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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0762
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5848-5857
Copyright © 2005 by The Endocrine Society

Response of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases Messenger Ribonucleic Acids to Ovarian Steroids in Human Endometrial Explants Mimics Their Gene- and Phase-Specific Differential Control in Vivo

Vassil Vassilev, Chrystel M. Pretto, Patricia B. Cornet, Denis Delvaux, Yves Eeckhout, Pierre J. Courtoy, Etienne Marbaix and Patrick Henriet

Cell Biology Unit (V.V., C.M.P., P.B.C., D.D., Y.E., P.J.C., E.M., P.H.), Christian de Duve Institute of Cellular Pathology, and Department of Pathology (E.M.), Université Catholique de Louvain, B-1200 Bruxelles, Belgium

Address all correspondence and requests for reprints to: Etienne Marbaix, Cell Biology Unit, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, avenue Hippocrate, 75, B-1200 Bruxelles, Belgium. E-mail: marbaix{at}cell.ucl.ac.be.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Cyclic remodeling and breakdown of the extracellular matrix, a unique feature of the human endometrium, depends on matrix metalloproteinases (MMPs). These enzymes are globally controlled by estradiol and progesterone or their withdrawal, but various MMPs and their tissue inhibitors (TIMPs) show distinct responses.

Objective and Design: To clarify the role of ovarian steroids in the differential regulation of MMP-1, MMP-3, MMP-7, MMP-8, MMP-10, TIMP-1, TIMP-2, and TIMP-3 mRNAs, we compared their variations in the cycling endometrium in vivo with their response to hormone addition or withdrawal in corresponding explants.

Results: Different patterns were identified in vivo according to the time frame (secretory vs. perimenstrual increase), sharpness (peak vs. progressive increase or decrease), and magnitude of the changes. In vivo ratios between early/midsecretory and perimenstrual phases ranged from more than 1000 (MMP-1, MMP-3, and MMP-10) to less than 10 (TIMPs). Differential response to ovarian steroids of the various MMPs and TIMPs mRNAs tested in cultured explants matched the same ranking and varied according to the phase at sampling. Remarkably, ovarian steroids repressed MMPs and TIMP-1 and TIMP-2 but, in secretory explants, increased TIMP-3 mRNA. Finally, in situ hybridization evidenced the major contribution of fibroblasts to the increase in MMP-8 mRNA at menstruation or in explants cultured without hormones.

Conclusions: Both phase- and gene-specific modulators finely tune in space, time, and amplitude the global control of MMPs and TIMPs mRNAs by estradiol and progesterone in the cycling human endometrium.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
MATRIX METALLOPROTEINASES (MMPS) play a key role in physiological and pathological extracellular matrix (ECM) remodeling, as illustrated in the human endometrium during normal menstruation and dysfunctional uterine bleeding (1, 2, 3). In addition, the cycling human endometrium offers a unique physiological example of alternative regulation of tissue breakdown and reconstruction. It is now established that ovarian steroids (estrogens and especially progesterone) control the production and activity of MMPs through a network of local regulators, including cytokines (reviewed in Refs.4, 5, 6). This network would preserve endometrial integrity during the proliferative and secretory phases, then cause at menstruation an abruptly amplified, yet spatially constrained degradation of the functional layer (7).

Menstrual-like ECM breakdown by MMPs and its global control by the ovarian steroids have been reproduced in endometrial explants (1, 8). Both in the menstrual endometrium in vivo and in explants from nonmenstrual endometria cultured without added hormones, various MMPs were found to be selectively produced by foci of stromal cells in areas of tissue breakdown. Addition of combined estradiol and progesterone to the explants blocked both MMPs release in the medium and ECM degradation (9, 10).

In previous studies, the mRNA expression pattern of several MMPs and their physiological tissue inhibitors (TIMPs) was determined in the cycling human endometrium in vivo by in situ hybridization (11, 12), Northern blotting (13), and RT-PCR (14, 15). Several MMP genes, including those of collagenase-1 (MMP-1), collagenase-2 (MMP-8), stromelysin-1 (MMP-3), and matrilysin-1 (MMP-7), as well as genes encoding TIMP-1, TIMP-2, and TIMP-3, were maximally expressed around menstruation. However, their expression profile significantly differed during the other phases of the cycle, leading to the distinction of several groups. MMP-1, MMP-8, and MMP-3 mRNAs were reported to be restricted to the menstrual phase, in contrast with MMP-7 mRNA, which remained abundant during the proliferative phase. TIMP-3 showed cyclic variations and reached maximal levels during the late secretory phase, unlike TIMP-1 and TIMP-2, which were reported to be expressed at high, but rather constant levels throughout the cycle.

