (24) and washed twice in RPMI. The separated single cells were used as an endometrial stromal cell fraction. The fraction of large clumps containing epithelial cells was incubated in a 10-cm dish (Corning, Inc., New York, 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 and subjected to the following RT-PCR and Northern blot analysis (8). For flow cytometrical analysis, the nonadherent cell clumps were collected again and further treated with 0.05% trypsin, 0.02% EDTA, and 0.005% deoxyribonuclease I, for 5 min, to obtain single cells (25).
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 ALCAM 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 Limited, Buckinghamshire, UK). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotide primers designed based on the sequence of human ALCAM cDNA (Table 1
) or with human S26 primers (sense primer, 5'-GGTCCGTGCCTCCAAGATGA-3': positions 8–27; antisense primer, 5'-TAAATCGGGGTGGGGGTGTT-3': positions 308–327) (26). 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.
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Northern blot analysis was performed as described previously (8). Ten micrograms of total RNA, derived from isolated endometrial epithelial and stromal cell fractions (proliferative phase, n = 1; secretory phase, n = 2), was separated by electrophoresis on a 1% agarose-formaldehyde gel and transferred to a nylon membrane (Hybond-N+; Amersham Pharmacia Biotech UK Limited). The membrane was incubated with prehybridization solution (Rapid Hybridization Buffer; Amersham Pharmacia Biotech UK Limited) for 30 min at 65 C, then hybridized with 32P-labeled ALCAM cDNA probe for 2 h at 65 C in the same solution. After hybridization, the membrane was washed in 2x standard saline citrate (SSC: 2 mM sodium citrate and 20 mM sodium chloride in distilled water, pH7.0) with 0.1% sodium dodecyl sulfate at room temperature for 15 min, and then in 0.2x SSC with 0.1% sodium dodecyl sulfate 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.
Flow cytometric analysis of human endometrial epithelial cells
Freshly isolated endometrial epithelial cells were analyzed by flow cytometry as described previously (27). A single-cell preparation of endometrial epithelial cells was sedimented and incubated with anti-ALCAM mAb (100 µg/ml, 10 µl), anti-CD9 mAb (100 µg/ml, 10 µl), anti-CD13 mAb (100 µg/ml, 10 µl), anti-CD45 mAb (100 µg/ml, 10 µl) or mouse IgG1 negative mAb (100 µg/ml, 10 µl) for 30 min at 4 C. After washing with Hanks’ balanced salt solution, the cell pellet was incubated with FITC-conjugated rabbit antimouse Ig (diluted 1:40, 20 µl), for 30 min at 4 C, in the dark. After washing with Hanks’ balanced salt solution, the cells were resuspended in the same solution, and viable cells were analyzed by flow cytometry (FACScalibur; Becton Dickinson and Co. Immunocytometry Systems Japan, Tokyo, Japan).
Nested RT-PCR analysis of mRNA expression of ALCAM and CD6 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 UK Limited). The resulting cDNA mixtures were subjected to 35 cycles of first-round PCR amplification with oligonucleotides from the ALCAM and CD6 cDNAs as primers (Table 1
). One-tenth-microliter aliquots of cDNAs, obtained from the first amplification, served as templates for a second DNA amplification reaction that used inner nested primers (Table 1). This second-round PCR was carried out 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. The nested PCR product corresponding to ALCAM was sequenced to confirm its identity.
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Results |
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In the normal endometrium, high levels of ALCAM were detected on the luminal surface and on glandular epithelial cells that expressed cytokeratin 7, both in the proliferative and secretory phases (Fig. 1). There was no staining observed in the endometrial stromal cells that expressed CD 13 molecule, a marker of endometrial stromal cells (28). There were no significant differences in the intensity of expression of ALCAM on epithelial cells between the proliferative and secretory phases.
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By flow cytometry, ALCAM was clearly detected on the cell surface of the majority of the isolated endometrial epithelial cells that expressed CD9 molecule, a marker of endometrial epithelial cells (23, 25), whereas CD13 molecule was hardly detected in this cell population (Fig. 2). The positive rate for ALCAM was 95.6 ± 2.2% (n = 3; cycle d 18, 20, and 21).
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Five pairs of endometrial cell fractions, from three specimens in the proliferative phase and two specimens (cycle d 20 and 24) in the secretory phase, were subjected to RT-PCR analyses. PCR products of the expected size of 566 bp for ALCAM were amplified from the cDNAs derived from the endometrial epithelial cell fraction. The sequence of cloned PCR product was identical to that encoding ALCAM.
