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Regular Article |
a Department of Animal Science, University of Missouri, Columbia, Missouri 65211
b Departments of Animal Sciences and Physiology, Michigan State University, East Lansing, Michigan 48824
c Department of Animal and Range Sciences and
d Cell Biology Center, Biotechnology Institute, North Dakota State University, Fargo, North Dakota 58105
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, corpus luteum function, female reproductive tract, ovary, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent enzymes that are inhibited by tissue inhibitors of metalloproteinases (TIMPs 1, 2, 3, and 4). MMPs 2 and 9 (gelatinase A and B, respectively) play a key roles in cellular migration and have been localized near the surface of angiogenic endothelial cells [3, 9]. MMP-14 concentrates proteolytic activity at the cell surface through activation of proMMP-2 [10, 11] and intrinsic proteolytic activity [12]. The extent of ECM remodeling depends upon the ratio of active MMPs to TIMPs [13]. Focusing MMP activity at the cell membrane allows cells to degrade specific portions of the ECM and thereby promote cellular migration, differentiation, GF availability, and angiogenesis, even in an environment with high extracellular concentration of TIMPs [14]. Membrane type MMPs (mt-MMPs), which can activate certain proMMPs including proMMP-2, appear to be resistant to TIMP-1 inhibition and may play an important role in the focalization of MMP activity at the cell surface [15].
We previously demonstrated the presence of TIMPs 1, 2, and 3 in corpora lutea from domestic livestock during luteal growth, development, and regression [16–18]. However, there are limited data on the characterization and quantitation of MMP activity in luteal tissue [19, 20]. We hypothesized that MMP expression and activity is highest during times of increased tissue remodeling, particularly during luteal development and regression, and that MMPs are localized to migratory cells within corpora lutea. The objective of these experiments was to determine the mRNA expression, localization, and activity of MMPs 2, 9, and 14 throughout the luteal phase.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents used in these experiments included T4 DNA ligase, restriction endonucleases, JM109 Escherichia coli strain (Promega Corp., Madison, WI), a sequenase version 2.0 DNA sequencing kit (U.S. Biochemical, Cleveland, OH), [
-32P]dATP (3000 Ci/mmol; Dupont/NEN, Wilmington, DE), nitrocellulose (Nitropure) transfer membranes (Micron Separations, Westboro, MA), nylon (Brightstar) transfer membranes and Northern Max kit (Ambion, Austin, TX), XAR-5 and XRP film (Eastman Kodak, Rochester, NY), pGEM EZ vector and sequencing oligonucleotides (Pharmacia Biotech, Piscataway, NJ), P.F.U. polymerase (Stragene, La Jolla, CA), isopropyl-ß-D-thiogalactoside (IPTG; Alexis Corp., San Diego, CA), Luria broth (Difco Laboratories, Detroit, MI), synthetic oligonucleotides (Gibco/BRL, Gaithersburg, MD), EnzChek Galatinase/Collagenase Assay Kit (Molecular Probes, Eugene, OR), and Vector Elite ABC reagent peroxidase kit and normal mouse and rabbit IgGs (Vector Laboratories, Burlingame, CA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.
Animal Care
All procedures involving animals were approved by the University of Missouri-Columbia Animal Care and Use Committee (protocol 3185). Mixed-breed ewes were placed with vasectomized rams and observed for estrous behavior twice daily. The first day of estrus was designated as Day 0.
Collection of Corpora Lutea and Sera
Corpora lutea and corresponding jugular serum were obtained from ewes (n = 5–10 per time point) on Days 2, 4, 10, and 15 postestrus. Ovaries containing corpora lutea were sliced into 2- to 4-mm sections for in situ zymography or fixed in 10% neutral buffered formalin for histological evaluation. The remaining luteal tissue was removed from the ovary, quartered, snap frozen in liquid nitrogen, and stored at -80°C until RNA or protein analysis.