Altogether, these observations pointed to a differential modulation of gene expression under the overall control by ovarian steroids. We recently used RT-PCR to investigate the control by these hormones of selected gene expression in endometrial explant cultures, by comparison with the corresponding cyclic variations in the endometrium in vivo (16, 17). These studies not only provided precise measurements of the effects of estradiol and progesterone on mRNA amounts in the cultured explants but (more importantly) evidenced differences in the response to culture and ovarian steroids according to the phase of the cycle at sampling, especially for the TGF-ß-related cytokine, Lefty-A (16). This prompted us to extend this combined approach to further dissect the respective contribution of ovarian steroids and specific modulators to the regulation of menstruation-associated MMPs and their inhibitors.

In the present study, we first measured mRNA amounts of MMP-1, MMP-3, MMP-7, MMP-8, MMP-10, TIMP-1, TIMP-2, and TIMP-3 in a collection of 59 endometrial samples representative of the whole menstrual cycle, to determine the chronology and to quantify the amplitude of their cyclic variations. This first part of the analysis complements previous studies by: 1) the precise timing of samples along the menstrual cycle; 2) the use of a highly sensitive, quantitative, and standardized technique allowing the calculation of ranges of variation; and 3) the analysis of all individual results, not only of sample groups. The variable contribution of combined estradiol and progesterone to individual gene regulation was next determined by comparing mRNA levels in vivo to those measured in corresponding explants cultured for 24 h in the absence or presence of the ovarian steroids. Finally, because MMP-8 could originate from stromal fibroblasts or infiltrating neutrophils, we localized its mRNA by in situ hybridization in endometria in vivo and in cultured explants.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Tissue collection and explant culture

The study was approved by the Ethical Committee of the Université Catholique de Louvain, in accordance with the Declaration of Helsinki of the World Medical Association. Normal endometrial tissue was obtained from 52 hysterectomy specimens and seven biopsies sampled at various phases of the menstrual cycle as described (16). Briefly, endometrium was gently scraped with a sterile surgical blade from hysterectomy specimens and put in ice-cold PBS; biopsies were immediately collected in ice-cold PBS. Part of the endometrial tissue was fixed in 4% formaldehyde, and another part was quickly frozen at –80 C in lysis buffer (SV Total RNA Isolation System; Promega Corp., Leiden, The Netherlands). Tissue was dated according to its histological appearance (18) and the clinical recording of the last menstrual period. An ideal menstrual cycle of 28 d was divided into five phases: proliferative (d 6–14), early secretory (d 15–18), midsecretory (d 19–22), late secretory (d 23–27), and perimenstrual (d 28–5). All patients were premenopausal (range, 25–55 yr old; median, 44 yr old), were not on hormonal treatment, underwent surgery for conditions unrelated to endometrial pathologies or to endometriosis, or had a biopsy for routine investigation of infertility. They gave informed consent.

The collection and culture of endometrial explants was performed as previously described (16). Briefly, tissue samples were cut in pieces of about 1mm/side with a sterile surgical blade and placed in tissue culture inserts (Millipore Corp., Bedford, MA). DMEM (Life Technologies, Inc., Merelbeke, Belgium), devoid of serum and phenol red, was placed in the lower chamber and renewed daily (300 µl or 1.2 ml in 12-mm and 30-mm inserts, respectively). Medium was without hormonal addition, or supplemented with water-soluble complexes of 1 nM 17ß-estradiol and 100 nM progesterone in 2-hydroxypropyl-ß-cyclodextrin (+EP; Sigma-Aldrich Corp., Bornem, Belgium). The encapsulating 2-hydroxypropyl-ß-cyclodextrin (Sigma-Aldrich) was added at 0.3 µM in the culture medium devoid of ovarian steroids. We previously reported that this system allows us to maintain explants in good conditions for up to 3 d and that the effects of estradiol and progesterone can be reversed during culture (1). After collection, all media were supplemented with 0.05 vol of 1 M Tris-HCl buffer (pH 7.5); 1% (vol/vol) Triton X-100; 0.1 M CaCl2; and 60 mM NaN3 and kept frozen at –20 C until biochemical analysis. At the end of the culture, explants were fixed or frozen at –80 C in lysis buffer until RNA extraction.

RNA extraction and reverse transcription

After extraction of total RNA using the SV Total RNA Isolation System (Promega), 200 ng was reverse-transcribed by using the Thermoscript RT-PCR System (Life Technologies, Inc., Paisley, UK) as described (16).

Oligonucleotide primers used for PCR amplification

Sequences of the specific oligonucleotide primers and Taqman probes used for the real-time PCR amplifications are listed in Table 1Go. Customized primers were obtained from Life Technologies or BioSource International (Camarillo, CA).


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  		TABLE 1. Primers and probes used for quantitative PCR

 
Preparation of standards for real-time PCR

Standards for quantitative PCR amplification were prepared by cloning purified DNA products, obtained after PCR amplification with the appropriate primers, into the pCR II-TOPO vector (TOPO TA cloning kit; Life Technologies). Plasmids were amplified in Escherichia coli XL-1 blue competent bacteria (Stratagene, Amsterdam, The Netherlands), purified, and quantified by spectrophotometry. Known amounts (1 µg) of plasmids were stored at –20 C as aliquots that were thawed only once and used immediately.

