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BOR - Papers in Press, published online ahead of print December 10, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.023499
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BIOLOGY OF REPRODUCTION 70, 1024–1032 (2004)
DOI: 10.1095/biolreprod.103.023499
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Membrane Type 1-Matrix Metalloproteinase (MMP)-Associated MMP-2 Activation Increases in the Rat Ovary in Response to an Ovulatory Dose of Human Chorionic Gonadotropin1

Misung Jo2, Lauren E. Thomas, Sarah E. Wheeler, and Thomas E. Curry, Jr

Department of Obstetrics and Gynecology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0298


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gonadotropins stimulate ovarian proteolytic enzyme activity that is believed to be important for the remodeling of the follicular extracellular matrix. Membrane type 1-matrix metalloproteinase (MT1-MMP) has been identified in vitro as an activator of pro-MMP-2 by forming a complex with tissue inhibitors of metalloproteinase-2 (TIMP-2). In the present study, the expression pattern of MT1-MMP mRNA and the role of MT1-MMP were examined in the ovary using the gonadotropin-treated immature rat model. Ovaries were collected at selected times after eCG or hCG. RNase protection assays revealed a transient increase in MT1-MMP mRNA beginning 4 h after hCG. High expression of MT1-MMP mRNA was localized to the theca-interstitial layer of developing and preovulatory follicles, while low expression was observed in the granulosa cell layer of developing follicles by in situ hybridization. The localization pattern of MT1-MMP mRNA was compared with TIMP-2 mRNA. Both MMP-2 and TIMP-2 mRNA were expressed in the theca layer of preovulatory follicles, showing a similarity to MT1-MMP mRNA expression. To further determine whether MT1-MMP activates pro-MMP-2 in the ovary, crude plasma membrane fractions from preovulatory ovaries were analyzed by gelatin zymography. In plasma membrane fractions, pro-MMP-2 increased around the time of ovulation. Upon incubation, pro-MMP-2 was activated with the highest levels of activation at 12 h post-hCG. The addition of MT1-MMP antibody or excess TIMP-2 to membrane fractions inhibited pro-MMP-2 activation. The increase in MT1-MMP mRNA may be an important part of the mechanism necessary for the efficient generation of active MMP-2 during the ovulatory process.

follicle, luteinizing hormone, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Throughout each reproductive cycle, the ovary undergoes dynamic morphological changes, which require constant yet extensive tissue remodeling. Matrix metalloproteinases (MMPs) have been suggested to be instrumental in this process of remodeling. MMPs play an essential role in degradation of the extracellular matrix (ECM) and, to date, more than 26 different MMPs have been identified (for review, see [1]). The activity of MMPs is regulated at multiple levels; the production and secretion of latent enzymes, the activation of the latent enzyme, and the inhibition of enzyme activity by specific inhibitors, such as tissue inhibitors of metalloproteinases (TIMPs). The TIMPs can bind to active or latent forms of MMPs in the extracellular space, thereby inhibiting the action of MMPs [2]. A similar paradigm for controlling ovarian MMP activity may exist. For instance, levels of mRNA for several MMPs are upregulated by hormonal signals associated with follicular development, ovulation, or luteal formation and regression [3, 4]. The stimulation of the MMP gene expression results in secretion of latent enzymes, which are converted to an active form in the ovary via proteinases such as plasmin [5, 6]. Additional regulation of the active or latent form of MMPs may occur in the ovarian extracellular space by the TIMPs [3]. Four distinct TIMPs have been identified to date, some of which have been found to be regulated by hormonal signals that parallel the change in expression of MMPs in the ovary [3, 4].

Of the various MMPs found in the ovary, gelatinases (MMP-2 and MMP-9) have been implicated as important players during follicular growth and ovulation because of their ability to degrade major constituents of basement membranes. There is experimental evidence of an increase in mRNA for gelatinases (MMP-2 and MMP-9) and pro-MMP-2 activity following eCG injection to immature rats or administration of hCG to eCG-primed rats [7–9]. The importance of MMP-2 and its activation in the ovulatory process is forthcoming from studies in which injection of bioactivity-neutralizing MMP-2 antibody into preovulatory follicles blocked ovulation in ewes [10] and administration of the leukotriene B4-receptor antagonist, ZK158252, to rat ovaries inhibits hCG-induced ovulation and MMP-2 activation [11]. However, nothing is known about the regulatory mechanisms by which progelatinases are activated in the ovary during these critical periods. Recently, the unique, yet important characteristics of membrane type 1-MMP (MT1-MMP) as a dual player in cell surface proteolysis have been identified. Not only can MT1-MMP cleave a variety of extracellular components, but it can activate pro-MMP-2 on the cell surface in vitro [12], providing an additional level of proteolytic regulation.

