Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ricke, W. A.
Right arrow Articles by Smith, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ricke, W. A.
Right arrow Articles by Smith, M. F.
Agricola
Right arrow Articles by Ricke, W. A.
Right arrow Articles by Smith, M. F.
Biology of Reproduction 66, 685-691 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Matrix Metalloproteinase Expression and Activity Following Prostaglandin F2{alpha}-Induced Luteolysis1

William A. Ricke3,a, George W. Smithb, and Michael F. Smith2,a

a Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211 b Departments of Animal Sciences and Physiology, Michigan State University, East Lansing, Michigan 48824


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Luteal tissue contains matrix metalloproteinases (MMPs) that cleave specific components of the extracellular matrix (ECM) and are inhibited by tissue inhibitors of metalloproteinases (TIMPs). We previously reported a decrease in luteal TIMP-1 within 15 min of prostaglandin F2{alpha} (PGF2{alpha})-induced luteolysis. An increase in the MMP:TIMP ratio may promote ECM degradation and apoptosis, as observed in other tissues that undergo involution. The objectives of these experiments were to determine whether 1) PGF2{alpha} affects expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), membrane type (mt)-1 MMP (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) PGF2{alpha} increases MMP activity during PGF2{alpha}-induced luteolysis in sheep. Corpora lutea (n = 3–10/time point) were collected at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h after PGF2{alpha} administration. Northern blot analysis confirmed the presence of all MMPs except MMP-9. Expression of mRNA for the above MMPs (except MMP-2) increased significantly (P < 0.05) by 30 min, and all MMPs increased significantly (P < 0.05) by 6 h after PGF2{alpha} administration. Expression of MMP-14 mRNA increased significantly (P < 0.05) by 15 min post-PGF2{alpha} and remained elevated through 48 h. MMP activity in luteal homogenates (following proenzyme activation and inactivation of inhibitors) was increased significantly (P < 0.05) by 15 min and remained elevated through 48 h post-PGF2{alpha}. MMP activity was localized (in situ zymography) to the pericellular area of various cell types in the 0-h group and was markedly increased by 30 min post-PGF2{alpha}. MMP mRNA expression and activity were significantly increased following PGF2{alpha} treatment. Increased MMP activity may promote ECM degradation during luteolysis.

corpus luteum, corpus luteum function, female reproductive tract, ovary, progesterone


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent enzymes that collectively degrade the proteinaceous components of the extracellular matrix (ECM) [1]. MMPs are typically secreted as zymogens and can bind tissue inhibitors of metalloproteinases (TIMPs) in a 1:1 stoichiometric ratio. Currently, there are over 22 members of the MMP family, and they have been subdivided into the following categories based upon substrate specificity: collagenases, gelatinases, stromelysins, and membrane type MMPs (mt-MMPs) [2]. Overexpression of MMPs can cause cells to exit the cell cycle [3], dedifferentiate, and undergo apoptosis, ultimately resulting in glandular involution [4–6].

In domestic ruminants luteolysis is dependent upon uterine secretion of prostaglandin F2{alpha} (PGF2{alpha}) [7] and is marked by loss of cell adhesion to ECM [8, 9], loss of capacity to synthesize progesterone [10], and apoptosis [11]. Ovine large luteal cells (LLCs) are the luteal cell type that possess receptors for and respond to PGF2{alpha} [12]. LLCs also are surrounded by a distinct basal lamina [13] and produce TIMP-1, -2, and -3 and plasminogen activator inhibitor 1 [14–17]. MMP-1, -2, -3, and -9 also have been localized to LLCs [17], and increased levels of active gelatinases were observed during prolactin-induced luteolysis in rats [18]. We hypothesized that PGF2{alpha}-induced luteolysis increases MMP expression and activity in ovine corpora lutea. The objectives of these experiments were to determine whether PGF2{alpha} administration 1) induced a change in expression of mRNA encoding fibrillar collagenases (MMP-1 and -13), gelatinases A and B (MMP-2 and -9), mt-MMP 1 (MMP-14), stromelysin (MMP-3), and matrilysin (MMP-7), and 2) increased MMP activity in ovine corpora lutea.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Reagents used in these experiments included the following: 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); [{alpha}-32P]dATP (3000 Ci/mmol; Dupont/NEN, Wilmington, DE); nitrocellulose (Nitropure) transfer membranes (micron Separations Inc., 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 (Stratagene, La Jolla, CA); PGF2{alpha} (Lutalyse; Upjohn Co., Kalamazoo, MI); Luria Broth (Difco Laboratories, Detroit, MI); synthetic oligonucleotides (Gibco/BRL, Gaithersburg, MD); EnzChek Gelatinase/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 noted.