Real-time PCR

Real-time PCR was performed according to the manufacturer’s recommendations, with the LightCycler-FastStart DNA Master for Hybridization Probes or for SYBR-Green (Roche Boehringer Mannheim Diagnostics, Mannheim, Germany), as appropriate, using a LightCycler (Roche Boehringer) or a MyIQ thermocycler (Bio-Rad Laboratories, Inc., Hercules, CA, for MMP-8 mRNA measurements). Aliquots of the RT products were subjected to PCR in a total vol of 20 µl, with 500 nM adequate paired primers, 200 nM Taqman probe (when applicable), and MgCl2 at the optimized concentration for each target gene (2 mM for ß-actin, 3 mM for TIMP-1 and TIMP-3, and 4 mM for MMP-1, MMP-3, MMP-7, MMP-8, and TIMP-2). A control PCR without template DNA was performed in each experiment. A standard curve was generated from serial 10-fold dilutions of the purified plasmid containing the appropriate cDNA. Real-time PCRs were performed as follows. After an initial denaturation at 95 C for 10 min, DNA was amplified by 40–45 cycles of 20 sec at 95 C, and either 15 sec at 60 C for MMPs and TIMPs or 20 sec at 67 C for ß-actin. MMP-8 cDNA was amplified through 40 cycles of 20 sec at 95 C and 30 sec at 65 C. Data were analyzed by using the LightCycler Software (version 3.5, Roche Boehringer) or the MyIQ System Software (version 1.0.410, Bio-Rad). Regression analyses of the threshold cycle values of the standard dilution series were used to determine the amplification efficiency. The absolute number of molecules in the experimental samples was determined by extrapolating the threshold cycle values from the standard curves. We considered that a PCR experiment was valid when: 1) no amplification was detected in the control without DNA template; 2) the slope of the logarithmic standard curve was around –3.3 (range, –3.1 to –3.5), to ensure full efficiency of the PCR [calculated as (10–1/slope)]; and 3) the amplification curve of the samples was parallel to those of the standards. The ß-actin values were used for standardization.

In situ hybridization

Part of the endometrial tissues and of explants was fixed in freshly prepared, phosphate-buffered 4% formaldehyde (pH 7.4), and embedded in paraffin. Five-micrometer-thick sections were deparaffinized, rehydrated, and treated for 30 min at 37 C with 1 µg/ml proteinase-K (Sigma-Aldrich) in 100 mM Tris-HCl (pH 8.0) and 50 mM EDTA. Sections were then acetylated for 10 min in 0.25% acetic anhydride and 0.1 M triethanolamine (pH 8.0), and prehybridized for 1 h at 50 C in the hybridization mixture containing 40% (vol/vol) formamide, 50% (wt/vol) dextran sulfate, 2% (vol/vol) Denhardt’s solution, and 2x sodium saline citrate (SSC) (1x SSC = 150 mM NaCl and 15 mM sodium citrate) in a humidified chamber. A mixture of two MMP-8 specific oligonucleotide probes [5'-AGGTAGAATGGATACAGTGATGGGAAA-3' and 5'-CCTTACTTCTAACCAAAATCTAGTTC-3' derived from (19) and (20), respectively] or of two control oligonucleotides with the complementary sequence was prepared by fluorescein-deoxyuridine 5'-triphosphate labeling of the 3'-ends (Gene Images 3'-oligolabeling module; Amersham Biosciences, Piscataway, NJ). The sections were then hybridized overnight at 50 C with the hybridization mixture supplemented with 1.5 ng/µl labeled probes and washed with decreasing concentrations of SSC (4x, 2x, and 0.1x). All washes were carried out at room temperature for 2 x 10 min. After inactivation of endogenous peroxidase with 0.3% H2O2 for 30 min at room temperature, nonspecific binding sites were blocked by incubating slides for 1 h in Tris buffer (50 mM Tris-HCl, pH 7.4, containing 10% normal goat serum and 1% BSA). The histological sections were incubated for 1 h at room temperature with 8 µg/ml rabbit antifluorescein antibodies (DakoCytomation, Glostrup, Denmark). After two washes in deionized water and two washes in 50 mM Tris-HCl (pH 7.4), slides were incubated for 1 h at room temperature with peroxidase-conjugated dextran molecules carrying antirabbit secondary antibodies (Envision; DakoCytomation). The peroxidase activity was revealed by incubating the slides for 10 min at room temperature with 0.5 mg/ml diaminobenzidine in 50 mM Tris-HCl (pH 7.4).