The MT1-MMP mediated pro-MMP-2 activation process has been demonstrated in vitro to require both active MT1-MMP and TIMP-2-bound MT1-MMP on the cell membrane [13–15]. In the rat ovary, MT1-MMP mRNA was localized via in situ hybridization to the granulosa and theca-interstitial cells of developing follicles during the hCG-induced preovulatory period [16] and the corpus luteum (CL) during pseudopregnancy [17]. Messenger RNA for both TIMP-2 and MMP-2 were also detected in the theca-interstitial layer of all follicles [16, 18]. Although these observations lead to the postulate that MT1-MMP may act as an endogenous activator of pro-MMP-2 in the ovary, little experimental evidence has been reported so far. In the present study, we hypothesized that MT1-MMP is upregulated in the preovulatory follicles during the preovulatory period, the time when pro-MMP-2 levels are increased, and this upregulation of MT1-MMP induces an increase in pro-MMP-2 activation in the rat ovary. To test this hypothesis, we examined the expression pattern of MT1-MMP mRNA during the gonadotropin-induced preovulatory period. Subsequently, we measured the pro-MMP-2 activity and membrane-associated activation of pro-MMP-2 in ovarian membrane fractions at selected time points during the gonadotropin-induced preovulatory period in the rat ovary.


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

Unless otherwise noted, all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). Molecular biological enzymes, primers, pCRII-TOPO Vector, and Trizol reagent were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA).

Animals

All animal procedures (protocol 00110M2000) were approved by the University of Kentucky Animal Care and Use Committee. Immature female Sprague Dawley rats (15 days old) were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN), and were provided with water and rat chow ad libitum and maintained on a 14L:10D cycle. The rats (21 days old) were injected with 10 IU eCG s.c. to stimulate follicular development. Forty-eight hours later, the rats were injected with 10 IU hCG s.c at 0900 h during the light cycle to induce ovulation and subsequent formation of CL. In this model [19], ovulation occurs approximately 14–16 h post-hCG administration.

To examine the pattern of expression of MT1-MMP mRNA and its role during follicular growth and the preovulatory period, animals were killed at 0, 24, and 48 h post-eCG, or at 4, 8, 12, and 24 h post-hCG administration (n = 3–4 animals per time point per experiment). Ovaries were collected, snap frozen, and stored at -70°C for later extraction of total RNA or preparation of crude plasma membranes. For in situ hybridization analysis, the ovaries were placed in optimal cutting temperature compound (VWR Scientific, Atlanta, GA) and stored at -70°C.

Subsequent luteal changes in the expression pattern of MT1-MMP mRNA were examined in ovaries collected from gonadotropin-treated rats on selected days during pseudopregnancy. Animals were killed on Days 1 and 2 (luteal formation), Days 4 and 8 (luteal maintenance), or Day 14 (luteal regression) after hCG treatment (n = 3 animals/time point). The ovaries were snap frozen and stored at -70°C for later isolation of total RNA or placed in optimal cutting temperature compound and stored at -70°C for in situ hybridization.

Generation of the Plasmid Containing Rat cDNA for MT1-MMP

A 542-base-pair rat cDNA fragment was generated by reverse transcriptase-polymerase chain reaction (RT-PCR). Briefly, total RNA (1 µg) isolated from rat preovulatory ovaries (8 h post-hCG) was reverse-transcribed at 42°C for 1 h using SuperScript II and Oligo dT primers. First strand cDNA samples were amplified using oligonucleotide primer pairs (5'-CATGGCGTCTGAAGAAGAAGA-3,' 5'-CAAGCATTGGGTGTTTGATG-3') based on the previously reported sequence of mouse MT1-MMP cDNA (GenBank accession number NM008608). Amplification consisted of preincubation at 94°C for 2 min before adding Taq polymerase and then 35 cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec. A PCR product of the predicted size was cloned into the pCRII-TOPO Vector. DNA sequences of the cloned rat partial MT1-MMP cDNA were determined using a Standard ABI kit (Macromolecular Structure Analysis Facility, University of Kentucky, KY).

Quantification of mRNA for MT1-MMP

Total RNA was extracted from ovaries collected during follicular development, the preovulatory period, and pseudopregnancy using TRIZOL reagent according to the manufacturer's protocol and quantified by spectrophotometry. Plasmids containing rat cDNAs for MT1-MMP and mouse cDNA for ribosomal protein L32 (kindly provided by Dr. O.K. Park-Sarge, University of Kentucky, Lexington, KY) were linearized with ScaI and EcoRI, respectively. Antisense riboprobes were transcribed using [{alpha}-32P] UTP (10 mCi/ml; DuPont New England Nuclear, Boston, MA) and SP6 or T7 RNA polymerase, respectively.