Animal Care

All procedures described where animals were used were approved by the University of Missouri-Columbia Animal Care and Use Committee (protocol no. 3185). Mixed-breed ewes were placed with vasectomized rams and observed for estrous behavior twice daily. The first day of estrus was denoted as Day 0.

Collection of Corpora Lutea

Corpora lutea (CL) were obtained from ewes (n = 3–10/time point) on Day 10 postestrus at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, or 48 h after a single i.m. injection of 15 mg PGF2{alpha}. Ovaries containing CL were sliced into 4-mm sections for in situ zymography. 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

The CL (100 mg/ml) were homogenized [19] 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 gelatin zymography. To evaluate MMP activity associated with the cellular pellet, 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 tested for gelatinolytic activity.

Generation of MMP cDNAs

Complementary DNA encoding ovine MMP-1 (363 base pairs [bp]), MMP-2 (743 bp), MMP-7 (321 bp), MMP-13 (404 bp), and MMP-14 (787 bp) were generated by the reverse transcription polymerase chain reaction (RT-PCR) using ovine luteal fibroblast RNA and primers corresponding to conserved sequences present in the reported nucleotide sequences for respective MMPs in other species. The ovine MMP PCR products were subsequently subcloned into a plasmid vector and subjected to fluorescent dye primer sequencing to confirm identity. The nucleotide sequence of the ovine MMP-1 cDNA (GenBank accession no. AF267156) was approximately 84% identical to the reported sequence of human MMP-1 [20]. The nucleotide sequence of the ovine MMP-2 cDNA (GenBank accession no. AF267159) was 89% and 86% identical to the reported sequences of human [21] and rat [22] MMP-2, respectively. The nucleotide sequence of the ovine MMP-7 cDNA (GenBank accession no. AF267158) was 82% identical to the reported sequence of human MMP-7 [23]. The nucleotide sequence of the ovine MMP-13 cDNA (GenBank accession no. AF267157) was approximately 89% and 85% identical to the reported sequences of human and mouse MMP-13 [24], respectively. The nucleotide sequence of the ovine MMP-14 cDNA (GenBank accession no. AF267160) was 92% and 90% identical to the reported sequences of human [25] and rat [26] MMP-14, respectively.

A 402-bp cDNA encoding bovine MMP-9 was generated by RT-PCR using bovine luteal RNA and primers designed from the reported sequence of bovine MMP-9 [27]. After subcloning into a plasmid vector, identity of the bovine MMP-9 cDNA (corresponding to nucleotides 459–861 of the bovine MMP-9 cDNA) [27] was confirmed by fluorescent dye primer sequencing.

Plasmids containing human MMP-3 cDNA were generously donated by Dr. Hideaki Nagase (Kansas City Medical Center, Kansas City, KS).

Labeling of cDNA Probes

Radiolabeled probes (MMP-1, -2, -3, -7, -9, -13, and -14 and glyceraldehyde-3-phosphate dehydrogenase) were synthesized through PCR as described previously [28]. 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 (dCTP), deoxyguanidine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP) with or without 100 µCi [{alpha}-32P]deoxyadenine triphosphate (dATP). 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 insert of proper size.

Northern Blot Analysis

Total RNA was extracted from CL (Tri-Reagent; Molecular Research Center, Cincinnati, OH). Approximately 5 µg of RNA from pooled (n = 5 ewes) Day 10 CL or millenium RNA molecular weight markers (Ambion) was electrophoresed in a 1% agarose gel and 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 CL (Tri-Reagent; Molecular Research Center) 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; Ambion). 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.

Gelatin Substrate 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% polyacrylamide gels containing 1 mg/ml porcine skin gelatin (175 bloom). Homogenates containing 600 µg (based on wet weights) of luteal homogenates 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 3 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 zones of digested gelatin. 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 for 2 h prior to gel electrophoresis. The addition of APMA causes formation of the active lower molecular weight form of MMPs [29]. Negative controls consisted of (12 mM) 1,10-phenanthroline (chelates Zn++ and Ca++) or addition of MMP inhibitor I (300 µM; Calbiochem, La Jolla, CA) to substrate buffer prior to incubation.

