Molecular Human Reproduction, Vol. 8, No. 8, 742-749,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
The significance of membrane type 1 metalloproteinase in structural involution of human corpora lutea
1 Department of Obstetrics and Gynecology, 2 Department of Pathology, Cancer Research Institute, Sapporo Medical University School of Medicine, South-1 West-16 Chuo-ku, Sapporo and 3 Department of Molecular Virology and Oncology, Kanazawa University, Takara-machi, Kanazawa, Ishikawa 920, Japan
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The expression of membrane type 1 (MT1) matrix metalloproteinase (MMP), MMP-2, and tissue inhibitors of MMP-1 and -2 during structural involution of the human corpus luteum was examined using immunohistochemistry, Northern blotting, Western blotting, gelatin zymography and in-situ hybridization techniques. The corpora lutea of 20 patients were investigated at the time of total hysterectomy and were obtained from five patients each in the early, mid- and late luteal phases and during gestation. Immunohistochemistry for MT1-MMP in corpus luteum showed that the protein appeared in granulosa lutein cells in the late luteal phase. Both the expression of MT1-MMP mRNA and the amount of the protein increased in the late luteal phase. In-situ hybridization showed that MT1-MMP mRNA was localized mainly in the cytoplasm of granulosa lutein cells. These results suggest that increased expression of MT1-MMP may be a major factor in remodelling of the human corpus luteum during structural luteolysis.
human corpus luteum/MMP-2/MTI-MMP/TIMP-1/TIMP-2
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Structural luteolysis has been defined as the regression of the tissue of the corpus luteum (CL) in ovaries. The dramatic degeneration of the CL during structural luteolysis in rats is histologically characterized as the cellular infiltration and proliferation of mesenchymal cells, and an increased number of cells with vacuoles in the cytoplasm and a pyknotic nucleus (Malven and Sawyer, 1966
). Irving-Rodgers et al. reported that bovine antral atresia is characterized by early destruction of the layers of the granulosa membrane closest to the antrum, whereas the most basal cells remain intact (Irving-Rodgers et al., 2001); numerous pyknotic nuclei are observed in the antrum close to the granulosa membrane. It has also been shown that the human CL might share many histological characteristics with that of the rat (Corner, 1956). Furthermore, as remodelling of the extracellular matrix (ECM) takes place in the course of the histological changes, there may also be involvement of matrix metalloproteinases (MMPs). Duncan et al. have shown that the expression of MMP-2, leading to degeneration of collagen type IV, increases in the late luteal phase (LLP) of human CL (Duncan et al., 1998). However, the activating factor of MMP-2 remains unclear. We have reported that the activation of MMP-2 is enhanced during prolactin-induced structural luteolysis (Endo et al., 1993) and that an increase in MT1-MMP activity occurs along with an increase in MMP-2 activity in GnRH agonist-induced structural luteolysis of rats (Goto et al., 1999).
MMPs are known to change their form from inactive to active and to possess proteinase activity, because specific proteinases are required for the processing of MMPs (Nagase, 1997). MMP-2 is secreted in the form of pro-MMP-2, for which MT1-MMP is an activator (Sato et al., 1994). It has been contended that pro-MMP-2 is activated on the cell membrane by MT1-MMP (Atkinson et al., 1995; Takino et al., 1995; Kinoshita et al., 1996) and that it causes the degradation of collagen types I, IV and V and elastin (Liotta et al., 1980; Birkedal-Hansen et al., 1993; Sato et al., 1994; Aimes and Quigley, 1995). Accordingly, since MMP-2 in particular is believed to be involved in the remodelling of the ECM in human CL, MT1-MMP may be a genuine key enzyme in structural luteolysis of human CL. Therefore, we investigated the patterns and sites of MT1-MMP and MMP-2 expression in the CL throughout the luteal phase and during gestation.
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Human CL
Specimens were obtained from 50 patients who were operated on in Sapporo Medical University Hospital at the time of total hysterectomy under informed consent based on the regulations of the Ethics Committee of Sapporo Medical University. This study was also approved by the Institutional Review Board of the university. The patients suffered from cervical cancer (15 cases), myoma uteri (20 cases) or adenomyosis (15 cases).
