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a Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, Ontario,Canada N1G 2W1
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-smooth muscle actin and type I procollagen by immunostaining. Gelatinase A (MMP-2), gelatinase B (MMP-9), TIMP-1, and TIMP-2 were present in EOSC-conditioned medium, and TIMP-3 in ECM of EOSC. Transforming growth factor ß significantly stimulated the activity of gelatinases A and B and TIMP-1 in conditioned medium from EOSC (p < 0.05). Phorbol 12-myristate 13-acetate also significantly stimulated the activity of gelatinases A and B and TIMP-1 in conditioned medium and of TIMP-3 in ECM (p < 0.05). Our results suggest that EOSC produce important components of the ECM remodeling machinery and, therefore, may play a role in the ECM remodeling during follicle growth in this species.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The equine ovary has unique anatomic features: the cortex is in the center, and the medulla portion is superficial [13]. In the process of growth, the selected follicle expands from 50 µm in diameter at the primordial follicle stage to 5–7 cm in diameter at maturity. At the same time, the follicle migrates to the fossa, the only site from which a mature oocyte can be released from the ovary [13]. Therefore, in this species, the ovarian stromal tissue surrounding growing follicles undergoes extensive tissue remodeling in favor of the expansion and emigration of the follicle. The purpose of this study is to examine whether equine ovarian stromal cells (EOSC) might be involved in this process by producing components of the ECM remodeling machinery.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Minimum essential medium (MEM), L-glutamine, minimum nonessential amino acid solution, penicillin, streptomycin, gentamycin, fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco-BRL (Burlington, ON, Canada). Bacterial collagenase type I, DNase I, phorbol 12-myristate 13-acetate (PMA), PMSF, 4-aminophenylmercuric acetate (APMA), and monoclonal human -smooth muscle actin (-SMA) antibody were purchased from Sigma Chemical Company (St. Louis, MO). Rabbit anti-human Von Willebrand factor was obtained from Dako Corporation (Carpinteria, CA). Rabbit antihuman type I procollagen was obtained from Cedar Lane (Hornby, ON, Canada). The reverse zymography kit, which contained crude gelatinase A and mouse purified TIMP-1, TIMP-2, and TIMP-3, was purchased from University Technologies International Inc. (Calgary, AB, Canada). Transforming growth factor (TGF) ß1 was purchased from R&D Systems Inc. (Minneapolis, MN).
Cell Culture
Ovaries from mature, cyclic mares (presence of mature corpus luteum indicated prior ovulation) were collected from a local abattoir (Barton Feeders, Owen Sound, ON, Canada) during the breeding season from April to September. The reproductive histories of the mares were not available. Ovaries were transported to the laboratory on ice in sterile Hanks' balanced salt solution (HBSS) with gentamycin (5 µg/ml). The outer surface of the ovaries was rinsed with 70% alcohol and washed twice with HBSS. The ovaries were bisected longitudinally within 4–6 h of collection. Eight ovaries judged as healthy from eight horses were chosen for the isolation of stromal cells. Stromal tissue from the apex (portion of the follicle oriented towards the ovarian fossa) of large follicles (3–4 cm in diameter) was collected and minced into 1-mm3 pieces. The tissue was digested by shaking in bacterial collagenase type I (125 U/ml) in serum-free MEM at 37°C for 3 h. During the last half hour of digestion, DNase I (50 U/ml) was added to the solution. After the tissue pieces were allowed to settle, crude debris was removed, and the supernatant containing EOSC was centrifuged at 250 x g. The supernatant was discarded, and the cell pellet was resuspended with HBSS. Viability of the EOSC was estimated by trypan blue dye exclusion. EOSC were grown in MEM supplemented with 10% (v:v) FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamycin (5 µg/ml) at 37°C in a 5% CO2 atmosphere. Collection of ovaries and tissues was performed over two seasons, and established EOSC were pooled together in order to have sufficient cells for the whole study and to ensure that cells used in each experiment were from the same pool. Finally, EOSC were aliquoted into cryovials at a density of 1 x 106/ml in media containing dimethylsulfoxide (10%, v:v) and FBS (20%, v:v) and were frozen and stored in a liquid nitrogen tank. Cells between passages 3 and 8 were used in these experiments.
