Molecular Human Reproduction, Vol. 5, No. 4, 303-310,
April 1999
© 1999 European Society of Human Reproduction and Embryology
CD9 is expressed on the cell surface of human granulosa cells and associated with integrin 
6ß1
1 Department of Gynecology and Obstetrics, Faculty of Medicine, 2 Institute for Frontier Medical Science, and 3 Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606, Japan
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The CD9 molecule is a 24–27 kDa cell surface glycoprotein which is reported to be involved in cell adhesion and migration. Recently, CD9 was shown to be associated with ß1-related integrins. We have previously found that integrin
6ß1 is expressed on human granulosa cells (GC) and regulates luteinization of GC in concert with its ligand laminin. In this study, we examined the expression of CD9 in human ovary and the relationship between CD9 and integrin 6ß1 in GC. By immunohistochemistry, CD9 was detected on GC in a small antral follicle of <1 mm in diameter. In growing follicles, CD9 was moderately expressed on both GC and theca interna cells (TI). The expression intensity of CD9 on GC increased in preovulatory follicles. In the early luteal phase, CD9 was expressed in both luteinizing GC and TI. The expression intensity on large luteal cells decreased in the mid-luteal phase. In the corpus luteum (CL) of pregnancy, CD9 continued to be expressed on large luteal cells, but not on small luteal cells. By flow cytometry, CD9 was detected on the cell surface in ~90% of the isolated GC from patients undergoing in vitro fertilization. The molecular weight of CD9 in the isolated GC was shown to be 26.5 kDa by Western blotting. CD9 mRNA was also detected in the isolated GC and CL by reverse transcription–polymerase chain reaction (RT–PCR). The proteins purified from GC by immunoaffinity chromatography using anti-integrin 6 monoclonal antibodies were shown by Western blotting to include CD9 as well as integrin ß1. These findings suggest that CD9 is a differentiation-related molecule of GC and TI and that it is associated with integrin 6ß1 on the cell surface of GC, suggesting that CD9 is implicated in the function of human GC in cooperation with integrin 6ß1.
CD9/granulosa cells/integrin 6ß?1/luteal cells
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To investigate new mechanisms of ovarian function, we raised several monoclonal antibodies (mAbs) against granulosa cells (GC) and luteal cells (Fujiwara et al., 1993a
, 1994a, 1996a). One of these mAbs, OG-1, reacted with an antigen expressed on the cell surface of human GC in medium- and large-sized follicles and corpora lutea (CL) of the early luteal phase (Fujiwara et al., 1993b). We purified the OG-1 antigen from the human placenta and partially sequenced the N-terminal amino acids, which revealed that the OG-1 antigen is identical to integrin 6 (CD49f). Human GC express integrin ß1 (CD29), but not integrin ß4 (CD104), indicating that integrin 6 expressed on GC forms a heterodimer with ß1 but not with ß4 (Honda et al., 1995). Since the ligand for integrin 6ß1 is laminin, we examined physiological functions of the interaction between integrin 6ß1 and laminin on luteinizing GC and found that laminin suppressed luteinization of GC via the interaction with integrin 6ß1, indicating that integrin 6ß1 is a new regulator of luteinization of GC during corpus luteum formation (Fujiwara et al., 1997). We also demonstrated integrin 5ß1 and its ligand, fibronectin, in the luteinizing CL (Honda et al., 1997).
Similarly, we raised another mAb, POG-2, which reacted with the cell surface of porcine GC. By means of analysis of partial amino acid sequence of the antigen purified from the porcine ovary, the POG-2 antigen was also determined to be a porcine homologue of integrin 6 (Fujiwara et al., 1995). In contrast to human ovary, integrin 6 was expressed on GC in the small follicles (1–2 mm in diameter) with maximal immunoreactivity in the porcine ovary. These stage-specific expression profiles suggested the involvement of integrin 6 in folliculogenesis (Fujiwara et al., 1996b). In both human and porcine ovaries, integrin 6ß1 was also expressed on the cell surface of GC located in the inner layers, which are not in contact with the basal lamina (Fujiwara et al., 1996b). In the mouse ovary, the same expression profile of integrin 6 was observed on the inner layers of GC in primary and secondary follicles. The systemic administration of anti-murine integrin 6 mAb, which partially blocks the interaction between integrin 6ß1 and laminin, enhanced follicular growth in immature mice when they were stimulated by exogenous gonadotrophins in vivo, suggesting the involvement of integrin 6ß1 in granulosa cell function or differentiation (Nakamura et al., 1997).
