Molecular Human Reproduction, Vol. 9, No. 6, 311-319,
June 2003
© 2003 European Society of Human Reproduction and Embryology
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Melanoma cell adhesion molecule (MCAM/CD146) is expressed on human luteinizing granulosa cells: enhancement of its expression by hCG, interleukin-1 and tumour necrosis factor-
Submitted on April 23, 2002; resubmitted on December 17, 2002. accepted on February 23, 2003
1 Department of Gynecology and Obstetrics and 2 Institute for Frontier Medical Science, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
3 To whom correspondence should be addressed. e-mail: fuji{at}kuhp.kyoto-u.ac.jp
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Melanoma cell adhesion molecule (MCAM) was originally reported to be involved in the invasion and progression of melanoma. It was also shown to be responsible for the attachment of cells to endothelial cells. In this study, we demonstrated by immunohistochemistry that immunoreactive MCAM was not expressed on granulosa cells in the pre-ovulatory follicle, but it was clearly detected in large luteal cells in corpora lutea from the mid-luteal phase of the menstrual cycle. Northern blotting analysis confirmed the expression of MCAM mRNA in corpus luteum. MCAM was weakly detected by immunocytochemical staining in human luteinizing granulosa cells isolated from patients undergoing IVF treatment. Its expression was found to be increased during time in culture of these cells. Flow cytometry and Northern blot analysis revealed that MCAM expression on luteinizing granulosa cells was enhanced when the cells were cultured for 5 days in the presence of hCG (1 IU/ml) or cytokines such as interleukin-1
(10 ng/ml) and tumour necrosis factor- (10 ng/ml). No significant difference of MCAM expression was observed between the cultures under normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. These results indicate that luteinizing granulosa cells express MCAM and that MCAM expression is regulated by LH/hCG and cytokines during luteinization. Since MCAM has been reported to mediate cellular interaction with endothelial cells, this molecule may play a role in neovascularization during corpus luteum formation in the human ovary. Key words: CD146/cytokines/granulosa cells/hCG/MCAM/monoclonal antibody
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Introduction |
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Melanoma adhesion molecule (MCAM) has been reported to be a membrane-bound glycoprotein that mediates cell adhesion. It is expressed in a relatively limited spectrum of normal human tissues and malignant neoplasms. MCAM expression in extravillous trophoblasts has been described (Shih and Kurman, 1996), leading to speculation that it plays an important role in implantation and placentation, although its biological role remains unclear.
There have been no reports demonstrating the expression of MCAM in the corpus luteum in the human ovary. In a previous report, it was concluded that MCAM is not expressed in the human ovary (Shih et al., 1998b). However, our preliminary immunohistochemical study showed that the human corpus luteum was stained with CHL1 monoclonal antibody (mAb). In previous studies we raised the CHL1 mAb in mice by immunizing mice against human extravillous trophoblasts. CHL1 was found to recognize MCAM (Higuchi et al., 2003).
During corpus luteum formation, endothelial cells migrate into regions containing luteinizing granulosa cells and form vascular networks among luteal cells. Recently, MCAM was shown to mediate cell–endothelial cell interaction (Xie et al., 1997). An accumulating body of evidence suggests that endothelial migration among luteinizing granulosa cells is promoted by the secretion of soluble angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) (Phillips et al., 1990; Yan et al., 1993; Reynolds et al., 1998). Several recent works demonstrated that the production of some angiogenic factors by human luteinizing granulosa cells was regulated by the oxygen concentration (Friedman et al., 1997; Koga et al., 2000).
In addition to MCAM, the expression of several molecules including extracellular matrix (ECM) such as fibronectin and collagen have been reported to change on luteinizing granulosa cells during corpus luteum formation (Amsterdam et al., 1989; Honda et al., 1997; Yamada et al., 1999). The expression of these molecules is regulated by gonadotrophin and/or cytokines. For example, the expression of low density lipoprotein receptor, integrin 5 and collagen type IV is enhanced by hCG in luteinizing granulosa cell cultures (Golos et al., 1986; Golos and Strauss, 1987; Honda et al., 1997; Yamada et al., 1999), whereas dipeptidyl peptidase IV and leukocyte functional antigen-3 are induced not by hCG but by inflammatory cytokines such as interleukin (IL)-1 and tumour necrosis factor (TNF)- (Fujiwara et al., 1994; Hattori et al., 1995). On the other hand, the expression of endothelin converting enzyme-1, which is a cell surface endopeptidase and activates proendothelin peptide, was promoted by hCG, IL-1 and TNF- (Yoshioka et al., 1998).