Immunocytochemistry
In order to characterize EOSC, -SMA, Von Willebrand factor, and type I procollagen were chosen as markers. We verified that these antibodies cross-react with equine antigens, using appropriate controls (cultured equine uterine smooth muscle cells for -SMA, cultured equine endothelial cells for Von Willebrand factor, and equine skin for type I procollagen). Cultured EOSC were immunostained as follows. Cells were cultured on sterile coverslips in MEM with 10% FBS overnight, then were rinsed with HBSS solution, dried completely at room temperature, and fixed in cold acetone for 10 min. The cells were incubated with antibodies to -SMC (diluted 1:800 for 1 h) or Von Willebrand factor (1:20 for 1 h) or type I procollagen (1:50 overnight). Color reactions were then performed using secondary antibodies conjugated with horseradish peroxidase or alkaline phosphatase (1:20 for 30 min) and appropriate substrates.
Preparation of Samples for Enzyme Assays
EOSC at a density of 5 x 105 viable cells were seeded into 60-mm Petri dishes. MEM supplemented with 10% (v:v) FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamycin (5 µg/ml) was changed every 48 h until the cells became confluent (6–8-day culture). The cells were cultured in serum-free MEM for another 24 h and then given various treatments. The conditioned medium was removed from Petri dishes and centrifuged at 250 x g to remove dead cells and debris. The supernatant was decanted and stored at -80°C. This conditioned medium was lyophilized and resuspended with ddH2O to produce a final solution that was 20-fold concentrated, to ensure that sufficient enzyme levels were present in samples for detection by zymography. The protein content in this concentrated conditioned medium was determined using a modified Bradford microassay (Bio-Rad Laboratories, Mississagua, ON, Canada), and samples were aliquoted in 500-µl vials and stored at -80°C until analysis.
ECM in Petri dishes was prepared as described previously [14]. Briefly, after collection of conditioned medium, the confluent cell monolayers were washed three times with PBS containing 5 mM EDTA/5 mM EGTA, then incubated at 37°C in the same solutions (5 ml) until the cells began to detach from the surface of the dishes. The dishes were washed several times with PBS until cells could no longer be found on culture dishes when inspected by phase contrast microscopy. The remaining ECM was washed three times with PBS, followed by ddH2O to remove the salts. Then, ECM was collected by scraping the dish bottom with a hard rubber spatula in 10 µl of 5-strength gel nonreducing loading buffer (Tris, 3%; glycerol, 50%; SDS, 20%) and 40 µl of ddH2O. The volume of collected buffer was adjusted with ddH2O to 50 µl, and samples were used immediately for reverse zymography or were stored at -80°C.
Gelatin Zymography
Active and latent forms of gelatinase A and B were detected by zymography, as described by Heussen and Dowdle [15]. In brief, samples were loaded with 5-strength nonreducing loading buffer in a ratio of 1:4 into SDS polyacrylamide (7.5% v:v) gel polymerized with 0.2% (w:v) calf skin gelatin. After electrophoresis, gels were washed with 2.5% Triton X-100 (2 x 30 min), then rinsed in ddH2O. After being washed in the incubation buffer (50 mM Tris, 5 mM CaCl2, 1µM ZnCl2, pH 8) for 30 min, gels were incubated with the incubation buffer at 37°C overnight. Gels were stained with 0.1% Coomassie brilliant blue G-250 dye for 30 min and destained in 5% methanol/7% glacial acetic acid. Since gelatinases A and B degrade gelatin present in the acrylamide gel, clear lysis bands indicate the absence of gelatin and the presence of gelatinases. In order to further identify the gelatinolytic activity in conditioned medium of EOSC, 10 mM EDTA (which chelates Zn2+ at the active site of MMPs and inhibits their activity [16]), or 1 mM PMSF (which inhibits serine/cysteine proteases but not MMPs [17]), was incorporated into the incubation buffer. APMA (1 mM), which converts precursor MMPs to active forms [17], was added to some samples before electrophoresis. Gels were photographed using a Gel Print 2000i apparatus (Bio/Can Scientific, Mississauga, ON, Canada), and digitized images were saved for further densitometric analysis.
Reverse Gelatin Zymography
TIMPs were analyzed with reverse zymography as described by Hampton et al. [14], using a commercially available kit (University Technologies International, Inc.). Briefly, 12% SDS polyacrylamide gels were made with 0.2% calf skin gelatin and crude gelatinase A. Samples were loaded with 5-strength nonreducing loading buffer at a ratio of 1:4. After electrophoresis, gels were washed with 2.5% Triton X-100 (2 x 30 min) and rinsed in ddH2O. Gels were washed in incubation buffer for 30 min, then incubated with incubation buffer at 37°C overnight. Gels were stained and destained as described above. The activity of TIMPs resulted in the presence of dark blue bands on a clear background. Purified mouse TIMP-1, TIMP-2, and TIMP-3 were run as standards. Gels were photographed using a Gel Print 2000i apparatus, and digitized images were saved for further densitometric analysis.