We had 50 subjects, including five with gestational CL in the past 4 years. All patients, aged 35–45 years, had regular menstrual cycles and had not received any hormone therapy during the preceding 6 months. We examined specimens from 45 patients and selected five typical specimens, corresponding to each luteal phase, using ultrasonography to decide the day of ovulation, serum progesterone, basal body temperature and histological dating according to Corner (Corner, 1956). We determined in this report that the early luteal phase (ELP) was at day 3 post-ovulation, the mid-luteal phase (MLP) was at days 4–11 post-ovulation, the LLP was at days 12–14 post-ovulation and gestational CL (GCL) was at weeks 5–6 of gestation. The pregnant patients who were >40 years old had huge uterine myomas and did not want to continue the pregnancy. Five 5- to 6-week-pregnant patients who had uterine myoma in the past 4 years visited our clinics.
CL were enucleated at the time of hysterectomy and we divided them into two pieces. One piece was used for histological studies (immunohistochemistry and in-situ hybridization), and was fixed in 4% paraformaldehyde/phosphate-buffered saline (PBS) and embedded in paraffin. The other piece was used for biochemical studies (zymography, Western blotting and Northern blotting), and was frozen by dry ice and stored at –80°C until use.
Materials
Materials were obtained as follows: Ultraspec RNA from Biotex Laboratories, Inc. (Houston, TX, USA), 3,3'-diaminobenzidine (DAB) from Katayama Chemical (Osaka, Japan), Nytran-Plus from Schleicher & Schuell (Keene, NH, USA), 32P-dCTP, Nick column, horse-radish peroxidase (HRP) and chemiluminescent reagents from Amersham Pharmacia Biotech (Buckinghamshire, UK), Prime-It II random primer labelling kits from Stratagen (La Jolla, CA, USA), rabbit serum of anti-human MT1-MMP, MMP-2, TIMP-1, TIMP-2 antibodies from Torrey Pines Biolabs (San Diego, CA, USA) (this anti-MMP-2 antibody recognizes both non-activated and activated MMP-2), anti-MT1-MMP antibody from Fuji Chemical Industries, Ltd (Toyama, Japan), biotinylated antibodies and Vectastain ABC Elite kit from Vector Laboratories (Burlingame, CA, USA), fetal calf serum (FCS) from Gibco (Grand Island, NY, USA), 3-amino-propyltriEthoxySilane (APS)-coated glass slides from Matsunami (Tokyo, Japan), STUF-solution from Serotec Ltd (Kidlington, Oxford, UK), Block Ace from Dainippon Pharmaceutical Co. (Osaka, Japan), digoxigenin RNA labelling kit, SP6 and T7 RNA polymerase, digoxigenin-UTP and DIG nucleic acid detection kit from Boehringer Mannheim (Mannheim, Germany), ISHR Starting Kit and in-situ hybridization reagents from Nippon Gene Co., Ltd (Toyama, Japan). Other agents were purchased from Sigma Chemical Co. (St Louis, MO, USA).
Immunohistochemistry
The tissues embedded in paraffin were sectioned 5 µm thick and mounted on APS-coated glass slides. The slides were deparaffinized in xylene and placed on a hot plate at 90°C, covered with STUF-solution for 10 min, and subsequently rinsed several times with PBS. Endogenous peroxidase activity was blocked by 0.6% H2O2 in methanol for 30 min at room temperature. Block Ace was then used for 30 min at room temperature. Primary antibodies were applied on the sections for 60 min at room temperature and then the sections were incubated with a biotinylated antibody for 30 min at room temperature. Thereafter, we used the ABC method with DAB as a substrate and haematoxylin was used for counterstaining. For the negative control, the slides were incubated without a primary antibody. The tissues were also stained with haematoxylin and eosin.