Recently, it was shown that the CD9 molecule, which was originally reported to be expressed by a pre-B cell line (Kersey et al., 1981), is associated with ß1-related integrins on the cell surface in the various cells (Rubinstein et al., 1994). Although physiological role of CD9 is unknown, recent work showed that anti-CD9 mAb induced proliferation, adhesion and migration of Schwann cells (Anton et al., 1995; Hadjiargyrou and Patterson, 1995) and that CD9 regulates the adhesion of pre-B cells to bone marrow fibroblasts (Masellis-Smith and Shaw, 1994). In addition, it was reported that CD9 in mouse brain is associated with integrin 6ß1, suggesting that CD9 co-operates in neurite outgrowth on laminin in vitro (Schmidt et al., 1996).
In this study, to investigate the involvement of CD9 in GC differentiation, we examined the expression of CD9 in human ovary by immunohistochemistry, flow cytometry, Western blotting and reverse transcription–polymerase chain reaction (RT–PCR). We also investigated the association of CD9 with integrin 6ß1 on the ovarian cells by Western blotting using immunoaffinity-purified proteins.
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Antibodies
Two mouse anti-human CD9 mAbs [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. Another anti-CD9 mAb (SYB-1) was a generous gift from Dr C.Boucheix (INSERM U268, Hôpital Paul Brousse, Villejuif, France). The mouse anti-human integrin ß1 mAbs (DF5, IgG1 class and P4C10, IgG1 class) were purchased from Affinity Research Products Ltd (Nottingham, UK) and Life Technologies Inc (Gaithersburg, MD, USA) respectively. In immunohistochemical and Western blot analyses, anti-trinitropheny(TNP) mouse mAb (unrelated mAb, IgG1 class) was used as negative controls (Tsujimura et al., 1990
). The mouse anti-human integrin 6 mAb (OG-1, IgG1 class) was produced in our laboratory (Fujiwara et al., 1993b, 1997; Honda et al., 1995). Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin (Dakopatts, Glostrup, Denmark) were used for second antibody in immunohistochemistry. Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulins (Dakopatts) were used as a second antibody in Western blotting.
Human ovaries
A small antral follicle (<1 mm in diameter, n = 1), growing follicles (4–12 mm in diameter, n = 5), preovulatory follicles (18–20 mm in diameter, n = 4), CL of the menstrual cycle (n = 25) were obtained from 35 patients, aged 30–45 years, who had regular menstrual cycles and underwent surgery for benign gynaecological disease. CL of pregnancy, ranging from 6 to 15 weeks, were obtained from five pregnant patients who underwent hysterectomy for the treatment of cervical cancer or uterine myoma. The fetal growth of all five patients was normal on ultrasonographic examination. Informed consent was obtained from each patient.
Follicles were morphologically evaluated by staining cryosections with haematoxylin and eosin (H&E). Follicles with GC having mitotic figures and regularly shaped nuclei, cytoplasm, and stratified layers were classified as growing follicles (Ryan, 1981). If judgement was difficult, the H&E-stained sections from the same samples, fixed with 10% formalin and embedded in paraffin wax, were used. The post-ovulatory date of the CL was evaluated according to the histological dating method of Corner (1956), using H&E-stained tissue sections of 10% formalin-fixed and paraffin wax-embedded samples. In this work, the term `CL day' was used according to his definition. For example, CL day 2 is the day after ovulation.