MMP Activity Assay

Luteal homogenates from each ewe were represented in each of 4 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. Prior to determination of enzymatic activity, luteal homogenates from groups 3 and 4 were reduced and alkylated to inactivate TIMPs [30]. 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 [30]. The process of reduction and alkylation (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, 50 µl of luteal extracts 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 absence of light. Samples were slowly shaken for 1 min and counted 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 CL

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 (20°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; Molecular Probes). Fluorescent gelatin substrate (100 µl of 0.03 mg/ml stock concentration) was added directly to tissue sections on individual slides. Slides were kept in a horizontal position, cover slipped, and incubated in a light-protected humidified chamber at 37°C for 1 h. Lysis of the substrate was assessed by examination under a fluorescent microscope. Controls consisted of 1,10-phenanthroline (5 mM), EDTA (50 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 by general linear model ANOVA [31], with day of the estrous cycle as the main effect. When the F-test was significant (P < 0.05), differences among means were evaluated by the Duncan multiple range test (mRNA expression) or the Fisher test (MMP activity) [31]. When variances were different as determined by the Bartlett test of equality of variance, data were log transformed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of mRNAs Encoding MMP-1, -2, -3, -7, -13, and -14

Total cellular RNA was isolated from CL at 0, 15, 30 min or 1, 2, 4, 6, 12, 24, and 48 h post-PGF2{alpha} administration (n = 3–10 animals/time point). Northern analysis confirmed that MMP-1, -2, -3, -7, -13, and -14 mRNAs were present in ovine luteal tissues, and transcripts were similar in size to those observed in other species (data not shown). MMP-9 was not detected by this procedure. Subsequent slot blot analysis demonstrated that expression of mRNAs encoding MMP-1, -2, -3, -7, -13, and -14 was biphasic (Fig. 1). Messenger RNA expression of all MMPs except MMP-2 increased (P < 0.05) by 15–30 min post-PGF2{alpha} administration relative to 0-h controls. Expression of all MMPs was increased (P < 0.05) compared with control levels by 6 h. MMP-14 mRNA was increased (P < 0.05) by 15 min and remained elevated throughout all time points evaluated.



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 1. Temporal expression of MMP-1, -2, -3, -7, -13, and -14 mRNA in ovine CL during PGF2{alpha}-induced luteolysis. Northern analysis confirmed that MMP-1, -2, -3, -7, -13, and -14 were present in ovine luteal tissue and that ovine MMP transcripts were similar in size to those observed in other species (data not shown). Messenger RNA was measured by slot-blot analysis. Filters contained 5 µg of total cellular mRNA per slot from Day 10 CL at 0, 0.25, 0.5, 1, 2, 4, or 6 h (left column) and at 0, 6, 12, 24, or 48 h (right column) post-PGF2{alpha} administration. Each blot was probed with 32P-labeled MMP-1 (A and B), MMP-2 (C and D), MMP-3 (E and F), MMP-7 (G and H), MMP-13 (I and J), or MMP-14 (K and L) cDNA. Levels of MMP transcripts were normalized relative to levels of glyceraldehyde-3-phosphate dehydrogenase mRNA. Data are expressed as mean ± SEM (n = 3–10 animals/time point); means having different superscripts are significantly different (P < 0.05)

Gelatin Substrate Zymography

Luteal homogenates collected on Day 10 postestrus at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h post-PGF2{alpha} contained gelatinases (Fig. 2). Gelatinolytic bands corresponding to Mr 72000 and 92000 likely represent latent forms of MMP-2 and -9, respectively. Gelatinolytic activity at lower molecular weights are indicative of active and intermediate forms of MMP-2 or MMP-9. Addition of 1,10-phenanthroline (12 mM) or synthetic MMP inhibitors (300 µM; Day 10 luteal homogenates) strongly inhibited gelatinolytic activity (data not shown). As expected, the addition of APMA (2 mM; activates proMMPs) increased the amount of active enzyme (data not shown).



View larger version (43K):
[in this window]
[in a new window]
  		FIG. 2. Identification of gelatinolytic activity (clear bands of digested gelatin) by gelatin substrate zymography. A representative zymogram demonstrates the presence of both proMMPs (arrows) and active or intermediate forms of MMP-2 and MMP-9 (arrowheads) collected at 0, 0.25, 0.5, 1, 2, 4, 6, 12, 24, or 48 h after PGF2{alpha} administration. Activity detected was positively attributed to MMPs after detection of proMMP activation by APMA or inhibition of gelatinolytic activity with a metal ion chelator (1,10-phenanthroline) or synthetic MMP inhibitors (data not shown)

MMP Activity Assays

Treatments (APMA and RA) were applied to luteal homogenates to allow determination of endogenous MMP activity (control = no treatment), endogenous plus activated MMP activities (APMA), MMP activity in the absence of TIMPs (RA), and endogenous 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; Fig. 3; data pooled over time).