Luteal Homogenates
Corpora lutea were homogenized as previously described [21] (100 mg/ml; wet weight basis) in 10 mM CaCl2 with 0.25% (v/v) Triton X-100 and centrifuged at 9000 x g for 30 min at 4°C. Following centrifugation, the supernatant was collected and frozen at -80°C until protein analyses could be performed. To evaluate MMP activity associated with the remaining pellet, a high-calcium Tris buffer (50 mM Tris-HCl, 100 mM CaCl2, 0.15 M NaCl, pH 7.4) was used to dissociate active MMPs bound to substrate. Pellets were resuspended in the original volume used for homogenization. The suspension was heated at 60°C for 6 min and centrifuged at 27 000 x g for 30 min at 4°C and frozen at -80°C until analyzed for gelatinolytic activity.
Assay of Progesterone
Concentrations of serum progesterone were determined with a solid phase RIA kit (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA) as previously described [22]. Serum was collected by jugular venipuncture prior to luteal tissue collection. All serum samples were analyzed in a single assay, and the within-assay coefficient of variation was <5.0%. Sensitivity of the assay was 0.05 ng/tube.
Generation of MMP cDNAs
Complementary DNAs encoding ovine MMP-2 (743 base pairs [bp]), ovine MMP-14 (787 bp), and bovine MMP-9 (402 bp) were generated by the reverse transcription polymerase chain reaction (PCR) from ovine luteal fibroblast RNA or bovine luteal RNA as previously described [17]. The nucleotide sequences of the ovine MMP-2 and MMP-14 cDNAs were submitted to GenBank (accession nos. AF267159 and AF267160, respectively).
Labeling of cDNA Probes
Radiolabeled probes (MMPs 2, 9, and 14 and glyceraldehyde-3-phosphate dehydrogenase) were synthesized by PCR as described previously [23]. Components of the PCR mixture consisted of KlenTaq and P.F.U. polymerases, vector-specific primers, 20 µM dATP, and 200 µM each of deoxycytidine triphosphate, deoxyguanidine triphosphate, and deoxythymidine triphosphate with or without 100 µCi [-32P]deoxyadenine triphosphate. Upon completion of PCR, cDNA was precipitated and resuspended in 100 µl of water, which was subsequently added to the prehybridization buffer (Ambion). Reactions without radiolabel were subjected to electrophoresis in a 1% agarose gel to verify amplification of the insert.
Northern Blot Analysis
Total RNA was extracted from corpora lutea (Tri-Reagent; Molecular Research Center, Cincinnati, OH). Approximately 5 µg of RNA from pooled (n = 5 ewes) Day 10 corpora lutea or millenium RNA molecular weight markers (Ambion) were electrophoresed in a 1% agarose gel and capillary transferred to a nylon membrane. Hybridization to labeled MMP probes was carried out with a commercially available kit according to the manufacturer's instructions (BrightStar System; Ambion).
MMP RNA Quantification
Total RNA was extracted from corpora lutea and transferred to a nylon membrane with a slot-blot apparatus (5 µg/slot, Bio-Dot SF; Bio-Rad Laboratories, Hercules, CA). Hybridization and detection were carried out with a commercially available kit according to the manufacturer's instructions (BrightStar System). Hybridization signal intensities were quantified by densitometry, and the target mRNA values were expressed relative to levels of glyceraldyde-3-phosphate mRNA for each sample.
Western Blot Analysis
Luteal homogenates (600 µg/lane based on wet weights) were subjected to one-dimensional SDS-PAGE and electrophoretically transferred to nitrocellulose paper. Nonspecific binding sites were blocked by incubating nitrocellulose strips (6 mm) in Tris-buffered saline and 0.05% Tween (TBST, pH 7.5) for 0.5 h. Nitrocellulose strips were incubated (2 h at 4°C) in TBST with rabbit anti-MMP-2 antisera (gift from Dr. Stetler-Stevenson), rabbit anti-MMP-9, or rabbit anti-MMP-14 polyclonal antibodies (Chemicon, La Jolla, CA) at 1:300 dilution. Negative controls consisted of the replacement of primary antibodies with the appropriate normal sera or normal IgG at the same concentrations as the primary antibodies. Strips were washed in TBST and incubated with goat anti-rabbit IgG conjugated to biotin at a 1:10 000 dilution in TBST. After rinsing three times in TBST, strips were incubated (0.5 h at 25°C) in an avidin-biotin-peroxidase complex (ABC) solution. Strips were rinsed three times in TBST, incubated in a peroxidase substrate, and photographed. The relative molecular mass of each band was determined in relationship to migration of prestained molecular weight markers.