Image Processing and Densitometric Analysis
Gels from zymography and reverse zymography were processed with a Gel Print 2000i apparatus. Digitized images of gelatinolytic bands of gelatinases A and B from gelatin zymography and gelatin bands corresponding to TIMP-1, TIMP-2, and TIMP-3 from reverse gelatin zymography were analyzed on a Macintosh computer equipped with the NIH software "Image" (version 1.60). The integration area of each band was measured, and values were expressed as a ratio of the control area from the same gel (no additional treatment; set to unity). Results were thereby standardized for each gel and expressed in dimensionless units. Results were obtained from 3 separate experiments for each condition.
Statistical Analysis
Densitometry results from different replicate gels were compared, and means were calculated for each experimental system. All data are presented as the mean ± standard error of the mean (SEM). The software "Statistix" (V4.1) was used for statistical analysis. The significance of the differences among the means within each experiment was determined by one-way ANOVA, followed by Duncan's multiple-range procedure. Differences were considered significant at p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Under phase contract microscopy, EOSC showed fibroblast-like morphology. The doubling time of EOSC in 10% FBS/MEM was about 20 h, and they could survive in serum-free medium for up to 7 days without any obvious ill effects (data not shown). The cells tended to form localized multilayered clumps of cells after they reached confluence. Most of the EOSC were positive for -smooth muscle cell actin and type I procollagen (Fig. 1). Cultures were negative for Von Willebrand factor, indicating that our cultures were not contaminated with endothelial cells from the stromal microvessels (data not shown).
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Characteristics of Gelatinase Activity
In conditioned medium collected from EOSC cultured without any additional treatment, two major bands indicating gelatinolytic activity were obtained, as detected by gelatin zymography. The gelatinolytic activity at approximately 72 kDa (putative progelatinase A) was predominant (Fig. 2, whereas gelatinolytic activity at approximately 92 kDa (putative progelatinase B) was weak (Fig. 3a). In the presence of 1 mM APMA, which activates latent progelatinases, two additional faint bands at 68 and 82 kDa, thought to represent activated gelatinase A and B, respectively, were detected. In the presence of 10 mM EDTA (which inactivates MMPs by chelating Zn2+), gelatinolytic activity at both 72 and 92 kDa was completely inhibited (Fig. 2, lane c); the presence of 1 mM PMSF (which is not capable of inhibiting MMPs) had no effect on the gelatinolytic activity in conditioned medium from EOSC (Fig. 2, lane d). In order to determine whether the enzymes were gelatin-specific, fibrinogen or -casein were used as substrates for zymography, instead of gelatin. For the substrates tested, the enzyme activity in EOSC samples was absent on fibrinogen (Fig. 2, lane e) and -casein zymograms (Fig. 2, lane f).
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Effects of Different Doses of TGFß or PMA on Gelatinase Production of EOSC
TGFß and PMA are involved in regulation of gelatinases in many in vitro systems. Conditioned medium of EOSC treated with TGFß (1–10 ng/ml) or PMA (5–20 ng/ml) for 48 h was examined by gelatin zymography in three experiments. An example of a gelatin zymogram from one experiment (Fig. 3) showed that both TGFß and PMA increased gelatinolytic bands corresponding to latent gelatinase A (72 kDa) and latent gelatinase B (92 kDa) activities (Fig. 3a). In addition, TGFß at a concentration of 5 ng/ml increased active gelatinase A activity, and PMA increased active gelatinase B activity. Densitometric analysis demonstrated that increasing concentrations of TGFß resulted in a significant increase of latent gelatinase A and latent gelatinase B activities (Fig. 3b). Densitometric analysis also showed that stimulation by TGFß at a dose of 5 ng/ml caused a 2.1-fold increase of gelatinolytic bands corresponding to latent gelatinase A activity compared to control values. Stimulation by TGFß at a dose of 10 ng/ml resulted in a 3.5-fold increase of gelatinolytic bands representing latent gelatinase B activity. The phorbol ester PMA significantly increased latent gelatinase A activity at the lowest concentration of 5 ng/ml and continued to cause significant further increases at 10 and 20 ng/ml. Phorbol ester PMA at 20 ng/ml caused a 2.2-fold increase in latent gelatinase A activity compared to that of control. Phorbol ester PMA also caused a significant increase in latent gelatinase B activity. However, the maximal stimulatory effects of a 4-fold increase in latent gelatinase B activity was induced by PMA at concentration of 5 ng/ml. With increased concentrations from 5 to 20 ng/ml, PMA induced active gelatinase B activity. Densitometric analysis for active gelatinase B activity (86 kDa) was not performed because of the lack of detectable active gelatinase B activity in the control samples.