Indirect immunohistochemical staining of frozen sections
Indirect immunofluorescence histochemistry was performed as described (Fujiwara et al., 1993a). Briefly, each specimen was embedded in OCT compound (Tissue-Tec, Miles Scientific, Naperville, IL, USA), snap-frozen in liquid nitrogen and stored at –80°C. Frozen tissues (7 µm thick) were cut 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 incubated with anti-CD9 (2 µg/ml) or anti-TNP (2 µg/ml) mAb for 30 min at room temperature. After washing in phosphate-buffered saline (PBS), they were incubated with FITC-conjugated rabbit anti-mouse second antibody (diluted 1:50) for 30 min at room temperature in the dark. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA, USA), and examined under a fluorescence microscope (Nikon, Tokyo, Japan) by two individuals within 6 h. The intensity of antigen expression was graded from – to +++ based on its fluorescence intensity (– = absence of staining; + = weak staining; ++ = medium staining; +++ = intense staining). If their judgements differed, the lower value was selected. Serial cryosections were also stained with HE after fixation in 95% ethanol.
Isolation of human luteinizing GC
Human GC were isolated from 50 patients, aged from 27–38 years old, undergoing in-vitro fertilization (IVF) as described previously (Fujiwara et al., 1994b). Briefly, patients receiving a gonadotrophin-releasing hormone analogue (buserelin acetate; Hoechst Marion Roussel Ltd, Tokyo, Japan) from the first day of the cycle, were stimulated with human menopausal gonadotrophin (Organon Japan Co Ltd, Tokyo, Japan) until the follicles reached maturity. Follicles were aspirated 36 h after the administration of human chorionic gonadotrophin (Mochida Pharmaceutical Co Ltd, Osaka, Japan). The follicular fluid was collected and centrifuged. The sedimented cells were resuspended, overlayered on lymphocyte separation medium (Organon Teknika Co, Tokyo, Japan) and centrifuged at 400 g for 20 min. The cells were collected from the interphase. The isolated GC were immediately analysed by flow cytometry or kept at –80°C for Western blotting, RT–PCR, and immunoaffinity chromatography.
Flow cytometrical analysis of human luteinizing GC
Flow cytometry proceeded as described (Fujiwara et al., 1994b). The isolated human GC were washed in Hanks' balanced salt solution (HBSS) with 0.1% bovine serum albumin (BSA, Nitta Gelatin Corp, Osaka, Japan) and 0.1% NaN3, sedimented by centrifugation and incubated with anti-CD9 mAb (TP82, 100 µg/ml, 10 µl), anti-integrin 6 mAb (OG-1, 100 µg/ml, 10 µl), anti-integrin ß1 mAb (P4C10, diluted 1:50 from ascites, 10 µl) or anti-TNP mAb (100 µg/ml, 10 µl) for 30 min at 4°C. After washing with HBSS, the cell pellet was incubated with FITC-conjugated rabbit anti-mouse Ig (diluted 1:40, 20 µl), for 30 min at 4°C in the dark. After washing in HBSS, the cells were resuspended in the same solution and viable cells were analysed by flow cytometry (FACScan, Becton Dickinson Immunocytometry Systems Japan, Tokyo, Japan). The ratio of contaminating monocytes, identified by the anti-CD14 mAb (Becton Dickinson, Lincoln Park, NJ, USA), was <3%.
Western blotting
The isolated GC (1.3x106) or endometrial tissue at secretory phase (0.2 g, wet tissue) were lysed in sample buffer [2 ml, 20 mM Tris–HCl pH 8.6, 1% sodium dodecyl sulphate (SDS), 20% glycerol, BPB] containing p-amidinophenylmethanesulphonylfluoride hydrochloride (Wako Pure Chemicals; 1 mM), leupeptin (Peptide Institute; 20 µg/ml), and pepstatin (Peptide Institute; 20 µg/ml), separated by 12% SDS–polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions, and electrically transferred onto a polyvinyl difluoridine fluoride (PVDF) membrane (Millipore Corp, Bedford, MA, USA) in a buffer containing 25 mM Tris–HCl, 192 mM glycine, 0.03% SDS and 20% methanol. The membranes were blocked with a blocking agent (Block Ace, Snow Brand Milk Products Co Ltd, Tokyo, Japan), washed in PBS three times and incubated with mAb solutions (diluted ascites 1:1400 in PBS containing 0.1% BSA for SYB-1, 0.2 µg/ml for anti-TNP mAb, and 0.2 µg/ml for anti-integrin ß1 mAb) for 2 h at room temperature. The membranes were washed several times with PBS, and incubated for 1 h with HRP-conjugated rabbit anti-mouse IgG (diluted 1:500 in PBS containing 0.1% BSA). After several washings, the binding of the antibodies was visualized by incubation with 0.2 mg/ml of 3,3'-diaminobenzidine tetrahydrochloride and 0.006% H2O2 in PBS.