Immunohistochemistry
Immunohistochemical procedures were performed as described previously [18] with an elite ABC kit. Tissue sections for immunolocalization of MMPs 2, 9, and 14 were deparaffinized in clearing agent and rehydrated through a series of decreasing concentrations of ethanol. Endogenous peroxidase activity was quenched in 3% H2O2 in methanol for 5 min. Nonspecific binding sites were blocked with a 1.5% (v:v) dilution of appropriate normal serum in PBS (pH 7.4). Each sample was subsequently treated with specific antibodies against MMPs 2, 9, or 14 as described for Western blots above. For negative controls, primary antibodies were replaced with respective normal sera or normal IgGs under the same conditions. Incubation with specific antibodies or their respective controls were carried out at 4°C for 18 h in a humidified chamber. After primary antibody incubation, sections were treated with a biotinylated secondary antibody (diluted 1:10 000 in PBS; 30 min at 25°C) and subsequently with an avidin-biotinylated horseradish peroxidase macromolecular complex (diluted 1:5000 in PBS; 30 min at 25°C). Primary staining was accomplished with addition of peroxidase substrates. Sections were observed via light microscopy and photographed.
Gelatin Zymography
To qualitatively identify the types and relative abundance of the gelatinases within luteal homogenates, gelatin substrate zymography was performed. Luteal homogenates from the different days of the estrous cycle were subjected to electrophoresis in 0.75-mm-thick 10% SDS-polyacrylamide gels containing 1 mg/ml porcine skin gelatin (300 bloom). Homogenates containing 400 µg (based on wet weights) were mixed with equal volumes of Laemmli sample buffer lacking ß-mercaptoethanol and were loaded onto the gel. After electrophoresis, SDS was eluted from the gels in three changes of 2% Triton X-100 for a total of 60 min at 37°C. Gels were incubated overnight at 37°C in substrate buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 7.4) and subsequently stained with Coomasie blue R250. Gelatin-degrading enzymes were qualitatively identified by their ability to digest gelatin, as demonstrated by clear bands. Prestained molecular weight markers were used for molecular weight estimation of the bands resolved by gel zymography. Preincubation of homogenates with p-aminophenylmercuric acetate (APMA; 2 mM) was performed at 37°C up to 4 h prior to gel electrophoresis. The addition of APMA causes formation of the active lower molecular weight form of MMPs [24]. Negative controls consisted of 12 mM 1,10-phenanthroline (chelates Zn++ and Ca++) or addition of MMP inhibitor I (300 µM; Calbiochem, LaJolla, CA) to substrate buffer prior to incubation. To rule out possible serum contribution of MMPs to luteal homogenates, serum (0.1–5 µl) from corresponding ewes was also tested for gelatinolytic activity.
MMP Activity Assay
Luteal homogenates from each ewe were represented in each of four groups. Group 1 consisted of no treatment (control), group 2 was treated with APMA, group 3 was treated with dithiothreitol and iodoacetamide, and group 4 was treated with a combination thereof. Before being assayed for enzyme activity, luteal homogenates from groups 3 and 4 were treated with a procedure (referred to as reduction and alkylation [RA]) that inactivated TIMPs [21]. Each supernatant was reduced with 2 mM dithiothreitol for 30 min at 37°C followed by alkylation with 5 mM iodoacetamide for 30 min at 37°C as described previously [21]. The process of RA destroys TIMPs within the homogenates. To evaluate activation of proMMPs, APMA was added to luteal homogenates prior to substrate addition.
Detection of MMP activity with an EnzChek Gelatinase/Collagenase Assay kit was conducted according to directions provided by the manufacturer (Molecular Probes). In a 96-well plate format, 50 µl of luteal homogenate (100 mg/ml wet weight basis) was added per well with 130 µl of substrate buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 7.4) and 20 µl of fluorescein-labeled gelatin substrate (1 mg/ml). Samples were incubated for 24 h at 25°C in the dark. Samples were then slowly shaken for 1 min and fluorescence was quantified in a fluorometer. Negative controls consisted of 1,10-phenanthroline (12 mM), EDTA (20 µM), synthetic MMP inhibitor (300 µM), excess gelatin (10 mg/ml), or boiling samples for 5 min. Assays were optimized for time of incubation and quantity of homogenate. Gelatinolytic activity was linear over time and with increasing amount of homogenate.