Immunolabeling

Histological sections were immunolabeled by mouse monoclonal antibodies specific for CD10 (clone 56C6, 2 µg/ml; BIOCARE MEDICAL, Walnut Creek, CA); CD45 (a mixture of clones PD7/26 and 2B11, 1 µg/ml; DakoCytomation), cytokeratin (clone CAM5.2, 1 µg/ml; Becton Dickinson and Co., San Jose, CA), or CD68 (clone KP-1, 4.5 µg/ml; DakoCytomation). For controls, the primary antibody was replaced by a mixture of nonrelevant antibodies raised against epitopes present in immature melanosomes (Pan Melanoma Cocktail, 4.5 µg/ml; BIOCARE MEDICAL). After removal of paraffin and inactivation of endogenous peroxidases with 0.3% H2O2 for 30 min at room temperature, slides were transferred in 10 mM sodium citrate buffer (pH 5.8) and incubated into a water bath at 98 C for 75 min to unmask the antigenic sites, except for cytokeratin. After blocking of the nonspecific binding sites by incubating the slides for 30 min into 50 mM Tris-HCl (pH 7.4) containing 10% normal goat serum and 1% BSA, sections were incubated overnight at 4 C with the primary antibody. Washes and staining were performed as for in situ hybridization, using peroxidase-conjugated dextran molecules carrying antimouse secondary antibodies (Envision; DakoCytomation).

Statistical analysis

Statistical significance was tested using the Wilcoxon rank-sum two-sample test or the Wilcoxon signed-rank matched-pairs test, as appropriate. Differences were interpreted as significant for P < 0.05 and assigned the following symbols: *, P < 0.05; **, P < 0.005; ***, P < 0.001; NS, not significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Variations of MMPs and TIMPs relative mRNA amounts in the cycling endometrium in vivo

We first analyzed in vivo variations of mRNA amounts normalized to that of ß-actin (hereafter referred to as relative mRNA amounts). As shown in Fig. 1Go, these values could vary considerably between patients, even for a given phase of the cycle, as best illustrated for MMP-1, MMP-3, MMP-8, and MMP-10 (notice the logarithmic scale, up to 5 logs between extremes during the proliferative phase). On the other hand, individual values for MMP-7, TIMP-1, TIMP-2, and TIMP-3 mRNAs showed much less variation (generally within 1 log for TIMPs).



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  		FIG. 1. Expression of MMPs and TIMPs mRNAs in the cycling human endometrium. Endometrial samples were dated according to histological criteria and clinical information. Dating begins by the clinical occurrence of vaginal bleeding, but the graph starts with d 15 (1 d after ovulation in the idealized cycle), to emphasize the perimenstrual phase (from d 28–5). Perimenstrual samples are represented by black-filled symbols and late secretory endometria by gray symbols. The amount of mRNA molecules of MMP-1, MMP-3, MMP-10, MMP-8, MMP-7, TIMP-1, TIMP-2, TIMP-3, and ß-actin was quantified by RT-PCR. The relative mRNA amounts (ratios of MMPs or TIMPs to ß-actin) are presented on the same logarithmic scale. For panel ranking, see Fig. 2Go. Not detected values (ND) are represented below the dotted line.

 
Along the menstrual cycle, mRNA amounts of all studied genes culminated around menstruation, with some gene products, such as those of MMP-7 and MMP-10, being 10–100 times more abundant than that of ß-actin, assuming equal amplification efficiency during the early uncontrollable steps of the PCR. However, the time frame, sharpness, and amplitude of the cyclic changes in mRNA expression varied significantly between genes. Some similarities were observed between groups of mRNAs, but grouping was not fully consistent for these three criteria.

With respect to time frame as first criterion, TIMP-3 was maximally expressed before menstruation, then its mRNA declined at the beginning of menses. Other gene products increased throughout the secretory phase and culminated at menstruation (best illustrated for MMP-10, a behavior shared by TIMP-1 and TIMP-2). Still other gene products peaked in the perimenstrual phase (MMP-7) or essentially appeared at that phase (MMP-1, MMP-3, and MMP-8). This chronology will be analyzed in more detail in the following section. Second, with respect to the rapidity of changes, some relative mRNA levels increased either sharply (MMP-1, MMP-3, MMP-8, MMP-7, and also TIMP-3) or progressively (MMP-10, TIMP-1, and TIMP-2). Third, with respect to level of expression, some mRNAs showed low minimal amounts and a large range of cyclic variation (MMP-1, MMP-3, MMP-10, and MMP-8); one gene product (MMP-7) showed high minimal amounts and an intermediate range of cyclic variation, and still others showed high minimal amounts and limited cyclic variation (TIMP-1, TIMP-2, and TIMP-3).

Thus, although several genes shared some characteristics, there was no unequivocal gene grouping. Therefore, the chronology and the amplitude of the in vivo variations in mRNA amounts will hereafter be presented separately.

Chronology of the various mRNA changes in vivo

The chronology of the changes for the various mRNAs will now be described, starting at ovulation, to focus on changes preceding, during, and after menstruation. MMP-10 and TIMP-1 relative mRNA levels progressively increased throughout the secretory phase. TIMP-2 expression pattern was similar, although changes were not significant. Relative mRNA levels of TIMP-3 increased more sharply, at the transition between the early/mid- and the late secretory phases. MMP-10 further increased at the end of the secretory phase, to peak during menses. In contrast with these secretory changes, MMP-1, MMP-3, MMP-7, and MMP-8 rose abruptly at the start of the perimenstrual phase, or just before for MMP-8.

