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Regular Article |
-Induced Luteolysis1
a Department of Animal Science, University of Missouri, Columbia, Missouri 65211
b Departments of Animal Science and Physiology, Michigan State University, East Lansing, Michigan 48824
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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administration would decrease expression of the tissue inhibitor of metalloproteinase (TIMP)-1, -2, and -3 and effectively increase the MMP:TIMP ratio, leading to glandular involution. In experiment 1, we tested the effects of PGF2 administration (Day 10 postestrus; Day 0 = estrus) on luteal TIMP-1, -2, and -3 mRNA and protein expression. Corpora lutea were collected at 0, 15, or 30 min or at 1, 2, 4, 6, 12, 24, and 48 h following PGF2 administration (n = 3–9 animals/time point). Following PGF2 administration, TIMP-1 mRNA levels decreased (P < 0.05) at 1 and 2 h relative to 0 h (controls), then increased to levels greater than controls at 4 and 6 h. In contrast, TIMP-2 and -3 mRNA levels did not decrease following PGF2 administration. The TIMP-1, -2, and -3 proteins were localized to large luteal cells (LLCs) within control (untreated) tissues. However, histodepletion of TIMP-1 within LLCs was evident within 30 min (earliest time point collected) following PGF2 injection and continued through 48 h. Luteal concentration of TIMP-1, as determined by RIA, was decreased (P < 0.05) by 15 min (earliest time point collected) following PGF2 administration and remained low through 48 h. In contrast, TIMP-2 and -3 immunolocalization was not altered by PGF2 administration. Experiment 2 was conducted to determine if PGF2 could initiate the preceding changes in TIMP-1 in early (Day 3) corpora lutea that can bind PGF2 but are refractory to its luteolytic effects. Serum concentrations of progesterone and luteal concentrations of TIMP-1 mRNA and protein were similar at 0 and 6 h after PGF2 injection on Day 3 postestrus. These data suggest that an early and sustained effect of PGF2 is the specific depletion of TIMP-1 within LLCs that are capable of responding to the luteolytic action of PGF2. This action may increase the MMP:TIMP-1 ratio, creating an environment that favors extracellular matrix degradation and, thereby, facilitates both functional and structural regression.
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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In sheep, TIMP-1, -2, and -3 and plasminogen activator inhibitor-1 have been localized primarily to large luteal cells (LLCs) [6–9]; therefore, LLCs may play an important role in luteal ECM remodeling. Ovine LLCs contain a prominent basal lamina. Basement membrane integrity is critical for maintaining a differentiated phenotype [10–13]. Disruption of the basal lamina can lead to cellular dedifferentiation and cell death [2]. If degradation of luteal ECM is an important component of the physiological response to prostaglandin (PG) F2, then it is logical that LLCs have the dual role of maintaining ECM homeostasis and of being responsive to PGF2. We hypothesized that altering the MMP:TIMP ratio by decreasing one or more TIMPs might lead to an environment that is conducive to increased ECM degradation and, thereby, facilitate luteolysis. The objective of this study was to determine if PGF2 would alter mRNA and protein expression of TIMP-1, -2, and -3 in corpora lutea that were responsive (Day 10) or unresponsive (Day 3) to PGF2.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Reagents, kits, and cells 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), [-32P]deoxyadenine triphosphate (dATP; 3000 Ci/mmol; Dupont/NEN, Wilmington, DE), nitrocellulose transfer membranes (Nitropure; Micron Separations, Inc., Westboro, MA), nylon transfer membranes (BrightStar; Ambion, Austin, TX) and northern Max kit (Ambion), XAR-5 film (Eastman Kodak, Rochester, NY), pGEM EZ vector and sequencing oligonucleotides (Pharmacia Biotech, Piscataway, NJ), P.F.U. polymerase (Stratagene, La Jolla, CA), PGF2 (Lutalyse; Upjohn Co., Kalamazoo, MI), isopropyl-ß-D-thiogalactoside (Alexis Corp., San Diego, CA), Luria Broth (Difco Laboratories, Detroit, MI), synthetic oligonucleotides (Gibco/BRL, Gaithersburg, MD), as well as Vector Elite ABC reagent peroxidase kit and normal mouse and rabbit IgGs (Vector, Burlingame, CA). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.