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 3. Main effect of treatment on gelatinolytic activity in ovine CL collected at different time points post-PGF2{alpha} administration. Luteal homogenates (0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h post-PGF2{alpha}) received the following treatments: no treatment (control; detected endogenous MMP activity), APMA (detected endogenous plus activated MMP activities), RA (detected endogenous MMP activities in the absence of TIMPs), and APMA and RA (detected endogenous and activated MMP activities in the absence of TIMPs). Treated homogenates were subsequently added to fluorescein-labeled gelatin substrate. Gelatinolytic activity in luteal homogenates on Day 10 at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 post-PGF2{alpha} were determined by fluorometry. Main effects of treatment (data pooled over time) are expressed as mean ± SEM (n = 20–40 animals/treatment); means having different superscripts are significantly different (*P < 0.05)

There was a significant increase in MMP activity (P < 0.05) after PGF2{alpha} administration. Unexpectedly, endogenous luteal MMP activity (untreated group) decreased at 4 h (P < 0.05) relative to 0 h (Fig. 4A). No changes were observed in MMP activity in the absence of TIMPs (RA group) at any time point (Fig. 4C). However, APMA increased MMP activity (P < 0.05) as early as 15 min post-PGF2{alpha} and gelatinolytic activity remained elevated throughout 48 h post-PGF2{alpha} (Fig. 4B). The combination of APMA and RA treatments increased gelatinolytic activity (P < 0.05) by 30 min post-PGF2{alpha} relative to controls (Fig. 4D). The increase in MMP activity was maintained throughout luteal regression. The gelatinolytic activities were presumed to be due primarily to MMPs, based on inhibition of fluorescence when homogenates were incubated with 1,10-phenanthroline, EDTA, synthetic MMP inhibitors, or subjected to heat denaturation (data not shown).



View larger version (36K):
[in this window]
[in a new window]
  		FIG. 4. Effect of treatment and time on gelatinolytic activity in CL collected at various time points post-PGF2{alpha} administration. Luteal homogenates (0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 h post-PGF2{alpha}) received the following treatments: no treatment (control; detected endogenous MMP activity), APMA (detected endogenous plus activated MMP activities), RA (detected endogenous MMP activities in the absence of TIMPs), and APMA and RA (detected endogenous and activated MMP activities in the absence of TIMPs). Treated homogenates were subsequently added to fluorescein-labeled gelatin substrate. Gelatinolytic activity in luteal homogenates on Day 10 was recorded at 0, 15, and 30 min and 1, 2, 4, 6, 12, 24, and 48 post-PGF2{alpha} in response to the following treatments: control (A), APMA (B), RA (C), and APMA/RA (D). Data are expressed as mean ± SEM (n = 5–10); means having different superscripts are significantly different from controls (*P < 0.05; **P < 0.10)

Localization of MMP Activity in Ovine CL

In situ detection of MMP activities demonstrated the presence of gelatinolytic enzymes that appeared to be primarily extracellular in location; however, some cytoplasmic and nuclear localization were visible. Luteal tissue from the control group (0 h) contained pericellular gelatinolytic activity associated with nearly all cells, including LLCs (Fig. 5A). A noticeable increase in fluorescence was observed at 30 min post-PGF2{alpha} (Fig. 5B); it was highest at 1 h (Fig. 5C) and remained high through 48 h. Moreover, increased fluorescence appeared to be pericellular in origin. Addition of a synthetic MMP inhibitor I (Fig. 5E), EDTA, 1,10-phenanthroline, and excess unlabeled gelatin all markedly decreased fluorescence. Addition of APMA increased gelatinolytic activity (Fig. 5F).



View larger version (94K):
[in this window]
[in a new window]
  		FIG. 5. In situ zymographic analysis of frozen sections of ovine CL. Analysis of MMP activity in 0 h controls (A), 30 min (B), 1 h (C), and 6 h (D) post-PGF2{alpha} administration reveals pericellular gelatinolytic activity. Intracellular fluorescence was also detected. Note increase in pericellular activity in CL collected 30 min, 1 h, and 6 h after administration of PGF2{alpha}. Activity was markedly reduced following addition of synthetic MMP inhibitor (E), 1,10-phenanthroline, recombinant ovine TIMP-1, EDTA, or excess unlabeled gelatin (data not shown). Addition of APMA (F) increased gelatinolytic activity


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In domestic ruminants, PGF2{alpha} induces luteal regression, which is necessary to maintain ovarian cyclicity. Luteal regression is characterized by decreased progesterone secretion and structural involution of the gland. Structural regression requires extensive tissue remodeling and is essential for the ovary to maintain its proper size. Alteration of the MMP:TIMP ratio can have a profound effect on tissue remodeling [5, 6]. Ovine LLCs are the primary luteal source of PGF2{alpha} receptors as well as a major source of MMPs [17] and TIMPs [14, 17]. If degradation of luteal ECM is an important component of the physiological response to PGF2{alpha}, then it is logical that LLCs have a dual role of maintaining ECM homeostasis and being responsive to PGF2{alpha}.