Preparation of RNA probe and in-situ hybridization
We employed oligonucleotide primers based on the human MT1-MMP cDNA sequence (Sato et al., 1994). In-vitro transcription and labelling with digoxigenin were simultaneously performed using SP6 and T7 polymerases for the single-strand antisense and sense probes respectively. The digoxigenin-labelled probe was 1242 bp of MT1-MMP cDNA. We used an ISHR Starting Kit and in-situ hybridization reagents and procedures were performed according to the manufacturer's protocol. The deparaffinized sections were subjected to proteinase treatment in a solution containing 5 µg/ml proteinase K at room temperature for 10 min. They were acetylated with 0.25% acetic anhydride in 0.1 mol/l triethanolamine (pH 8.0), and subsequently rinsed twice with 4x standard saline citrate (SSC) at room temperature for 10 min. The slides were then incubated with 50% formamide/2xSSC at 42°C for 30 min. The sections were covered with an in-situ hybridization mixture [50% formamide, 0.6 mol/l, 10 mmol/l Tris–HCl, pH 7.6, 1 mmol/l EDTA, 0.2 mg/ml tRNA, 0.25% sodium dodecyl sulphate (SDS), 1x Denhardt's solution, 10% dextran sulphate and digoxigenin-labelled RNA probes] and hybridized at 50°C for 36 h. After hybridization, the slides were washed with 50% formamide/2xSSC at 42°C for 20 min, three times, and incubated in NTE buffer (10 mmol/l Tris–HCl, pH 8.0, 0.5 mol/l NaCl, 1 mmol/l EDTA) with RNase A solution (10 mg/ml RNase A) at 37°C for 30 min. After incubation in NTE buffer for 5 min, the slides were washed at 42°C in 0.1xSSC for 20 min three times. For the detection of the signal, we used a DIG-Nucleic Acid Detection Kit. The slides were incubated with 1% blocking reagent in TN buffer (100 mmol/l Tris–HCl, pH 7.5, 150 mmol/l NaCl) at room temperature for 30 min. After washing twice with TN buffer, an anti-digoxigenin polyclonal antibody conjugated with immunogold was applied for 60 min and the slides were twice washed with TN buffer for 15 min. Thereafter, they were incubated with colour-substrate solution at room temperature for 24 h in a dark room and subsequently counterstained with methyl green.
Northern blotting
The total RNA of CL was extracted using the Ultraspec RNA isolation system. RNA (20 µg/lane) was electrophoresed on 1% agarose/formaldehyde gel (100 V; 2 h) and transferred onto nylon membranes in 20xSSC (3 mol/l sodium chloride, 0.3 mol/l trisodium citrate) overnight and then fixed with a UV linker. Filters were prehybridized at 42°C for 4 h in 50% formamide, 5xSSC NaCl, 0.75 mol/l; sodium citrate, 75 mmol/l final ph 7.0), 5xDenhardt's solution (Ficoll, 0.5%; polyvinylpyrrolidone, 0.5%; bovine serum albumin, 0.5%), 0.1% SDS, and 100 g of denatured salmon sperm DNA per ml. A 32P-labelled cDNA probe was then added to the same prehybridization solution except that it contained 2.5% dextran sulphate and hybridized overnight at 42°C. The following probes were used for Northern blotting: a 1.2 kb of Hind III and EcoRI fragment of MT1-MMP cDNA, a 1.5 kb EcoRI and BamHI fragment of MMP-2 cDNA, a 0.6 kb ClaI and BamHI fragment of TIMP-1 cDNA, and a 0.7 kb EcoRI and BglII fragment of TIMP-2 cDNA. A 450 bp ribosomal protein, L38, cDNA was used as an internal control (Kuwano et al., 1991). Human probes were radiolabelled with 32P-dCTP using Prime-It II random primer labelling kits. Labelled probes were purified through a Nick column before hybridization. Filters were washed in 2xSSC containing 0.1% SDS at room temperature twice for 15 min, and twice with 0.2xSSC containing 0.1% SDS at 65°C for 15 min. They were exposed to Fuji RX X-ray film at –70°C for 1–2 days.
Western blotting
Luteal extracts (20 µg of protein) were separated by 12.5% SDS–PAGE under reducing conditions and transferred to nitrocellulose membranes using an electroblotting apparatus. Non-specific binding sites were blocked by immersing the membrane overnight at room temperature in PBS containing 0.05% Tween 20 (T–PBS) and 5% skim milk on an orbital shaker, and washing five times with T–PBS. The membrane was incubated with the primary antibody (1/1000) for 90 min at room temperature in a humidified chamber and washed five times with T–PBS. The membrane was then incubated with an HRP-conjugated antibody (1:5000) at room temperature in a humidified chamber and washed five times with T–PBS. The membrane was finally incubated with a chemiluminescence reagent, and exposed to X-ray film.
Gelatin zymography of luteal extracts
As previously described (Endo et al., 1993), the samples of CL were homogenized (100 mg wet weight/ml of PBS containing 0.2% Triton X-100) with a Teflon glass tissue grinder on ice (15 strokes). Homogenates were centrifuged (12 000 g; 4°C for 20 min) and the supernatant was collected for protein assay and gelatin zymography. The luteal extracts (40 µg of protein) were subjected to electrophoresis in 10% polyacrylamide gels containing 1 mg/ml gelatin. Samples were diluted in non-reducing sample buffer.