In the present study, therefore, we investigated: (i) MCAM protein and mRNA expression in the human ovary using CHL1, another commercially available anti-MCAM mAb, and Northern blot analysis; and (ii) the effect of oxygenic tension, hCG and various cytokines on MCAM expression using cultures of human luteinizing granulosa cells.
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Tissue samples
Pre-ovulatory follicles (n = 5) and corpora lutea (CL) from the early (n = 10) and mid-luteal phases (n = 10) of the menstrual cycle were obtained from 25 women, aged between 25 and 43 years. They had undergone unilateral ovarian cystectomy or oophorectomy and contralateral wedge resection to treat benign ovarian tumours. All the women had a history of regular menstrual cycles (28–30 days) and their ovulatory basal body temperature charts were consistent with normal luteal phase length. Term placentae (n = 3) were obtained from normal deliveries. For RNA isolation, tissues were immediately frozen in liquid nitrogen and stored at –80°C. For immunohistochemistry, each specimen was embedded in OCT compound (Tissue-Tec, Miles Scientific, USA), snap-frozen in liquid nitrogen, and stored at –80°C. The corpus luteum day was re-evaluated according to histological dating, using haematoxylin and eosin-stained tissue sections that were fixed with 10% formalin and embedded in paraffin (Corner, 1956). The migration of endothelial cells and the size of luteal cells were used for this classification. In the present study, the term ‘CL day’ was used according to his definition. For example, CL day 2 is the day after ovulation.
Ethical approval for this study was granted by the Ethical Committee of Kyoto University Hospital. Informed consent for the use of these tissues in this study was obtained from all donors.
Antibodies
Mouse mAb CHL1 (IgG1 class), which recognizes human MCAM/CD146, a commercially available anti-human MCAM/CD146 mouse mAb (clone S-Endo1, IgG1 class; Alexis Biochemicals, USA) or rabbit anti-human 3ß-hydroxysteroid dehydrogenase (3ß-HSD) polyclonal antibody (pAb) (Oxygene, USA) were used in the immunohistochemical study. The production and characterization of mouse mAb CHL1 is detailed in another paper (Higuchi et al., 2003). Briefly, CHL1 was raised in our laboratory by immunizing chorion laeve from fetal membrane into mice. cDNA clones encoding the CHL1 antigen were recovered from a chorion laeve cDNA library by mAb panning of transfected COS7 cells. One of the recovered cDNA clones was named pCHL1-12 which showed the cell surface expression of the CHL1 antigen on COS7 cells as detected by flow cytometry. Plasmid clone pCHL1-12 was subjected to DNA sequencing, and comparison of the nucleotide sequences with the EMBL/GenBank databases showed that clone CHL1 was identical to the human melanoma cell adhesion molecule (MCAM/CD146 antigen) (Lehmann et al., 1989). In addition, the immunohistochemical staining profiles of placenta with CHL1 and a commercially available anti-human MCAM/CD146 mouse mAb (clone S-Endo1) were similar.
For the immunochemistry and flow cytometry, an anti-trinitrophenyl (TNP) mouse mAb (unrelated mAb, IgG1) (Tsujimura et al., 1990) was used as the negative control. Fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin pAb (Dakopatts, Denmark), rhodamine-conjugated goat anti-mouse immunoglobulin pAb (Santa Cruz Biotechnology Inc., USA) or FITC-conjugated swine anti-rabbit immunoglobulin pAb (Dako, Denmark) were used as the secondary antibody.