After this general pre- or early menstrual increase in MMPs and TIMPs gene expression, mRNA levels quickly returned to minimal levels during (for TIMP-3) or at the end of the menstrual phase (for all other gene products, except for MMP-7 mRNA, which slowly decreased during the proliferative phase). Thus, TIMP-3 mRNA clearly stood out among all tested gene products for its decrease between early (d 28–1) and late (d 4–5) perimenstrual phase. MMP-1, MMP-3, MMP-10, and MMP-8 mRNAs were most dispersed in proliferative endometria.

Amplitude of the various mRNA changes in vivo

To correlate the amplitude of the changes in the various mRNA levels with the cyclic variations in circulating estradiol and progesterone concentrations, individual values were then pooled in the four following populations so that medians could be compared: early and midsecretory (increasing concentrations of both steroids), late secretory (decreasing concentrations of both steroids), perimenstrual (minimal steroid concentration), and proliferative (essentially impregnation by estradiol). Table 2Go compares the median levels between all consecutive cycle phases, with the significance of changes. Whereas MMP-10 mRNA gradually increased during the secretory phase, sharp and strong (over 2 logs) increases in MMP-1, MMP-3, and MMP-8 mRNAs occurred at the beginning of menstruation (see perimenstrual/late secretory ratio), with reciprocal decreases at its end (proliferative/perimenstrual ratio). The amplitude of perimenstrual increase and postovulation decrease of MMP-7 mRNA, and that of late secretory increase and menstrual decrease of TIMP-3 mRNA, was intermediate (around 20 times). Finally, TIMP-1 and TIMP-2 increases and decreases were smaller than 1 log. For all but TIMP-3 mRNAs (see in particular MMP-7), medians were lower in early/midsecretory than in proliferative endometria, suggesting a general inhibition of all but one of the genes studied upon increase in circulating progesterone (acting in combination with estradiol), and an opposite stimulatory effect on TIMP-3.


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  		TABLE 2. Changes in median relative mRNA amounts between consecutive cycle phases in vivo

 
For a ranking, Fig. 2Go is focused on the changes in relative mRNA amounts between endometria with the highest concentration of ovarian steroids (early/midsecretory phase) and those with the lowest concentration (perimenstrual phase). The early/midsecretory median was over 3 logs lower than the perimenstrual one for MMP-1, MMP-3, and MMP-10 (and most likely MMP-8, because the early/midsecretory median for MMP-8 was below the level of detection). On the opposite, MMP-9 (see Ref.17), MMP-7 and especially TIMP-1, TIMP-2, and TIMP-3 mRNA levels were much more stable throughout the cycle: their early/midsecretory medians were, respectively, less than 2 logs and less than 1 log lower than the perimenstrual ones, reflecting, in part, the persistence of high mRNA amounts in nonperimenstrual samples (0.1–0.01, relative to ß-actin mRNA).



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  		FIG. 2. Changes in MMPs and TIMPs mRNA amounts between early/midsecretory and perimenstrual endometria in vivo. Ratios of median relative mRNA amounts for MMPs and TIMPs in early/midsecretory (ES-MS) endometria, compared with perimenstrual endometria, are presented on a logarithmic scale. Values for MMP-9 mRNA were derived from Ref.17 , with new additional samples, and are presented solely for the purpose of comparison. Genes are ranked according to increasing ratio values. The ratio for MMP-8 was calculated between late secretory and perimenstrual medians because the ES-MS median is under the limit of detection. The actual ratio is thus less than 4 x 10–3, as indicated by the question mark. *, P < 0.05; **, P < 0.005; ***, P < 0.001.

 
Altogether, the differences between analyzed mRNAs in the time frame, sharpness, and amplitude of their cyclic variations in vivo pointed to a differential modulation of their overall control by ovarian steroids, thanks to additional gene- and phase-specific factors.

Effects of ovarian steroids on the relative mRNA amounts in endometrial explants

The hypothesis of such modulators was tested by measuring mRNAs amounts after culture of explants collected from the different phases, in the presence or absence of ovarian steroids for 24 h (Fig. 3Go). For each gene and patient, relative mRNA amounts measured in explants were compared with corresponding values in the noncultured sample from the same patient. To parallel the in vivo analysis, paired ratios were then grouped for proliferative, early/midsecretory, or late secretory samples (Table 3Go). Perimenstrual endometria were not used for this analysis because they were previously shown not to respond to ovarian steroids (9).



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  		FIG. 3. Effects of ovarian steroids on MMPs and TIMPs mRNA concentration in endometrial explant culture. Relative mRNA amounts were measured in explants from proliferative (PROLIF), early/midsecretory (ES-MS), and late secretory (LS) endometria after culture for 24 h in the absence (–H, filled symbols) or in the presence of 1 nM estradiol and 100 nM progesterone (EP, open symbols). Notice that logarithmic scales for the various mRNAs are used with constant spacing for 1-log increments but with variable ranges.