Gelatin zymography of membrane fractions
Isolation of the crude luteal membrane fraction was carried out by a previously described method (Luborsky et al., 1984). Briefly, samples of CL were homogenized in 2 ml Tris buffer containing 0.25 mol/l sucrose with a Teflon glass tissue grinder at 45 g on ice. Homogenates were filtered through nylon mesh (42 µm) and centrifuged at 120 g for 10 min. The supernatant was centrifuged (10 000 g at 4°C for 30 min), and the pellet was stored at –80°C until use. We examined the activity of MMP-2 processing of this fraction as previously described (Cao et al., 1995). Crude luteal membrane fractions (20 µg) were mixed with 1 µl FCS and incubated for 2 h at 37°C. The reaction was terminated by the addition of the sample buffer and the mixture was analysed by gelatin zymography.
Densitometric analysis
Bands of gelatinase activity and Western blots were analysed using NIH Image (Version 1.61) and those of Northern blots were analysed with a BAS 2000 Bio-Imaging Analyser (FUJI, Tokyo, Japan). Radioactivity was adjusted to the band of L38 (ribosomal protein) RNA.
Statistics
The data were evaluated by one-way analysis of variance, followed by Scheffe's F-test post-hoc analysis, and by the unpaired Student's t-test. P < 0.05 was considered significant.
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Expression and localization of MT1-MMP and MMP-2 in human CL
To examine the expression and localization of MT1-MMP and MMP-2 in human CL, immunohistochemistry and in-situ hybridization were performed respectively. Immunohistochemistry for MT1-MMP revealed that the protein was strongly stained in both granulosa lutein and thecal lutein cells in the LLP and moderately stained in the MLP (Figure 1g
). In addition, in-situ hybridization revealed that MT1-MMP mRNA was localized mainly in the cytoplasm of granulosa lutein cells (Figure 2a). The intensity of MT1-MMP staining in the LLP was stronger than that of MT1-MMP in the ELP, MLP or GCL (Figure 1e–h). In the ELP and GCL the granulosa lutein and thecal lutein cells were faintly stained. Granulosa lutein cells with or without MT1-MMP protein coexisted in the CL of the MLP (Figure 1f). Immunohistochemistry for MMP-2 revealed that its staining pattern was very similar to that of MT1-MMP. MMP-2 was strongly stained in both granulosa lutein and the thecal lutein cells in the LLP (Figure 1k). Positive staining was mainly found in the cytoplasm of the granulosa lutein and thecal lutein cells. The intensity of MMP-2 staining in the LLP was stronger than that of MMP2 in the ELP, MLP and GCL (Figure 1i–l).
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Expression of MT1-MMP, MMP-2, TIMP-1 and TIMP-2 proteins
The production of MT1-MMP, MMP-2, TIMP-1 and TIMP-2 proteins in CL was examined by Western blotting. As shown in Figure 3a,b
, the amount of MT1-MMP in the LLP was 1.4-, 1.2- and 2.8-fold greater than that of MT1-MMP in the ELP, MLP and GCL respectively, and the amount of 72 kDa MMP-2 in the LLP was 2.8-, 1.9- and 2.4-fold greater than that of 72 kDa MMP2 in the ELP, MLP and GCL respectively. Furthermore, the amount of 67 kDa MMP-2 in the LLP was 2.4-, 1.9- and 2.8-fold greater than that of 67 kDa MMP2 in the ELP, MLP and GCL respectively. TIMP-1 and TIMP-2 proteins, which are physiological inhibitors of MMPs, were also examined using Western blot analysis. As shown in Figure 3a,b, the amount of TIMP-1 protein in the LLP was 53, 26 and 86% of that in the ELP, MLP and GCL respectively. The expression of TIMP-1 in the GCL was significantly decreased compared with that of TIMP-1 in the ELP and MLP, but not significantly different from that of TIMP-1 in the LLP. In addition, the amount of TIMP-2 in the LLP was 6.6-, 3.3- and 1.6-fold greater than that of TIMP-2 in the ELP, MLP and GCL respectively. The amount of TIMP-2 in the GCL was much greater than that of TIMP-2 in the ELP and MLP.