Isolation of human luteinizing granulosa cells
Human luteinizing granulosa cells were obtained from 14 patients aged 25–39 years who had undergone treatment for IVF as previously described (Fujiwara et al., 1994). Briefly, patients receiving a GnRH analogue (buserelin acetate; Aventis Forma Ltd, Japan), beginning on the first day of the cycle, were hyperstimulated with pure FSH (Serono Japan, Co., Japan) or hMG (Organon Japan Co. Ltd, Japan) until the follicles reached maturity. Follicles were aspirated 36 h after the administration of hCG (Mochida Pharmaceutical Co. Ltd, Japan). The aspirated follicular fluid was centrifuged, and the resuspended granulosa cells were overlaid on Ficoll-Hypaque and centrifuged at 400 g for 30 min. Granulosa cells were collected from the interphase, washed twice with phosphate-buffered saline (PBS) and subjected to culturing, immunocytochemistry, flow cytometry or RNA isolation.
Informed consent for the use of granulosa cells in this study was obtained from all donors.
Immunohistochemical examination of MCAM expression in human ovaries
Indirect immunofluorescence histochemistry was performed as previously described (Fujiwara et al., 1992). Frozen tissues were sliced to 7 µm thickness using a cryostat microtome (Cryocut 1800; Reichert-Jung, Germany), immediately air-dried on Neoplene-coated glass slides (Nisshin EM, Japan), and fixed in acetone at –20°C for 5 min. The slides were incubated with mouse mAb CHL1 (5 µg/ml, diluted in culture medium), anti-human MCAM/CD146 mouse mAb (clone S-Endo1, 5 µg/ml) or anti-TNP mouse mAb (5 µg/ml) for 40 min at room temperature. After the slides had been washed in PBS, they were incubated with FITC rabbit anti-mouse Ig antibody (diluted 1:40) for 40 min at room temperature in the dark. The slides were washed, mounted with mounting agent (Perma Fluor Aqueous Mounting Medium; Immunon, USA), and examined under a fluorescence microscope (Nikon, Japan). The staining intensity of MCAM expression was further classified into four grades (–, absence of staining; +, weakly positive; ++, moderately positive; +++, strongly positive). To reduce bias in the visual assessment, the staining intensity of large luteal cells in the sole sample derived from corpus luteum on day 7 was defined as moderately positive intensity and was used for standard controls to assess fluorescence intensity throughout the immunohistochemical analyses. Two individuals carried out these assessments.
For indirect double immunofluorescence staining, the frozen sections were incubated for 30 min at room temperature with CHL1 mAb (5 µg/ml) or anti-TNP mAb (5 µg/ml). After the slides had been washed in PBS, they were incubated with rhodamine-conjugated goat anti-mouse immunoglobulin pAb (diluted 1:40) for 30 min at room temperature in the dark. The washed slides were then reacted with rabbit anti-human 3ß-HSD pAb (diluted 1:20) for 30 min, and incubated with FITC-conjugated swine anti-rabbit immunoglobulin pAb (diluted 1:20) for 30 min. The slides were washed, mounted with Perma Fluor Aqueous Mounting Medium, which reduces fluorescence fading, and then examined under a confocal laser scanning microscope (Carl Zeiss Inc., Germany).
Indirect immunofluorescence staining of human isolated granulosa cells
Isolated granulosa cells were immediately centrifuged, attached to glass slides using a cytospin, air-dried, and then fixed with acetone at –20°C. These slides were indirectly stained using CHL1 and anti-TNP mAb as described above. Three independent experiments were performed.
Flow cytometric analysis and immunofluorescence staining of cultured human luteinizing granulosa cells
Isolated human granulosa cells were resuspended in culture medium consisting of Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum (Flow Laboratories, USA) penicillin (100 IU/ml) and streptomycin (100 µg/ml). The cells were collected after mild washing, suspended in culture medium at a density of 3x105 cells/ml and incubated in 6-well plates (Corning, USA) at 3 ml/well or in 8-chamber culture slides at 300 µl/well (Lab-Tec; Nunc Inc., USA) in the presence or absence of recombinant hCG (1 IU/ml, Rhoto Pharmaceutical Co. Ltd, Japan), recombinant human IL-1 (10 ng/ml; Dainippon Pharmaceutical Co. Ltd, Japan) or recombinant human TNF- (10 ng/ml; Dainippon). On the next day (day 2) and day 4, the medium was replaced by fresh medium with or without recombinant hCG, IL-1 or TNF-. These experiments were repeated five times. Where indicated in the text, the isolated human granulosa cells were incubated under 1% oxygen concentration for 1 day and were then divided into two groups that were incubated under 1% or 20% oxygen concentration for an additional 2 days. These experiments were repeated eight times.