RNA isolation
The isolated GC and CL were immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. Total RNAs of these tissues was isolated using a commercial kit (TRIzol, Gibco BRL, Gaithersburg, MD, USA).
RT–PCR analysis of CD9 mRNA in the human GC and CL
Total RNAs from GC and CL (5 µg) were reverse-transcribed with random primers by 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 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 product was electrophoresed on a 1% agarose gel, and amplified bands were detected by ethidium bromide staining.
Western blotting and silver staining of the integrin 6-associated proteins purified from GC
Integrin 6 and the associated proteins were purified by immunoaffinity chromatography as previously described with several changes (Honda et al., 1995). Briefly, OG-1 mAb was conjugated with activated gels (Affi-Gel 10, Bio Rad; 2 mg IgG/ml gel). The isolated GC (2.2x107) were homogenized in a Polytron A (Kinematica AG, Switzerland) in 40 mM phosphate buffer, pH 7.3, containing 150 mM NaCl, 1 mM CaCl2 1 mM MgCl2 1% 3,3-cholamidopropyl-dimethylammonio-1-propanesulphonic acid (CHAPS, Wako Pure Chemicals, Osaka, Japan), and protease inhibitors p-amidinophenylmethanesulphonylfluoride hydrochloride (0.25 mg/ml), leupeptin (20 µg/ml), and pepstatin (20 µg/ml). After centrifugation (9000 g; 20 min), the concentration of CHAPS in the lysate was reduced by dilution to 0.3%. The lysate was passed through a column of anti-TNP mAb-conjugated gel (2 mg/ml IgG per gel) to remove non-specifically bound compounds, then incubated with OG-1 mAb-conjugated gel (100 µl gel) or anti-TNP mAb-conjugated gel at 4°C for 2 h. After extensive washing of the gel, the antigen was eluted with SDS sample buffer at 100°C for 2 min. The eluted proteins were separated by 12% SDS–PAGE under non-reducing conditions, and electrically transferred onto a PVDF membrane. The blotted proteins were stained using anti-CD9 mAb (SYB-1), anti-integrin ß1 mAb (DF5), and anti-TNP mAb as described above. In some experiments, the separated proteins by 12% SDS–PAGE were stained with a silver stain kit (Wako Pure Chemicals).
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Immunohistochemical analysis of CD9 antigen expression in human ovary
In an antral follicle of <1 mm in diameter, CD9 was detected on GC, but not on theca interna cells (TI) (data not shown). In growing follicles (4–12 mm in diameter), CD9 antigen was moderately expressed on GC and moderately or intensely on TI (Figure 1
and Table I). In preovulatory follicles (18–21 mm in diameter), CD9 was moderately or highly expressed on GC and TI (Figure 2).
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In CL in the early luteal phase (CL days 2–5, n = 7), CD9 was moderately expressed on GC/large luteal cells, whereas it was weakly detected on TI/small luteal cells (Figure 3
). In CL in the mid-luteal phase (CL days 6–9, n = 12), CD9 was weakly detected on large luteal cells and weakly to highly expressed on small luteal cells (Figure 4). In the late luteal phase (CL days 12–14, n = 6), CD9 was moderately or weakly detected on large luteal cells and weakly expressed on small luteal cells (data not shown).
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In CL of pregnancy (n = 5), CD9 was expressed on large luteal cells in weak intensity, but not on small luteal cells (data not shown). These immunohistochemical analyses were carried out using two anti-CD9 mAbs, clone TP82 and ALB6, and showed the similar profiles of CD9 expression. The expression of CD9 was also observed in the vessels. The expression profiles of CD9 are summarized in Table I
. Similar expression profiles of integrin 6 on GC/large luteal cells have been reported (Fujiwara et al., 1993b; Honda et al., 1995).
Detection of CD9 on the cell surface of the freshly isolated GC
By indirect immunofluorescence staining, expression of CD9 antigen was observed on the cell surface of freshly isolated GC (data not shown). The staining pattern of CD9, integrin 6 or integrin ß1 was uniform around the surface of GC. Flow cytometry showed that percentage positivity of CD9, integrin 6, integrin ß1 on GC was 88.9 ± 5.8%, 86.4 ± 6.5%, and 84.7 ± 9.6% respectively (mean ± SD, n = 3, Figure 5).
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Western blotting analysis of CD9 and integrin ß1 on GC and endometrium
By Western blotting with anti-CD9 mAb (SYB-1), CD9 was detected as a single 26.5 kDa protein band from the lysate of GC, which was compatible with the reported molecular mass of the CD9 antigen (Figure 6
: lane 1) (Kersey et al., 1981). Integrin ß1 was also detected with anti-integrin ß1 mAb (DF5) as a single protein band at 175~180 kDa in the GC (Figure 6: lane 3). On the other hand, integrin ß1 from the endometrium was detected as a single protein band at 110 kDa (Figure 6: lane 2).
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RT–PCR analysis of CD9 mRNA expression in GC and CL
The expression of CD9 mRNA was observed in GC and CL (Figure 7
). The nucleotide sequence of the PCR product, 473 bp in length, from GC was analysed by DNA sequencing and confirmed to be identical to that of CD9 cDNA (Boucheix et al., 1991).
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Silver staining and Western blotting of the integrin
6-associated proteins purified from GCThe integrin
6 with the associated proteins were purified from GC by immunoaffinity chromatography using OG-1 mAb (anti-integrin 6), or anti-TNP mAb as the negative control. This procedure was repeated three times. The visualization of proteins by silver staining after SDS–PAGE showed that the purified proteins using OG-1 mAb were detected as specific bands at 175–180, 145, 110–115, 59, and 26.5 kDa respectively (Figure 8). The main band, 145 kDa, corresponded to integrin 6 as reported by Fujiwara et al. (1993b), and the others could be concluded to be the proteins co-purified with integrin 6.
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By the Western blotting analysis of the proteins obtained by the purification as above, anti-CD9 mAb (SYB-1) reacted with a 26.5 kDa protein and anti-integrin ß1 mAb (DF5) reacted with a 175–180 kDa protein respectively (Figure 9
).
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Discussion |
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By immunohistochemistry, we have demonstrated that CD9 was expressed on GC in the follicles and GC/large luteal cells in the CL of the menstrual cycle and pregnancy. The intensity of CD9 expression on GC was highest in the preovulatory follicles and decreased after ovulation. Weak expression of CD9 on large luteal cells continued in the CL of early pregnancy. These expression profiles of CD9 on GC/large luteal cells resembled those of integrin
6 (Fujiwara et al., 1993b). CD9 was also expressed on TI in the growing and preovulatory follicles and on small luteal cells in the CL of menstrual cycle, but not detected in the CL of pregnancy. Using isolated GC from patients undergoing IVF treatment, CD9 was shown to be present on the cell surface of ~90% of GC. The expression of CD9 in GC and CL was confirmed by Western blotting, showing that the molecular mass of CD9 in GC is 26.5 kDa in accordance with the previous report. RT–PCR also demonstrated that CD9 mRNA is expressed in GC and CL. Thus, we concluded that CD9 is stage-specifically expressed on GC and TI in the human ovary and is a differentiation-related cell surface molecule of these cells.