Animal Care
All procedures in which animals were used were approved by the University of Missouri 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. In experiments 1 and 2, luteolysis was induced following a single i.m. injection of 15 mg of PGF2.
Assay of Progesterone
Serum concentrations of progesterone were determined with a solid-phase RIA kit (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA) as previously described [14]. Serum was collected by jugular venipuncture before corpus luteum collection on Day 10 from 0 to 48 h following PGF2 administration (15 mg; i.m.). On Day 3, serum was collected at 0 or 6 h following PGF2 administration. All serum samples were analyzed in a single assay, and the within-assay coefficient of variation was less than 5.0%.
Collection of Corpora Lutea
Corpora lutea were collected from ewes (n = 3–12 per treatment) on Day 10 postestrus at 0, 15, or 30 min or at 1, 2, 4, 6, 12, 24, or 48 h following PGF2 administration (experiment 1) or on Day 3 at 0 or 6 h following PGF2 administration (experiment 2). Ovaries containing corpora lutea were immediately sliced into 4-mm sections, fixed in 10% (v/v) neutral buffered formalin, and embedded in paraffin for immunocytochemical studies. 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 [15]. Briefly, corpora lutea were homogenized (100 mg/ml) in 10 mM CaCl2 with 0.25% (v/v) Triton X-100 and centrifuged at 9000 x g for 30 min at 4°C. The supernatant was frozen at -80°C until assayed for TIMP-1 by RIA (see TIMP-1 RIA below).
Cloning of Ovine TIMPs
Ovine TIMP-1 and -2 cDNA have been previously described [8, 16]. The TIMP-3 cDNA (408 base pairs) was amplified from a Day 10 ovine corpus luteum cDNA library by polymerase chain reaction (PCR) and, subsequently, subcloned into a plasmid vector and subjected to fluorescent dye primer sequencing to confirm identity.
Labeling of cDNA Probes
Radiolabeled probes for ovine TIMP-1, -2, and -3 and glyceraldehyde-3-phosphate dehydrogenase were synthesized through PCR as described previously [17]. Briefly, 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]dATP. On completion of the PCR reaction, cDNA was precipitated and resuspended in 100 µl of water, which was subsequently added to the prehybridization buffer (Ambion). Reactions without radiolabel were visualized in a 1% (w/v) agarose gel to verify amplification of proper transcript size.
Northern Blot Analysis
Each TIMP probe was validated by Northern blot analysis before use in a slot-blot detection system. Total RNA was extracted from corpora lutea (Tri-Reagent; Molecular Research Center, Cincinnati, OH). Briefly, luteal tissue was homogenized in 1 ml of Tri-Reagent and stored at room temperature (RT) for 5 min. Following the 5-min incubation, 0.1 ml of bromochloropropane was added and thoroughly mixed. Samples were incubated at RT for approximately 15 min and centrifuged (4°C) at 12 000 x g for 15 min. The aqueous phase was collected and mixed with 0.5 ml of isopropanol and incubated at RT for 10 min before centrifugation (4°C) at 12 000 x g for 8 min. The resulting RNA pellets were washed in 1 ml of 75% (v/v) ethanol, followed by centrifugation (4°C) at 7500 x g for 5 min. The RNA pellet was dried and reconstituted in RNase-free water. Integrity of RNA was verified following gel electrophoresis.
Approximately 5 µg of total cellular RNA from pooled Day 10 corpora lutea (n = 5 ewes) or millennium RNA molecular weight markers (Ambion) were electrophoresed in a 1% (w/v) agarose gel and transferred by capillary action to a nylon membrane. Hybridization to labeled TIMP-1, -2, and -3 probes was carried out with a commercially available kit (BrightStar) according to the manufacturer's instructions. Briefly, each TIMP probe was denatured by boiling for 10 min before addition of probes to the hybridization buffer. Prehybridization and hybridization occurred at 42°C for 1 h or overnight, respectively, and blots were washed (15 min per wash) in 2x, 1x, and 0.1x SSC (1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate). Blots were subsequently exposed to XAR-5 film for 8 h (TIMP-1 and -3) or 12 h (TIMP-2).