Characteristics of TIMPs Produced by EOSC
Reverse gelatin zymography revealed that cultured EOSC secreted three factors that inhibited gelatinase activity (Fig. 4a). Two inhibitors in conditioned medium were approximately 29 and 22 kDa in size, while the third, in ECM, was approximately 24 kDa in size. In SDS-PAGE gels without gelatin and gelatinase, EOSC-conditioned medium or ECM did not contain visible proteins in the range of 21–30 kDa (Fig. 4b). Therefore, these three bands in Figure 4a represented undegraded gelatin; thus gelatinases added into the gels as part of the reverse zymography protocol were inhibited by these three factors. The molecular masses of two inhibitors in conditioned medium were very close to those of mouse TIMP-1 and TIMP-2; the molecular mass of the third factor bound to ECM was similar to that of mouse TIMP-3 (Fig. 4c). These three inhibitors remained partially active at pH 4.5 (Fig. 4d) or when heated to 90°C for 70 min (Fig. 4e).
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Effects of Different Doses of TGFß or PMA on TIMP Production by EOSC
The effects of increasing concentrations of TGFß (1–10 ng/ml) or PMA (5–20 ng/ml) on TIMP-1 and TIMP-2 activity in conditioned medium were assessed by reverse gelatin zymography. EOSC were treated with TGFß or PMA for 48 h. A representative reverse gelatin zymogram shows two bands representing TIMP-1 and TIMP-2 activity in conditioned medium of EOSC (Fig. 5a). Densitometric analysis showed that TGFß at 5 and 10 ng/ml or PMA at all concentrations tested significantly increased TIMP-1 activity over control values in conditioned medium of EOSC (Fig. 5b). Maximum increases of 3.6- and 3.3-fold in TIMP-1 activity were caused by TGFß at 10 ng/ml and PMA at 20 ng/ml, respectively, compared to control values. TGFß at concentrations of 5 and 10 ng/ml, and PMA at concentrations of 5 and 10 ng/ml also caused slight increases in TIMP-2 activity. However, these increases were not statistically significant compared to control values. In ECM, TGFß had no significant effect on TIMP-3 activity even at the concentration of 10 ng/ml. Phorbol ester PMA at 20 ng/ml caused a significant increase of TIMP-3 activity at 5, 10, and 20 ng/ml (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-SMA. Bovine ovary stromal cells assume a fibroblast-like morphology in vitro [18]. Other authors have reported -SMA-positive cells in the stroma of human ovaries [19, 20]. While the EOSC can form multilayered aggregations under crowded culture conditions, EOSC cells in this study did not display the classic "hill and valley" morphology highly characteristic of smooth muscle cells in vitro which is seen in our cultures of equine uterine smooth muscle cells. Although EOSC are strongly positive for type I procollagen immunostaining and have a fibroblast-like morphology, they showed neither the classic spindle shape and motile morphology, nor the contact inhibition of "typical" connective tissue fibroblasts [21]. On the basis of these characteristics, we postulate that the EOSC are most likely myofibroblasts, a cell type first identified in 1971 [22] and reported to express characteristics of both smooth muscle cells and fibroblasts [23, 24]. Myofibroblasts are typically found in connective tissues undergoing remodeling [25]. In addition to their contraction function, myofibroblasts have been found to take part in tissue remodeling by producing several proteolytic enzymes, such as urokinase-type plasminogen activator and type-1 plasminogen activator inhibitor by myofibroblasts of human breast cancer [26], and TIMPs by human skin fibroblasts [24]. Thus, myofibroblasts in equine ovary may take part in the matrix remodeling required for follicle growth. Histological and biochemical changes have been observed at the follicular apex in several other species. An electron microscope study of rabbit ovaries documented the disappearance of the collagen bundles in the apex wall of preovulatory follicles [27]. Degradation of ECM in this apical region in sheep ovaries was significantly enhanced in comparison to ECM degradation in basal tissue of ovarian follicles [28]. In rabbits, tissue at the follicular apex contains more MMP-1, demonstrated by immunohistochemistry, than do other areas of the follicle wall [29]. These results indicate that cells within the follicle apex are possibly more important for follicle expansion, migration, and ovulation than cells elsewhere in the follicle wall.