Results from the present study support the hypothesis that PGF2{alpha}-induced luteolysis is accompanied by increased MMP expression and activity within ovine luteal tissue. We previously demonstrated a rapid decrease in luteal TIMP-1 concentration (including that of LLCs) following administration of a luteolytic dose of PGF2{alpha} in sheep [17]. Consequently, PGF2{alpha}-induced luteolysis may cause a rapid increase in the MMP:TIMP-1 ratio within luteal tissue. An increase in luteal MMP activity may be responsible for the loss of cell adhesion that has been reported in ovine CL during luteolysis [8]. An increase in MMP-2 expression has been reported in rat CL undergoing structural luteolysis [18]. In addition, in human granulosa lutein cells, treatment with hCG decreased MMP-2 and increased TIMP-1 production in vitro [32]. In nonhuman primates, luteolysis was accompanied by decreased TIMP-1 expression, and administration of hCG (luteal rescue) decreased luteal MMP-2 expression [33, 34]. Consequently, luteal rescue in humans and nonhuman primates may be associated with stabilization of ECM-luteal cell contacts via a decrease in the MMP:TIMP ratio. These data implicate the importance of the MMP:TIMP ratio in the involution of the gland.

There was a biphasic pattern of luteal MMP expression following PGF2{alpha} administration. Expression of MMP-1, -3, -7, -13, and -14 but not MMP-2 increased within 15–30 min of PGF2{alpha} injection, and all of the MMPs increased by 6 h post-PGF2{alpha}. Although MMP-2 expression was not increased early after PGF2{alpha} administration, there was an increase in MMP-14, which activates proMMP-2 [35, 36]. Gelatinolytic activity (APMA and APMA/RA groups) increased by 15 min and remained elevated through 48 h post-PGF2{alpha} injection. We predicted that MMP activity would be increased in the control homogenates (endogenous MMP activity); however, the only groups in which MMP activity increased were the APMA and APMA/RA groups. The increase in MMP activity within the preceding luteal homogenates is most likely due to the production of latent forms of gelatinases (primarily proMMP-2 and proMMP-9). The lack of increased MMP activity within control and RA groups may not accurately represent MMP activity changes occurring during luteolysis because active enzymes and inhibitors are mixed within luteal homogenates. Therefore, to determine the in situ activity associated with luteolysis, we evaluated PGF2{alpha}-treated CL with an in situ zymographic technique. Although MMP activity was not increased at any time within control or RA homogenates, in situ zymography detected increased MMP activity from intact luteal tissue by 30 min post-PGF2{alpha}. The activity was highest at 1 h post-PGF2{alpha} and remained high thereafter. This technique may be a valid new method for qualitative and quantitative analysis of MMP activity.

The rapid increase in MMP expression and activity may be associated with the decrease in luteal progesterone secretion and with structural regression. Dissociation of ovine CL with bacterial collagenase induced changes in the expression of PGF2{alpha} receptor, LH receptor, low-density lipoprotein receptor, steroidogenic acute regulatory protein, and 3ß-hydroxy steroid dehydrogenase within ovine luteal cells. These changes were similar to those detected within luteal tissue following PGF2{alpha} injection in vivo (G.D. Niswender, personal communication). These data suggest that increased ECM degradation is associated with biochemical changes that occur during luteolysis.

In other cells or tissues, MMP degradation of integrin-mediated cell-ECM contacts preceded loss of cellular function and structural involution [4, 5]. The ECM supports maintenance of a differentiated phenotype. Disruption of the ECM leads to dedifferentiation and ultimately apoptosis and (or) involution of the gland [4–6]. Structural regression of the CL occurs by apoptosis [11], and separation of cells from the ECM may be an important part of this process. Endothelial cells in ovine CL detach from the ECM [8] and undergo apoptosis during involution of the gland [9]. In the present study, MMP activity was localized to the pericellular region of various luteal cell types, and MMP activity (in situ) was increased by 30 min post-PGF2{alpha} administration.

In mammary cells, an increase in ECM degradation was associated with increased interleukin-1ß-converting enzyme and apoptosis. Transgenic pregnant mice that overexpressed MMP-3 underwent premature involution of the mammary gland as a result of apoptosis [5, 6]. Conversely, crosses of these mice with transgenic mice that overexpressed TIMP-1 resulted in mice with normal mammary gland involution [6]. These data indicate that alterations in the MMP:TIMP ratio can have a profound effect on timing of mammary gland involution. Similar mechanisms may act on luteal tissue at the time of luteolysis.