In Situ Detection of Gelatinolytic Enzymes in Corpora Lutea
Luteal tissue was embedded in tissue-freezing medium without fixation and stored at -80°C. Frozen sections (12 µm) of luteal tissue were mounted on positively charged microscope slides and prewarmed (25°C) in substrate buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 7.4) for 5 min prior to the addition of fluorescein-labeled gelatin substrate (EnzChek Gelatinase/Collagenase Assay Kit). Fluorescent gelatin substrate (100 µl of 0.03 mg/ml solution) was added directly to tissue sections on individual slides. Slides were kept in a horizontal position, coverslipped, and incubated in a light-protected humidified chamber at 37°C for up to 2 h. Lysis of the substrate was assessed by examination under a fluorescent microscope. Controls consisted of EDTA (50 µM), 1,10-phenanthroline (5 mM), synthetic MMP inhibitor I (300 µM), recombinant ovine TIMP-1 (10 µM), excess unlabeled gelatin (10 mg/ml), and APMA (2 mM).
Statistics
Data were analyzed with a general linear models ANOVA, with day of estrous cycle as the main effect [25]. When the F-test was significant (P < 0.05), differences among means were evaluated with a Duncan multiple range test. When variances were different as determined with the Bartlett test of equality of variance, data were log transformed and reanalyzed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum concentrations of progesterone increased (P < 0.05) at each time interval from Day 2 to Day 10 postestrus (Day 0 = estrus) and declined (P < 0.05) on Day 15 relative to Day 10 (Fig. 1).
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Expression of MMP mRNA in Ovine Luteal Tissue
Specificity of DNA probes for slot blot analysis was confirmed by Northern analysis. MMPs 2 and 14 cDNA probes hybridized to luteal mRNA transcripts of 3.1 and 4.5 kilobases in length, respectively (Fig. 2A). The sizes of these transcripts are consistent with the size of MMPs 2 and 14 transcripts in other species [26]. Messenger RNA encoding MMP-9 was not detectable by Northern analysis. Relative levels of MMP-2 mRNA increased on Day 10 compared with Days 2 and 4 (Fig. 2B). Expression of MMP-14 mRNA was highest (P < 0.05) on Day 4 postestrus relative to all other days (Fig. 2C).
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Immunodetection of MMPs 2, 9, and 14
Specificity of antibodies for immunolocalization experiments was verified by Western blot analysis (data not shown). In representative immunoblots of luteal homogenates, multiple bands of protein representing latent and active forms of MMPs 2, 9, and 14 were identified. The observed molecular weight values of 72 000 and 66 000 (proMMP-2 and MMP-2) and 63 000 and 52 000 (proMMP-14 and MMP-14) are consistent with the molecular weight values for other species [26]. Western blot analysis for MMP-9 within luteal homogenates demonstrated the presence of a single band (Mr = 92 000) consistent with the size of latent MMP-9. Substitution of primary antibodies with normal rabbit sera or rabbit IgGs (negative controls) indicated no nonspecific binding to luteal homogenates.
The cellular localization of MMPs 2, 9, and 14 within ovine corpora lutea collected on Days 2, 4, 10, and 15 of the estrous cycle was examined by immunohistochemistry. MMP-2 was localized to large luteal cells (LLCs) in mature corpora lutea (Fig. 3). Cells were judged to be LLCs if they met the criteria as outlined by Niswender and Nett [27] (>22 µm in diameter and large centrally located nucleus). In addition to LLCs, MMP-2 was localized to putative endothelial cells. Endothelial cells were identified based on the morphological characteristics of endothelial cells (<10 µm in length, thin or curved cytoplasm or nuclei). An increase in the number of immunopositive putative endothelial cells was observed between Days 10 (Fig. 3C) and 15 (Fig. 3D). Substitution of primary antibodies with normal rabbit sera (negative control) demonstrated that binding of MMP-2 antisera to luteal cells was specific (Fig. 3, E–H).