MMP RNA Quantification
Total RNA was extracted from corpora lutea (Tri-Reagent) as described above and transferred to a nylon membrane with a slot-blot apparatus (5 µg/slot; Bio-Dot SF, Bio-Rad Laboratories, Hercules, CA). Two independent slot-blot analyses (0–6 and 0–48 h) were conducted. Hybridization to labeled TIMP-1, -2, and -3 probes was carried out with a commercially available kit according to the manufacturer's instructions (BrightStar) as described above. The prehybridization and hybridization conditions were identical to the previously discussed conditions for Northern blot analysis. Hybridization signal intensities were quantified by densitometry (Bio-Rad Model GS 700 Imaging Densitometry; Molecular Analyst, Molecular Bioscience Group, Hercules, CA), and the target mRNA values were expressed relative to glyceraldyde-3-phosphate dehydrogenase RNA for each sample. For each TIMP probe, signal intensities increased linearly with increasing amounts of ovine luteal total cellular RNA.
Western Blot Analysis
Luteal homogenates (600 µg/lane wet wt) were subjected to one-dimensional SDS-PAGE and electrophoretically transferred to nitrocellulose paper. Nonspecific binding sites were blocked by incubating nitrocellulose strips in Tris-buffered saline and 0.05% (v/v) Tween (TBST; pH 7.5) for 0.5 h. Nitrocellulose strips were incubated (2 h at 4°C) in TBST with rabbit anti-ovine TIMP-1 antisera, rabbit anti-human TIMP-2 antisera, TIMP-3 polyclonal affinity-purified antibodies (Chemicon, La Jolla, CA), or TIMP-3 monoclonal antibodies (Oncogene Sciences, Boston, MA) at a 1:300 dilution. Negative controls consisted of 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 immunoglobulin conjugated to biotin at a 1:10 000 dilution in TBST. After rinsing in TBST (3x), strips were incubated (0.5 h at 20°C) in an avidin-biotin-peroxidase complex 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 [6, 18] with an Elite ABC kit. Briefly, tissue sections for immunolocalization of TIMP-1, -2, and -3 were deparaffinized in clearing agent and rehydrated through a series of decreasing concentrations of ethanol. 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 either specific antiserum (1:100 dilution) against TIMP-1 or polyclonal affinity-purified rabbit IgGs against TIMP-2 or -3 (Chemicon). For negative controls, primary antibodies were substituted with respective normal sera (1:100 dilution) or normal IgGs (1:100 dilution) under the same conditions. Incubation with specific antibodies or their respective controls was 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 and 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. Each section was evaluated independently by two individuals to determine whether histodepletion of TIMP-1, -2, and -3 occurred following PGF2-induced luteolysis.
TIMP-1 RIA
Concentration of TIMP-1 was determined in triplicate 0.1-µl aliquots of luteal homogenates using a competitive equilibrium RIA established and validated in our laboratory [19]. Luteal homogenates or ovine TIMP-1 standards (25 pg to 10 ng) were incubated with 16 000–18 000 cpm radio-iodinated ovine TIMP-1 and a 1:15 000 dilution of an antiserum generated in rabbits against ovine TIMP-1. Serial dilutions of luteal homogenates exhibited displacement curves that were parallel to the displacement curve derived using the ovine TIMP-1 standard. The mean within-assay coefficient of variation was less than 5%.
Statistics
Data were analyzed by general linear models [20] analysis of variance, with time following PGF2 administration as the main effect. When the F-test was significant (P < 0.05), differences among means were evaluated by the Duncan multiple-range test. When variances were different as determined by the Bartlett test of equality of variance, data were first log-transformed. Serum concentrations of progesterone were analyzed by analysis of variance for repeated measures.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A significant decrease (P < 0.05) in serum concentrations of progesterone was first observed at 6 h and again at 24 h following PGF2 administration on Day 10 of the estrous cycle (Fig. 1).
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Expression of mRNA Encoding TIMP-1, -2, and -3 in Ovine Luteal Tissue
Total cellular RNA was isolated from corpora lutea on Day 10 at 0, 15, or 30 min or at 1, 2, 4, 6, 12, 24, or 48 h following PGF2 administration (n = 3–10 animals/time point). Northern blot analysis confirmed that TIMP-1, -2, and -3 mRNAs were present in ovine luteal tissues and that the size of the ovine TIMP-1, -2, and -3 transcripts was similar to the sizes previously published by our laboratory for sheep [8, 16, 21].