MMPs may promote apoptosis through mechanisms other than anoikis (apoptosis caused by loss of cell anchorage). MMPs have been implicated in cleaving membrane-bound cytokines, including tumor necrosis factor {alpha} (TNF{alpha}) [37] and FasL [38, 39]. MMPs also can cleave plasminogen, forming the potent antiangiogenesis factor angiostatin [40, 41], which may aid in the demise of CL. Increasing the solubility of the proapoptotic agents may facilitate luteolysis in sheep. This assumption is supported by the fact that addition of TNF{alpha} to porcine luteal cell cultures decreased progesterone and increased MMP secretion [42].

In addition to promoting ECM degradation, certain MMPs have additional functions. MMP-2 cleaves fibroblast growth factor (FGF) receptors [43], and FGF-2 increases progesterone production in luteal cell culture [44]. The proliferative capabilities of FGFs have also been demonstrated in CL of a variety of species, including sheep [45, 46]. FGF-2 is antiapoptotic [47] and promotes a differentiated phenotype [48]. Therefore, the functionality of FGFs may be compromised through MMP-dependent inactivation of FGF receptors. Decreased proliferation, dedifferentiation, and progesterone production and increased apoptosis are all hallmarks of luteolysis.

The ECM is a key regulator of both induction and maintenance of a differentiated phenotype. Increased ECM degradation leads to dedifferentiation and ultimately apoptosis [4]. Our results support the hypothesis that administration of PGF2{alpha} to sheep increases luteal MMP expression and activity. Increased MMP activity, and possibly ECM degradation, precedes the decrease in progesterone, thereby leaving open the possibility that MMPs have a role in functional and structural regression. Elevated MMP activity was sustained throughout luteolysis (48 h) and likely helps facilitate structural changes associated with CL.


   FOOTNOTES
 
First decision: 25 June 2001.

1 This work is a contribution from the Missouri Agricultural Experiment Station, Journal Series No. 13108, and was supported by USDA grants 98-35203-6282 (to M.F.S.) and 98-35203-6226 (to G.W.S.). Back

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 Back

3 Current address: Department of Anatomy and Obstetrics/Gynecology, University of California, San Francisco, CA 94143 Back

Accepted: October 15, 2001.