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MMP-9 was localized primarily to LLCs in mature corpora lutea (Fig. 4). Replacement of primary antibodies with normal rabbit IgGs (negative control) demonstrated that binding of MMP-9 antibodies to luteal cells was specific (Fig. 4, E–H). MMP-14 was localized to several unidentified cell types across all days evaluated (Fig. 5). Immunopositive cells had characteristics of fibroblasts, endothelial cells, or small luteal cells on all days evaluated (Fig. 5, A–D); however, precise identification of specific cell types was not possible. LLCs were also positive (albeit with less intensity, relative to other cells). Substitution of primary antibodies with normal rabbit IgG (negative control) verified that MMP-14 antibodies were specific in ovarian tissues (Fig. 5, E–H).
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Gelatin Substrate Zymography
Luteal homogenates collected on Days 2, 4, 10, and 15 postestrus contained gelatinases, and addition of 1,10-phenanthroline (zinc ion chelator) or a synthetic MMP inhibitor strongly inhibited gelatinolytic activity (Fig. 6A). Gelatinolytic bands corresponding to Mr 72 000 and 92 000 likely represent latent forms of MMP-2 and 9, respectively. As expected, the addition of p-aminophenylmercuric acetate (APMA; activates proMMPs) increased the amount of active enzyme (Fig. 6A). Gelatinolytic activity at Mr 62 000 and 66 000 or 82 000 and 88 000 likely represent active and intermediate forms of MMP-2 or MMP-9, respectively. Jugular sera collected from corresponding ewes demonstrated little to no gelatinolytic activity (data not shown).
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MMP Activity
Treatments (APMA and the process of RA) were applied to luteal homogenates to allow determination of 1) endogenous MMP activity in the presence of inhibitors (control; no treatment), 2) endogenous plus activated MMP activities in the presence of inhibitors (APMA), 3) endogenous MMP activity plus MMP activity in the absence of TIMPs (RA), and 4) endogenous MMP activity plus activated MMP activities in the absence of TIMPs (APMA and RA). As expected both APMA and RA increased MMP activity (P < 0.0001) relative to the control group, and the combination (APMA/RA) had an additive effect (P < 0.0001; data not shown).
There was a main effect of treatment (P < 0.0001) but not for day of the estrous cycle, and the treatment x day of the estrous cycle interaction was significant (P < 0.05). Therefore, the effects of each treatment on MMP activity over days of the cycle were analyzed. Luteal MMP activity in the absence of TIMPs was increased (P < 0.05) on Days 4 and 10 postestrus (Fig. 6B, RA). However, there were no observed changes in MMP activity over time for the control, APMA, or APMA and RA treatments. Gelatinolytic activities were presumed to be due primarily to MMPs, based on inhibition of fluorescence when homogenates were incubated with 1,10-phenanthroline, EDTA, or synthetic MMP inhibitors or were denatured by heat (data not shown).
In Situ Localization of MMP Activity in Ovine Corpora Lutea
In situ detection of MMP activities revealed that gelatinolytic activity was localized primarily to the extracellular space, although cytoplasmic and nuclear localization were also visible. During luteal formation (Day 2), gelatinolytic activity was detected in multiple cell types within cells that appeared to be of thecal origin (Fig. 7A). Later in luteal development (Day 4), gelatinolytic activity appeared pericellularly in all cell types (Fig. 7B). Luteal tissue collected during the middle luteal phase (Day 10) had the most intense gelatinolytic activity; pericellular proteolysis was occurring in nearly all cells, including LLCs (Fig. 7C). By Day 15, a slight decrease in fluorescence (relative to Day 10) and distinct cell outlines were observed (Fig. 7D). Negative controls for the in situ zymography included EDTA (Fig. 7E), 1,10-phenanthroline, synthetic MMP inhibitors, and excess unlabeled gelatin, all of which completely inhibited or markedly decreased fluorescence. Addition of APMA increased the amount of fluorescence detected in Day 10 corpora lutea (Fig. 7F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gelatinolytic activity (as determined by gelatin zymography) is present within ovarian extracts from cattle [20], pigs [19], and pseudopregnant rats [21]. Increased gelatinase activity occurs during luteal development in a number of different species [20, 21, 35]. The increase in gelatinase expression and activity is consistent with the hypothesized role of gelatinases in cellular migration [3], angiogenesis [9], and biochemical and morphological changes associated with developing luteal cells. Both gelatinase activity and MMP-14 expression are associated with a rise in progesterone (P4) on Day 4 of the estrous cycle.