On day 4 or 6, the cultured granulosa cells in 6-well plates were collected after trypsin and EDTA treatment and were washed in Hanks’ balanced salt solution (HBSS) with 0.1% bovine serum albumin and 0.1% NaN3. The granulosa cells were sedimented by centrifugation and incubated with CHL1 mAb (100 µg/ml) or anti-TNP mAb (100 µg/ml) for 30 min at 4°C. After washing in HBSS, the cell pellet was incubated with FITC-conjugated rabbit anti-mouse Ig 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 (FACScalibur; Becton Dickinson Immunocytometry Systems Japan, Japan). Fluorescence intensity of 5000 viable cells that were gated as luteinizing granulosa cells by cell size and granularity were evaluated in each flow cytometric experiment. The ratio of contaminating monocytes, identified by their staining with anti-CD14 mAb (Becton Dickinson Labware, USA), was <3%.
On days 2 and 6, the granulosa cells cultured in the 8-chamber slides were washed gently three times with PBS, thoroughly dried, and fixed with acetone at –20°C. The slides were indirectly stained using CHL1 and anti-TNP mAb as described above. Three independent experiments were performed.
RNA isolation and Northern blot analysis
Total RNA from two human placentae, two pre-ovulatory follicles, six corpora lutea and cultured granulosa cells on day 6 obtained from five patients were isolated by the TRIzol method. Northern blot analysis was performed as previously reported (Espey et al., 2000). In brief, 1.2% agarose in 6.7% formaldehyde and 1 mol/l HEPES/NaPO4 was used for electrophoresis of 15 µg of total RNA from each sample and the RNA in agarose gels was blotted onto nylon membranes by standard procedures. cDNA for MCAM radiolabelled with [32P]dCTP was then hybridized to the blots (Higuchi et al., 2003). The blots were stripped and then reprobed with S26 cDNA as previously reported (Higuchi et al., 1995). The intensity of the bands was quantified and analysed by a National Institutes of Health image software program. The data were expressed as a ratio of MCAM densitometric intensity divided by the corresponding intensity of S26.
Statistics
Data are shown as means ± SEM. The rate of MCAM-positive cells and the bands of MCAM mRNA in the five experiments with granulosa cell cultures in the presence or absence of hCG, IL-1 or TNF- was analysed by analysis of variance, followed by Scheffé’s F-test. The rate of MCAM-positive cells in the eight experiments with granulosa cell cultures under hypoxic or normoxic conditions was analysed by a two-tailed paired t-test. The bands of MCAM mRNA in corpora lutea obtained in the early luteal and mid-luteal phase of the menstrual cycle were analysed by an unpaired t-test.
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The expression of MCAM protein in human ovaries
In pre-ovulatory follicles (18–20 mm, n = 5), high expression of MCAM was detected on the endothelial cells of the vessels in the theca layer, but not on the granulosa cells or the theca interna cells (Figure 1B). In the corpora lutea obtained in the early luteal phase (n = 8), MCAM was weakly or moderately expressed on the luteinizing granulosa/large luteal cells (Figure 1E). In corpora lutea obtained during the mid-luteal phase (n = 8), MCAM was moderately expressed on the large luteal cells (Figure 1H). The expression of MCAM was not detected on the small luteal cells, but it was strongly detected on the endothelial cells. Double staining showed that MCAM is clearly expressed on the 3ß-HSD-positive luteal cells (Figure 2C and F).
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The expression of MCAM on isolated human granulosa cells was very weakly detected by immunocytochemical staining (n = 3, data not shown). These expression profiles are summarized in Table I. The staining profiles for MCAM expression using CHL1 and anti-MCAM mAb (S-Endo1) were almost similar (Figure 3).