Following slot-blot analysis, there appeared to be a biphasic pattern of mRNA expression for TIMP-1, -2, and -3 (Fig. 2). A trend was observed for levels of TIMP-1 mRNA to increase at 15 min following PGF2 administration. The TIMP-1 levels subsequently decreased (P < 0.05) at 1 and 2 h relative to earlier time points, including 0 h (Fig. 2). However, by 4 and 6 h following PGF2 administration, TIMP-1 expression was increased (P < 0.05) relative to other time points. Thereafter, expression of TIMP-1 mRNA was not different from controls. Expression of TIMP-2 mRNA was increased (P < 0.05) at 15–30 min, returned to basal levels by 1 h, and was higher (P < 0.05) from 2 to 48 h (Fig. 2) following PGF2 administration. Relative expression of TIMP-3 mRNA was increased at 15 min, decreased to basal levels by 30 min, and increased from 2 to 6 h following PGF2 administration (Fig. 2).
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Immunolocalization of TIMP-1, -2, and -3 in Ovine Corpora Lutea
TIMP-1 localization
Antibodies directed toward TIMP-1, -2, or -3 were used in Western blot analysis to confirm the presence of TIMP proteins within corpora lutea and to verify the specificity of antibodies used for immunohistochemistry. Antibodies detected a single band at Mr 30 000 and 21 000 for TIMP-1 and -2, respectively, as previously reported by our laboratory [8, 16]. For TIMP-3, a band was detected at 24 000 and at 48 000 [21]. The TIMP-1 was localized to LLCs in the control group (0 h) (Fig. 3A). However, by 30 min following PGF2 injection (earliest time point evaluated), a marked decrease in immunolocalization of TIMP-1 was observed in LLCs (Fig. 3B). Immunohistodepletion was sustained through 48 h following PGF2 administration (Fig. 3, B–H). Substitution of primary antisera with preimmune sera (negative control) indicated no specific binding to ovarian tissues (Fig. 3I). No cross-reactivity with other proteins has been observed during Western blot or immunoprecipitation analyses. The MMPs do not appear to alter detection of TIMP-1; displacement characteristics of the ovine TIMP-1 standard were not affected by addition of 10 molar excess of recombinant human stromelysin.
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TIMP-2 localization Tissue inhibitor of metalloproteinase-2 was localized to LLCs at 0 h (Fig. 4, A–C), and at no time (0–48 h) did the intensity of TIMP-2 staining decrease relative to controls. Substitution of primary antisera with normal rabbit sera (negative control) indicated no specific binding to ovarian tissues (Fig. 4, G–I).
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TIMP-3 localization
Tissue inhibitor of metalloproteinase-3 was localized to LLCs and other cell types and/or to ECM at 0 h (Fig. 4, D–F). At no time (0–48 h) did the intensity of TIMP-3 immunoreactivity decrease relative to controls, nor were there changes in TIMP-3 localization following PGF2 injection. Substitution of primary antibodies with normal rabbit IgG (negative control) indicated no specific binding to ovarian tissues (Figure 4, G–I).
Luteal Concentrations of TIMP-1
Because changes in TIMP-1 based on immunocytochemistry are not quantitative, RIA was utilized to determine if luteal concentrations of TIMP-1 decrease following PGF2 administration. Luteal concentrations of TIMP-1 decreased (P < 0.05) by 15 min following administration of a luteolytic dose of PGF2 (Fig. 5). The decrease (P < 0.05) in luteal TIMP-1 remained low throughout all time points evaluated. As pointed out earlier, the decrease in TIMP-1 is unlikely to result from binding to MMPs, because addition of a 10 molar excess of recombinant human stromelysin did not alter displacement characteristics of the ovine TIMP-1 standard.