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Gelatinase activity in extracts of luteal tissue of human CL
Overall gelatinase activity in extracts of luteal tissue was examined by gelatin zymography (Figure 4a,b
). The major gelatinase activity in luteal extracts was more prominent in bands of 62 than 68 kDa. Treatment with EDTA/orthophenanthroline eliminated the gelatinase activity in luteal extracts (data not shown). Pretreatment of luteal extracts with aminophenyl mercuric acid, which is a procedure known to activate latent collagenase, resulted in a marked shift of the 68 kDa band to the 62 kDa band (data not shown). Although not all MMPs could be detected by zymography, we suspected that the major MMP in luteal extracts was MMP-2, present predominantly as the latent (68 kDa) form with a small amount of activated MMP-2 (62 kDa). The results of zymography showed that the amount of what appeared to be activated MMP-2 in the LLP was 4.1-, 2.8- and 1.9-fold greater than that of activated MMP-2 in the ELP, MLP and GCL respectively. However, the expression of what appeared to be the latent form of MMP-2 in the LLP was not significantly different from that of the latent form of MMP-2 in the ELP, MLP and GCL. The differences in molecular weight between Western blotting and gelatin zymography were probably due to the differences between reduced and non-reduced samples.
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Gelatinase activity was also detected at
97 kDa (latent form of MMP-9), and the expression of MMP-9 in the MLP was less than in the ELP, LLP and GCL. However, there was no gelatinase activity at 88 kDa (active form of MMP-9).
Pro-MMP-2 processing activity in the human CL was examined by using plasma membrane fractions of CL. Plasma membrane fractions of CL were incubated with FCS as a source of procollagenase as reported previously (Cao et al., 1995). As shown in Figure 5a,b, the membrane fraction of CL in LLP demonstrated that its induction of what appeared to be the active form of MMP-2 was 5.3-, 6.4- and 4.5-fold greater than in the ELP, MLP and GCL respectively.
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Expression of MT1-MMP, MMP-2, TIMP-1 and TIMP-2 mRNAs in human CL
As shown in Figure 6a,b
, MT1-MMP mRNA expression in the LLP was 3.1-, 2.5- and 2.7-fold greater than in the ELP, MLP and GCL respectively, and the expression of MMP-2 mRNA in LLP was 5.3-, 3.5- and 4.9-fold greater than in the ELP, MLP and GCL respectively. As also shown in Figure 6a,b, the expression of TIMP-1 mRNA in the LLP was decreased to 73 and 59% of the expression in the ELP and MLP respectively. However, the expression of TIMP-1 mRNA in the GCL was not significantly different from that of TIMP-1 mRNA in the ELP, MLP and LLP. Furthermore, the expression of TIMP-2 mRNA in the LLP was 5.4-, 2.8- and 3.8-fold greater than in the ELP, MLP and GCL respectively. In addition, the expression of TIMP-2 mRNA in the GCL was greater than that in the ELP, but was not significantly different from that in the MLP.
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It is known that MT1-MMP and MMP-2 are involved in structural luteolysis in rats (Endo et al., 1993
; Goto et al., 1999). The present study focused on the expression and regulation of MMP during human luteolysis and compared samples with data for the CL during gestation.
MMPs are known to be involved in the regression of the CL. We have reported that MMP-2 is one of the MMPs that might play a central role in structural luteolysis (Endo et al., 1993). We later reported that MT1-MMP in the membrane fraction of the rat CL could activate MMP-2 and cause remodelling of the CL during the process of structural luteolysis (Goto et al., 1999). In the present study, we found that the expression of MT1-MMP in the human CL increases in the LLP in comparison with that in the ELP and MLP. In addition, zymography revealed that the processing activity of pro-MMP-2 in human CL was higher in the LLP than in the ELP and MLP. An examination of the localization of MT1-MMP mRNA and protein showed that they were observed mainly in the cytoplasm of granulosa lutein cells. These findings demonstrated that MT1-MMP is produced by luteal cells. The expression of MMP-2, especially the activated form of MMP-2, was also found to be greater in the LLP than in the ELP and MLP. Duncan et al. have reported that MMP-2 mRNA is localized more in the thecal layer and not found in the granulosa cell layer (Duncan et al., 1998). The present work disagrees with their immunohistochemical results. Though the reason for the difference is not clear, it may depend on the difference between mRNA and protein of MMP-2 or on the properties of the antibodies employed. Our immunohistochemical study also did not detect the MMP-2 activities in immune cells that Duncan indicated in his review (Duncan, 2000).