MMPs 2, 9, and 14 are likely produced by ovine luteal tissue; proteins were detected in luteal tissue from all days evaluated. Putative latent and activated forms of MMP-2 and MMP-9 gelatinolytic activity (Mr = 72 000 and 62 000, and 92 000 and 82 000, respectively) were detected in luteal homogenates by gelatin substrate zymography, and gelatin activity was inhibited in the presence of the chelator 1,10-phenanthroline or synthetic MMP inhibitors. The addition of APMA to luteal homogenates activated both zymogens, as demonstrated by gelatin zymography. In addition, Western blot analysis and immunohistochemical analysis demonstrated the presence of all three proteins. Northern blot analysis verified the presence of mRNA corresponding to the correct size for MMPs 2 and 14. The inability to detect mRNA encoding MMP-9 may have been due to small quantities of mRNA at the time points evaluated.
We previously localized TIMPs 1 [18], 2 [16], and 3 [17] and plasminogen activator inhibitor-1 (unpublished data) within ovine LLCs. The observation that MMPs 2 and 9 were primarily localized to LLCs suggests that this cell type may have a central role in controlling luteal ECM remodeling. Accumulating evidence indicates that LLCs play a key regulatory role in ovine corpora lutea [17, 18]. Although LLCs represent only 20% of the total number of luteal cells, they make up >50% of the luteal volume. Moreover, LLCs are estimated to secrete approximately 80% of the progesterone secreted by the ovary and contain prostaglandin F2 receptors [27]. LLCs are generally believed to be derived from granulosa cells and contain a distinct basal lamina [36]. Remodeling of the basal lamina (or basement membrane) has important effects on differentiation, exit from the cell cycle, apoptosis, and overall gene expression in mammary epithelial cells [2, 6, 37, 38]. Therefore, expression of enzymes that regulate basal lamina turnover in LLCs may be important in regulating luteal function. MMP-2 expression and gelatinase activity are highest on Day 10 when P4 was significantly higher than at all other time points.
Localization of protease activity on the surface of cells may promote basal lamina turnover, cellular migration, differentiation, angiogenesis, and GF bioavailability. MMP-14 is a member of the newly discovered class of membrane-bound MMPs and may help mediate ECM remodeling near the cell surface. Gelatinolytic activity can be localized to the plasma membrane by activation of proMMP-2 through formation of a trimolecular complex with TIMP-2. During activation, TIMP-2 initially binds to MMP-14 and subsequently binds proMMP-2 through its carboxy terminal hemopexin domain. Binding to TIMP-2 localizes proMMP-2 to the cellular surface in close proximity to MMP-14, where the propiece of latent MMP-2 can be cleaved through an additional mt-MMP, yielding an active enzyme. Messenger RNA and protein expression for each of the components of the trimolecular complex (proMMP-2, TIMP-2, and MMP-14) were detected in ovine luteal tissue [16, 17]. These findings imply that in addition to individual proteolytic activities associated with MMPs 2 and 14, a potent cell surface activation mechanism for MMP-2 appears likely within ovine corpora lutea. Other means of localizing MMP activity to the cell surface include active mt-MMPs, secretion of active MMPs free from TIMP, and binding of integrin. However, the role of such mechanisms in luteal function was outside the scope of the present study.