Received: May 22, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Smith M, McIntush E, Ricke W, Kojima F, Smith G. Regulation of ovarian extracellular matrix remodeling by metalloproteinases and their tissue inhibitors: effects on follicular development, ovulation and luteal function. J Reprod Fertil 1999; 54:367-381
  2. Nagase H, Woessner JF Jr. Matrix metalloproteinases. J Biol Chem 1999; 274:21491-21494[Free Full Text]
  3. Boudreau N, Werb Z, Bissell MJ. Suppression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle. Proc Natl Acad Sci U S A 1996; 93::3509-3513[Abstract/Free Full Text]
  4. Boudreau N, Sympson CJ, Werb Z, Bissell MJ. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 1995; 267:891-893[Abstract/Free Full Text]
  5. Sympson C, Talhouk R, Bissell M, Werb Z. The role of metalloproteinases and their inhibitors in regulating mammary epithelial morphology and function in vivo. Perspect Drug Discov Des 1994; 2::401-411[CrossRef]
  6. Alexander CM, Howard EW, Bissell MJ, Werb Z. Rescue of mammary epithelial cell apoptosis and entactin degradation by a tissue inhibitor of metalloproteinases-1 transgene. J Cell Biol 1996; 135::1669-1677[Abstract/Free Full Text]
  7. McCracken JA, Glew ME, Scaramuzzi RJ. Corpus luteum regression induced by prostaglandin F2-alpha. J Clin Endocrinol Metab 1970; 30:544-546[Medline]
  8. Nett TM, McClellan MC, Niswender GD. Effects of prostaglandins on the ovine corpus luteum: blood flow, secretion of progesterone and morphology. Biol Reprod 1976; 15:66-78[Abstract]
  9. Modlich U, Kaup FJ, Augustin HG. Cyclic angiogenesis and blood vessel regression in the ovary: blood vessel regression during luteolysis involves endothelial cell detachment and vessel occlusion. Lab Invest 1996; 74:771-780[Medline]
  10. Niswender G, Nett T. The corpus luteum and its control in infraprimate species. In: Knobil E, Neill JD (eds.), The Physiology of Reproduction, 2nd ed. New York: Raven Press; 1994: 781–816
  11. Juengel JL, Garverick HA, Johnson AL, Youngquist RS, Smith MF. Apoptosis during luteal regression in cattle. Endocrinology 1993; 132::249-254[Abstract]
  12. Fitz TA, Mayan MH, Sawyer HR, Niswender GD. Characterization of two steroidogenic cell types in the ovine corpus luteum. Biol Reprod 1982; 27:703-711[CrossRef][Medline]
  13. Farin CE, Moeller CL, Sawyer HR, Gamboni F, Niswender GD. Morphometric analysis of cell types in the ovine corpus luteum throughout the estrous cycle. Biol Reprod 1986; 35:1299-1308[Abstract]
  14. McIntush EW, Pletz JD, Smith GD, Long DK, Sawyer HR, Smith MF. Immunolocalization of tissue inhibitor of metalloproteinases-1 within ovine periovulatory follicular and luteal tissues. Biol Reprod 1996; 54:871-878[Abstract]
  15. Smith GW. Local regulators of corpus luteum function. Columbia, MO: University of Missouri; 1996. Dissertation
  16. Smith GW, McCrone S, Petersen SL, Smith MF. Expression of messenger ribonucleic acid encoding tissue inhibitor of metalloproteinases-2 within ovine follicles and corpora lutea. Endocrinology 1995; 136:570-576[Abstract]
  17. Ricke W. Extracellular remodeling in ovine corpora lutea. Columbia, MO: University of Missouri; 2000: 185. Dissertation
  18. Endo T, Aten RF, Wang F, Behrman HR. Coordinate induction and activation of metalloproteinase and ascorbate depletion in structural luteolysis. Endocrinology 1993; 133:690-698[Abstract]
  19. Nothnick WB, Edwards DR, Leco KJ, Curry TE Jr. Expression and activity of ovarian tissue inhibitors of metalloproteinases during pseudopregnancy in the rat. Biol Reprod 1995; 53:684-691[Abstract]
  20. Goldberg GI, Wilhelm SM, Kronberger A, Bauer EA, Grant GA, Eisen AZ. Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein. J Biol Chem 1986; 261:6600-6605[Abstract/Free Full Text]
  21. Collier IE, Wilhelm SM, Eisen AZ, Marmer BL, Grant GA, Seltzer JL, Kronberger A, He CS, Bauer EA, Goldberg GI. H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J Biol Chem 1988; 263:6579-6587[Abstract/Free Full Text]
  22. Marti HP, McNeil L, Davies M, Martin J, Lovett DH. Homology cloning of rat 72 kDa type IV collagenase: cytokine and second-messenger inducibility in glomerular mesangial cells. Biochem J 1993; 291:441-446
  23. Muller D, Quantin B, Gesnel MC, Millon Collard R, Abecassis J, Breathnach R. The collagenase gene family in humans consists of at least four members. Biochem J 1988; 253:187-192[Medline]
  24. Henriet P, Rousseau GG, Eeckhout Y. Cloning and sequencing of mouse collagenase cDNA. Divergence of mouse and rat collagenases from the other mammalian collagenases. FEBS Lett 1992; 310:175-178[CrossRef][Medline]
  25. Will H, Hinzmann B. cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment. Eur J Biochem 1995; 231:602-608[Medline]
  26. Okada A, Bellocq JP, Rouyer N, Chenard MP, Rio MC, Chambon P, Basset P. Membrane-type matrix metalloproteinase (MT-MMP) gene is expressed in stromal cells of human colon, breast, head and neck carcinomas. Proc Natl Acad Sci U S A 1995; 92:2730-2734[Abstract/Free Full Text]
  27. Baylis HA, Megson A, Hall R. Infection with Theileria annulata induces expression of matrix metalloproteinase 9 and transcription factor AP-1 in bovine leucocytes. Mol Biochem Parasitol 1995; 69:211-222[CrossRef][Medline]
  28. Ealy AD, Alexenko AP, Keisler DH, Roberts RM. Loss of the signature six carboxyl amino acid tail from ovine interferon-tau does not affect biological activity. Biol Reprod 1998; 58:1463-1468[Abstract/Free Full Text]