A low level of expression of TIMP-1 was observed in the LLP. TIMP-1 is known to bond stoichiometrically to almost all MMP active sites and the activity of MMPs is thereby irreversibly inhibited (Umenishi et al., 1991; Willenbrock and Murphy, 1994). Therefore, the increased expression of TIMP-1 in the ELP and MLP would be expected to inhibit MMP-2, while the drop in TIMP-1 expression in the LLP could contribute to the maintenance of high activity of MMP-2. O'Sullivan et al. have reported induction of TIMP-1 in vitro by HCG (O'Sullivan et al., 1997). One could speculate that TIMP-1 expression is possibly decreased without HCG in the LLP. In addition, in pregnancy, HCG may cause an increase in expression of TIMP-1. In contrast with TIMP-1, TIMP-2 was much more strongly expressed in the LLP than in the ELP and MLP. Although TIMP-2 is known to exert a strong inhibitory action on MMP-2 (Stetler-Stevenson et al., 1989), it is also involved in the activation of pro-MMP-2. Previously it had been reported that TIMP-2 forms an MT1-MMP/TIMP-2 complex or MT1-MMP/TIMP-2/pro-MMP-2 ternary complex and that these complexes are related to activation of pro-MMP-2 (Strongin et al., 1995; Imai et al., 1996; Sato et al., 1997; Werb, 1997; Butler et al., 1998; Kinoshita et al., 1998). Therefore, the increased expression of TIMP-2 in the LLP was involved in the high activation of pro-MMP-2 by making those complexes. However, Duncan et al. reported no significant changes in the expression of TIMP-1 and TIMP-2 in human CL occurring throughout the luteal phase (Duncan et al., 1998); the causes of the discrepancy between this finding and our results are unclear at present.
As the CL regresses, microenvironmental changes around the luteal cells are major characteristics in structural luteolysis. The term `remodelling' is often used to describe changes in the ECM as well as in the cells. Collagens are major components of the ECM in the CL. There are many reports of basal lamina around large luteal cells, suggesting the presence of collagen type IV collagen and laminin within the CL (Deane et al., 1966; Farin et al., 1986; O'Shea, 1987; Kenny et al., 1989; Leardkamolkarn and Abrahamson, 1992). Although a structural basement membrane does not exist in the CL, type I and IV collagens are known to be abundant in rat CL (Matsushima et al., 1996). In addition, the amount of collagen is increased in the LLP in the ruminant CL (Luck et al., 1995; Zhao and Luck, 1995). The accumulation of collagens is recognized in degenerating CL, and might constitute the corpus albicans at the end stage of the destruction resulting from the interaction between cells and the ECM. The mature corpus albicans is a well circumscribed structure with the convoluted borders composed of densely packed collagen fibres with occasional admixed fibroblasts (Clement, 1987).
The organization of the CL is a dynamic process in which cells and ECM can reconstruct the tissue in ovaries, and it is cyclically regulated by hormones. The human CL is completed at 3–7 days after ovulation and, thereafter, it produces both estrogen and progesterone for 7–11 days. In the presence of implantation, the CL continues the synthesis of both estrogen and progesterone for 8–10 weeks via the stimulation of HCG produced by the placenta. In the present study, expression of MT1-MMP, MMP-2 and TIMP-2 mRNAs in the GCL was as low as in the MLP. It has been reported that MMP-2 production in cultured granulosa cells is suppressed by HCG (Stamouli et al., 1996). Furthermore, proMMP-2 synthesis in cultured endometrial cells is known to be inhibited by progesterone, and the hormone also attenuates the expression of MT1-MMP mRNA (Zhang et al., 2000). Irwin et al. reported that a withdrawal of progesterone in stromal cell cultures results in the induction of MMP-2 (Irwin et al., 1996). From these reports, it appears that withdrawal of progesterone might up-regulate MT1-MMP expression and that MT1-MMP could stimulate pro-MMP-2 activation. Therefore, the low expression of MT1-MMP in the GCL seems to be related to the low activation of pro-MMP-2. In addition, since an increase of the active form of MMP-2 may trigger degeneration of the CL by dissolving the ECM, it is reasonable that the expression of the active form of MMP-2 is lower in the GCL than in the LLP.
We have studied the factors involved in the remodelling of the ECM in the CL during the menstrual cycle and present evidence of the importance of MT1-MMP and MMP-2 in degeneration of the CL. We hypothesize that the increased expression of MMP-2 may be activated by MT1-MMP in the LLP and may be a major cause of the remodelling in human CL during structural luteolysis.
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We thank our colleagues T.Kiya, T.Goto, N.Akutagawa and M.Iwasaki for expert technical assistance. We also thank Professor Harold R.Behrman and Professor Ylikorkara A. for their careful reading of our manuscript.
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4 To whom correspondence should be addressed. E-mail: endot{at}sapmed.ac.jp
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References |
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Submitted on December 20, 2001; accepted on May 10, 2002.
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