EOSC secreted enzymes of approximately 72 and 92 kDa into conditioned medium, and these enzymes possessed gelatinolytic activity. Conditioned medium of EOSC treated with APMA, a substance used in vitro to activate latent forms of gelatinases, contained two extra enzymes of approximately 68 and 82 kDa. The gelatinolytic activity normally observed in conditioned medium from EOSC was completely abolished in the culture medium by inclusion of EDTA, which chelates with Zn2+ at the active site of MMPs and leads to the inhibition of MMP activities [16]. PMSF, a serine/cysteine protease inhibitor but not an MMP inhibitor [17], did not inhibit gelatinolytic activity in EOSC-conditioned medium. The enzyme activity was not found when fibrinogen or -casein was used as the substrate for zymography, suggesting that the enzymes in conditioned medium causing gelatinolytic bands were gelatin-specific. Taken together, the evidence supports our conclusion that the gelatinolytic activity at 72 kDa represents progelatinase A, while that at 92 kDa represents progelatinase B. The gelatinolytic activities at 68 and 82 kDa represent active gelatinases A and B, respectively.
Two proteins with inhibitory activity on gelatinases were found in EOSC-conditioned medium and had approximate molecular masses of 29 and 22 kDa, similar to mouse TIMP-1 and TIMP-2, respectively. A third inhibitor of 24 kDa was found exclusively in ECM and also had the same molecular mass as mouse TIMP-3. All these proteins were heat- and acid-resistant, consistent with the TIMP characteristics described by Cawston and coworkers [30]. Taken together, the evidence supports our conclusion that the two inhibitors in conditioned medium represent TIMP-1 and TIMP-2, and the inhibitor in ECM represents TIMP-3. To our knowledge, this is the first report showing that equine cells synthesize gelatinases and TIMPs in vitro. Our study also demonstrates that ovarian stromal cells may play roles in tissue remodeling during follicle growth, as reported for thecal and granulosa cells [4–8, 31].
In this study, gelatinases and TIMPs were identified in conditioned medium of EOSC simultaneously. Such a phenomenon is demonstrated in other ovarian cell types. In the ovary, theca cells, granulosa cells, and corpora lutea synthesize both gelatinases and TIMPs simultaneously [4,5,31]. It is believed that coexpression of MMPs and TIMPs is required to maintain proteolytic homeostasis and provide localized control of ECM degradation in the ovary [32].
TGFß is thought to be an intraovarian factor involved in follicle development. Studies demonstrate that TGFß is present in human ovarian stromal, granulosa, and theca cells [33], and porcine theca cells [34] during follicle growth. Evidence shows that TGFß may act on the target cells in an autocrine/paracrine manner [34, 35]. On the other hand, TGFß is also involved in ECM remodeling in other tissues [36, 37]. Our study suggests that TGFß may play a role in ECM remodeling during equine follicle growth by regulating MMPs and TIMPs.
In general, TGFß is considered to increase accumulation of ECM by down-regulating gelatinase expression and/or by stimulating TIMPs [2]. However, different effects of TGFß on gelatinases or TIMPs have been reported. Several studies show that TGFß stimulated the production of both gelatinases A and B [38, 39], and one study showed that TGFß increased production of mRNA and protein for gelatinase A and TIMP-1 in cultured human glomerular mesangial cells [36]. Therefore, it seems that TGFß, in certain circumstances, may accelerate ECM remodeling by stimulating both MMPs and TIMPs. During follicle maturation, acceleration of ECM remodeling in stromal tissue would facilitate follicle expansion.
Graham and coworkers [40] demonstrated that TGFß was able to up-regulate levels of TIMP-2 mRNA in normal human trophoblasts. Other investigators reported that TGFß had inhibitory or no effects on TIMP-2 mRNA expression in different systems [41]. In primary bovine and human articular chondrocytes, TGFß increased TIMP-3 mRNA in a dose-dependent manner that required message transcription and de novo protein synthesis [42]. In the present study, TGFß failed to stimulate increased TIMP-3 activity in ECM produced by EOSC.