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Effects of PGF2 on Progesterone Secretion and TIMP mRNA and Protein Expression on Day 3 of the Estrous Cycle
Neither serum concentrations of progesterone nor luteal concentrations of TIMP-1 were decreased by 6 h following PGF2 administration on Day 3 postestrus (Fig. 6A). Therefore, corpora lutea at this time were unresponsive to the luteolytic action of PGF2. Furthermore PGF2 administration did not alter luteal TIMP-1, -2, and -3 mRNA levels in samples collected at 6 h following administration of PGF2 on Day 3 postestrus (Fig. 6B). Finally, no evidence was observed to indicate immunohistodepletion of TIMP-1 in LLCs following PGF2 administration on Day 3 postestrus (Fig. 6C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the uterine luteolysin, and are estimated to produce more than 80% of total luteal progesterone [23]. Large luteal cells appear to have a central role in luteal ECM remodeling, as demonstrated by the presence of TIMP-1, -2, and -3 and plasminogen activator inhibitor-1 within this cell type [6–9]. In addition, we have previously localized MMP-1, -2, -3, and -9 to LLCs [7]. If localized ECM degradation has a role in luteolysis, then LLCs may have the dual role of maintaining ECM homeostasis and responding to the uterine luteolysin.
In this study, PGF2 induced a rapid decrease in TIMP-1 protein expression within ovine corpora lutea. Alterations in the expression of MMPs and TIMPs have been hypothesized to be associated with structural, but not with functional, regression of corpora lutea in rats [24] and pigs [25]. However, in the present study, the decrease in luteal concentrations of TIMP-1 (15 min) and the immunohistodepletion of TIMP-1 (30 min) preceded the decrease in serum concentrations of progesterone and the reported decrease in luteal blood flow (2 h) [26]. Although further investigation is necessary, alteration of the MMP:TIMP ratio in corpora lutea may have implications for the decrease in progesterone as well as involution of the gland during luteolysis.
Decreased luteal concentrations of TIMP-1 may be associated with the ability of PGF2 to induce luteolysis. Administration of PGF2 to domestic ruminants does not induce luteolysis during the early luteal phase. Although functional PGF2 receptors and signal transduction mechanisms are present in developing ovine corpora lutea [27, 28], the acquisition of luteolytic capacity is not established until after Day 4 postestrus [27]. The decreased luteal concentrations of TIMP-1 on Day 10, but not on Day 3, suggest an association between PGF2-induced depletion of TIMP-1 and the acquisition of luteolytic capacity.
The mechanisms that mediate luteal TIMP-1 depletion are unknown. Within ovine LLCs, TIMP-1 has been localized to secretory granules that are capable of undergoing exocytosis [6]. Prostaglandin F2-induced luteolysis is accompanied by rapid exocytosis of secretory granules [29, 30] and may induce rapid secretion of TIMP-1 from LLCs. Alternatively, TIMP-1 may be destroyed by mechanisms associated with luteolysis. Increased polyubiquitin mRNA expression occurs within 2 h of PGF2 administration [31] and may promote targeted proteolysis of a number of proteins, including TIMP-1 [32]. Production of superoxide radicals occurs relatively soon after the initiation of luteolysis [33–36]. Peroxynitrite (generated by the reaction of superoxide 
with nitric oxide) inactivated TIMP-1 [37] and may be produced during PGF2-induced luteolysis. Another potential mechanism for the destruction of TIMP-1 is via neutrophil elastase-mediated extracellular proteolysis [38]. However, timing of neutrophil infiltration within 30 min of PGF2 administration has not been evaluated. The preceding mechanisms may explain rapid and/or sustained decreases in luteal TIMP-1.
Depletion of TIMP-1 following PGF2-induced luteolysis may alter the MMP:TIMP ratio within luteal tissue to facilitate ECM degradation and, thus, contribute to functional and/or structural regression. Induction of luteolysis in primates was accompanied by decreased expression of TIMP-1 mRNA (by 12 h), and administration of hCG decreased luteal MMP-2 expression [39, 40]. In addition, hCG decreased MMP-2 and increased TIMP-1 expression by human luteinizing granulosa cells in vitro [41]. These observations link changes in the MMP:TIMP ratio to luteal rescue or luteal regression, respectively. Moreover, maintaining a differentiated phenotype and functional gland may be influenced by this ratio [2, 12, 13]. Although the specific mechanisms associated with luteal rescue and luteolysis in humans and primates are unknown, stabilization of ECM-luteal cell contacts may enhance progesterone secretion and promote luteal cell survival.