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The expression of MCAM mRNA in human ovaries
A 3.3 kbp-specific band of MCAM mRNA was clearly detected by Northern blot analysis of mRNA isolated from term placentae, pre-ovulatory follicles and corpora lutea in the early and mid-luteal phases of the menstrual cycle (Figure 4A). The MCAM mRNA expression was significantly increased (P < 0.01) from the stage of the corpus luteum in early luteal phase through that in mid-luteal phase, in accord with the immunohistochemical results described above (Figure 4B, C).
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Cell surface expression of MCAM on cultured human luteinizing granulosa cells
Although the MCAM expression on isolated luteinizing granulosa cells was very weak, during the 5 day culture an increase of the expression on luteinizing granulosa cells was observed by immunocytochemical staining (n = 3) (Figure 5A, C). Flow cytometric analysis showed that the rates of MCAM-positive cells in the hCG, IL-1
and TNF--treated groups were significantly higher than that in the non-treated control group (59.5 ± 3.7, 73.2 ± 3.2 and 72.5 ± 3.3 versus 35.2 ± 4.4%, P < 0.01). In the TNF--treated group, the mean fluorescence intensity was significantly higher than that of the control group (88.2 ± 14.1 versus 42.6 ± 5.6, P < 0.05). The increasing tendency of mean fluorescence intensity was also observed in the hCG and IL-1 groups, but their changes were not significant (63.8 ± 6.1 and 62.4 ± 4.1 respectively) (n = 5) (Figure 6A).
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In contrast, MCAM expression was not affected by culturing under hypoxic conditions (1% oxygen concentration) compared with normoxic conditions (the rates of MCAM-positive cells, 30.5 ± 5.4 versus 35.6 ± 3.5%; and the mean fluorescence intensity, 47.1 ± 2.8 versus 53.5 ± 3.5) (n = 8, data not shown).
Expression of MCAM mRNA in cultured luteinizing granulosa cells
A 3.3 kbp-specific band of MCAM mRNA was clearly detected by Northern blot analysis of the mRNA isolated from cultured luteinizing granulosa cells. The expression of MCAM mRNA was significantly enhanced in cultured granulosa cells in the presence of hCG or TNF- compared with the control group. An increasing tendency of MCAM mRNA was also observed in the IL-1 group, but its change was not significant (n = 5) (Figure 6B, C).
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Discussion |
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MCAM is a transmembrane glycoprotein which functions as a Ca2+-independent cell adhesion molecule involved in heterophilic cell–cell interactions. It is also known as CD146, MUC18, A32 antigen and S-Endo-1 (Lehmann et al., 1989). MCAM was originally described as a marker of malignant potential in melanoma and was reported to play a role in melanoma invasion and metastasis (Shih et al., 1994). MCAM is also abundantly expressed on endothelial cells throughout the vascular tree. In endothelial cells, the engagement of MCAM was reported to lead to an outside-in signalling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin (Anfosso et al., 1998). Shih and Kurman (1996) first reported that human extravillous trophoblasts express MCAM and they speculated that this molecule plays an important role in implantation and placentation by regulating the invasiveness of extravillous trophoblasts (Shih et al., 1998a). Recently, we showed that MCAM expression on trophoblasts was induced by soluble factors derived from decidual tissues and that cAMP was involved in this induction (Higuchi et al., 2003).
Previously Shih et al. (1998b) immunohistochemically screened MCAM expression in various organs and reported that MCAM was not detected in human ovary. However, in the present study, immunohistochemical analysis using CHL1 showed that MCAM was expressed on the human corpus luteum and luteinizing granulosa cells. Shih et al. did not describe whether they used ovarian tissues contain follicles and/or corpora lutea. Considering the rarity of encountering corpora lutea in human ovarian samples, there has not yet been any conclusive information on MCAM expression in human corpora lutea. However, in this study similar expression profiles using another anti-MCAM antibody, S-endo1, confirmed MCAM expression in corpus luteum. In addition, Northern blot analysis showed that MCAM mRNA was expressed on the corpus luteum and on cultured granulosa–lutein cells. Furthermore, double staining clearly showed that MCAM is expressed on the surface of 3ß-HSD-positive large luteal cells. Based on these findings and the results of flow cytometry, we conclude that luteinizing granulosa/large luteal cells express MCAM on their surface.