Phorbol ester PMA has been used to examine regulatory mechanisms in many studies of proteolytic enzymes, although it is a nonphysiological factor. It has been shown that PMA can induce the production of gelatinase B through a 12-O-tetradecanoylphorbol-13-acetate-responsive element in the promoter region of the gelatinase B gene [43] and can activate gelatinase A through protein kinase C-dependent mechanisms [43–46]. In the present study, PMA led to increased levels of progelatinase B and of a form of lower molecular size (86 kDa), which represents activated gelatinase B. The induction and activation of progelatinase A was also observed in this study. Since the active gelatinase A at 68 kDa could not be separated from progelatinase A at 72 kDa by gelatin zymography in our study using 7.5% acrylamide gels, it was not possible to quantify the relative levels. Thus, in our study, PMA not only stimulated production but also activated both progelatinases A and B. The reason for this is not clear. However, similar to that in other cell types, MMP production by EOSC involves protein kinase C-dependent mechanisms, and the equine gene most likely contains a 12-O-tetradecanoylphorbol-13-acetate-responsive element in its promoter region [43–45]. Phorbol ester PMA also induces the mRNA expression and protein synthesis of TIMP-1 and TIMP-2 [37, 44], but PMA is not reported to affect TIMP-3 at the mRNA and protein level in these studies. Our results are consistent with these previous reports.
In conclusion, cultured EOSC are able to synthesize gelatinases and TIMPs, and their activity is regulated by TGFß and PMA. This suggests that stromal cells in the ovary may actively take part in the ECM remodeling during follicle growth and ovulation in mares. Further studies are required to elucidate whether EOSC are also involved in matrix remodeling in vivo and whether this activity can be regulated by hormones and other growth factors involved in follicle growth.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 519 767 1450; bcoomber{at}uoguelph.ca
Accepted: August 17, 1998.
Received: March 9, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-SM actin) in normal human ovaries, in ovarian stromal hyperplasia and in ovarian neoplasms. Arch B Cell Pathol 1989; 57:55–61.This article has been cited by other articles:
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  D. Rosairo, I. Kuyznierewicz, J. Findlay, and A. Drummond Transforming growth factor-{beta}: its role in ovarian follicle development Reproduction, December 1, 2008; 136(6): 799 - 809. [Abstract] [Full Text] [PDF]
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 A. E. Starr, T. Dan, K. Minhas, P. E. Shewen, and B. L. Coomber Potential Involvement of Gelatinases and Their Inhibitors in Mannheimia haemolytica Pneumonia in Cattle Infect. Immun., August 1, 2004; 72(8): 4393 - 4400. [Abstract] [Full Text] [PDF]
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 F.-C. Ke, L.-C. Chuang, M.-T. Lee, Y. J. Chen, S.-W. Lin, P. S. Wang, D. M. Stocco, and J.-J. Hwang The Modulatory Role of Transforming Growth Factor {beta}1 and Androstenedione on Follicle-Stimulating Hormone-Induced Gelatinase Secretion and Steroidogenesis in Rat Granulosa Cells Biol Reprod, May 1, 2004; 70(5): 1292 - 1298. [Abstract] [Full Text] [PDF]
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 T. E. Curry Jr. and K. G. Osteen The Matrix Metalloproteinase System: Changes, Regulation, and Impact throughout the Ovarian and Uterine Reproductive Cycle Endocr. Rev., August 1, 2003; 24(4): 428 - 465. [Abstract] [Full Text] [PDF]
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T. E. Curry Jr., L. Song, and S. E. Wheeler Cellular Localization of Gelatinases and Tissue Inhibitors of Metalloproteinases During Follicular Growth, Ovulation, and Early Luteal Formation in the Rat Biol Reprod, September 1, 2001; 65(3): 855 - 865. [Abstract] [Full Text] [PDF]
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T. E. Curry Jr and K. G. Osteen Cyclic Changes in the Matrix Metalloproteinase System in the Ovary and Uterus Biol Reprod, May 1, 2001; 64(5): 1285 - 1296. [Abstract] [Full Text]
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 G Manandhar, R. Moreno, C Simerly, K Toshimori, and G Schatten Contractile apparatus of the normal and abortive cytokinetic cells during mouse male meiosis J. Cell Sci., January 12, 2000; 113(23): 4275 - 4286. [Abstract] [PDF]
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