CD9 is a 24–27 kDa cell surface glycoprotein expressed on a variety of tumours and normal human cells, and is a member of the tetraspan family of four transmembrane domain-containing proteins that are thought to be involved in the regulation of cell growth (Gil et al., 1992). In several cells, CD9 was demonstrated to be associated with ß1-related integrins, such as integrin 3ß1 (Nakamura et al., 1995), 4ß1 (Rubinstein et al., 1994), or 6ß1 (Schmidt et al., 1996), on the cell surface. Other studies showed that anti-CD9 mAbs caused a rise in intracellular calcium and enhanced tyrosine phosphorylation in platelets and Schwann cells (Favier et al., 1989; Yatomi et al., 1993; Hadjiargyrou and Pattersonet al., 1995). In platelets, intracytoplasmic signalling pathways mimic those caused by integrin ß3 (gpIIb/IIIa), suggesting that CD9 has a role in integrin-associated cell signalling (Slupsky et al., 1989, 1997). We also observed that anti-CD9 mAb (ALB6) enhanced migration of a human choriocarcinoma-derived cell line, BeWo cells, and that integrin 5ß1 is involved in the effect of the anti-CD9 mAb on BeWo cell migration (Hirano et al., 1999).
We previously purified integrin 6 and its associated proteins from isolated GC by immunoaffinity chromatography using OG-1 mAb. The purified proteins were shown to consist of 125 and 145 kDa proteins under non-reducing conditions (Fujiwara et al., 1993b). In the present study, a different lysis procedure for GC was used to keep the integrin 6 complex associated. That is, the homogenization of GC was performed by Polytron A, not by sonication, and the modified lysis buffer contained Ca2+ and Mg2+ without EDTA. By silver staining SDS–PAGE gels of the purified proteins using OG-1 mAb, several proteins, which were not detected in the previous study, were revealed to be associated with integrin 6. The molecular mass of the main protein was 145 kDa and those of the other proteins were 175–180, 110–115, 59, and 26.5 kDa (Figure 8). Of these, the only protein which was observed in the present and previous studies is the 145 kDa protein, which has a similar molecular mass to integrin 6 under non-reducing conditions. Thus, we concluded that the 145 kDa protein is integrin 6 in GC. The following Western blotting analysis revealed that the 26.5 kDa protein is CD9, indicating that CD9 is one of the proteins associated with integrin 6. Unexpectedly, anti-integrin ß1 mAb (DF5) reacted with the 175–180 kDa protein, but not with the 110–115 kDa protein, although the reported molecular mass of integrin ß1 under non-reducing conditions was 110–120 kDa. The molecular mass of integrin ß1 detected by Western blotting in the whole GC lysate was also 175–180 kDa, which has not been previously reported, as far as we know. Since integrin ß1 is known to be expressed in the human endometrium, we also detected integrin ß1 in the endometrial tissue by Western blotting with the same mAb as a positive control, indicating that the molecular mass of integrin ß1 in the endometrium is 110 kDa which is compatible with the reported one. Using several anti-integrin ß1 mAbs, we further confirmed the presence of integrin ß1 on GC isolated from IVF-treated patients. Since the 175–180 kDa protein is associated with integrin 6, we concluded that this protein is integrin ß1 and that the molecular mass of integrin ß1 associated with integrin 6 on GC is 175–180 kDa. These findings indicate the association of integrin 6ß1 with CD9 in GC, suggesting that CD9 is concerned with integrin 6ß1 functions including the regulation of GC luteinization (Fujiwara et al., l997). CD9 is also detected on large luteal cells in CL of pregnancy that did not express integrin 6ß1 (Fujiwara et al., 1993b). Additionally, CD9 was clearly detected on TI in the follicles and small luteal cells in CL, which lack integrin 6ß1 expression. Therefore, it is reasonable to speculate that CD9 associates with other ß1-related integrins in those cells.
Recently, other tetraspan molecules such as CD63, CD81, and CD82 were shown to be associated with the integrin ß1 family (Rubinstein et al., 1996). As well as tetraspan molecules, it was recently reported that surface molecules on spermatozoa were associated with integrin ß1 on oocytes during gamete fusion (Zhong et al., 1998). Although little is known about the function of the tetraspan molecules, their major feature is association with other surface proteins. Human leukocyte antigen (HLA)-DR was also reported to be associated with these tetraspan molecules including CD9 (Rubinstein et al., 1996). Previously, we demonstrated that HLA-DR is a differentiation-related molecule of human GC, which is expressed on large luteal cells in CL of menstrual cycle and early pregnancy (Fujiwara et al., 1993a). Thus, it is possible that CD9 is connected with various cell surface molecules expressed on ovarian cells and plays a role in ovarian physiology by modulating the function of these cell surface molecules.
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Acknowledgments |
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The authors are grateful to Mrs Yumiko Tomita for technical assistance in flow cytometric analysis. The authors are also grateful to Dr Tetsuro Honda and Dr Toshihiro Higuchi for valuable advice in technology and discussion. This work was supported in part by Grants-in-Aid for Scientific Research (no. 09671673, 09671674, 09671676).
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4 To whom correspondence should be addressed
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References |
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Anton, E.S., Hadjiargyrou, M., Patterson, P.H. and Matthew, W.D. (1995) CD9 plays a role in Schwann cell migration in vitro. J. Neurosci., 15, 584–595.[Abstract]
Boucheix, C., Benoit, P., Frachet, P. et al. (1991) Molecular cloning of the CD9 antigen. A new family of cell surface proteins. J. Biol. Chem., 266, 117–122.
Corner, G.W. (1956) The histological dating of the human corpus luteum of menstruation. Am. J. Anat., 98, 377–401.[ISI][Medline]
Favier, R., Lecompte, T., Morel, M.C. et al. (1989) Calcium rise in human platelets elicited by anti-CD9 and -CD41 murine monoclonal antibodies. Thromb. Res., 55, 591–599.[ISI][Medline]
Fujiwara, H., Ueda, M., Imai, K. et al. (1993a) Human leukocyte antigen (HLA)-DR is a differentiation antigen for human granulosa cells. Biol. Reprod., 49, 705–715.[Abstract]
Fujiwara, H., Maeda, M., Ueda, M. et al. (1993b) A differentiation-related molecule on the cell surface of human granulosa cells. J. Clin. Endocrinol. Metab., 76, 956–961.[Abstract]
Fujiwara, H., Ueda, M., Fukuoka, M. et al. (1994a) A new monoclonal antibody (POG-1) detects a differentiation antigen of porcine granulosa and thecal cells and indicates heterogeneity of theca-stromal cells. Endocrinology, 134, 1132–1138.[Abstract]
Fujiwara, H., Fukuoka, M., Yasuda, K. et al. (1994b) Cytokines stimulate dipeptidyl peptidase-IV expression on human luteinizing granulosa cells. J. Clin. Endocrinol. Metab., 79, 1007–1011.[Abstract]
Fujiwara, H., Ueda, M., Takakura, K. et al. (1995) A porcine homolog of human integrin 6 is a differentiation antigen of granulosa cells. Biol. Reprod., 53, 407–417.[Abstract]
Fujiwara, H., Ueda, M., Hattori, N. et al. (1996a) A differentiation antigen of human large luteal cells in corpora lutea of the menstrual cycle and early pregnancy. Biol. Reprod., 54, 1173–1183.[Abstract]
Fujiwara, H., Maeda, M., Honda, T. et al. (1996b) Granulosa cells express integrin 6: possible involvement of integrin 6 in folliculogenesis. Horm. Res., 46 (Suppl. 1), 24–30.
Fujiwara, H., Honda, T., Ueda, M. et al. (1997) Laminin suppresses progesterone production by human luteinizing granulosa cells via interaction with integrin 6ß1. J. Clin. Endocrinol. Metab., 82, 2122–2128.
Gil, M.L., Vita, N, Lebel-Binay. S. et al. (1992) A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line. Cloning and preliminary functional studies. J. Immunol., 148, 2826–2833.[Abstract]
Hadjiargyrou, M. and Patterson, P.H. (1995) An anti-CD9 monoclonal antibody promotes adhesion and induces proliferation of Schwann cells in vitro. J. Neurosci., 15, 574–583.[Abstract]
Hirano, T., Higuchi, T., Katsuragawa, H. et al. (1999) CD9 is involved in invasion of human trophoblast-like choriocarcinoma cell line BeWo cells. Mol. Hum. Reprod., 5, 168–174.
Honda, T., Fujiwara, H., Ueda, M. et al. (1995) Integrin 6 is a differentiation antigen of human granulosa cells. J. Clin. Endocrinol. Metab., 80, 2899–2905.