The RNase protection assays (RPAs) were carried out using Ambion RPA II kit (Ambion Inc., Austin, TX) according to the manufacturer's protocol. Briefly, samples of total RNA (n = 3 individual samples/time point) were hybridized with excess amounts of 32P-radiolabeled antisense riboprobes for rat MT1-MMP and mouse L32 for 12–15 h at 42°C. Radioactivity in each band was quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The relative levels of mRNA for MT1-MMP were normalized to the ribosomal protein L32 by dividing the band intensity of mRNA for MT1-MMP by the band intensity of the corresponding L32 in each sample lane.

Northern blot analyses were carried out as described previously [20]. Briefly, 10–15 µg of total RNA (n = 3 individual samples/time point) were separated by electrophoresis, capillary-transferred to a nylon membrane (0.2 µm pore size, Nytran N; Schleicher and Schuell Inc., Keene, NH) and cross-linked to the membrane by baking in a vacuum oven at 80°C for 2 h. The membrane was hybridized with 1 x 106 cpm 32P-labeled antisense riboprobe first for MT1-MMP mRNA and later for L32 mRNA per ml of NorthernMax hybridization buffer (Ambion Inc.) at 65°C for at least 16 h. Excess probe was washed with a stringent buffer (0.1 x SSC, 0.1% SDS) twice at 65°C for 60 min. The membrane was exposed to a phosphorimaging plate and quantified with a PhosphorImager (Molecular Dynamics). The relative levels of mRNA for MT1-MMP were calculated as described above.

In Situ Hybridization of mRNA for MT1-MMP, TIMP-2, MMP-2, and LH Receptor

Ovaries collected from immature gonadotropin-treated rats were sectioned at 10 µm and mounted on Probe On Plus slides (Fisher Scientific). In situ hybridization was carried out as described previously [18]. Briefly, riboprobes for MT1-MMP, TIMP-2, MMP-2, and LH receptor were synthesized using rat cDNA for MT1-MMP, TIMP-2 (kindly provided by Dr. Kevin Leco, University of Western Ontario, London, ON), MMP-2 (a gift from Dr. Dylan Edwards, University of East Anglia, Norwich, England), and LH receptor (kindly supplied by Dr. Joanne Richards, Baylor University, Houston, TX). Plasmids were linearized using the appropriate restriction enzymes. The antisense and sense riboprobes were synthesized using the corresponding linearized plasmid and labeled with [{alpha}-35S]UTP (10 mCi/ml; DuPont New England Nuclear, Boston, MA) and appropriate RNA polymerases. The sections were hybridized overnight with 1 x 106 cpm 35S-labeled riboprobes/slide in a humidified chamber at 55°C. The next day, the slides were washed and treated with RNase A (0.1 mg/ml) for 30 min at 45°C. Tissue sections were washed again at high stringency, dried, dipped in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), and exposed at 4°C for 3–6 wk. To visualize the hybridized riboprobes, slides were developed with Kodak D19 and counterstained with hematoxylin solution. Tissues were examined with an Eclipse E800 Nikon microscope (Nikon Corp., Melville, NY) under bright- and darkfield optics. One ovary from each of three animals was used for in situ hybridization. At least eight sections/ovary were analyzed for each antisense probe, making a total of at least 24 tissue sections analyzed for each time point. A sense riboprobe, used as a control for nonspecific binding, was included for each ovary and each time point to correspond with the different antisense riboprobes.

Preparation of Plasma Membrane Fractions

Plasma membrane fractions were prepared according to the method described previously [21] with slight modification. Briefly, ovaries were homogenized in 40 mM Tris buffer (pH 7.4) containing 0.25 M sucrose using a Dounce homogenizer at 4°C. Homogenates were filtered through nylon mesh (42 µM; Spectrum Laboratories, Inc., Rancho Dominguez, CA) and centrifuged at 700 x g for 20 min at 4°C. The supernatant was collected and centrifuged again at 100 000 x g for 1 h at 4°C to pellet the crude plasma membrane fractions. The pellet was resuspended in Tris buffer and stored at -70°C. The protein concentration of the plasma membrane fractions was determined using the Bradford method [22].

Gelatin Zymography

Plasma membrane fractions (3 µg protein/lane) were either subjected directly to gelatin zymography or incubated at 4°C or 37°C for 12 h in the absence or presence of mouse anti-human MT1-MMP monoclonal antibody (Ab-4; Oncogene Research Products, Boston, MA), TIMP-2 (human recombinant TIMP-2; Oncogene Research Products), or aminophenylmercuric acetate (APMA) in Tris incubation buffer (50 mM Tris/HCl, 5 mM CaCl2, pH 8.0) and then used for gelatin zymography. Mouse immunoglobulin (Ig) G (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or TIMP-3 (mouse recombinant TIMP-3; Oncogene Research Products) was added to the plasma membrane fractions during the incubation to serve as controls for MT1-MMP antibody or TIMP-2 treatments, respectively. Plasma membrane fractions were diluted with nonreducing sample buffer (final concentration, 1% SDS and 5% glycerol) and electrophoresed in 7.5% polyacrylamide gels containing 1 mg/ml of gelatin at 20 mAmp for 4 h. After electrophoresis, the gel was washed in 2% Triton X-100 for 2 h to elute the SDS, rinsed briefly in Tris incubation buffer (50 mM Tris/HCl, 5 mM CaCl2, pH 8.0), and then incubated for at least 24 h at 37°C in Tris incubation buffer. Subsequently, the gels were stained with Coomassie Brilliant Blue R250 dye. Gelatin-degrading enzymes were identified by their ability to digest the gel. The digested band densities for gelatinases were determined using MetaMorph Image analysis software (Universal Imaging Corp., West Chester, PA).

Statistical Analyses

All data are presented as means ± SEM. One-way analysis of variance (ANOVA) was used to test differences in levels of MT1-MMP mRNA and gelatinase activities across time of tissue collection, or gelatinase activities following the incubation of ovarian membrane fractions with APMA, MT1-MMP antibody, or TIMP-2. If ANOVA revealed significant effects of time of tissue collection or treatment, the means were compared by Tukey test, with P < 0.05 considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Levels of mRNA for MT1-MMP During Follicular Growth, the Preovulatory Period, and Pseudopregnancy

To examine whether the levels of MT1-MMP mRNA are regulated during follicular development, the ovulatory process, and luteal development, total RNA isolated from ovaries of gonadotropin-treated rats at selected time points was analyzed by RPA or Northern blot analysis. RPA revealed a transient increase in the levels of mRNA for MT1-MMP. The level increased at 4 h after hCG injection (about twofold higher at 4 and 8 h post-hCG compared with the values of pre-hCG; Fig. 1, A and B).



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  		FIG. 1. A) Autoradiograph of a representative RNase protection assay demonstrating the protected fragments of mRNA for MT1-MMP and ribosomal protein L32 in gonadotropin-stimulated rat ovaries collected at selected times after eCG or hCG injection. B) Relative levels of mRNA for MT1-MMP in gonadotropin-stimulated rat ovaries collected at selected times after eCG or hCG injection (mean ± SEM; n = 4 animals/time point). Relative levels of MT1-MMP mRNA were normalized to the L32 band in each sample. Bars with no common superscripts are significantly different (P < 0.05). C) Autoradiograph of a representative Northern blot demonstrating the expression of mRNA for MT1-MMP and ribosomal protein L32 in gonadotropin-stimulated rat ovaries collected at selected days of pseudopregnancy

Northern blot revealed a single transcript (~4.2 kilobases) for the MT1-MMP gene in total RNA isolated from ovaries obtained at different stages of luteal development (Fig. 1C). The levels of mRNA for MT1-MMP did not show any significant changes throughout pseudopregnancy (P > 0.05).

Localization of mRNA for MT1-MMP During Follicular Growth, the Preovulatory Period, and Pseudopregnancy

To determine the cellular localization of mRNA for MT1-MMP in the rat ovary, in situ hybridization was performed. In ovaries from gonadotropin-treated rats, MT1-MMP mRNA was found to be highly expressed in the theca layer of developing follicles and surrounding stroma tissue (Fig. 2). MT1-MMP mRNA was also detected in the granulosa cell layer of growing follicles. However, its level of expression appeared to be lower compared with that of the theca layer of the same follicles. Of particular interest was the observation that the expression of MT1-MMP mRNA appeared to decline in the granulosa cell layer of certain large preovulatory follicles in ovaries collected at 8 and 12 h post-hCG compared with that of adjacent small follicles (Fig. 2, E and F), while the thecal expression of MT1-MMP mRNA appeared to remain the same or be higher than that of previous time points. Upon ovulation, mRNA for MT1-MMP was localized in newly forming CL (Fig. 2H).



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  		FIG. 2. In situ hybridization analysis of MT1-MMP mRNA during follicular development and the preovulatory period in the rat ovary. Representative bright field (A, C, E, and G) and dark field (B, D, F, and H) photomicrographs are depicted. Ovaries were collected at 48 h after eCG (0 h post-hCG; A and B), 8 h post-hCG (C and D), 12 h post-hCG (E and F), and 24 h post-hCG (G and H). Arrows in E and F indicate granulosa cell expression of MT1-MMP mRNA in small follicles. Arrow heads indicate follicles that lack granulosa cell expression of MT1-MMP mRNA. Gc, Granulosa cells; T, theca layer; F, follicle; CL, forming corpus luteum. A–H, original magnification x60

Subsequent changes in the localization pattern of MT1-MMP mRNA in luteal tissue were also examined in rat ovaries collected at specific stages of luteal development during gonadotropin-induced pseudopregnancy. In ovaries collected during early luteal formation, a strong hybridization signal for MT1-MMP mRNA was observed in developing CL (Fig. 3B). During the middle (Fig. 3D) and late luteal (Fig. 3F) stages of pseudopregnancy, the mRNA for MT1-MMP was localized uniformly throughout the mature or regressing CL and the expression is relatively low compared with that of adjacent follicles.



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  		FIG. 3. In situ hybridization analysis of MT1-MMP mRNA during pseudopregnancy in the rat ovary. Representative bright field (A, C, and E) and dark field (B, D, and F) photomicrographs are shown. Ovaries were collected at 2 days post-hCG (A and B), 8 days post-hCG (C and D), and 14 days post-hCG (E and F). CL, Corpus luteum; F, follicle. A–F, original magnification x30

Comparison of MT1-MMP, MMP-2, and TIMP-2 mRNA Expression in Preovulatory Rat Ovaries

Because MT1-MMP has been reported to activate pro-MMP-2 by forming a complex with TIMP-2 on the plasma membrane in vitro [13], we compared the expression pattern of MT1-MMP mRNA with that of MMP-2 and TIMP-2 mRNA in serial sections of ovarian tissue from gonadotropin-treated rats. To differentiate the developmental stage of each follicle, LH receptor mRNA expression was also examined in the adjacent section of the same ovary. As shown in representative photomicrographs (Fig. 4), similarities were observed among the expression patterns of mRNA for TIMP-2, MMP-2, and MT1-MMP. As expected, abundant expression of MT1-MMP mRNA was localized in the theca layer of growing follicles. Likewise, mRNAs for both MMP-2 and TIMP-2 were localized in the theca layer of those follicles and stroma tissue. One of the intriguing observations was the fact that MT1-MMP mRNA detected in granulosa cells of growing follicles disappeared in certain large follicles after hCG injection, while the other follicles maintained its expression (denoted in Fig. 4D). In those follicles that continued to express MT1-MMP mRNA in the granulosa cell layer, there was concurrent expression of LH receptor mRNA in the same granulosa cell compartment (Fig. 4E).



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  		FIG. 4. In situ hybridization analysis of mRNAs for TIMP-2 (B), MMP-2 (C), MT1-MMP (D), and LH receptor (E) in the rat ovary. The representative bright field (A) and dark field (B, C, D, and E) photomicrographs in serial sections of an ovary collected at 8 h after hCG injection are illustrated. Arrows indicate follicles that express mRNAs for both LH receptor and MT1-MMP in the granulosa cell layer. An arrowhead indicates a follicle that does not express both the LH receptor and MT1-MMP mRNA. A–E, original magnification x30

Activation of pro-MMP-2 in the Crude Plasma Membrane Fraction from Preovulatory Rat Ovaries

To investigate if MT1-MMP is involved in cell surface activation of ovarian pro-MMP-2, crude plasma membrane fractions from immature rat ovaries obtained at selected times after eCG or hCG injection were analyzed by gelatin zymography. Gelatin zymography revealed the presence of several gelatinase activities in crude plasma membrane fractions (Fig. 5A). Among them, digested regions of approximately 105, 72, and 62 kDa, corresponding to pro-MMP-9 (pro-gelatinase B), pro-MMP-2 (pro-gelatinase A), and active MMP-2, respectively, were detected. The pro-MMP-2 gelatinolytic activity was the most abundant. Densitometric quantification revealed significant increases in gelatinolytic activities of pro-MMP-2 and pro-MMP-9 at 12 h post-hCG and 8 h post-hCG, respectively, but no changes in levels of active MMP-2 were detected across time of tissue collection (Fig. 5B).



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  		FIG. 5. A) Representative gelatin zymograph demonstrating digested fragments by gelatin-degrading enzymes in crude plasma membrane fractions isolated from ovaries collected at selected times after eCG or hCG injection. HT1080 cell culture medium (50 µl) was used as a positive control as well as a marker for specific enzymes. Equal amounts of protein were loaded in each lane. B) Densitometric analysis (density values of digested band; mean ± SEM) of pro-MMP-9, pro-MMP-2, and active MMP-2 in crude plasma membrane fractions isolated from ovaries collected at selected times after eCG or hCG injection (n = 4 animals/time point). Bars with no common superscripts among the specific MMP subclasses are significantly different (P < 0.05)

To determine if MT1-MMP is proteolytically functional in processing pro-MMP-2 to its active form, the crude plasma membrane fractions were incubated at 37°C for 12 h. The incubation resulted in a shift in the digested region of 72 kDa to a predominant zone of 62 kDa, indicating the conversion of pro-MMP-2 to the active form (Fig. 6A). Densitometric quantification of the gelatin zymography showed a significant increase in levels of active MMP-2 at 12 h post-hCG compared with the time of eCG (0 h eCG) administration through 4 h after hCG injection (Fig. 6B).



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  		FIG. 6. A) Representative gelatin zymograph demonstrating digested fragments by gelatin degrading enzymes in crude plasma membrane fractions isolated from ovaries collected at selected times after eCG (e) or hCG (H) injection. Equal amounts of protein from crude plasma membrane fractions incubated at 4°C (C) or 37°C (I) were loaded in each lane. B) Densitometric analysis (density values of digested band; mean ± SEM) of active MMP-2 in crude plasma membrane fractions after incubation at 37°C for 12 h. Membrane fractions were isolated from ovaries collected at selected times after eCG or hCG injection (n = 4 animals/time point). Bars with no common superscripts are significantly different (P < 0.05)

To further examine if the activation of pro-MMP-2 in the plasma membrane fraction is mediated by adjacent TIMP-2-free MT1-MMP, the plasma membrane fractions were incubated with MT1-MMP antibody or human recombinant TIMP-2. As shown in Figure 7, when the plasma membrane fractions were incubated with 100 µM APMA, a chemical activator for gelatinases, both pro-MMP-2 and pro-MMP-9 were activated. However, the addition of MT1-MMP antibody to plasma membrane fractions during the incubation period inhibited the conversion of pro-MMP-2 to the active form in a dose-dependent manner. When a low dose (50 ng/20 µl) of MT-MMP antibody was added to plasma membrane fractions during the incubation period, pro-MMP-2 was converted to the active form, whereas a high dose of MT1-MMP antibody (150 or 300 ng/20 µl) inhibited the conversion of pro-MMP-2 to the active form. Likewise, treatments with TIMP-2 (10, 20, or 30 ng/20 µl) inhibited the activation of pro-MMP-2 in plasma membrane fractions. Neither TIMP-2 nor MT1-MMP antibody had any effect on pro-MMP-9 activity in the plasma membrane fraction. To confirm that the inhibitory effect of the MT1-MMP antibody and TIMP-2 on the MMP-2 activation process is specific, the plasma membrane fraction was incubated with normal mouse IgG (50–300 ng/20 µl) or mouse recombinant TIMP-3 (10–60 ng/20 µl). Neither mouse IgG nor TIMP-3 had any inhibitory effect on the activation of pro-MMP-2 in ovarian membrane fractions (Fig. 7C).



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  		FIG. 7. A) Representative gelatin zymograph demonstrating digested fragments by gelatin-degrading enzymes in membrane fractions isolated from ovaries collected at 12 h after hCG injection. Membrane fractions were incubated without (I) or with MT1-MMP antibody (50, 150, or 300 ng/20 µl; order of 1–3), TIMP-2 (10, 20, or 30 ng/20 µl; order of 1–3), or APMA (100 µM) at 37°C for 12 h. The membrane fractions incubated at 4°C were loaded as a control (C). Equal amounts of protein were loaded in each lane. B) Densitometric analysis (density values of digested band; mean ± SEM) of pro-MMP-2 and active MMP-2 in crude plasma membrane fractions after incubation with various treatments (n = 4 animals/time point). Bars with no common superscripts among the specific MMP subclasses are significantly different (P < 0.05). C) A representative gelatin zymograph showing digested fragments in the membrane fraction incubated with MT1-MMP antibody (150 ng [1]), mouse IgG (150 ng [1] or 300 ng [2]), TIMP-2 (10 ng [1] or 20 ng [2]) or recombinant TIMP-3 (20 ng [1] or 30 ng [2]).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows dynamic changes in the levels of mRNA for MT1-MMP, as well as the cellular localization pattern of MT1-MMP mRNA during the processes of follicular development, ovulation, and luteinization. Furthermore, this study demonstrates for the first time that both the levels of MT1-MMP mRNA and the cell surface activation of pro-MMP-2 increase around the time of ovulation. We also provide evidence that this activation occurs via a complex of pro-MMP-2 with membrane-bound MT1-MMP and TIMP-2 in rat ovarian membrane fractions, demonstrating at least one functional role for MT1-MMP in rat ovaries during the preovulatory period.

Unlike most of the secreted MMPs, MT1-MMP contains a cell membrane-spanning domain that confers its function to the extracellular surface of the cell membrane and a cytoplasmic domain, which may be important in intracellular signaling [23]. In addition to intrinsic proteolytic enzyme activity for various ECM components [24, 25], MT1-MMP is known to be critical for the focal recruitment of other pro-MMPs, such as pro-MMP-2 and pro-MMP-13, and the subsequent activation of these enzymes on the cell surface [12]. MMP-2 was reported to be increased during the late stage of follicular development and/or the preovulatory period in the ovary of several species including rodents, cattle, and primates [3]. These observations can be interpreted that MMP-2 has a crucial role in follicular development and ovulation. However, this gelatinase is secreted as an inactive zymogen that needs to be activated [26, 27]. This activation has been shown to occur in other tissues, mainly by membrane-bound MT1-MMP [28, 29]. The activation of pro-MMP-2 has been demonstrated to require both active MT1-MMP and TIMP-2-bound MT1-MMP on the cell membrane [13–15]. The TIMP-2 serves as an intermolecular bridge between pro-MMP-2 and MT1-MMP. An adjacent TIMP-2-free MT1-MMP then cleaves the propeptide of pro-MMP-2, generating an activated form of MMP-2. Therefore, the efficient activation of pro-MMP-2 in vivo requires both focal cellular localization of MT1-MMP, TIMP-2, and pro-MMP-2 and an optimal concentration balance among these three components.

In the present study, MT1-MMP mRNA was predominantly expressed in the theca layer of growing and preovulatory follicles, while low expression was observed in the granulosa cell layer of some follicles. Moreover, this localization pattern of MT1-MMP mRNA was similar to that of the other components of the trimolecular complex of the MMP-2 activation machinery in that both MMP-2 and TIMP-2 mRNA were present in the theca cell layer of the follicles. These results are consistent with a previous report showing a similar localization pattern between MT1-MMP and MMP-2 mRNA in the rat ovary during the preovulatory period, but provide further information on the focal cellular localization of all three components of the complex during the preovulatory period.

One of the intriguing findings in this study was the dynamic changes in levels of mRNA for MT1-MMP and its expression pattern in preovulatory follicles following hCG treatment. For instance, although the levels of MT1-MMP mRNA in the whole ovary increased shortly after hCG injection, the expression of MT1-MMP mRNA disappeared in the granulosa cell layer of certain preovulatory follicles, while some follicles maintained MT1-MMP mRNA in the granulosa cell layer. This transient decrease in MT1-MMP mRNA expression in the granulosa cell layer was also reported in rodent ovaries [16, 30], although in the whole mouse ovary the levels of MT1-MMP mRNA did not change after hCG injection. Careful comparison of adjacent tissue sections by the present in situ hybridization study extends these earlier findings to reveal a positive correlation between MT1-MMP and LH receptor mRNA expression in the granulosa cell layer. These data can be interpreted that expression of MT1-MMP mRNA in granulosa cells of rat preovulatory follicles may be downregulated in response to hCG treatment, as shown for LH receptor mRNA. The physiological significance of this temporal downregulation of MT1-MMP mRNA in the granulosa cell layer of periovulatory follicles is yet to be determined.

The current study revealed that the levels of MT1-MMP mRNA increase during the preovulatory period in the rat ovary. Previously, we reported that levels of mRNA for MMP-2 began to increase at 4 h after hCG injection to eCG-primed immature rats and reached maximal levels at 12 h post-hCG, whereas levels of TIMP-2 mRNA remained constant throughout the preovulatory period [9]. Taken together, these findings indicate that the simultaneous upregulation of MT1-MMP and MMP-2 after the LH surge may be an important part of the mechanism necessary for the efficient generation of active MMP-2, which in turn plays an important role in the degradation of the follicular wall during the ovulatory process in the rat ovary. This proposal is in agreement with previous reports showing an increase in gelatinolytic activity in rat ovarian extracts [8] and follicular fluid of the ewe [31] and pig [32] collected during the periovulatory period. However, both ovarian extracts and follicular fluids exhibited predominantly latent forms of gelatinases by gelatin zymography. These authors suggested that the latent form of these enzymes may convert to an active form at the specific site of action, which, as supported by the present findings, may occur through the action of MT1-MMP.

To verify the proposal of site-specific activation of progelatinases, we isolated crude plasma membrane fractions from the gonadotropin-primed immature rat and analyzed them by gelatin zymography. The rationale for such experiments was that, if MT1-MMP forms a complex with TIMP-2 and pro-MMP-2 on the cell surface, ovarian plasma membrane fractions would exhibit pro-MMP and/or active MMP-2 activity. Indeed, both pro-MMP-2 and active MMP-2 were present in crude plasma membrane fractions and the levels of pro-MMP-2 increased close to the time of ovulation (12 h post-hCG). Moreover, upon incubation, the latent form of MMP-2 in the plasma membrane fractions was converted to the active form with the highest levels at 12 h post-hCG. Previously, our laboratory has reported the lack of active gelatinase activity in rat ovarian extracts when incubated at 37°C in the absence of APMA, a chemical activator for gelatinases [8]. This lack of active enzyme may be explained by the fact that the ovarian extracts did not contain plasma membranes. Further support for plasma activation of pro-MMP-2 comes from the current finding that MT1-MMP antibody inhibited the activation of pro-MMP-2 in ovarian plasma membrane fractions. Taken together, these results provide compelling evidence that pro-MMP-2 is activated by MT1-MMP on the ovarian cell membrane and further demonstrate that the membrane-associated activation of pro-MMP-2 increases at the time of ovulation.

To further test whether the activation of pro-MMP-2 is mediated by TIMP-2-free MT1-MMP on the cell surface, the crude membrane fractions were incubated with recombinant human TIMP-2. The addition of TIMP-2 inhibited the activation of pro-MMP-2, suggesting that this activation process was highly sensitive to the concentration balance between TIMP-2 and other components of the complex in ovarian membrane fractions. Consistent with this finding is the hypothesis that, if TIMP-2 is present in excess relative to MT1-MMP on the cell surface, all the MT1-MMPs are occupied with TIMP-2 and, although pro-MMP-2 binding occurs, no free MT1-MMP remains to initiate the activation process. This proposal has been substantiated in cell free kinetic studies [13] and in vivo and in vitro studies using TIMP-2 deficient mice [33–35]. However, current findings provide the first experimental evidence for direct, yet collaborative actions of TIMP-2 and MT1-MMP on the activation of pro-MMP-2 in the ovary, and further indicate that the LH surge may increase the efficiency of the pro-MMP-2 activation via increasing the concentration of MT1-MMP, while maintaining constant levels of TIMP-2.

Interestingly, the membrane fraction also exhibited pro-MMP-9 enzyme activity that began to rise at 8 h after hCG injection. This is the first report showing a significant increase in pro-MMP-9 activity during the preovulatory period in the rat ovary. Similar increases in the pro-MMP-9 content were observed in mouse ovarian extracts after hCG administration, although the levels appeared to be the highest at 4 h and declined by 12 h post-hCG [30, 36]. In the present study, neither incubation nor addition of TIMP-2 or MT1-MMP antibody had any significant effect on pro-MMP-9, indicating that the membrane-associated activation is specific for pro-MMP-2. Moreover, the presence of pro-MMP-9 activity in ovarian plasma membrane fractions implies that pro-MMP-9 may be engaged with peptides that act as receptor-like molecules on the plasma membrane of the rat ovary. Indeed, MMP-9 was reported to interact with the cell surface hyaluronan receptor CD44 in mouse mammary carcinoma and human melanoma cells [37]. In the ovary, CD44 mRNA and protein were detected in porcine cumulus cells and cumulus-oocyte complex, respectively, and the levels of mRNA for CD44 were increased by hCG [38]. At present, whether pro-MMP-9 is anchored to ovarian plasma membranes by interacting with CD44 or unknown molecules is not clear. However, the present finding indicates that MMP-9 may play a role during the ovulatory process in the rat ovary.

Following ovulation, the ruptured follicle is transformed into the CL by extensive tissue remodeling and neovascularization, both of which are severely impaired in MT1-MMP-deficient mice [29, 39]. In the present study, we used the gonadotropin-induced pseudopregnant rat model to examine the level and localization of mRNA for MT1-MMP throughout the luteal period. MT1-MMP mRNA was highly expressed in the newly forming and developing CL. Previously, Liu et al. [16] reported that MT1-MMP mRNA is constitutively expressed in the CL from adult pseudopregnant rat ovaries, while MMP-2 mRNA is detected only in developing CL. Our preliminary in situ localization data from naturally cycling rat ovaries showed that, in the newly forming CLs that express high levels of MT1-MMP mRNA, the localization pattern of MT1-MMP mRNA is similar to that of pro-MMP-2 and TIMP-2 mRNA (T.E. Curry, Jr., unpublished results). These observations indicate that MT1-MMP may serve as an activator of pro-MMP-2 in the early stage of luteal formation. Active MMP-2 has been known to play a critical role in angiogenesis [40, 41]. In addition to MMP-2, MT1-MMP was also reported to be directly involved in angiogenesis through its ability to degrade ECM components and various cell surface molecules (for review, see [42]). Therefore, MT1-MMP may play an important role in angiogenesis and the tissue remodeling process during the early CL development in the rat ovary by acting directly on the extracellular matrix or indirectly through activating pro-MMP-2.

In conclusion, the upregulation of MT1-MMP gene expression in the rat preovulatory ovary corresponds to the increase in gelatinase activity and membrane-associated activation of pro-MMP-2 around the time of ovulation. Moreover, the present study provides experimental evidence that the increase in membrane-associated activation of pro-MMP-2 results from the coordinated action and optimized concentration balance among all three components of the activation machinery, MT1-MMP, TIMP-2, and pro-MMP-2.


   ACKNOWLEDGMENTS
 
The authors would like to thank Drs. O.K. Park-Sarge, K. Leco, D. Edwards, and J. Richards for providing plasmids containing cDNAs for L32, TIMP-2, MMP-2, and LH receptor respectively, and Drs. W.B. Nothnick and C.M. Komar for critical reading of the manuscript.


   FOOTNOTES
 
1 This work was supported by grants from the NIH NCRR P20 RR 15592 and the Lalor Foundation. Back

2 Correspondence: Misung Jo, Department of Obstetrics and Gynecology, Chandler Medical Center, 800 Rose Street, Room MS 331, University of Kentucky, Lexington, KY 40536-0298. FAX: 859 323 3761; mjo2{at}uky.edu Back

Received: 22 September 2003.

First decision: 14 October 2003.

Accepted: 24 November 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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