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Ovary |
Department of Obstetrics and Gynecology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0298
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, follicle-stimulating hormone, luteinizing hormone, metalloproteinases, ovary, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Throughout the reproductive cycle, matrix metalloproteinases (MMPs) play important roles in ovarian extracellular matrix (ECM) remodeling [10, 11]. The MMPs are a family of proteolytic enzymes capable of degrading a wide variety of ECM components, thereby regulating connective tissue remodeling and modulation of cell-matrix interaction [12, 13]. So far, more than 25 MMPs have been described [10, 12, 14]. These MMPs share common domain structures and have wide and often overlapping substrate specificities. The MMPs are broadly grouped into the collagenases, the gelatinases, the stromelysins, and the membrane-type MMPs, with a number of other MMPs that fall outside these four classes [10, 12, 14].
A recently discovered member of the MMP family is MMP-19 [15, 16]. In addition to the typical MMP domain structure common to other MMP family members, MMP-19 displays a series of unique features that distinguish it from other MMP subfamilies. For example, MMP-19 exhibits a unique intron/exon distribution that differs from that of other MMPs. This enzyme has been shown to lack various structural traits of other MMP subfamilies (e.g., the fibronectin-like repeats of gelatinases) but to possess a unique insertion of five Glu residues in the propeptide domain and an additional Cys residue in the catalytic domain [15]. Therefore, MMP-19 is classified as the first member of a new MMP subfamily [15]. Recent studies have revealed the expression of MMP-19 mRNA in a variety of normal adult human tissues, including placenta, ovary, lung, pancreas, spleen, and intestine [15], as well as constitutive expression of MMP-19 mRNA in arthritic and traumatic synovial membranes [17]. Therefore, MMP-19 may play a role in ECM remodeling that occurs in all these tissues. Previous experimental data demonstrated that purified catalytic domains of MMP-19 were capable of hydrolyzing aggrecans [18] and basement membrane components, including type IV collagen, laminin, and fibronectin [16], suggesting that MMP-19 is a potent proteolytic enzyme capable of degrading a broad range of ECM components.
In the ovary, MMP-19 mRNA expression is transiently induced in granulosa and theca-interstitial cells of periovulatory follicles after administration of hCG to eCG-primed immature mice [19]. Although this observation of an up-regulation of MMP-19 mRNA suggests a possible role of MMP-19 in connective tissue degradation during the ovulatory process, little is known about MMP-19 expression during other ovarian reproductive phases or in the ovaries of other species. Furthermore, nothing is known about the regulatory mechanisms of MMP-19 mRNA expression in the ovary. Therefore, in the present study, we examined the spatiotemporal expression of MMP-19 mRNA during both the follicular and the luteal phase using eCG/hCG-primed immature rat ovaries as well as ovaries from naturally cycling adult rats. We also determined the hormonal regulation of MMP-19 mRNA expression during the periovulatory period through in vitro studies on rat follicular cells obtained before the LH surge.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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, culture media, and Trizol were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA).
Animals
All animal procedures were approved by the University of Kentucky Animal Care and Use Committee. In the present study, both gonadotropin-treated immature female rats and sexually mature adult female rats exhibiting regular, 4-day estrous cycles were used.
Immature female Sprague Dawley rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN), were provided with water and rat chow ad libitum, and were maintained on a 14L:10D photoperiod. The rats were injected with 10 IU of eCG s.c. at 22 days of age to stimulate follicular development and 48 h later with 10 IU of hCG s.c. to induce ovulation and subsequent formation of CL. In this model [20], ovulation occurs approximately 14–16 h post-hCG administration. Animals were killed at 0 (i.e., at the time of eCG administration), 12, 24, or 48 h post-eCG or at 4, 8, 12, 24, or 48 h post-hCG administration (n = 4 animals/ time point). Rats were also killed on Days 1 and 2 (24 and 48 h post-hCG, respectively; luteal formation), Days 4 and 8 (luteal maintenance when progesterone production is maximal), or Days 16 and 21 (luteal regression) after hCG treatment (n = 4 animals/time point) to study subsequent luteal changes. In this gonadotropin-induced rat model, the ovulated follicles transformed into CL that remained functional for 9 ± 1 days and then regressed [21]. Ovaries were collected, snap-frozen, and stored at –70°C for later extraction of total RNA or were placed in OCT (VWR Scientific, Atlanta, GA) and stored at –70°C until sectioned and processed for in situ hybridization.
Adult female Sprague Dawley rats (body weight, 150–180 g; age, 2– 3 mo) were purchased from Harlan Sprague Dawley and housed as described above. Stages of the estrous cycle were determined by daily examinations of vaginal cytology, and only animals showing at least two consecutive 4-day cycles were used for the experiment. Rats were killed at 1600, 2000, and 2400 h on proestrus and at 0400 h on estrus. In this colony of rats, the peak of the LH surge was at 1600 h on proestrus. Ovaries were collected, placed in OCT, and stored at –70°C until sectioned and processed for in situ hybridization.
Isolation and Culture of Granulosa and Theca-Interstitial Cells
To isolate granulosa cells, ovaries collected from eCG-primed immature rats (48 h post-eCG) were processed as described previously [22]. Briefly, granulosa cells were isolated by follicular puncture. The cells were pooled, pelleted by centrifugation at 600 x g for 5 min, and resuspended in defined medium consisting of Dulbecco modified Eagle medium-Ham F-12 medium supplemented with 1% BSA, 0.01% pyruvic acid, 0.22% bicarbonate, 0.05 mg/ml of gentamicin, and ITS (insulin, transferin, and selenium). The cells were distributed at a density of approximately 1 x 106 viable cells per milliliter of defined medium and cultured in the absence or presence of various treatments for 0, 6, 12, 24 or 48 h at 37°C in a humidified atmosphere of 95% air:5% CO2. At the end of each time of culture, cells were collected and snap-frozen for later isolation of total RNA.