Honda, T., Fujiwara, H., Yamada, S. et al. (1997) Integrin 5 is expressed on human luteinizing granulosa cells during corpus luteum formation, and its expression is enhanced by human chorionic gonadotrophin in vitro. Mol. Hum. Reprod., 3, 979–984.
Kersey, J.H., Tucker, W.L., Abramson, C.S. et al. (1981) A human leukemia-associated and lymphohemopoietic progenitor cell surface structure identified with monoclonal antibody. J. Exp. Med., 153, 726–731.
Masellis-Smith, A. and Shaw, A.R.E. (1994) CD9-regulated adhesion. Anti-CD9 monoclonal antibody induce pre-B cell adhesion to bone marrow fibroblasts through de novo recognition of fibronectin. J. Immunol., 152, 2768–2777.
Nakamura, K., Iwamoto, R. and Mekada, E. (1995) Membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) and diphtheria toxin receptor-associated protein (DRAP27)/CD9 form a complex with integrin 3ß1 at cell-cell contact sites. J. Cell. Biol., 129, 1691–1705.
Nakamura, K., Fujiwara, H., Higuchi, T. et al. (1997) Integrin 6 is involved in follicular growth in mice. Biochem. Biophys. Res. Commun., 235, 524–528.[ISI][Medline]
Rubinstein, E., Le Naour, F., Billard, M. et al. (1994) CD9 antigen is an accessory subunit of the VLA integrin complexes. Eur. J. Immunol., 24, 3005–3013.[ISI][Medline]
Rubinstein, E., Le Naour, F., Lagaudriere-Gesbert, C. et al. (1996) CD9, CD63, CD81, and CD82 are components of a surface tetraspan network connected to HLA-DR and VLA integrins. Eur. J. Immunol., 26, 2657–2665.[ISI][Medline]
Ryan, R.J. (1981) Follicular atresia: some speculations of biochemical markers and mechanisms. In Schwartz, N.B. and Hunzicker-Dunn, M. (eds), Dynamics of ovarian function. New York, Raven Press, USA, pp. 1–11.
Schmidt, C., Kunemund, V., Wintergerst, E.S. et al. (1996) CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the 6/ß1 integrin and the neural adhesion molecule L1. J. Neurosci. Res., 43, 12–31.[ISI][Medline]
Slupsky, J.R., Seehafer, J.G., Tang, S.-C. et al. (1989) Evidence that monoclonal antibodies against CD9 antigen induce specific association between CD9 and the platelet glycoprotein IIb–IIIa complex. J. Biol. Chem., 264, 12289–12293.
Slupsky, J.R., Cawley, J.C., Kaplan, C. and Zuzel, M. (1997) Analysis of CD9, CD32, and p67 signalling: use of degranulated platelets indicates direct involvement of CD9 and p67 in integrin activation. Br. J. Haematol., 96, 275–286.[ISI][Medline]
Tsujimura, K., Park, Y.H., Miyama-Inaba, M. et al. (1990) Comparative studies on FcR (FcRII, FcRIII and FcR) functions of murine B cells. J. Immunol., 144, 4571–4578.[Abstract]
Vincent, S., Marty, L. and Fort, P. (1993) S26 ribosomal protein RNA: an invariant control for gene regulation experiments in eucaryotic cells and tissues. Nucleic Acids Res., 21, 1498.
Yatomi,Y., Ozaki, Y., Satoh, K. and Kume, S. (1993) Anti-CD9 monoclonal antibody elicits staurosporine inhibitable phosphatidylinositol 4,5-bisphosphate hydrolysis, phosphatidylinositol 3,4-bisphosphate synthesis, and protein-tyrosine phosphorylation in human platelets. FEBS Letts., 322, 285–290.[ISI][Medline]
Zhong, J.Y., Wolf, J.P., Jouannet, P. and Bomsel, M. (1998) Human gamete fusion can bypass ß1 integrin requirement. Hum. Reprod., 13, 682–689.
Submitted on July 20, 1998; accepted on January 6, 1999.
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