BIOLOGY OF REPRODUCTION 61, 1309–1316 (1999)
Collagenase and Gelatinase Messenger Ribonucleic Acid Expression and Activity During Follicular Development in the Rat Ovary 1 Robert G. Cooke III,3,4 Warren B. Nothnick,4 Carolyn Komar,4 Patrick Burns,5 and Thomas E. Curry, Jr.2,4 Department of Obstetrics and Gynecology4 and the Department of Animal Science,5 University of Kentucky, Lexington, Kentucky 40536-0293 ABSTRACT Metalloproteinases are members of a family of proteinases that remodel the extracellular matrix throughout the body. To test the hypothesis that metalloproteinases are regulated by gonadotropin-induced changes during follicular growth, rats were injected with eCG (20 IU, s.c.), and ovaries and serum were collected at the time of eCG administration (0 h) and at 6, 12, 24, 36, or 48 h later for analysis of metalloproteinase mRNA expression, metalloproteinase activity, and steroidogenesis. Serum estradiol levels increased from 18.9 pg/ml at 0 h to 503.8 pg/ml at 48 h. Analysis of mRNA expression was performed for collagenase-3, 72-kDa gelatinase, and 92-kDa gelatinase (n 5 3–4). For collagenase-3, eCG stimulated a 32-fold increase in collagenase-3 mRNA at 48 h after eCG injection as compared to that in ovaries collected at the time of eCG administration (i.e., 0-h control). The mRNA levels for 72-kDa gelatinase were 2.8-fold compared to 0 h at 36 h after eCG treatment and returned to control levels by 48 h after gonadotropin treatment. Levels of the 92-kDa mRNA expression peaked at 24 h (4.2-fold compared to 0 h) and returned to control levels by 36 h. Gel zymography revealed 3 gelatinolytic bands corresponding to the gelatinases of approximately 72 kDa, 92 kDa, and 105 kDa. Analysis of metalloproteinase activity as the degradation of collagen or gelatin per ovary showed an increase in gelatinolytic and collagenolytic activity between 12 and 48 h after eCG treatment. In summary, these findings demonstrate that the gonadotropin induction of folliculogenesis results in changes in the metalloproteinases that may be responsible for extracellular matrix remodeling associated with follicular growth.
INTRODUCTION
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes involved in connective tissue remodeling throughout the body. The MMP family consists of over 20 structurally related enzymes and includes 4 major classes: the collagenases, the gelatinases, the stromelysins, and the membrane-associated metalloproteinases [1–3]. Collagenases predominantly cleave fibrillar collagen, altering the solubility and stability of the collagen molecule and making it susceptible to further degradation by other proteinases, including the gelatinases. There are two forms of gelatinase, a 72-kDa enzyme and a 92-kDa enzyme, referred to as gelatinase A or MMP-2 and gelatinase B or MMP-9, respectively. Both of the gelatinases are capable of degrading collagen fragments and other important components of basement membranes such as type IV collagen, laminin, and fibronectin [1, 2]. The stromelysins act on a wide vaAccepted July 12, 1999. Received February 18, 1999. 1 This work was supported by NIH HD23195. 2 Correspondence: Thomas E. Curry, Jr., Department of Obstetrics and Gynecology, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536-0293. FAX: 606 323 1931; e-mail:
[email protected] 3 Current address: Robert Cooke, Eastover OB-GYN Associates, 1822 Brunswick Ave., Charlotte, NC 27208.
riety of substrates and degrade proteins such as gelatin, proteoglycan, laminin, and fibronectin [1, 2]. Metalloproteinases in the newest class, known as the membrane-type metalloproteinases, are bound to the cell membrane and act to regulate proteolysis as well as inhibitor action at the cell surface [2]. In the ovary, it has been postulated that MMPs play a critical role in extracellular matrix remodeling associated with ovulation as well as the development and regression of the corpus luteum. During follicular rupture, administration of hCG in the periovulatory rat induces an increase in ovarian collagenase and gelatinase expression as well as activity [4–9]. It is hypothesized that the increase in MMPs leads to the enzymatic degradation of the apical follicular connective tissue, allowing follicular rupture and oocyte release [7–9]. Such a hypothesis is supported by the findings that administration of synthetic metalloproteinase inhibitors results in an inhibition of ovulation [10, 11]. After ovulation, the development of the corpus luteum is characterized by tissue reorganization with an ingrowth of fibroblasts and theca blood vessels together with cellular differentiation of granulosa and theca cells from the ruptured antral follicle. The role of metalloproteinases in extracellular matrix remodeling during luteal formation has recently begun to receive attention. Nothnick and colleagues demonstrated that collagenase and gelatinase activity remain elevated following ovulation during development of the corpus luteum in the rat [12]. In cattle, gelatinase activity is increased during luteal formation [13] and during the transition of granulosa cells to luteal cells [14]. During luteal regression, there is an increase in the activity of the 72-kDa gelatinase [15] as well as an increase in ovarian collagenase mRNA expression [12]. Thus, evidence exists to support the postulate that MMPs play an integral role in the development and regression of the corpus luteum. During folliculogenesis, extensive cellular proliferation and angiogenesis occur that are associated with growth of the primordial follicle to the graafian follicle [16, 17]. We hypothesized that MMPs would be involved in the extracellular matrix remodeling that occurs during follicular growth. In support of this postulate is the observation that mRNA expression levels for the tissue inhibitors of metalloproteinases (TIMPs), a family of related proteins that inhibit the activity of metalloproteinases, change in association with gonadotropin-induced follicular development [18]. To test our hypothesis, we induced follicular development in immature rats and measured collagenase and gelatinase mRNA expression and activity at varying time points during follicular development. MATERIALS AND METHODS Materials Equine CG was purchased from Sigma Chemical Co. (St. Louis, MO). Nylon membranes (Nytran) for Northern
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analysis were obtained from Schleicher and Schuell (Keene, NH); [a-32P]dCTP was acquired from New England Nuclear (Boston, MA). Coat-A-Count kits for RIA of estradiol were purchased from Diagnostic Products (Los Angeles, CA). Animals
Immature Sprague-Dawley female rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and kept in environmentally controlled conditions under the supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee. Rats were maintained on a 14L:10D cycle and provided water and rat chow ad libitum. Between 0900 and 1000 h on Day 23, rats were injected s.c. with 20 IU of eCG to induce follicular development. The animals were killed at the time of eCG administration (0 h) and 6, 12, 24, 36, or 48 h after injection of eCG. After death, the ovaries were removed, cleaned, weighed, and snap frozen for measurement of either collagenase and gelatinase mRNA expression, gelatinase activity, or prostaglandin F2a (PGF2a) concentrations as described below. Serum was collected for measurement of estradiol by RIA. Northern Analysis
RNA was isolated from the ovarian samples by the method of Chomczynski and Sacchi [19] using an acid guanidinium thiocyanate-phenol-chloroform extraction procedure as routinely performed in our laboratory [12]. RNA samples (20 mg/lane) were electrophoresed through a 1.0% agarose gel containing 2.2 M formaldehyde and were transferred to a nylon membrane (Nytran). Complementary DNA probes for murine 72-kDa gelatinase, murine 92-kDa gelatinase (supplied by Dr. Dylan Edwards, University of East Anglia, Norwich, England), and rat 18S rRNA were prepared by isolation of excised fragments and randomprimer labeling to a specific activity of 1.0 3 109 cpm using [a-32P]dCTP. The membranes were hybridized overnight at 428C in accordance with the recommendations of the manufacturer. Membranes were hybridized with the various gelatinase cDNAs, allowed to decay, and then hybridized with a cDNA probe for the 18S rRNA. The resulting blots were visualized by autoradiography and analyzed with a laser densitometer to calculate the relative mRNA content. Gelatinase expression was normalized to the 18S ribosomal RNA to account for slight differences in sample loading or transfer to the Nytran membrane. RNase Protection Assay
Levels of mRNA for rat collagenase-3 were quantitated by an RNase protection assay according to Ambion’s RPAII kit instructions (Ambion, Austin, TX). Collagenase-3 mRNA was determined by an RNase protection assay, rather than by Northern analysis as for the gelatinases, because of low levels of abundance of this MMP. The cDNA probes for rat collagenase-3 (MMP-13, supplied by Dr. L Matrisian, Vanderbilt University, Nashville, TN) and the ribosomal protein L32 (supplied by Dr. O.-K. Park-Sarge, University of Kentucky, Lexington, KY) were linearized, and 32P-labeled antisense probes were transcribed using a Maxiscript kit (Ambion). Total RNA (6 mg) from each of the different time points was analyzed. Protected fragments were visualized by electrophoresis through a 5% acrylam-
ide/8 M urea gel. Relative levels of mRNA for collagenase3 were calculated by normalizing the intensity of the protected collagenase fragment to that of the L32 internal control using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Total RNA from periovulatory ovaries collected at 12 h after hCG administration were used as a positive control. Collagenase and Gelatinase Assays
Collagenase and gelatinase were extracted as previously described for ovarian metalloproteinases [5, 7]. Ovaries were homogenized in 10 volumes of 10 mM CaCl2 with 0.25% Triton X-100 and centrifuged at 9000 3 g for 30 min at 48C. The supernatant (i.e., Triton extract) was saved and assayed as described below. The pellet was resuspended in 10 volumes of high-calcium Tris buffer (50 mM TrisHCl, 100 mM CaCl2, 0.15 M NaCl, pH 7.4). The suspension was heated at 608C for 6 min and centrifuged at 27 000 3 g for 30 min at 48C. Extraction with high calcium and heating was used to dissociate metalloproteinases bound to substrate [5, 7]. The supernatant (i.e., heat extract) was saved and processed as described below. The Triton extract contained enzymes that were not bound to substrate, whereas the heat extract contained MMPs that were bound to endogenous substrate in vivo. The protein concentration of the Triton and heat extracts was determined by the method of Bradford [20]. Before being assayed for enzyme activity, aliquots of the Triton and heat extracts were reduced and alkylated to inactivate metalloproteinase inhibitors [7]. The Triton and heat extracts with and without reduction and alkylation were dialyzed overnight against assay buffer (50 mM TrisHCl, 10 mM CaCl2, 0.15 M NaCl, 0.05% polyoxyethylene ether [Brij 35], 0.02% NaN3, pH 7.5) before determination of collagenase and gelatinase activity. Collagenase and gelatinase activity was determined by measuring the digestion of tritium-labeled type I collagen or gelatin, respectively, after incubation with the ovarian sample as previously described [5, 7]. Briefly, aliquots of the ovarian extracts (100 ml) were incubated with the 3Hlabeled substrate for 18 h at 378C. Assays were performed in the presence of 1 mM aminophenylmercuric acetate (APMA) to examine the total enzyme activity (i.e., latent and active forms). APMA is an organomercurial compound that is widely used to activate precursor or inactive forms of metalloproteinases [21]. At the end of the incubation period, the intact labeled collagen or gelatin was pelleted and separated from the degradation fragments by trichloroacetic acid precipitation and centrifugation (13 600 3 g, 5 min). A 100-ml aliquot of the supernatant was placed in liquid scintillation vials containing Aquasol scintillant (New England Nuclear-Dupont, Boston, MA) and counted. The enzyme activity is reported as counts per minute (cpm) of radiolabeled substrate digested per milligram of tissue, which was calculated as ([cpm in ovarian fraction] 2 [cpm in buffer blank]) where the buffer blank represents 100 ml of the assay buffer run in a manner identical to that for the ovarian extracts, or activity was expressed as the cpm of radiolabeled substrate digested per ovary. Gel Zymography
Gel zymography was performed to qualitatively identify the types and relative abundance of enzymes responsible for the measured gelatinolytic activity [7]. Ovarian extracts
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MMPs AND FOLLICULAR DEVELOPMENT TABLE 1. Changes in ovarian weight, serum estradiol, and ovarian PGF2a concentrations following eCG administration.
Treatment
Ovarian weight (mg) (n 5 9)
0 6 12 24 36 48
7.9 9.2 11.9 16.2 23.9 30.1
h (vehicle) h h h h h
6 6 6 6 6 6
0.5 0.6 0.6 2.0* 1.9* 1.9*
Estradiol (pg/mL) (n 5 5) 18.9 12.6 15.5 99.9 177.0 503.8
6 6 6 6 6 6
PGF2a (pg/mg tissue) (n 5 3)
3.9 1.0 1.7 14.2 15.1* 34.2*
110 200 153 150 140 113
6 6 6 6 6 6
17 35 22 32 36 20
PGF2a (pg/ovary) (n 5 3) 913 1960 1760 2505 3248 3243
6 6 6 6 6 6
131 322 236 392* 535* 478*
* Significantly different from 0 h vehicle treated rats, P , 0.05. Values represent the mean 6 SEM for 3 separate experiments in duplicate (PGF2a concentrations), 5 separate experiments in duplicate (estradiol concentrations), or 9 separate experiments (ovarian weight).
from the varying time points were electrophoresed in 10% polyacrylamide gels containing 1 mg/ml of gelatin. Extracts containing 20 mg of protein were lyophilized as necessary and resuspended in 30 ml of Laemmli sample buffer lacking b-mercaptoethanol. SDS-PAGE was carried out at 15 mAmp per slab gel for 5–8 h under nonreducing conditions. After electrophoresis, SDS was eluted from the gels in 2 changes of 2% Triton X-100 for a total of 45–60 min at 378C. Gels were incubated overnight at 378C in substrate buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 8.0) and subsequently stained with Coomassie Blue R250. Gelatin-degrading enzymes were qualitatively identified by their ability to digest the gel. Rainbow molecular weight markers (Amersham, Arlington Heights, IL; now Amersham Pharmacia Biotech, Piscataway, NJ) and a positive control, conditioned medium from HT1080 cells [22], were used to allow final molecular weight estimation of the bands resolved by gel zymography. Estradiol and PGF2a RIA
Serum estradiol-17b levels were determined by RIA with Coat-A-Count kits (Diagnostic Products). The CoatA-Count kits are direct, solid-phase 125I RIA kits. Serum (100 ml) and 1.0 ml of 125I-labeled estradiol were added to the precoated antibody tubes and incubated at room temperature for 3 h. Samples were decanted and then counted with a gamma counter, and estradiol concentration was determined. The sensitivity of the assay was 6 pg/ml. Intraand interassay coefficients of variation were 5.4% and 9.7%, respectively. For determination of ovarian PGF2a concentrations, whole ovaries were homogenized in 0.5 ml of homogenization buffer (saline containing 1026 M indomethacin), acidified with formic acid, and extracted with ethyl acetate. Extracts were reconstituted in 1 ml of 0.01 M PBS containing 0.1% gelatin, diluted 1:20, and assayed for PGF2a by RIA as described previously for the ewe [23] and previously validated in our laboratory for rat ovarian tissue [24]. Intra- and interassay coefficients of variation were 8.8% and 15.5%, respectively. All samples were run in duplicate, and the sensitivity of the assay was 10 pg/tube. Experimental Design
Each experiment was designed to test the changes in ovarian PGF2a content, serum steroid concentrations, enzyme activity, and mRNA levels for the MMPs during follicular growth. To normalize the variability between different experimental replicates for the mRNA quantitation, data are expressed as the ratio of the metalloproteinase mRNA to an internal control (18S or L32). Each experimental replicate represents tissue generated from a separate group of
rats collected at the identified time points. For the Northern blot analyses and RNase protection assays, one animal (i.e., 2 ovaries) was used for each time point. For the enzyme activity assays, ovaries were pooled from several animals to yield approximately 100 mg of tissue for each time point. Thus for each time point, the ovaries were pooled from approximately the following number of animals to yield a n 5 1: 0 h 5 6 animals, 6 h 5 5 animals, 12 h 5 4 animals, 24 h 5 3 animals, 36 h and 48 h 5 2 animals. Differences in ovarian PGF2a content, serum steroid concentrations, enzyme activity, and mRNA levels between different times were tested with a one-way ANOVA. Student-NeumanKeuls procedure was used for post hoc group comparisons. A P value , 0.05 was considered significant. RESULTS
Hormonal Simulation of Ovarian Follicular Development
There was an increase in both ovarian weight and serum estradiol after eCG administration (Table 1), confirming hormonal induction of follicular growth. Administration of eCG stimulated approximately a 3.8-fold increase in ovarian weight and a 26-fold increase in serum estradiol at 48 h after gonadotropin treatment. Administration of eCG did not change ovarian levels of PGF2a when expressed as PGF2a/mg tissue at any of the time points examined but did increase the overall ovarian content of PGF2a 24 h after gonadotropin stimulation (Table 1). Collagenase mRNA Expression and Activity During Follicular Development
RNase protection assay for collagenase-3 mRNA demonstrated a protected fragment of the expected size of 273 base pairs (bp). There was no change in collagenase-3 mRNA expression within the first 24 h after eCG administration (Fig. 1, A and B). At 36 h after eCG treatment, collagenase-3 mRNA increased to 4.4-fold compared to the 0-h value to reach maximal levels at 48 h (32-fold induction compared to 0 h). Collagenase activity was determined by a highly sensitive assay that quantitates the breakdown of 3H-labeled type I collagen [5]. Triton extracts contained basal collagenase activity irrespective of whether or not the extract was reduced or alkylated to remove metalloproteinase inhibitors (data not shown). Collagenase activity in heat extracts that were not treated (no reduction and alkylation) or extracts that were reduced and alkylated was unchanged throughout follicular development when expressed as counts of 3Hlabeled collagen released per milligram of tissue (Fig. 2). However, when expressed as counts of 3H-labeled collagen released per ovary, there was an increase in collagenase
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FIG. 1. Changes in collagenase-3 mRNA during follicular development in the rat. Ovaries were collected at varying time points after eCG treatment for determination of mRNA expression by RNase protection assay as described in Materials and Methods. A) Detection of the protected fragment of collagenase-3 and the ribosomal protein L32. The depicted blot is a representation of 2 separate sets of samples. B) The relative changes in collagenase-3 mRNA expression normalized to the L32 band during follicular development. Data are expressed as the mean 6 SEM for a total of 4 experiments. Asterisks indicate significant differences from the 0-h time point (P , 0.05).
FIG. 3. Northern analysis of 72-kDa gelatinase mRNA during follicular development in the rat. Ovaries were collected at varying time points after eCG, and total RNA was isolated and analyzed for 72-kDa gelatinase mRNA content by Northern analysis as described in Materials and Methods. A) Detection of a transcript of approximately 3.1 kb, consistent with the transcript size of 72-kDa gelatinase, and the hybridization of the constitutively expressed 18S ribosomal transcript (approximately 1.9 kb) in 3 separate sets of samples (n 5 3). B) The relative changes in 72-kDa gelatinase expression normalized to the 18S band during follicular development. Data are expressed as the mean 6 SEM for a total of 3 experiments (n 5 3), and the depicted blot represents all of the samples analyzed. Asterisks indicate significant differences from the 0-h time point (P , 0.05).
activity at 48 h after eCG treatment in heat extracts that were reduced and alkylated (Fig. 2). Gelatinase mRNA Expression and Activity During Follicular Development
FIG. 2. Collagenase activity in extracts of ovaries collected during follicular development. Ovaries were collected at varying time points after eCG treatment, and collagenase activity was assessed in ovarian extracts as described in Materials and Methods. Data are presented as the counts of 3H-labeled collagen released (cpm) per milligram of tissue in extracts with no treatment or after reduction and alkylation (RA, left ordinate), or as the counts of 3H-labeled gelatin released per ovary in extracts with no treatment or after reduction and alkylation (right ordinate). Data represent the mean 6 SEM for a total of 3 experiments performed in duplicate. Asterisks indicate significant differences from the 0-h time point (P , 0.05).
Northern analysis of the 72-kDa and 92-kDa gelatinases revealed a single transcript of approximately 3.1 kilobases (kb) and 2.8 kb in size, respectively, throughout follicular development (Figs. 3A and 4A). The 72-kDa mRNA peaked at 36 h (2.8-fold induction compared to the control, i.e., 0 h) and had returned to control levels by 48 h (Fig. 3, A and B). The levels of the 92-kDa mRNA peaked at 24 h (4.2-fold induction compared to 0 h) and had returned to control levels by 36 h (Fig. 4, A and B). Gelatinase activity was determined by a highly sensitive assay that quantitates the breakdown of 3H-labeled gelatin [7]. Triton extracts contained basal gelatinase activity irrespective of whether or not the extract was reduced or alkylated to remove metalloproteinase inhibitors (data not shown). Heat extracts that were not reduced or alkylated showed no changes in gelatinase activity throughout follicular development when expressed as counts of 3H-labeled
MMPs AND FOLLICULAR DEVELOPMENT
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FIG. 5. Gelatinase activity in extracts of ovaries collected during follicular development. Ovaries were collected at varying time points after eCG treatment, and gelatinase activity was assessed in ovarian extracts as described in Materials and Methods. Data are presented as the counts of 3H-labeled gelatin released (cpm) per milligram of tissue in extracts with no treatment or after reduction and alkylation (RA), or as the counts of 3H-labeled gelatin released per ovary in extracts with no treatment or after reduction and alkylation. Data represent the mean 6 SEM for a total of 3 experiments performed in duplicate. Asterisks indicate significant differences from the 0-h time point (P , 0.05).
FIG. 4. Northern analysis of 92-kDa gelatinase mRNA during follicular development in the rat. Ovaries were collected at defined time points after eCG administration; total RNA was isolated, and Northern analysis for the 92-kDa gelatinase mRNA was performed. A) The detection of transcripts for the 92-kDa gelatinase (approximately 2.9 kb) and the 18S ribosomal protein. B) The relative changes in 92-kDa gelatinase expression normalized to the 18S band during follicular development. Data are expressed as the mean 6 SEM for a total of 3 experiments (n 5 3), and the depicted blot is a representation. Asterisks indicate significant differences from the 0-h time point (P , 0.05).
gelatin released per milligram of tissue (Fig. 5). However, reduced and alkylated heat extracts exhibited an approximate doubling of gelatinolytic activity within 12 h after eCG treatment before returning to pre-eCG levels (i.e., 0h values, Fig. 5). When gelatinase activity was analyzed as counts of H3-labeled gelatin released per ovary, there was an increase in activity at 24 h after eCG in extracts that were reduced and alkylated, and the activity remained elevated through 48 h after gonadotropin treatment (Fig. 5). Gel zymography was performed to qualitatively identify the gelatinases present in the ovary. Triton and heat extracts contained activity corresponding to gelatinases of 72 kDa, 92 kDa, and 105 kDa, with the 72-kDa gelatinolytic band being the most prominent (Fig. 6). In extracts activated with APMA there was a corresponding shift in the molecular size of the 72-kDa gelatinase to a predominant zone of gelatinolytic activity at approximately 62 kDa (Fig. 6), which is consistent with the activated form of the enzyme [1]. The 105-kDa gelatinase has been described in the rodent as being highly homologous to the human 92-kDa gelatinase [25].
FIG. 6. Representative gelatin zymogram from the Triton and heat extracts of ovaries collected during follicular development. Gel zymography was performed as described in Materials and Methods, and samples were treated as follows: no treatment, Triton extract (A); reduced and alkylated, heat extract (B); or reduced and alkylated and incubated with APMA to activate the enzymes, heat extract (B). Gelatinolysis is indicated by the white zones of degradation in the gelatin matrix. Numbers on the right correspond to molecular size of the gelatinolytic region. HT1080: fibrosarcoma cell line, 1 control.
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DISCUSSION
Follicular growth requires extensive cellular proliferation and remodeling of the extracellular matrix as the follicle differentiates from a primordial follicle to a large graafian follicle [16, 17]. Follicular maturation begins with proliferation of the granulosa cells, ultimately resulting in a 20-fold increase in the volume of granulosa cells during follicular growth in the rat. The granulosa cells secrete a mucopolysaccharide-rich fluid that coalesces to form the antral cavity in the secondary, tertiary, and graafian follicles. During follicular development, the theca cells differentiate from the ovarian stroma and are separated from the granulosa cells by a basement membrane. Unlike the granulosa cells, the theca layer is well vascularized and contains circumferential collagen bundles. The resultant mature follicle is approximately 400-fold larger than the initial primordial follicle and rests in an extracellular environment of collagen, laminin, and fibronectin [16, 17, 26]. The marked increase in cellular proliferation and accumulation of follicular fluid results in an increase in ovarian weight as noted in the present study. Changes in steroidogenesis during differentiation of the follicle result in high circulating levels of estradiol. In our experiment, estradiol increased approximately 25-fold and peaked at 503 pg/ml within 48 h after eCG administration, validating hormonal induction of follicular development. The present findings demonstrate that gonadotropin induction of folliculogenesis results in stimulation of the metalloproteinases concomitant with the changes in extracellular matrix remodeling associated with follicular growth. Specifically, both collagenase and gelatinase mRNA levels were elevated during the time period when follicular growth and expansion occur, although variations in the pattern of mRNA expression were observed. For collagenase, the increase in mRNA expression occurs 48 h after eCG treatment. For the gelatinases, the increase in the 72-kDa gelatinase mRNA and the 92-kDa gelatinase mRNA occurs earlier. This period from 24 to 48 h represents a time of marked follicular growth as reflected by the massive increase in the number of granulosa cells, the concomitant increase in estradiol production, the presence of developing preovulatory follicles, and the rapid rise in ovarian weight as the follicles grow. We hypothesized that follicular growth would require remodeling of the ovarian extracellular matrix, and the data presented support a role for collagenase and the gelatinases in these follicular connective tissue changes. Further support for the metalloproteinases in follicular growth is forthcoming from immunohistochemical observations of the 72-kDa gelatinase in granulosa, theca, and interstitial cells, as well as the presence of the 92-kDa gelatinase in theca cells and interstitial tissue at 48 h after eCG administration [27]. Similarly, collagenase activity has been noted to increase with increasing follicular size in goats, which Garcia and colleagues [28] proposed would regulate normal follicular maturation and atresia to achieve the appropriate number of ovulatory follicles. In the present study, expression of mRNA for collagenase-3, 72-kDa gelatinase, and the 92-kDa gelatinase increased after eCG injection. There was, however, a discordance between the metalloproteinase mRNA expression and the corresponding metalloproteinase activity. For example, the marked induction of collagenase-3 mRNA at 48 h after eCG administration was not accompanied by a change in collagenase activity when normalized to activity
per milligram of tissue. Similarly, there was an increase in mRNA expression of the 72-kDa and 92-kDa gelatinases without a concomitant increase in gelatinolytic activity per milligram of tissue. This disparity between enzyme mRNA expression and enzyme activity may result from the intricate regulation of metalloproteinases that is rigorously controlled at multiple levels including 1) transcriptional regulation of metalloproteinase mRNA, 2) translation of synthesized mRNA into latent enzyme, 3) secretion of latent metalloproteinases, 4) activation of latent enzymes, and 5) inhibition of enzyme activity in the extracellular matrix by metalloproteinase inhibitors. To circumvent the potential problem of quantitating latent enzyme activity, activity in the current study was determined in extracts from intact ovaries that had been treated with APMA to activate the latent collagenase or gelatinase. The possibility exists that during the extraction and isolation of the metalloproteinases, enzymes are exposed to substrate or metalloproteinase inhibitors, either of which would complex with the enzyme and inhibit enzymatic degradation of the labeled substrates used in the present assays. Although the ovarian extracts were treated to inactivate metalloproteinase inhibitors, the possibility exists that not all of the enzyme activity was recovered following reduction and alkylation. Another discordance observed in the present study was the findings with collagenase-3 mRNA. Previous reports have noted an absence of collagenase-3 mRNA expression in ovaries collected at the time of hCG administration as well as up to 72 h after hCG treatment by Northern blot analysis [29]. In the present study, collagenase-3 mRNA was present at 48 h after eCG, corresponding to the 0-h hCG time point