  29. Woessner JF Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991; 5:2145-2154[Abstract]
  30. Nothnick WB, Keeble SC, Curry TE Jr. Collagenase, gelatinase, and proteoglycanase messenger ribonucleic acid expression and activity during luteal development, maintenance, and regression in the pseudopregnant rat ovary. Biol Reprod 1996; 54:616-624[Abstract]
  31. Statistical Analysis System. SAS/STAT User's Guide, version 6, 4th ed. Cary, NC: Statistical Analysis Systems Institute, Inc.; 1989
  32. Stamouli A, O'Sullivan MJ, Frankel S, Thomas EJ, Richardson MC. Suppression of matrix metalloproteinase production by hCG in cultures of human luteinized granulosa cells as a model for gonadotrophin-induced luteal rescue. J Reprod Fertil 1996; 107:235-239[Abstract/Free Full Text]
  33. Duncan WC, Illingworth PJ, Fraser HM. Expression of tissue inhibitor of metalloproteinases-1 in the primate ovary during induced luteal regression. J Endocrinol 1996; 151:203-213[Abstract/Free Full Text]
  34. Duncan WC, McNeilly AS, Illingworth PJ. The effect of luteal "rescue" on the expression and localization of matrix metalloproteinases and their tissue inhibitors in the human corpus luteum. J Clin Endocrinol Metab 1998; 83:2470-2478[Abstract/Free Full Text]
  35. Sato H, Takino T, Okada Y, Cao J, Shinagawa A, Yamamoto E, Seiki M. A matrix metalloproteinase expressed on the surface of invasive tumour cells. Nature 1994; 370:61-65[CrossRef][Medline]
  36. Strongin AY, Collier I, Bannikov G, Marmer BL, Grant GA, Goldberg GI. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 1995; 270:5331-5338[Abstract/Free Full Text]
  37. Gearing AJ, Beckett P, Christodoulou M, Churchill M, Clements J, Davidson AH, Drummond AH, Galloway WA, Gilbert R, Gordon JL. Processing of tumour necrosis factor-alpha precursor by metalloproteinases. Nature 1994; 370:555-557[CrossRef][Medline]
  38. Tanaka M, Suda T, Haze K, Nakamura N, Sato K, Kimura F, Motoyoshi K, Mizuki M, Tagawa S, Ohga S, Hatake K, Drummond AH, Nagata S. Fas ligand in human serum. Nat Med 1996; 2:317-322[CrossRef][Medline]
  39. Kayagaki N, Kawasaki A, Ebata T, Ohmoto H, Ikeda S, Inoue S, Yoshino K, Okumura K, Yagita H. Metalloproteinase-mediated release of human Fas ligand. J Exp Med 1995; 182:1777-1783[Abstract/Free Full Text]
  40. O'Reilly M, Wiederschain D, Stetler-Stevenson W, Folkman J, Moses M. Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance. J Biol Chem 1999; 274::29568-29571[Abstract/Free Full Text]
  41. Patterson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem 1997; 272:28823-28825[Abstract/Free Full Text]
  42. Pitzel L, Ludemann S, Wuttke W. Secretion and gene expression of metalloproteinases and gene expression of their inhibitors in porcine corpora lutea at different stages of the luteal phase. Biol Reprod 2000;; 65:1121-1127
  43. Levi E, Fridman R, Miao HQ, Ma YS, Yayon A, Vlodavsky I. Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1. Proc Natl Acad Sci U S A 1996; 93:7069-7074[Abstract/Free Full Text]
  44. Grazul-Bilska AT, Redmer DA, Jablonka-Shariff A, Biondini ME, Reynolds LP. Proliferation and progesterone production of ovine luteal cells from several stages of the estrous cycle: effects of fibroblast growth factors and luteinizing hormone. Can J Physiol Pharmacol 1995; 73:491-500[Medline]
  45. Grazul-Bilska AT, Redmer DA, Killilea SD, Zheng J, Reynolds LP. Initial characterization of endothelial mitogens produced by bovine corpora lutea from the estrous cycle. Biochem Cell Biol 1993; 71::270-277[Medline]
  46. Ricke W. Cellular growth of porcine corpora lutea throughout the estrous cycle. Fargo, ND: North Dakota State University; 1995. Thesis
  47. Yasuda T, Grinspan J, Stern J, Franceschini B, Bannerman P, Pleasure D. Apoptosis occurs in the oligodendroglial lineage and is prevented by basic fibroblast growth factor. J Neurosci Res 1995; 40:306-317[CrossRef][Medline]
  48. Aharoni D, Meiri I, Atzmon R, Vlodavsky I, Amsterdam A. Differential effect of components of the extracellular matrix on differentiation and apoptosis. Curr Biol 1997; 7:43-51[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Drug Metab. Dispos.Home page
H. Rachmawati, C. Reker-Smit, M. N. Lub-de Hooge, A. van Loenen-Weemaes, K. Poelstra, and L. Beljaars
Chemical Modification of Interleukin-10 with Mannose 6-Phosphate Groups Yields a Liver-Selective Cytokine
Drug Metab. Dispos., May 1, 2007; 35(5): 814 - 821.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R. Wu, K. H. Van der Hoek, N. K. Ryan, R. J. Norman, and R. L. Robker
Macrophage contributions to ovarian function
Hum. Reprod. Update, March 1, 2004; 10(2): 119 - 133.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
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]

Home page
 Vet PatholHome page
S. J. Yan and E. A. G. Blomme
In Situ Zymography: A Molecular Pathology Technique to Localize Endogenous Protease Activity in Tissue Sections
Vet. Pathol., May 1, 2003; 40(3): 227 - 236.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ricke, W. A.
Right arrow Articles by Smith, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ricke, W. A.
Right arrow Articles by Smith, M. F.
Agricola
Right arrow Articles by Ricke, W. A.
Right arrow Articles by Smith, M. F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS