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a Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232
b Reproductive Sciences, Oregon Regional Primate Research Center, Beaverton, Oregon 97006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The MMPs are a multigene family of enzymes that require zinc for their activation. Secreted as proenzymes, the MMPs undergo activation to a catalytically active form that may be inhibited by specific endogenous tissue inhibitors of metalloproteinases (TIMPs; reviewed in [5]). The MMP family has been subdivided based on substrate specificities, although the rapid addition of new members to the family has prompted alternative classifications based on structural domains [6]. In combination, members of the MMP family can degrade all components of the extracellular matrix.
MMP and TIMP expression levels are highly regulated during the menstrual cycle. The mRNAs for interstitial collagenase (IC), stromelysin-1 (STR1), stromelysin-2 (STR2), stromelysin-3 (STR3), matrilysin (MAT), gelatinase A (GELA), gelatinase B (GELB), and TIMP-1 have all been localized in the human endometrium at the time of menstruation (see [4] for review). The mRNA for MAT is localized to epithelial cells, while the other MMPs are detected in the stromal cells [7, 8]. The localization of IC at the periphery of shedding endometrial fragments and along arterioles [9] implicates this enzyme in particular in the breakdown of the endometrial matrix that occurs during the menstrual process. In women, many MMPs are undetectable by in situ hybridization and Northern blot analysis at times other than during menstruation, but STR2 and GELB can be detected in the late secretory phase, MAT and STR3 during the proliferative phase, and GELA throughout the entire menstrual cycle [7, 8]. TIMP-1 and TIMP-2 are expressed at relatively constant levels throughout the entire menstrual cycle in women, and TIMP-3 is expressed in the mid-secretory phase of the cycle [4]. Using synthetic MMP inhibitors and explant cultures in vivo, Marbaix and his collaborators have provided experimental evidence strongly implicating MMPs in the degradation of endometrial tissue during menstruation [10].
In vitro studies with cultured endometrial cells have indicated that several MMPs may be regulated by steroid hormones, cytokines, and growth factors [4]. Endometrial cells that are subjected to P4 withdrawal, the physiologic stimulus for menstruation, increase their metalloproteinase production and secretion [11]. Interleukin-1
(IL-1) and tumor necrosis factor alpha (TNF) are stimulators of IC, STR1, and GELB, but not GELA, secretion [12]. The addition of P4 to either endometrial explants or isolated stromal cells leads to the decline of MMP expression [13, 14]. P4-mediated down-regulation of MAT expression in endometrial epithelial cells is the result of a paracrine effect of transforming growth factor ß (TGFß) produced by stromal cells [15]. These in vitro studies suggest that P4 may down-regulate, either directly or indirectly, MMP levels in the cycling endometrium.
A more complete understanding of the regulation and role of endometrial MMPs in vivo requires a system in which precise control of hormonal conditions can be achieved. Studies of ECM remodeling in the human endometrium during the cycle historically have utilized biopsies taken during the natural cycle. However, a closely timed examination of MMP expression before, during, and after menstruation under precisely controlled hormonal conditions in ovariectomized women in the presence or absence of E2 has not been done. In the present study, we induced artificial cycles in ovariectomized rhesus monkeys and removed uteri at key times before, during, and after menstruation under controlled hormonal conditions. Expression and localization of a variety of endometrial MMPs were determined by Northern analysis and immunocytochemistry. Some of these data have been presented in a preliminary report [16]. The current study extends that report and provides new immunocytochemical and biochemical information on zone-specific regulatory gradients. The results provide new insights into the regulation of MMP expression in the primate endometrium.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animal care and handling were provided by the veterinary staff of the Oregon Regional Primate Research Center (ORPRC) in accordance with the National Institutes of Health Policy on the care and use of animals. Thirty-one sexually mature rhesus macaques (Macaca mulatta) were ovariectomized. Each animal was rested for 4–6 wk after ovariectomy before receiving further treatment.