 

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  		TABLE 3. Effect of 24-h explant culture and ovarian steroids on mRNA amounts

 
As summarized in Table 3AGo, relative mRNA amounts of all tested genes increased upon 24 h of culture without added ovarian steroids (filled symbols in Fig. 3Go) to reach values similar to noncultured menstrual samples (compare with corresponding filled symbols in Fig. 1Go). This induction was spectacular, exceeding 2 logs for MMP-10 and MMP-8 mRNAs and reaching 4 logs for MMP-1 and MMP-3 mRNAs, but was less pronounced (around 1 log) for MMP-7 and the three TIMPs mRNAs.

Surprisingly, all relative mRNA amounts also increased upon explant culture in the presence of estradiol and progesterone (albeit to a much lesser extent than in the absence of the hormones, except for TIMP-3; compare A and B in Table 3Go) and irrespective of the analyzed phase. This observation indicates that the mere addition of ovarian steroids, although sufficient to prevent ECM breakdown (1, 10), did not fully reproduce, in the explants, their in vivo inhibitory effect on gene expression.

In general, when comparing paired values in treated and untreated cultured explants, ovarian steroids significantly repressed the expression of all MMPs, as well as that of TIMP-1 and TIMP-2, but not of TIMP-3 (Fig. 4Go). This inhibition was most prominent in proliferative and late secretory explants and less pronounced or absent (for TIMP-1 and TIMP-2) in early/midsecretory explants (Table 3CGo). The more limited repression by the ovarian steroids at that phase was essentially due to the lower values reached upon culture in the absence of hormones, when compared with proliferative explants.



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  		FIG. 4. Effect of ovarian steroids on mRNA amounts. The figure shows the geometric mean ratios of relative mRNAs amounts for MMPs and TIMPs in explants cultured in the presence (+EP), compared with the absence (–H), of added ovarian steroids. Explants are from endometria sampled at all proliferative and secretory phases of the cycle. For the purpose of comparison, values are presented on a logarithmic scale and ranked as in Fig. 2Go. Values for MMP-9 are derived from Ref.17 , with new additional samples. *, P < 0.05; ***, P < 0.001.

 
TIMP-3 was a noticeable exception. In proliferative explants, it was not significantly inhibited by the ovarian steroids. Moreover, TIMP-3 mRNA levels were significantly increased by the addition of the steroids in nine of 11 secretory samples. This unique induction by ovarian steroids in vitro is in agreement with our in vivo observation that TIMP-3 mRNA level was the only case of a higher median for early/midsecretory than for proliferative biopsies (see Table 2Go). Future work is needed to verify whether this induction is due to estradiol, progesterone, or their combination.

Cellular origin of MMP-8 mRNA

Because MMP-8 was originally reported to be exclusively produced by neutrophils, the occasional detection of MMP-8 mRNA in a minority of nonperimenstrual samples in vivo could reflect the presence of infiltrating inflammatory cells. Moreover, inflammatory cells are abundant at menstruation, when we noted the systematic occurrence of large amounts of MMP-8 mRNA. However, neutrophil MMP-8 is synthesized during cell maturation in bone marrow and stored in specific granules until release in inflammatory sites, where neutrophils are not expected to contain large amounts of MMP-8 mRNA (21). MMP-8 was more recently shown to be also produced by mononuclear fibroblast-like cells, for example articular chondrocytes and rheumatoid synovial membrane fibroblasts in vivo (22, 23) and odontoblasts in organ culture (24). The large increase in MMP-8 mRNA upon culture of nonperimenstrual explants, where inflammatory cells are barely detected, rather suggested a similar production of MMP-8 by resident endometrial cells, such as stromal fibroblasts. We therefore determined the cellular origin of MMP-8 mRNA by combining in situ hybridization with immunolabeling for cell type markers in adjacent serial sections (Fig. 5Go).



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  		FIG. 5. Cellular origin of MMP-8 mRNA. Cellular localization of MMP-8 mRNA in vivo and in explant culture. MMP-8 antisense (A–C, M, N, Q) or sense control (K) probes were hybridized to serial sections of: a noncultured menstrual endometrium (A–L), a noncultured proliferative endometrium (M), and a proliferative endometrium cultured for 24 h in the absence (N–P) or presence (Q–S) of estradiol and progesterone. Immunolabeling for CD10 (fibroblasts, D–F, O, R), CD45 (leukocytes, G–I, P, S), cytokeratin (epithelial cells, J), CD68 (macrophages, L), or epitopes present in immature melanosomes (negative control, not shown) was performed in adjacent sections. B–C, E–F, and H–I show progressive enlargements of areas in A, D, and G, respectively. Notice that most cells expressing MMP-8 mRNA are also labeled for CD10. Bars, 50 µm.

 
A strong signal for MMP-8 mRNA was found to colocalize with stromal fibroblasts in menstrual endometria in vivo (compare Fig. 5Go, A–C, with D–F) as well as in proliferative explants cultured 24 h in the absence of hormones (compare Fig. 5Go, N with O). Conversely, MMP-8 mRNA signal was dramatically reduced, if not undetectable, in proliferative endometria before culture (Fig. 5MGo) or after 24 h of explant culture in the presence of the ovarian steroids (Fig. 5QGo). Leukocytes were present in all samples tested, in higher proportions in menstrual endometria (Fig. 5Go, G–I) than in nonperimenstrual endometria, before (not shown) or after explant culture in the absence (Fig. 5PGo) or presence (Fig. 5SGo) of ovarian steroids. Although it cannot be ruled out that neutrophils release MMP-8 in the menstruating endometrium, our in situ hybridization data clearly demonstrate that stromal fibroblasts are the major cell type expressing MMP-8 mRNA in noncultured menstrual endometria as well as in explants cultured in the absence of ovarian steroids. Fibroblast-derived MMP-8 is directly produced in situ, as a rapid response to ovarian steroids withdrawal, exactly as the other fibroblast-derived MMPs MMP-1 and MMP-3.


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
During the reproductive life, human endometrium expresses many MMPs and TIMPs, which are involved in the menstrual and abnormal breakdown of the tissue (1, 2, 3, 4, 25). However, some MMPs are also variably expressed during the nonmenstrual phases of the cycle (11, 15), at which time their physiological role is less clear, although they are likely to participate to several key features of the proliferative and secretory phases, such as reepithelialization and tissue regeneration, angiogenesis, and stromal decidualization. For such roles, MMPs expression and activation could be more limited in time and in space than at menstruation. In contrast, TIMPs are expected to be largely expressed during nonmenstrual phases to prevent inappropriate lysis of the tissue. In particular, TIMP-3 is thought to limit invasion of the decidualized mucosa by trophoblast at implantation (25). The purpose of this study was to test whether a differential response of MMPs and TIMPs to ovarian steroids in the cycling human endometrium could explain their different time course and expression levels. This hypothesis was investigated by quantitative RT-PCR in a well-characterized collection of human endometrial samples and in corresponding explants cultured with or without addition of ovarian steroids.

Our measurements in the noncultured samples are in excellent agreement with reported expression patterns (Refs.11 ,15 , and reviewed in Ref.4) and nicely correlate with those observed for MMP-1, MMP-3, and MMP-7 mRNAs upon withdrawal of a progesterone-releasing implant in ovariectomized monkeys (26). Moreover, these results provide new important information on the cyclic variations of these genes. The precise timing of samples along the menstrual cycle led to the discrimination between abrupt (within 1 or 2 d) and progressive changes in mRNA expression. The use of a highly sensitive technique with a large dynamic range evidenced the striking contrast between weakly (TIMPs) and strongly inhibited genes (MMP-1, MMP-3, and MMP-10). High levels of TIMPs mRNAs, maintained throughout the cycle and steadily increasing during the secretory phase, suggest that the corresponding proteins were abundantly produced before menses but overwhelmed by MMPs at menstruation. Moreover, high levels of TIMP-3 during the implantation window are in support of its proposed key role at this early stage of embryonic development (27, 28). Conversely, the physiological relevance of the very low mRNA amounts of most MMPs detected in nonperimenstrual endometrial extracts remains to be clarified. For example, MMP-1 and MMP-3 mRNAs were previously detected by in situ hybridization in a minority (about 1 of 4) of proliferative endometria (11). The absence of detection in the other samples could result from the lower sensitivity of in situ hybridization, compared with RT-PCR, or indicate the existence of MMP-expressing cell foci outside the analyzed fields.

The distinctive behavior of MMP-10 and MMP-7 deserves comment. Very high values for MMP-10 mRNA in late secretory samples, to further increase as a menstrual peak, agree with an in situ hybridization study showing the occurrence of MMP-10 mRNA in stromal foci of late secretory endometria and throughout the stroma at menstruation (11). MMP-10 therefore appears as a special case of menstruation-associated MMP, with steadily increasing expression throughout the secretory phase, to reach particularly high levels at menses. Finally, we confirm that MMP-7 mRNA is still expressed at high levels during the early proliferative phase (almost as abundant as during menses) and does not decrease as much as MMP-1 and MMP-3 mRNAs during the secretory phase (15, 29, 30).

We feel that the comparison between quantitative changes in expression profiles in vivo and in cultured explants from the same samples upon hormone addition or withdrawal is a major contribution of this study. Comparison of Figs. 2Go and 4Go shows that the relative extent of changes between the various mRNAs identified in the cycling human endometrium in vivo matched that resulting from the presence or absence of ovarian steroids in the cultured explants, especially proliferative ones. Although the control by ovarian steroids in explants was quantitatively less effective than in vivo, a parallel was evident, suggesting that the differential hormone control on MMPs and TIMPs is directly modulated through gene-specific coactivators and/or corepressors and does not depend on other circulatory factors or on extended priming by ovarian steroids.

