Molecular Human Reproduction, Vol. 5, No. 2, 162-167,
February 1999
© 1999 European Society of Human Reproduction and Embryology
CD9 is expressed in extravillous trophoblasts in association with integrin 
3 and integrin 5
1 Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, 2 Institute for Virus Research, and 3 Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto, 606, Japan
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Abstract |
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The CD9 molecule is a 24–27 kDa cell surface glycoprotein, which may be related to Schwann cell migration and adhesion. In this study, we examined the expression of CD9 in human extravillous trophoblasts, which invade into the endometrium during implantation and placentation. CD9 was detected immunohistochemically on the extravillous trophoblasts in the cell columns of first trimester placentae, but not on villous trophoblasts. In the second and third trimester, CD9 was highly expressed on the extravillous trophoblasts in the basal plate of placentae, and in the chorion laeve in the fetal membrane of term placentae. The molecular mass of CD9 in the chorion laeve was shown to be 27 kDa by Western blotting. The mRNA of CD9 was also detected in the chorion laeve by reverse transcription–polymerase chain reaction (RT–PCR). Proteins were purified from chorion laeve by affinity chromatography with anti-integrin
3 and 5 monoclonal antibodies and Western blotting, revealed that CD9 was associated with both integrins. These findings indicate that CD9 is a differentiation-related molecule present in the extravillous trophoblasts. Since it is associated with integrin 5 which has been proposed to regulate trophoblast invasion, CD9 may be implicated in trophoblast invasion at the feto–maternal interface. CD9/differentiation/extravillous trophoblast/integrin/monoclonal antibody
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Introduction |
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Recently, it has been demonstrated that the cluster of differentiation (CD) antigen 9 molecules, originally reported to be expressed on a pre-B cell line (Kersey et al., 1981
), is associated with ß1-related integrins on the cell surface (Rubinstein et al., 1994). Therefore, we examined the immunohistological expression of CD9 in the reproductive organs, including the endometrium during early pregnancy. We found that CD9 is expressed on the extravillous trophoblasts in the cell columns of first trimester placentae, which then differentiate into invading extravillous trophoblasts. Trophoblast invasion is one of the most important steps of implantation and placentation. Furthermore, many elements of trophoblast invasion resemble the events that occur during malignant tumour cell invasion (Aplin, 1991; Tabibzadeh and Babaknia, 1995). Unlike malignant tumour cells, trophoblast invasion is confined spatially to the uterus and temporally to early pregnancy. However, the regulatory mechanisms which prevent the extravillous trophoblasts from further invasion are not well understood. Since CD9 was reported to be related to Schwann cell migration and adhesion (Anton et al., 1995; Hadjiargyrou and Patterson, 1995), it is important to determine whether CD9 is physiologically involved in trophoblast invasion. Thus, we used immunohistochemistry to examine the expression of CD9 on the extravillous trophoblasts localized at the feto–maternal interface during the pregnancy.
Extravillous trophoblasts in the feto–maternal interface have been reported to express integrin 3 and integrin 5, which may be involved in trophoblast invasion (Damsky et al., 1994; Irving and Lala, 1995). Therefore, we also examined the possible association of CD9 with these integrins.
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Materials and methods |
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Tissues
First trimester human placentae (6–10 weeks gestation, n = 5) were obtained from patients who had undergone legal abortions. The gestational age was calculated from the date of the last menstrual period and, where necessary, was adjusted by taking into account ultrasonic measurements of the gestational sac and the fetal crown–rump length. Second trimester placentae (15–21 weeks gestation, n = 5) were also obtained following legal abortions. Term placentae (n = 5) and term fetal membranes (n = 5) were obtained at normal deliveries. Informed consent for the use of these tissues in this study was obtained from all donors.
Antibodies
Two mouse anti-human CD9 monoclonal antibodies (mAb) [TP-82 and ALB-6, immunoglobulin (Ig) G1 class] were purchased from Nichirei Co Ltd (Tokyo, Japan) and Cosmo Bio Co Ltd (Tokyo, Japan) respectively (Boucheix et al., 1983; Higashihara et al., 1985). Another anti-CD9 mAb (SYB-1, IgG1) was a generous gift from Dr C.Boucheix, INSERM U268, Hospital Paul Brousse (Rubinstein et al., 1994). The mouse anti-cytokeratin mAb (NCL-5D3, IgG2a) was obtained from Cosmo Bio Co. Ltd (Angus et al., 1987). Mouse anti-human integrin 3 (11G5, IgG1) and 5 (SAM 1, IgG2b) were purchased from Serotec (Oxford, UK) and mouse anti-human integrin ß1 (K20, IgG2a) was obtained from DAKO A/S (Glostrup, Denmark) (Morimoto et al., 1985). In the immunohistochemical and Western blot analyses, anti-trinitrophenyl (TNP) mouse mAb (unrelated mAb, IgG1 class) was used as a negative control (Tsujimura et al., 1990). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulins (Dakopatts, Glostrup, Denmark) were used as secondary antibodies for the immunohistochemistry. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulins (Dakopatts) were used as secondary antibodies in Western blotting.
Immunohistochemistry
Cryosections of the tissues were prepared as previously described, with slight modifications (Honda et al., 1995). Briefly, fresh tissues were embedded in an OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL, USA), snap-frozen in liquid nitrogen and stored at –80°C. The frozen tissues were cut into 7 µm thick sections using a cryostat microtome (Histostat; Reichert-Jung, Heidelberg, Germany). The sections were immediately and thoroughly air-dried on Neoplene (Nisshin EM Co Ltd, Tokyo, Japan) coated glass slides, and then fixed with acetone at –20°C. The cryosections were incubated with the anti-CD9 mAb (5 µg/ml), anti-integrin 3 (5 µg/ml), anti-integrin 5 (5 µg/ml), anti-integrin ß1 (5 µg/ml), anti-cytokeratin (5 µg/ml), or the anti-TNP mAb (5 µg/ml) for 60 min at room temperature. The antibodies were diluted with Roswell Park Memorial Institute (RPMI) culture medium (GIBCO, Grand Island, NY, USA) containing 10% fetal calf serum (FCS; Dainippon Pharmaceutical Co., Osaka, Japan) and 0.1% NaN3. After washing in phosphate-buffered saline (PBS), the slides were incubated with the FITC-conjugated rabbit anti-mouse immunoglobulins (diluted 1:40) for 30 min at room temperature in the dark. The slides were washed extensively, mounted with an anti-fade agent (Perma Fluor Aqueous Mounting Medium; Immunon, Pittsburgh, PA, USA), and then examined by fluorescence microscopy (Nikon, Tokyo, Japan). Some serial sections were also stained with haematoxylin and eosin prior to examination under a light microscope.
RNA isolation
Human chorion laeve tissue of term fetal membrane was immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. The total RNA of these tissues was isolated using a commercial kit (TRIzol; Gibco BRL, Gaithersburg, MD, USA).
RT–PCR analysis of CD9 mRNA in the human chorion laeve
Total RNA (5 µg) from chorion laeve were reverse-transcribed with random primers using a commercial kit (First Strand cDNA Synthesis Kit; Pharmacia Inc, Piscataway, NJ, USA). The resulting cDNA mixtures were subjected to 30 cycles of PCR amplification with oligonucleotides from the human CD9 cDNA as primers (Boucheix et al., 1991) (sense primer 5'-ACTGTTCTTCGGCTTCCTCT-3': position 321–340; antisense primer 5'-AAAATCCCAAAAATCTTCAT-3': position 774–793) or with human S26 primers (Vincent et al., 1993) (sense primer 5'-GGTCCGTGCCTCCAAGATGA-3': position 8–27; antisense primer 5'-TAAATCGGGGTGGGGGTGTT-3': position 308–327). After PCR amplification, 10 µl from each PCR reaction was electrophoresed on a 1% agarose gel, and amplified bands were detected by ethidium-bromide staining. The resultant PCR fragment was cloned into a pBluescript SK(–) plasmid. DNA sequencing was performed using a commercial kit (BcaBEST Dideoxy Sequencing Kit, Takara, Ohtsu, Japan).
Western blotting
Chorion laeve tissue (10 mg, wet weight) was lysed in sample buffer [20 mM Tris–HCl pH 8.6, 1% sodium dodecyl sulphate (SDS), 20% glycerol, Bromophenol Blue], and the lysed proteins were separated under non-reducing conditions by 12% SDS–polyacrylamide gel electrophoresis (PAGE). The bands were then electrically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, USA) in a buffer containing 25 mM Tris–HCl, 192 mM glycine and 20% methanol. The membranes were soaked in a blocking solution, PBS containing 1% bovine serum albumin (BSA) for 3 h at room temperature, and were then incubated for 1 h with the anti-CD9 mAb (SYB-1, diluted ascites 1:10 000 in PBS containing 0.1% BSA) or the control mAb (anti-TNP, 1 µg/ml in PBS containing 0.1% BSA). The membranes were washed several times with PBS, and incubated for 1 h with HRP-conjugated rabbit anti-mouse IgG (diluted 1:1000 in PBS containing 0.1% BSA). After several washes, the binding of the antibodies was visualized by incubation with 0.5 mg/ml of diaminobenzidine and 0.02% H2O2 in PBS.
Affinity chromatography and Western blotting
Chorion laeve (0.5 g, wet weight) was homogenized in 5 ml of 40 mM phosphate buffer, pH 7.3, containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1% 3-[13-cholamidopropyl)dimethylammonio]-1-propane-sulphate (CHAPS), 0.2 mg/ml phenylmethylsulphonyl fluoride hydrochloride (Wako Pure Chemicals, Osaka, Japan), 10 µg/ml leupeptin (Peptide Institute Inc, Osaka, Japan), and 10 µg/ml pepstatin (Peptide Institute Inc). After centrifugation (11 000 g for 30 min), the supernatant was passed at 4°C through a column containing 10 ml of anti-TNP-conjugated Affigel 10 (Bio-Rad Laboratories, Hercules, CA, USA; 2 mg IgG/ml gel) which caused certain compounds to bind non-specifically with low affinity. The non-bound fraction was incubated with 0.2 ml of anti-integrin 3 or 5-conjugated Affigel 10 (80 µg IgG/ml gel) at 4°C for 2 h. After extensively washing the gel, the antigen and its associated protein were eluted with SDS buffer. After 12% SDS–PAGE (under non-reducing conditions) they were transferred onto a PVDF membrane. The membranes were stained with anti-CD9 mAb (SYB-1) or the control mAb (anti-TNP) as described above.
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Results |
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Immunohistochemical analysis of CD9 expression in human placentae
In chorionic villi of the first trimester, weak CD9 expression was observed in the extravillous trophoblasts of the cell columns, whereas CD9 was only minimally present on the invading interstitial trophoblasts in the decidua (Figure 1B
). In contrast, CD9 expression was not detected in the villous cytotrophoblasts or in the syncytiotrophoblasts (Figure 1B). CD9 was also weakly expressed in the stromal cells of the floating villi as previously reported (Morrish et al., 1991).
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In the second and third trimester placentae, the extravillous trophoblasts in the basal plate expressed cytokeratin strongly. In these cells, intense CD9 immunostaining was also detected (Figures 1E and 2B
). In the floating villi, CD9 was expressed in the stromal cells, but not in the villous cytotrophoblasts or in the syncytiotrophoblasts.
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In the fetal membranes at term pregnancy, CD9 was strongly expressed in the amniotic epithelial cells and the extravillous trophoblasts of the chorion laeve, but not in the decidual cells (Figure 2E
). Immunohistochemical analyses, using the two anti-CD9 mAb clones TP-82 and ALB-6, showed similar profiles of CD9 expression.
Integrins 3, 5, and ß1 were expressed on the extravillous trophoblasts in the basal plates of the second and third trimester placentae as described previously (Korhonen et al., 1991), and these integrins were also expressed on the chorion laeve in the fetal membrane (data not shown).
RT–PCR analysis of CD9 mRNA expression in the chorion laeve of term fetal membrane
The expression of CD9 mRNA was observed in the chorion laeve of term fetal membrane, (Figure 3, lanes 2 and 3). The nucleotide sequence of the PCR product, 473 bp in length, in the chorion laeve was analysed by DNA sequencer and confirmed to be identical to that of CD9 cDNA as previously reported (Boucheix et al., 1991). The expected PCR product of S26 were also detected in the chorion laeve.
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Western blotting analysis of CD9 protein expression in the chorion laeve of term fetal membrane
Using Western blotting, the antigen in the chorion laeve of term placenta was detected as a single 27 kDa protein band, which was compatible with the reported molecular mass of CD9 (Rubinstein et al., 1994
) (Figure 4, lane 2).
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Association of CD9 with integrin
3 and 5 in the chorion laeve of term fetal membraneFollowing Western blotting analysis of the proteins purified with anti-integrin
3 or 5 mAb, CD9 was detected as a single 27 kDa protein band (Figure 5), which is compatible with that observed in the whole lysate of the chorion laeve in Figure 4.
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Discussion |
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Human trophoblasts are believed to differentiate into two main cell lineages: villous and extravillous cells. Villous trophoblasts ultimately cover all of the chorionic villi of the placenta, their function being to transport oxygen and nutrients from the mother to the fetus. Since the villi are bathed in blood, villous trophoblasts are in contact with the maternal cells present in the circulation. In contrast, extravillous trophoblasts migrate deeply into the uterine mucosa, and thus encounter the many types of maternal cells present in the endometrium (Loke and King, 1995
). Extravillous trophoblasts are believed to be derived from the cell column of the anchoring villi, and differentiate into interstitial trophoblasts in the placental bed. In non-placental sites, they constitute the chorion laeve.
In this study, we have demonstrated the intense expression of CD9 in the extravillous trophoblasts of the placental bed and chorion laeve using immunohistochemistry, RT–PCR and Western blotting. In addition, CD9 was weakly expressed on the cell column of the chorion. On the other hand, it was not expressed on villous trophoblasts as previously reported (Morrish et al., 1991). Based on this expression profile, we concluded that CD9 is a differentiation-related molecule and may be involved in the functions of the extravillous trophoblasts.
CD9 was initially considered to be specific to acute lymphoblastic leukaemia cells (Kersey et al., 1981). This antigen was also expressed on a variety of tumours and normal human cells, including pre-B cells, activated T cells, platelets and Schwann cells (Jennings et al., 1990; Masellis-Smith et al., 1990; Miyake et al., 1991; Anton et al., 1995). Although its physiological role is unknown, recent studies have shown that anti-CD9 mAb induces the migration of Schwann cells (Anton et al., 1995), and that CD9 regulates the adhesion of pre-B cells to bone marrow fibroblasts (Masellis-Smith and Shaw, 1994), suggesting its involvement in cell adhesion and migration. Since extravillous trophoblasts are able to invade the endometrium, we postulated a possible role for CD9 in extravillous trophoblast invasion.
Recently, it has been reported that ß1-related integrins, especially integrin 5ß1, are involved in trophoblast invasion, which has been proposed to be regulated by the switching of integrin expression (Damsky et al., 1992, 1994; Burrows et al., 1996). We demonstrated immunohistochemically that extravillous trophoblasts in the placental bed express integrins 3ß1 and 5ß1, as described by Korhonen et al. (1991), and that chorion laeve also express these integrins. Since CD9 is known to be associated with ß1-related integrins in other cells (Rubinstein et al., 1994; Nakamura et al., 1995), CD9 in the extravillous trophoblasts may modulate the function of ß1-related integrins. Therefore, we investigated whether or not CD9 expressed on the extravillous trophoblast is associated with these integrins. Western blotting analysis of the affinity-purified proteins using anti-integrin 3 and 5 mAb demonstrated that both integrin 3 and 5 in the chorion laeve were associated with CD9, suggesting a functional relationship between CD9 and these integrins.
Ikeyama et al. (1993) found that transfecting the CD9 gene into several cell lines suppressed cell motility and metastasis. In addition, the expression of CD9 in breast cancer appeared to be inversely correlated with its metastatic potential (Miyake et al., 1995). These studies suggest that CD9 is involved in the suppression of cell invasion. Although similarities between trophoblast invasion and tumour cell invasion have been investigated extensively (Yagel et al., 1988; Fisher et al., 1989; Librach et al., 1991; Burrows et al., 1995), the factors which limit trophoblast invasion within the uterus and which cause these invading trophoblasts to revert into their quiescent, non-invasive state are largely unknown. Damsky et al. (1992, 1994) reported that the expression of integrin 5 on extravillous trophoblast is maintained during invasion process, suggesting that integrin 5 is involved in trophoblast invasion. On the other hand, CD9 was initially detected on the cell column, after which its expression on invading interstitial trophoblasts decreased. However, a high level of expression of CD9 was observed on the extravillous trophoblasts in the basal plate of second and third trimester placentae and chorion laeve, which have already ceased invasion. Combined with the above hypothesis of a suppressive role for CD9 in tumour invasion, the dynamic changes in CD9 expression observed in this study suggest its inhibitory regulation of extravillous trophoblast invasion.
In conclusion, we have demonstrated immunohistochemically that CD9 is a differentiation-related molecule present in human extravillous trophoblasts. It was highly expressed on the extravillous trophoblasts that have ceased invasion. In addition, in non-invasive extravillous trophoblasts, CD9 was associated with integrin 5, which has been reported to regulate trophoblast invasion. Thus, CD9 may be involved in the regulation of extravillous trophoblast invasion at the fetomaternal interface. Further investigation into its role in extravillous trophoblasts may provide further clarification of the protective mechanisms against both excessive trophoblast invasion and malignant tumour invasion.
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Acknowledgments |
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This work was supported in part by Grants-in-Aid for Scientific Research (no. 09671673, 09671674, 09671676).
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Notes |
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4 To whom correspondence should be addressed
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Submitted on June 18, 1998; accepted on November 5, 1998.
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