MCAM protein was not detected on the granulosa cells in pre-ovulatory follicles by immunohistochemistry. On the other hand, large luteal cells in the mid-luteal phase clearly expressed this molecule. The intensity of immunohistochemical staining for MCAM expression on the luteinizing granulosa cells seemed to increase during corpus luteum formation. Similarly, although the granulosa cells obtained from patients undergoing IVF treatment very weakly expressed this molecule, immunocytochemical staining showed that the expression of MCAM gradually increased during 5 days culture. In addition, the expression of MCAM mRNA increased from the pre-ovulatory follicle stage through the mature corpus luteum stage in accordance with the immunohistochemical results. These findings indicate that MCAM expression on luteinizing granulosa cells is up-regulated during corpus luteum formation and suggest that increased expression of MCAM on granulosa cells is positively correlated with the luteinization process, implying that MCAM is a cell surface marker of granulosa cell luteinization.
It is well known that luteinization of granulosa cells is induced by LH/hCG hormones. Therefore, using granulosa cell cultures, the effect of hCG on MCAM expression was examined by flow cytometry and Northern blot analysis. HCG was found to enhance MCAM expression on granulosa–lutein cells. This suggests that the LH surge just prior to ovulation is the initial trigger of induction of MCAM expression. Recent studies have shown that agents amplifying intracellular cAMP levels can increase the expression of MCAM in a glioma cell line (Rummel et al., 1996) and that the cAMP-responsive element is a major transcriptional activator of MCAM expression in the majority of melanoma cell lines (Mintz-Weber and Johnson, 2000). These reports are compatible with our results since LH/hCG elevates intracellular cAMP levels in luteinizing granulosa cells.
It has been proposed that MCAM is involved in neovascularization (Shih et al., 1997). A MCAM-transfected melanoma-derived cell line was reported to show increased attachment to human endothelial cells, suggesting that MCAM is responsible for cell attachment to endothelial cells, although the responsible ligand(s) are still unknown (Xie et al., 1997). bFGF, VEGF and angiogenin have been reported to be related to neovascularization during corpus luteum formation. The production of VEGF and angiogenin was induced by hypoxic stress in cultures of human luteinizing granulosa cells (Friedman et al., 1997; Koga et al., 2000). However, this study showed that the expression of MCAM on cultured luteinizing granulosa cells was not affected by lowering the oxygen concentration to 1%. This suggests that MCAM expression is not regulated by oxygenic tension.
On the other hand, MCAM expression was significantly induced by TNF-, which is known to be an angiogenic and inflammatory cytokine (Ferrara, 2000; Kim et al., 2002). Similar promoting effects on MCAM expression were also observed by treatment with another inflammatory cytokine, IL-1. Although these cytokines have been reported to stimulate other angiogenic factors such as VEGF and angiopoietin (Jung et al., 2001; Scott et al., 2002), there are few reports about their enhancing effects on MCAM expression. In fact, we have previously observed that neither IL-1 nor TNF- promote MCAM expression on choriocarcinoma cell line JEG3 cells (Higuchi et al., 2003). However, in the present study the inducing effects of IL-1 and TNF- on MCAM were demonstrated. After ovulation, the basement membrane is destroyed and immune cells and endothelial cells rapidly invade the luteinizing granulosa cell layer in a process resembling tissue inflammation (Espey, 1980). Additionally, it is suggested that the invading immune cells that can produce several cytokines are one of the regulatory factors in corpus luteum formation (Bukulmez et al., 2000). Based on these findings, we speculate that such inflammatory reactions among the luteinizing granulosa cells, where IL-1 and TNF- may be produced, play a role in the angiogenic process during corpus luteum formation, not only by promoting the production of soluble angiogenic factors but also by regulating MCAM expression to induce the attachment of the luteinizing granulosa cells to the endothelial cells.
In conclusion, we have demonstrated that MCAM, which was recently shown to mediate cell–endothelium adhesion, is expressed on human granulosa–large luteal cells. The increase of MCAM expression during luteinization suggests its involvement in human corpus luteum formation, especially in neovascularization.
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Acknowledgements |
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We thank Mrs Mizuho Ohshima for her technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research (No. 12470342, 13557140, 136(1709, 136(1710).
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