However, there were differences in the hormonal control of TIMP-3 mRNA, compared with the other mRNAs, between proliferative and late secretory endometria. Because both phases are characterized by low concentrations of progesterone, this suggests the involvement of phase-specific factors that could locally regulate gene expression in endometria primed by estradiol and progesterone, by analogy to other decidualization markers such as prolactin and IGF binding protein-1. Increased TIMP-3 expression was previously observed as a result of progesterone-induced in vitro decidualization of human endometrial stromal cells (31). This predicts important differences between two comparisons in the endometrial explants: proliferative endometria cultured for 24 h in the presence of high concentrations of progesterone combined with estradiol are subject to abrupt hormonal addition and would lack secretory phase-specific factors that require prolonged priming by progesterone; conversely, secretory explants cultured in the absence of ovarian steroids are subject to severe hormone withdrawal but could still retain endogenous hormones and/or express the secretory phase-specific factors. Differences in MMP production by endometrial stromal cells on ovarian steroids addition or withdrawal have been previously reported (32, 33).

The similarity in hormone response within each of the four groups: 1) MMP-1, MMP-3, and MMP-10; 2) MMP-9 and MMP-7; 3) TIMP-1 and TIMP-2 vs. (4) TIMP-3 pointed to common transcriptional controls. We tested this possibility in silico by using the Genomatix GEMS launcher software tool, to compare the proximal promoters (–500 to +100 base pairs) of MMPs and TIMPs genes included in this study (settings by default: core matrix similarity 0.75, optimized matrix similarity, and transcription factors sites common to 90% of the input sequences). This in silico analysis revealed that, besides the presence of a TATA-box, genes encoding MMP-1, MMP-3, MMP-10, MMP-8, and MMP-7 contain several common binding sequences, including activator protein 1 (AP-1), CCAAT/enhancer-binding protein (CEBP) and E26 transformation specific (Ets) transcription factors. These sequences are, however, organized in a different order in the promoters of MMP-1, MMP-3, and MMP-10, as compared with those of MMP-8 and MMP-7, thereby opening the possibility for a differential tuning between these genes. In contrast, genes encoding MMP-9 and TIMP-3 contain no consensus TATA-box and none or few of the above-mentioned binding sequences.

Of note, no progesterone receptor consensus sequence seems to be present in any of the genes studied. This is entirely consistent with the recent report that suppression by progesterone of genes containing AP-1 binding sequences occurs through indirect inhibitory interaction with Jun and Fos (34). In support of this hypothesis, significant progesterone inhibition in proliferative explants was found in all genes containing AP-1 binding site(s) (all tested genes except TIMP-3). The TIMP-3 promoter contains several binding sites for Sp1 and nuclear factor 1 (NF1) (35), which factors mediate progesterone receptor A-induced activation of ADAMTS-1 promoter (36)

In conclusion, the comparison of the effects of the ovarian steroids on MMPs and TIMPs mRNAs in cultured explants with corresponding in vivo variations confirms that these hormones are powerful quantitative brakes and reveals their need to act in concert with additional gene-specific and/or phase-specific local factors. This network prevents a general proteolytic activity but allows a local ECM remodeling in nonmenstrual tissues and ensures adequate sequential production of the multiple partners involved in massive menstrual tissue breakdown. Local factors can operate as inhibitors amplifying the response to ovarian steroids, or as inducers turned on upon ovarian steroids withdrawal. These two hypotheses are not mutually exclusive, and multiple regulatory pathways must coexist, for example to account for the maintenance of high expression of MMP-7 during the proliferative phase.


   Acknowledgments
 
We thank Drs. Y. Christiane, M. Donnay, J. Donnez, E. Longueville, V. Malvaux, J. C. Verougstraete, and their colleagues for providing endometrial tissues; Dr. P. Goubau for providing access to laboratory equipment; and P. Camby, D. Dubois, A. Noel, and Y. Marchand for skillful technical help or expert secretarial assistance.


   Footnotes
 
This work was supported by grants from the Belgian "Fonds de la Recherche Scientifique Médicale" (3.4555.02 to E.M.), from the "Fonds Spécial de Recherche" of the Université Catholique de Louvain (to V.V. and E.M.), from Interuniversity Attraction Poles and Concerted Research Actions (to P.J.C.), and from Organon (to E.M.). P.B.C. was a Research Fellow and P.H. is a Research Associate of the Belgian "Fonds National de la Recherche Scientifique". C.M.P. is recipient of a fellowship from the Belgian "Fonds pour la formation à la Recherche dans l’Industrie et dans l’Agriculture".

First Published Online July 28, 2005

Abbreviations: AP-1, Activator protein 1; ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; NS, not significant; SSC, sodium saline citrate.

Received April 8, 2005.

Accepted July 14, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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