Corpus luteum development and luteolysis are accompanied by extensive remodeling of the ECM, which can modulate specific cellular processes including luteinization, cellular migration, angiogenesis, apoptosis, and gene expression. During early luteal development (Day 2), mRNA expression and gelatinolytic activity was lower relative to those on Days 4 and 10, which suggests a reduced role for gelatinases during early luteal development. However, MMP activity was localized to the pericellular region of cells that appeared to be of thecal origin. This finding is consistent with a role of MMPs and ECM degradation during the processes of cellular migration and angiogenesis. The ability of small luteal cells, fibroblasts, immune cells, and endothelial cells to degrade ECM and intersperse with LLCs is essential for luteal development. This finding presents a paradox; TIMP-1 expression is high during luteal formation and development, a time when cellular migration is high. Localizing MMPs to the cell surface may provide a mechanism for actively degrading ECM in the presence of high concentrations of TIMPs. Furthermore, MMP-14 activity is not inhibited by TIMP-1 [15], making ECM degradation possible when local concentrations of TIMP-1 are elevated.
An increase in MMP mRNA expression and activity during the early to middle luteal phase (Day 4) was unexpected because this is not a time normally associated with extensive luteal tissue remodeling. The increase in MMP expression was consistent with an increase in gelatinolytic activity, as measured in luteal homogenates and visualized via in situ zymography. Secreted gelatinase activity was highest in bovine corpora lutea (Day 4 postestrus) [20], and an increase in secreted MMPs was observed from early to middle luteal phase corpora lutea in the pig [19]. These data suggest that increased gelatinase activity may play a role in luteal remodeling during the middle luteal phase. Indeed, rapid turnover of basal lamina of LLCs has been observed in midluteal phase ovine corpora lutea (H. Sawyer, unpublished data). Type IV collagen is a preferred substrate for MMPs 2 and 9 and a major component of basement membrane. GFs that promote luteal development and function (i.e., fibroblast growth factor, insulin-like growth factor, and vascular endothelial cell growth factor) are known to bind ECM and components of the basal lamina. Moreover, numerous studies have demonstrated the ability of MMPs to release GFs bound to ECM [39, 40]. Consequently, the luteal ECM may provide a mechanism for concentrating, protecting, and modulating the activity of GFs.
Luteolysis has been associated with separation of cells from the ECM. Increased ECM degradation or cell separation from ECM results in apoptosis [6, 37, 38], and this type of apoptosis has been termed anoikis [41]. In regressing bovine or ovine corpora lutea, endothelial cells appear to be the first cells to undergo separation from ECM and apoptosis [42, 43]. In mammary epithelial cells, MMP-3 appears to be an important mediator of cell separation from ECM. In the present study, immunolocalization of MMP-2 and in situ gelatinolytic activity to putative endothelial cells was evident in Day 15 corpora lutea, thus implicating gelatinases as potential mediators of cell separation and death.
The presence of MMP mRNA, protein, and activity in Day 15 corpora lutea is not surprising because most tissues undergoing involution need to degrade the ECM to accommodate structural remodeling. However, MMPs have more recently been implicated in other roles that may promote involution. These proapoptotic roles include cleavage of membrane-bound tumor necrosis factor [44] and Fas ligand [45, 46], inactivation of fibroblast growth factor receptors [47], and conversion of plasminogen to the potent apoptotic agent angiostatin [48, 49]. Collectively, these data suggest a proapoptotic role during luteal regression.
Our results indicate that MMPs 2, 9, and 14 are produced by ovine corpora lutea throughout the estrous cycle. The necessary components for pericellular localization of gelatinolytic activity are present throughout the ovine luteal phase and thus may facilitate processes important to luteal development, maintenance, and regression. LLCs appear to be a primary regulator of ECM remodeling and may be responsible for overall homeostasis of luteal ECM. Increased gelatinolytic activity associated with mature corpora lutea may facilitate key functions, including GF availability and ultimately increased progesterone synthesis.
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FOOTNOTES |
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1 This work was supported by USDA 98-35203-6282 (M.F.S.) and USDA 98-35203-6226 (G.W.S.) and is a contribution from the Missouri Agricultural Experiment Station Journal Series, no. 13151. 
2 Correspondence: Michael F. Smith, Department of Animal Science, 160 Animal Sciences Center, University of Missouri, Columbia, MO 65211. FAX: 573 884 7827; smithmf{at}missouri.edu
3 Current address: Departments of Anatomy and Obstetrics and Gynecology, University of California, San Francisco, CA 94143
Accepted: November 6, 2001.
Received: May 30, 2001.
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