Northern blot analysis of ALCAM mRNA expression in endometrium
Northern blot analysis showed ALCAM mRNA at 4.7 kbp, which is compatible with the reported size (29), was detected in the RNAs derived from endometrial epithelial cell fraction in the proliferative phase (n = 1) and in the secretory phase (n = 2, cycle d 20 and 24), but was not observed in the RNAs derived from the endometrial stromal cell fraction isolated from the same specimen (Fig. 3).
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By nested RT-PCR analysis using ALCAM primers, a PCR product of 279 bp was detected by ethidium-bromide staining in the cDNA derived from five of six blastocysts examined (Fig. 4). The size of this product clearly differed from that derived from PCR amplification of genomic DNA (4400 bp). The sequence of this product was identical to that encoding ALCAM. Using similar methods, the RT-PCR products corresponding to CD6 could not be detected in this study (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because ALCAM is well known to mediate homophilic (ALCAM-ALCAM) or heterophilic (CD6-ALCAM) cell-to-cell interaction, we examined whether human embryos express ALCAM or CD6. By nested RT-PCR, cDNA products that correspond to ALCAM cDNA sequence were detected in five of six blastocysts examined, whereas such was not observed in mRNA samples derived from embryos in the eight-cell stage or morula stage. The expression of ALCAM protein on the cell surface region of the human blastocysts was immunocytochemically observed by our preliminary experiments (data not shown). These findings indicate that human blastocysts acquire ALCAM expression during their developmental process, suggesting the physiological significance of the ALCAM-ALCAM interaction with maternal endometrial luminar epithelial cells when blastocysts hatch from the zona pellucida.
Recently, it was reported that ALCAM-mediated homophilic cell-to-cell interaction is associated with lateral homooligomerization of ALCAM on the cell surface using the membrane-proximal C-type domains C2C3. However, the aminoterminal domain V1 is critically involved in homophilic ALCAM-mediated cell-to-cell interaction. These oligomerizated ALCAMs were reported to interact with counter ALCAMs expressed on the opposite cells. By this cooperation of oligomerization with ligand binding, ALCAM-ALCAM interaction is considered to induce the formation of a tight and bilayered ALCAM network between the cells (15). In this regard, ALCAM may be an important molecule that anchors human blastocysts to maternal endometrium.
Although the precise mechanisms have not been clarified, ALCAM-ALCAM interaction was proposed to regulate functions and differentiation of some stem cells. Recently, ALCAM was reported to be expressed on hematopoietic stem cells and endothelial progenitors (30, 31). When ALCAM-ALCAM-mediated interaction between stromal cells and hematopoietic cells was interrupted, not only hematopoietic development, but also maintenance of hematopoietic stem cells, were inhibited, showing its important role in hematopoiesis. In addition, they also demonstrated that ALCAM-ALCAM-mediated interaction among endothelial cells is necessary for tubal formation in angiogenesis (31). Furthermore, it was reported that ALCAM-positive mesenchymal stem cells in the perichondrium, which can differentiate into osteoblasts, adipocytes, chondrocytes, and stromal cells, play an important role in formation of bone marrow by regulating recruitment of osteoclasts and vessels through ALCAM-mediated cell-to-cell interaction (32). Based on these reports, it is reasonably speculated that the estimated ALCAM-ALCAM interaction between blastocysts and endometrial epithelial cells is one of the important candidates that induce differentiation in human embryos when they first encounter maternal tissues. The developing stage-dependent expression of ALCAM on the embryo may reflect its physiological significance.
In conclusion, this study demonstrated that ALCAM is expressed on human endometrial epithelial cells and blastocysts. The differentiation stage-specific expression of ALCAM on the embryo suggests that ALCAM-ALCAM interaction is involved in the initial step of human embryo implantation.
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Acknowledgments |
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Footnotes |
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Abbreviations: ALCAM, Activated leukocyte cell adhesion molecule; CD, cluster of differentiation; FITC, fluorescein isothiocyanate; IVF, in vitro fertilization; mAb, monoclonal antibody.
Received November 30, 2002.
Accepted March 12, 2003.
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) function of murine B cells. J Immunol 144:4571–4578[Abstract]
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