All macaques then received s.c. implants of a 3-cm Silastic capsule (0.34 cm i.d.; 0.46 cm o.d.; Dow Corning, Midland, MI) packed with crystalline E2 (Steraloids, Inc., Wilton, NH). After 2 wk, a 6-cm Silastic capsule (0.34 cm i.d.; 0.46 cm o.d) packed with crystalline P4 (Steraloids, Inc.) was implanted, and both implants remained for 2 additional weeks. This sequential E2/P4 regimen produced an endometrium typical of the follicular phase during the first 2 wk of E2 treatment and a secretory endometrium typical of the luteal phase during the second 2 wk of E2 plus P4 treatment [17]. Menstruation was induced by removal of the P4 implant.
In this study, three different hormonal states were established: an extended follicular phase, a luteal phase, and a spayed condition. To create an extended follicular phase, menstruation was induced by withdrawal of the P4 implant while the E2 implant remained in place for 28 days. The uterus was removed from two animals each on Days 1, 2, 3, 4, 5, 6, 8, 10, 14, 21, and 28 after P4 implant removal. For the luteal phase group, P4 implants were reintroduced into four animals on Day 14 of the follicular phase, and uteri were removed from 2 animals each on Days 7 and 14 of the induced luteal phase, equivalent to Days 21 and 28 of the menstrual cycle. In the spayed group, both the E2 and P4 implants were removed and uteri were taken from 2 animals each on Days 3, 5, 10, and 14 after implant removal. One animal was sampled on Day 2 after removal of E2 and P4. Tissues from this animal were processed solely for histology and immunocytochemistry.
Figure 1 illustrates the above treatment and tissue sampling protocol. Days 1–6 of the induced follicular phase are known as the luteal-follicular transition (LFT; [18]), a period that includes premenstrual regression, menstruation, and the onset of endometrial regeneration. This experimental design permits analysis of MMP expression before, during, and after menstruation in the presence and absence of E2, and during combined E2 plus P4 treatment. Comparisons among groups can reveal the effects of E2 versus the effects of P4 on MMP expression.
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These experiments were performed in two sets. In the first set, one animal per time point from all hormonal treatments was sampled and analyzed. At a later time, a second set of animals was similarly treated, sampled, and analyzed. Most time points were represented by samples from two animals.
RNA Extraction and Northern Blot Analysis
Endometrium was separated from myometrium by dissection with fine scissors, frozen in liquid nitrogen, and stored at -80°C for RNA extraction. Endometrial tissue was homogenized in a guanidinium thiocyanate-acid phenol solution, and total RNA was extracted as described by Chomczynski and Sacchi [19]. Ten micrograms of total cellular RNA was electrophoretically separated on a 1.2% agarose-formaldehyde gel, transferred to a nitrocellulose membrane (MSI, Westboro, MA), and hybridized with 32P-labeled, random-primed cDNA probes (specific activity approximately 1.5 x 109 cpm/µg) under conditions of high stringency (hybridization: 50% formamide and 5-strength SSC [single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate] at 42°C; wash: 0.1-strength SSC at 50°C). Blots were stripped by boiling in 0.01-strength SSC for 10 min and rehybridized with another MMP-specific probe or with a cDNA probe specific for constitutively expressed cyclophilin RNA to control for equal loading [20]. Values for MMP expression were determined densitometrically with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA), equalized with cyclophilin expression, and normalized to the percentage of the most intense signal. The following human MMP cDNA fragments were labeled using the Random Primed DNA Labeling Kit (Boehringer Mannheim, Indianapolis, IN), and [32P]CTP (DuPont NEN, Boston, MA) to generate cDNA probes for Northern analysis: the 815-base pair (bp) (+1 to +815) MAT cDNA, the 350-bp (+1210 to +1560) STR1 cDNA, the 530-bp (+1050 to +1580) STR2 cDNA, the 467-bp (+1127 to +1594) STR3 cDNA, the 540-bp (+1195 to +1735) IC cDNA, the 900-bp (+852 to +1751) GELB cDNA, the 327-bp (+1064 to +1391) MT-MMP cDNA, the 677-bp (+40 to +717) TIMP-1 cDNA, and the 614-bp (+340 to +954) TIMP-2 cDNA. The 1.7-kilobase EcoRI fragment (+654 to +2,326) of GELA was kindly provided by K. Tryggvason, University of Oulu, Oulu, Finland. The MMP cDNA probes for this study have been previously shown to be specific for the indicated transcripts [21, 22].
Statistical Analysis of MMP mRNA Induction
Because the two sets of tissue samples were assayed by Northern analysis several months apart, and because radiolabeled probes used in the hybridizations had different specific activities, there was considerable variation in the two densitometry data sets. However, the pattern of hormonally induced change in each MMP transcript was essentially the same in each set. To assess the statistical significance of MMP mRNA induction after P4 withdrawal, densitometric values for specific time periods during the artificial menstrual cycle were pooled. These periods were Days 2 + 3 (early LFT), Days 5 + 6 (late LFT), Days 21 + 28 (extended follicular phase), and Days 21 + 28 (luteal phase). The results are presented in Table 1. To determine whether there were significant differences in MMP mRNA patterns in the absence versus the presence of E2, densitometric values for Days 3–5 (menstruation), and Days 10 + 14 (postmenstruation) of the extended follicular phase group were compared with the same times for the spayed group, and the results are presented at the appropriate point in the text. All values were tested by Multifactorial ANOVA [23], with data set and hormone treatments as factors. When the ANOVA revealed significant main effects, or first-order interactions, significant differences between groups were identified with Fisher's Protected Least-Significant Difference Test [23].
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Immunocytochemistry (ICC)
General procedures We reported that microwave stabilization increased antigen retention and markedly enhanced morphological preservation of cryosections [24]. Therefore, all fresh tissues were microwaved for 7 sec before being embedded in Tissue Tek II Optimal Cutting Temperature mixture (O.C.T.; Miles Inc., Elkhart, IN), frozen in liquid propane, and cryosectioned at 5–7 µm. We also reported that freeze-substitution of frozen sections with absolute acetone at -80°C for several days improved morphology and antigen retention [18]. In this study, we combined microwaving and freeze-substitution procedures with some of the MMP antibodies and also used different fixatives with different antibodies. These choices were based on preliminary trials in which we ascertained the optimal technique for each antibody with rhesus monkey tissues.
When freeze-substitution was used, cryosections were mounted on Super Frost Plus slides (Fisher Scientific, Pittsburgh, PA), placed in cold (-80°C) absolute acetone, and then stored at -80°C. After 2–4 days, the slides were placed in 2% paraformaldehyde in phosphate buffer at pH 7.3 for 15 min at room temperature, placed next in 85% ethanol + 1.5% polyvinylpyrrolidone (PVP) at 4°C, and then rinsed several times in 0.37% glycine in PBS/PVP at 4°C to eliminate aldehyde groups. After several aqueous rinses, slides bearing cryosections were microwave-irradiated again for 2 sec. Sections were then incubated with blocking serum (20 min; 4°C) and then with primary antibody (2 µg/ml) overnight at 4°C. After rinsing and immersion in blocking serum again, sections were incubated with second antibody for 30 min at room temperature (25°C). Slides were rinsed, incubated with ABC solution (Vector Labs., Burlingame, CA) for 60 min at room temperature, and then treated for 10–15 min with 3,3' diaminobenzidine/4 HCl, 0.025% in Tris buffer (Dojindos DAB; Wako Chemicals, Richmond, VA) and 0.03% H2O2 (Fisher Scientific). They were rinsed several times and then treated with 0.026% OsO4 for 1 min; rinsed; postfixed with 2% paraformaldehyde-0.2% picric acid; counterstained with hematoxylin; dehydrated by treating (5 min each) with 30%, 50%, 70%, 85%, 95% (twice), and 100% (twice) ethanol, cleared with xylene, and mounted with Permount (Fisher Scientific).
Antibodies Mouse monoclonal IgG1 antibodies against human GELA (AB-3), STR1 (AB1), and TIMP-1 (AB1) were obtained from Calbiochem (Oncogene Research Products, Cambridge, MA). Anti-MAT was a mouse monoclonal IgG1 antibody provided by Dr. Roy Nagle, University of Arizona, Tucson. The anti-MAT was provided as a culture supernatant that was diluted 1:1 before use. Monoclonal mouse IgG1 against Escherichia coli (trp E; Oncogene, Catalog #OB01) was used as a control antibody for the primary mouse monoclonal antibodies. In all cases, staining with the control antibody was negative. The immunocytochemical procedures for GELA, STR1, and TIMP-1 were exactly as described above. For MAT, no freeze-substitution was used. Instead, slides bearing freshly prepared cryosections were microwaved for 2 sec and then immediately fixed in 2% paraformaldehyde-0.2% picric acid for 10–15 min, then in 85% ethanol + 1.5% PVP at 4°C, and then processed as described above.
Evaluations Two or three blocks of frozen endometrium from each animal were cryosectioned, and at least 2 slides from each block were processed. All slides were examined by two observers (R.M.B. and O.D.S.), and areas to be photographed were selected as representative by R.M.B. Photomicrographs were made on Kodak Technical Pan film (Eastman Kodak, Rochester, NY) and developed in Technidol (Eastman Kodak).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the extended follicular phase group, one day after P4 withdrawal, there was an overall shrinkage of the endometrium and compaction of the stroma. On Day 2 of P4 withdrawal, there was extensive tissue fragmentation accompanied by hemorrhage and sloughing, and external bleeding was usually apparent on Days 3–6. However, the erosion and fragmentation was confined to the uppermost third of the endometrium. Although the lower functionalis and basalis showed no signs of fragmentation, the glandular epithelial cells in these regions, especially in the basalis, underwent extensive apoptotic cell death during Days 2–6 as previously described [1]. Mitotic activity began around Day 5, but it was restricted to the glands in the uppermost regions of the surviving functionalis. By Day 8 there was increased sacculation of the deeper glands at the junction of the functionalis and basalis. Apoptosis was prevalent in this region on Days 6–8, and remodeling of the sacculated glands appeared complete by Days 10–14, at which time the endometrium consisted of straight, nonsacculated tubular glands penetrating a moderately loose stroma. These histological patterns were maintained through Days 21–28 of the extended follicular phase.
In the spayed group, the tissue breakdown patterns were identical to those evident in the extended follicular phase group. Fragmentation, hemorrhage, and sloughing were restricted to the upper zones, while apoptosis occurred primarily in the lower zones. Around Days 5–6 the luminal surface healed over and bleeding ceased, just as when E2 was present. However, in the absence of E2, there was little or no mitotic activity in the glands or stroma. Instead, the endometrium steadily atrophied.
In the luteal phase group, there was typical progestational transformation of the endometrium through Days 21–28 marked by stromal cell hypertrophy, increased edema, spiral artery proliferation, glandular sacculation, and proliferation of the basalis, all as previously described [1].
Expression of MMP mRNA in Endometrium
The mean percentage of mRNA inductions (± SE) for each of the MMPs are graphed in Figure 2. Transcripts for GELB and TIMP-2 were not detected in any of the samples tested in this study.
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In general, the transcripts for the observed MMPs and TIMP-1 were expressed at their highest levels during the LFT in both the follicular phase and spayed groups, and were clearly associated with menstruation. All MMP mRNAs were up-regulated by Days 2–3 of the LFT, as compared to the levels on Days 21–28 of the luteal phase. This elevation was statistically significant for MAT, STR3, STR1, STR2, and IC; but GELA, MT-MMP, and TIMP1 mRNAs showed only a trend to higher expression after P4 withdrawal (Table 1).
The data also revealed two patterns of MMP down-regulation, namely, "transient" versus "sustained." Transient expression was exemplified by STR1, STR2, and IC. The expression of these three MMPs peaked around Days 2–3 and then rapidly dropped to minimal transcript levels by Days 5–6 in both the extended follicular and spayed groups (Fig. 2, Table 1). The addition of P4 had no effect on STR1, STR2, and IC as their expression did not differ significantly between Days 21 and 28 of the luteal phase versus Days 21 and 28 of the extended follicular phase (Table 1). Sustained expression was exemplified by GELA, MT1-MMP, TIMP-1, MAT, and STR3. These transcripts were maximal during the LFT but declined more gradually, reaching low levels of expression by Days 21–28 of the extended follicular phase, in the absence of P4 (Fig. 2A). These declines were significant for MAT, STR3, and TIMP1, but the decreases in GELA and MT1-MMP mRNA levels were not significant (Table 1). The addition of P4 lowered MAT and STR3 expression significantly further on Days 21–28 of the luteal phase compared to the same days of the extended follicular phase (Table 1). It should be noted that the major decline in MAT and STR3 transcript expression levels, and presumably the activity of these two enzymes, occurred in the absence of P4 (Fig. 2, Table 1). GELA and MT-MMP mRNA levels appeared to be unaffected by P4 treatment.
In the spayed group, expression of the transient MMP mRNAs rose and fell quickly, exactly as in the presence of E2, but there was a trend for some of the sustained MMP mRNAs and the TIMP-1 transcripts to remain elevated in the absence of E2 (Fig. 2A). A specific comparison of the mean induction of the sustained MMP mRNAs on Days 10–14 in the extended follicular phase versus Days 10–14 in the spayed group showed that this trend was significant for GELA (22.3 ± 5.7 vs. 66.0 ± 8.2, p < 0.05), STR3 (22.1 ± 4.4 vs. 51.7 ± 18.8, p < 0.05), and TIMP-1 (5.5 ± 2.2 vs. 45.5 ± 18.3, p < 0.01) mRNAs, though not for MAT or MT1-MMP mRNAs.
ICC of MMPs and TIMP-1
In general, there was good consistency between the ICC results and the Northern analyses, with only modest variation between animals. Previous in situ hybridization studies had localized MAT to the glandular epithelial cells in the human endometrium [7]. Our immunocytochemical studies confirmed this localization and added new information on the pattern of MAT expression in the macaque endometrium. On Day 28 of the luteal phase, MAT staining was nondetectable (Fig. 3a). In the follicular phase and spayed groups, intense staining was first detected 2 days after P4 withdrawal, consistent with the Northern blot data. However, the signal was primarily evident in the glandular epithelium below the major zone of fragmentation and was restricted to the upper portions of these glands, with minimal staining in the basalis zone (Fig. 3, b and c). From Days 3–6, the staining intensity increased in the upper glands. However, by Day 8, there was a remarkable shift of the MAT signal from the upper to the deeper glands of the basalis (Fig. 3e). By Day 10 (Fig. 3f), MAT staining was confined to the deepest glands, where it remained weak but detectable through Days 14, 21, and 28 of the extended follicular phase (data not shown). Some MAT staining was seen in the lumen of the glands, an indication that MAT may be secreted luminally. In the spayed group (Fig. 4), MAT staining followed essentially the same pattern of expression, including the shift from functionalis to basalis, as in the extended follicular phase group.
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As an example of an MMP that showed a transient pattern of expression by Northern blotting, STR1 protein was localized by ICC. Two days after P4 withdrawal in both the follicular phase and spayed groups, there was a dramatic increase in staining intensity strictly confined to the upper fragmenting endometrial zones (Fig. 5, a, d, and e). The positive cells were located in clumps among the fragmenting glands, and staining was very intense just subjacent to the basement membranes of the glands, around spiral arteries (Fig. 5, d and e), and within small groups of stromal cells at the very uppermost regions of the endometrium. Some widely dispersed cells were positive for STR1 throughout the stroma of all zones of the endometrium around this time (data not shown). The identity of these widely scattered cells was not established in this study.
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During Days 2–5, the STR1-positive stromal cells became fewer in number and were restricted to the outermost regions of the endometrium. After the surface healed over on Day 5, positively stained stromal cells were evident in groups beneath the luminal epithelium (Fig. 5b), but by Day 6 staining was nondetectable. No staining was evident in either follicular phase or spayed groups by the 14th day (Fig. 5, c and f), nor was there any STR1 staining in the luteal phase group. This transient expression of STR1 protein staining was consistent with the transient expression of the mRNA of this MMP.
GELA was detectable by ICC in all zones under all hormonal conditions. For example, GELA staining was clearly evident in the stromal cells of all zones on Day 28 of the luteal phase (Fig. 6a). However, two days after P4 withdrawal, there was a dramatic increase in staining intensity that was restricted to the stromal cells of the uppermost fragmenting endometrial zones (Fig. 6, b and c). Similar up-regulation of GELA in the uppermost zones occurred in the spayed group (Fig. 6, e and f). During the extended follicular phase, the intensity of the signal greatly diminished over time to a baseline level (Fig. 6d), but in the spayed group, intense staining for GELA persisted in the upper regions through Day 14 (Fig. 6f), consistent with the higher expression of GELA mRNA levels in this group. Higher magnification confirmed that in both groups, GELA staining was intense in the cytoplasm of stromal cells, negative in glandular epithelial cells (Fig. 7a), and very strong in the walls of spiral artery fragments (Fig. 7b).
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TIMP-1 staining was minimal on Day 28 of the luteal phase (Fig. 8a) and became very strong within the upper fragmenting zones two days after P4 withdrawal in both the follicular phase (Fig. 8, b and c) and spayed groups (Fig. 9a) Staining was strikingly evident in the smooth muscle and perivascular stroma of the spiral arteries (Figs. 8, b and c, 9a, and 10a) as well as in large numbers of stromal cells in the fragmenting and sloughing endometrial regions. Staining was especially intense around the basement membranes of gland fragments (Figs. 8b, 9a, and 10b) and small veins in the sloughing regions. A clear gradient of expression was evident, as all stromal staining was confined to the upper third of the endometrium. Even the spiral arteries showed this gradient, as the portions of the spiral arteries located in the sloughing zones showed much more intense TIMP-1 staining than did the arterial segments in the deeper zones (Figs. 8, b and c, 9a, and 10a). During Days 3–6, the intensity of the TIMP-1 staining in the upper regions diminished, but by Days 8–10 TIMP-1 staining had shifted to the deeper glands in parallel with the shift of MAT to these regions (Fig. 8, d–f, and Fig. 9, b and c). TIMP-1 staining diminished but remained detectable in the basalis through Days 14–28 (Fig. 8f) of the extended follicular phase. In the luteal phase, there was some further diminution of TIMP-1 staining in the basalis by Day 28 (Fig. 9a), but there also was a moderate return of TIMP-1 staining to the spiral arteries (not shown). This modest arterial staining in the luteal phase was consistent with the trend for a slight increase in TIMP-1 mRNA in the luteal phase (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern analysis indicated that expression of the transient MMP mRNAs declined extremely rapidly. Therefore, we considered the possibility that cells expressing these MMPs were simply lost from the endometrium in the sloughing process. ICC revealed, however, that many stromal cells expressing STR1 were detectable within the endometrium just beneath the epithelial surface after sloughing had ceased, and these cells stopped expressing STR1 within a short time after the surface had healed. While some cells expressing transient MMPs are undoubtedly lost during sloughing, the remaining cells turn off their MMP expression much more rapidly than the sustained MMPs. The different mechanisms that control expression of the transient versus sustained MMPs are unknown.
Does E2 play any role in MMP regulation? The pattern of up-regulation of both the transient and sustained MMPs was the same in the follicular phase and spayed groups, indicating that E2 played no role in this up-regulation. The transient MMPs also declined in a similar manner in the follicular phase and spayed groups, indicating that E2 played no role in their decline. However, the expression of the sustained MMPs GELA, STR3, MT1-MMP, and TIMP1 tended to remain elevated on Day 14 when E2 was absent, whereas these declined to very low levels by Day 14 when E2 was present.
Because the endometrium undergoes a regressive process of atrophy and shrinkage in the absence of E2, the continued expression of certain MMPs in the spayed group is probably more related to the continued dissolution of the ECM than to any direct inhibitory action of E2 on MMP synthesis.
The dramatic shift of expression of MAT from the upper to the lower glands, and of TIMP-1 from the arteries and stroma to the deeper glands, was also independent of E2 action since both shifts occurred similarly in the follicular phase and spayed groups. Clearly, MMP expression and endometrial remodeling after menstruation involve functionalis-basalis gradients and zone-specific regulatory mechanisms that are essentially E2 independent, and it seems unlikely that E2 has any direct effects on MMP expression in primate endometrium.
What is the role of P4 in MMP regulation in vivo? Many in vitro studies of MMP regulation in human endometrial stromal and glandular epithelial cells agree that P4 can suppress expression of GELA, GELB [11, 13], IC [27], STR1, and STR3 [14]. P4 has been shown to suppress MAT indirectly through TGFß [15]. However, in vivo, after the pronounced up-regulation of MMPs by P4 withdrawal in the rhesus macaque endometrium, many of the MMP transcripts declined either abruptly (STR1, STR-2, IC) or gradually (MAT, STR3, GEL A) in the absence of P4. Although the addition of P4 after Day 14 suppressed the expression of MAT and STR3 (in agreement with several of the above in vitro studies), the bulk of the decline in these transcripts occurred well before P4 was added back. Therefore, it seems unlikely that relief of direct P4 suppression of MMP expression can be the only factor responsible for the dramatic rise in these MMPs when P4 falls at the end of the cycle. In particular, relief of P4 suppression cannot explain the rise of the transient MMPs since they were essentially nondetectable within 5 days of P4 withdrawal, 9 days before P4 levels rose.
Our current working hypothesis is based on Markee's classic observations [28] that P4 withdrawal induces a local endometrial injury or anoxic insult that is confined to the upper regions of the endometrium. Markee reported constriction of the spiral (but not the basilar) arteries after P4 withdrawal and described periods of vasodilation with recurrent vasoconstriction that could lead to vascular reperfusion injury with subsequent anoxia in the upper zones. Such anoxia may lead to local increases in various cytokines such as IL-1, TNF [12], and vascular endothelial growth factor (VEGF) [29]. P4 is also known to suppress prostaglandin production [30], and prostaglandins have been shown to induce MMPs in several systems [31]. MMPs are elevated in fresh endometrial explants, presumably because of the injury associated with surgical biopsy [32]. In addition, there is an extreme stromal compaction that occurs immediately after P4 withdrawal, and as the intercellular space becomes vanishingly small the cells contact each other closely. Regulation of MMP expression, particularly the expression of STR1 and IC, has been associated with changes both in cell shape and in cell contacts involving interactions with matrix components [33–36].
As the endometrium heals, angiogenesis would occur in the living endometrial zones, perhaps stimulated by VEGF that had been up-regulated by anoxia. Circulation would be renewed in the surviving regions, hypoxia would diminish, noxious factors would decline, and the lowering of these various MMP stimulators would lead to reductions in MMP expression, all in the absence of P4. A comprehensive analysis of the mechanism underlying menstruation must also include the role of the inflammatory leukocytes, such as mast cells and eosinophils, which invade the endometrium before menstruation begins and may play important roles in the cascade of events that lead to MMP activation [37]. The above views provide a reasonable explanation for the initial restriction of MMP expression to the upper regions of the endometrium and their decline in the absence of P4. If MMPs were elevated simply by relief of P4 suppression, one would expect MMP up-regulation in all zones because the P4 receptor is present in stromal cells throughout all endometrial zones [1].
We conclude that although relief of suppression of some MMPs, especially MAT and STR3, occurs on P4 withdrawal, such relief does not explain the dramatic up-regulation of the rest of the MMPs, as these are not suppressed by P4 in vivo. The shift of expression of MAT and TIMP-1 from the upper to lower zones occurs in the absence of both E2 and P4 after the anoxia has subsided, and is associated with glandular remodeling. While the underlying mechanism for this shift is not understood, it is clear that hormone-independent, zone-specific regulatory factors are involved. The spiral arteries themselves are key players, as they are intense foci of MMP expression. Courtoy's laboratory [9] has thoughtfully pointed to the close association of IC expression with the basement membranes of glandular fragments and vascular elements as evidence that complex regulatory processes involving paracrine interactions are at work in the intact endometrium during menstrual breakdown. We concur, and we conclude that continued research is needed to explain the in vivo mechanisms regulating the expression, localization, and function of endometrial MMPs.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Robert M. Brenner, Reproductive Sciences, Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006. FAX: 503 690 5563; rmbrenner{at}compuserve.com
Accepted: July 27, 1998.
Received: April 28, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 V. Vassilev, C. M. Pretto, P. B. Cornet, D. Delvaux, Y. Eeckhout, P. J. Courtoy, E. Marbaix, and P. Henriet 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 J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5848 - 5857. [Abstract] [Full Text] [PDF]
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A. Germeyer, A. E. Hamilton, L. S. Laughlin, B. L. Lasley, R. M. Brenner, L. C. Giudice, and N. R. Nayak Cellular Expression and Hormonal Regulation of Neuropilin-1 and -2 Messenger Ribonucleic Acid in the Human and Rhesus Macaque Endometrium J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1783 - 1790. [Abstract] [Full Text] [PDF]
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T. Collette, C. Bellehumeur, R. Kats, R. Maheux, J. Mailloux, M. Villeneuve, and A. Akoum Evidence for an increased release of proteolytic activity by the eutopic endometrial tissue in women with endometriosis and for involvement of matrix metalloproteinase-9 Hum. Reprod., June 1, 2004; 19(6): 1257 - 1264. [Abstract] [Full Text] [PDF]
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 W. B. Nothnick, X. Zhang, and H.-E Zhou Steroidal Regulation of Uterine Edema and Tissue Inhibitors of Metalloproteinase (TIMP)-3 Messenger RNA Expression Is Altered in TIMP-1-Deficient Mice Biol Reprod, February 1, 2004; 70(2): 500 - 508. [Abstract] [Full Text] [PDF]
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F. Goffin, C. Munaut, F. Frankenne, S. Perrier d'Hauterive, A. Beliard, V. Fridman, P. Nervo, A. Colige, and J.-M. Foidart Expression Pattern of Metalloproteinases and Tissue Inhibitors of Matrix-Metalloproteinases in Cycling Human Endometrium Biol Reprod, September 1, 2003; 69(3): 976 - 984. [Abstract] [Full Text] [PDF]
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 T. E. Curry Jr. and K. G. Osteen The Matrix Metalloproteinase System: Changes, Regulation, and Impact throughout the Ovarian and Uterine Reproductive Cycle Endocr. Rev., August 1, 2003; 24(4): 428 - 465. [Abstract] [Full Text] [PDF]
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