The pattern of expression of TIMP-1 mRNA and protein differed following PGF2-induced luteolysis. Although concentrations of TIMP-1 protein decreased rapidly and remained low following PGF2 administration, TIMP-1 mRNA expression decreased by 1 h and increased by 4 h following PGF2 administration relative to the control group. A biphasic pattern of mRNA expression was detected for both TIMP-2 and -3, in which mRNA expression increased within 15 min, returned to baseline by 30 min (TIMP-3) to 1 h (TIMP-2), and increased by 2 h following PGF2 administration. The preceding pattern of expression is almost identical to the pattern of mRNA expression of the early response genes, c-fos and c-jun, in bovine corpora lutea following PGF2-induced luteolysis [42]. The preceding gene products form a heterodimer (AP-1), and genes encoding for MMPs and TIMPs are reported to have AP-1 sites in the upstream regulatory regions [43].
Although histodepletion of TIMP-1 occurred following PGF2-induced luteolysis, no evidence was found to indicate histodepletion of TIMP-2 or -3. Localization of TIMP-2 and -3 to LLCs confirmed that histodepletion of these two proteins did not occur within this luteal cell type after PGF2 administration. Therefore, luteolysis appeared to be associated with a specific and rapid decrease of luteal TIMP-1.
Reportedly, TIMP-2 and -3 have activities, in addition to MMP inhibitory activity, that have been associated with tissue involution, including activation of proMMPs [3, 4] and facilitating apoptosis [2]. Gelatinolytic activity can be increased through the activation of proMMP-2 via mt-MMPs. Activation of proMMP-2 occurs through formation of a trimolecular complex with TIMP-2 [3, 4]. During activation, TIMP-2 initially binds to MMP-14. Subsequently, TIMP-2 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 MMP-2 molecule. Luteal tissue contains all three components of the activation cascade, and increased gelatinase activity has been reported within rat corpora lutea following the induction of luteolysis [25]. Consequently, TIMP-2 may increase activation and, thus, focalize gelatinolytic activity near the cellular surface. An increase in ECM degradation resulting in cell death has been defined as "anoikis" [44].
Corpora lutea undergo apoptosis during PGF2-induced luteolysis [45]. Tissue inhibitor of metalloproteinase-3 can enhance cell death (i.e., apoptosis) through inhibiting the proteolytic processing and shedding of tumor necrosis factor (TNF) receptors [46–48]. Tumor necrosis factor and its receptors are present within ovine corpora lutea and increase after PGF2 administration [49–52]. Increasing TNF receptor stability may facilitate luteolysis. Addition of TNF to porcine luteal cells in culture decreased progesterone secretion and increased production of MMP-1, -2, and -9 [25]. Therefore, TNF may activate the apoptotic cascade and/or increase MMP production to promote ECM degradation. Increased TIMP-3 expression may therefore stabilize TNF receptors and promote luteolysis through a TNF-dependent pathway.
These data provide evidence for a rapid and sustained decrease in luteal TIMP-1 during PGF2-induced luteolysis in sheep. The depletion of TIMP-1 preceded a decrease in serum concentrations of progesterone, and the preceding effect did not occur in corpora lutea that were unresponsive to the luteolytic action of PGF2. Depletion of TIMP-1 within LLCs may be associated with increased ECM degradation during luteolysis. Collectively, PGF2 may facilitate luteolysis through altering expression of TIMPs in a manner that is conducive for involution of the gland.
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FOOTNOTES |
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1 Supported by U.S. Department of Agriculture grants 98-35203-6282 (M.F.S.) and 98-35203-6226 (G.W.S.). Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 13, 107. 
2 Correspondence: Michael F. Smith, Department of Animal Sciences, 160 Animal Science Center, Columbia, MO 65211. FAX: 573 884 7827; smithmf{at}missouri.edu
3 Current address: Department of Anatomy, University of California at San Francisco, San Francisco, CA 94122
4 Current address: Bethyl Labs, P.O. Box 850, Montgomery, TX 77356
Accepted: December 3, 2001.
Received: April 16, 2001.
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  X. Hou, E. W. Arvisais, C. Jiang, D.-b. Chen, S. K. Roy, J. L. Pate, T. R. Hansen, B. R. Rueda, and J. S. Davis Prostaglandin F2{alpha} Stimulates the Expression and Secretion of Transforming Growth Factor B1 Via Induction of the Early Growth Response 1 Gene (EGR1) in the Bovine Corpus Luteum Mol. Endocrinol., February 1, 2008; 22(2): 403 - 414. [Abstract] [Full Text] [PDF]
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