of Balbin and colleagues [29]. We have experienced difficulty in consistently detecting collagenase-3 mRNA by Northern analysis; this was the rationale for using the more sensitive RNase protection assay. The difference between the two studies, therefore, may relate to the different means of mRNA detection. For the metalloproteinase inhibitors, two major classes are generally distinguished: serum borne and tissue derived (reviewed in [1, 2]). The serum-borne inhibitors include the macroglobulins, such as a2-macroglobulin, which have broad proteolytic susceptibility and are present in the ovary [1, 3, 30–32]. The second group of inhibitors are locally produced, specifically inhibit MMPs, and are referred to as tissue inhibitors of metalloproteinases or TIMPs [1–3, 32]. The mRNA expression for one of these inhibitors, TIMP1, is increased within 6 h after an eCG stimulus [18], implicating the TIMPs in the gonadotropin-induced changes associated with follicular growth. The increase in TIMP-1 mRNA could result in an increase in the inhibitor in the extracellular space to control the extent and location of metalloproteinase action during remodeling of the follicle. Homogenization of the ovary may allow TIMPs in one ovarian compartment to interact with metalloproteinases produced in a separate compartment. To address this question, current studies are exploring the cellular localization of enzyme and inhibitor activity during follicular growth. The stimulus for collagenase and gelatinase mRNA expression during follicular development is uncertain. The peak mRNA expression of 72-kDa and 92-kDa gelatinase occurs at 24 and 36 h, respectively, while collagenase-3 mRNA reaches maximal expression at 48 h after eCG. This increase in metalloproteinase mRNA expression occurs 24– 48 h after the initial eCG stimulus, yet such a time frame for FSH action is not unprecedented. For example, phosphorylation of FSH-induced messenger systems in granu-
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losa cells such as cAMP response element binding protein takes place 18–48 h after eCG treatment [33], and induction of granulosa cell a inhibin mRNA is observed at 48 h after addition of FSH [34]. Alternatively, the increases in gelatinase and collagenase mRNA expression are concordant with the initial rise in serum estradiol. This suggests a possible stimulatory role for estradiol, which has been reported to regulate metalloproteinases in other tissues, in the regulation of follicular metalloproteinase mRNA expression. For example, dilatation of the cervix at parturition is mediated by an estrogen-induced degradation of type I collagen by collagenase [35]. Similarly, estrogen treatment of human endometrial tissue in organ culture maintained secretion of metalloproteinases [36]. There is, however, limited information regarding estradiol regulation of the ovarian MMP system. Puistola and coworkers [37] reported that addition of estradiol to human granulosa-luteal cell cultures increased the content as well as activity of the 72-kDa gelatinase in conditioned culture medium. In light of these previous findings that estradiol is able to modulate MMPs in reproductive tissues, and the present observations that elevated levels of estradiol are temporally associated with an increase in mRNA for collagenase-3 and the gelatinases, the role of estradiol in the regulation of follicular MMPs deserves further consideration. Prostaglandins (PGs) could provide additional regulatory control for metalloproteinase production. In the present study, ovarian PGF2a was examined on the basis of previous reports that gonadotropins induce preovulatory PG production and that these PGs regulate metalloproteinase activity around the time of ovulation. There is a body of literature demonstrating a gonadotropin induction of PGs during the ovulatory process (reviewed in [38]). Although the mechanism(s) by which PGs may regulate follicular rupture is unknown, local PG production may impact ovarian proteolysis associated with apical connective tissue degradation and oocyte extrusion [38]. In support of this postulate are reports that administration of indomethacin, which inhibits PG synthase, results in a diminution of the preovulatory increase in collagenase activity [6, 39]. Present findings that ovarian PGF2a levels do not change following eCG treatment could be interpreted to suggest that PGF2a does not play a role in the increase in gelatinase and collagenase mRNA during follicular growth. In summary, these findings demonstrate changes in the metalloproteinases during folliculogenesis in the rat. Follicular growth requires extensive remodeling of the granulosa and theca components of the follicle as well as the surrounding ovarian stroma. We propose that gonadotropin induction of these enzymes may remodel the extracellular matrix associated with follicular growth and that follicular remodeling requires the coordinated action of MMPs and their inhibitors. ACKNOWLEDGMENTS The authors would like to thank Ms. Sarah Wheeler for her assistance in the preparation of the figures for this manuscript. The provision of the cDNA probes for the murine gelatinases by Dr. Dylan Edwards at University of East Anglia, the rat collagenase-3 by Dr. Lynn Matrisian at Vanderbilt University, and the ribosomal protein L32 by Dr. Park-Sarge at the University of Kentucky is gratefully acknowledged.
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