Theca-interstitial cells were isolated as previously described [23] with slight modification. Briefly, residual ovaries obtained after expelling granulosa cells by puncturing follicles were minced and incubated in an enzyme mixture of collagenase type II and deoxyribonuclease I to digest ECM components and free DNA released from damaged cells, respectively, for 15 min at 37°C. The dispersed ovarian cells were subjected to Percoll density gradient centrifugation. The enriched theca-interstitial cell phase was aspirated and then washed with McCoy 5A medium. The cells were plated at a density of 1 x 106 viable cells per milliliter of McCoy 5A medium supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin sulfate, and 2 mM L-glutamine and then cultured in the absence or presence of various treatments for 0, 6, 12, 24, or 48 h at 37°C in a humidified atmosphere of 95% air:5% CO2. At the end of each time period of culture, cells were collected and snap-frozen for later isolation of total RNA.
Generation of the Plasmid Containing cDNA for Rat MMP-19
A 584-base pair rat cDNA fragment was generated by reverse transcription-polymerase chain reaction. Briefly, total RNA (1 µg) isolated from rat preovulatory ovaries (12 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'-GCCACTGGAGAAAGAAGCAC-3', 5'-CATCATGGCATCCACTTCAC-3') based on the previously reported sequence of rat MMP-19 (GenBank accession no. XM_222317). Amplification consisted of a preincubation at 94°C for 5 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 polymerase chain reaction product of the predicted size was cloned into the pCRII-TOPO Vector. The DNA sequences of the cloned rat partial MMP-19 cDNA were determined using a Standard ABI kit (Macromolecular Structure Analysis Facility, University of Kentucky, Lexington, KY).
Quantification of mRNA for MMP-19
Total RNA was extracted from ovaries collected during follicular growth, the periovulatory period, and the luteal period as well as from cultured granulosa and theca-interstitial cells using Trizol reagents according to the manufacturer's protocol and then quantified by spectrophotometry. Northern blot analyses were carried out as described previously [24]. Briefly, 5–10 µg of total RNA were separated by electrophoresis, capillary-transferred to a nylon membrane (pore size, 0.2 µm; Nytran N; Schleicher & Schuell, Inc., Keene, NH), and cross-linked to the membrane by baking in a vacuum oven at 80°C for 2 h. Plasmids containing rat cDNAs for MMP-19 and mouse cDNA for ribosomal protein L32 (kindly provided by Dr. O.K. Park-Sarge, University of Kentucky) were linearized with NcoI and EcoRI, respectively. Antisense riboprobes were transcribed using [
-32P]UTP (10 mCi/ml; Perkin-Elmer Life Sciences, NEN, Boston, MA) and SP6 or T7 RNA polymerase, as appropriate. Northern membranes were hybridized with 1 x 106 cpm 32P-labeled antisense riboprobe for MMP-19 mRNA and L32 mRNA per milliliter of Ultrahyb hybridization buffer (Ambion, Inc., Austin, TX) at 68°C for at least 16 h. Excess probe was removed by washing with a stringent buffer (0.1x SSC [1x SSC: 0.15 M sodium chloride and 0.015 M sodium citrate], 0.1% SDS) twice at 68°C for 60 min. The membrane was exposed to a PhosphorImaging plate and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The relative levels of mRNA for MMP-19 were normalized to L32 mRNA levels by dividing the band intensity of MMP-19 mRNA by the band intensity of L32 mRNA in each lane.
In Situ Hybridization of mRNA for MMP-19
Ovaries collected from gonadotropin-treated immature rats or naturally cycling adult rats were sectioned (thickness, 10 µm) and mounted on Probe On Plus slides (Fisher Scientific). In situ hybridization was carried out as described previously [25]. Briefly, plasmids containing cDNA for rat MMP-19 were linearized with BamHI and NcoI to generate sense and antisense riboprobes for MMP-19, respectively. Linearized plasmids were labeled with [-35S]UTP (10 mCi/ml; MP Biomedicals, Inc., Costa Mesa, CA) 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 0.1 mg/ml of RNase A 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 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 dark-field optics. One ovary from each of three animals was used for in situ hybridization. At least eight sections per ovary were analyzed for each antisense probe, making a total analysis of at least 24 tissue sections for each time point. A sense riboprobe, used as a control for nonspecific binding, was included for each ovary and each time point.
Radioimmunoassay
Concentrations of androstenedione were determined in culture media by using Coat-A-Count Direct Androstenedione kit (Diagnostic Products, Los Angeles, CA). Assay sensitivity was 0.04 ng/ml for androstenedione. The intraassay coefficient of variation was 5.5%.
Statistical Analyses
All data are presented as the mean ± SEM. One-way ANOVA was used to test differences in levels of MMP-19 across time of tissue collection, time of culture, or among treatments in cultures. If ANOVA revealed significant effects of time of tissue collection or treatment, the means were compared by the Duncan test, with a value of P < 0.05 considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern blot analyses revealed a single transcript (
3.3 kilobases) for MMP-19 in total RNA isolated from ovaries of gonadotropin-treated immature rats obtained at selected time points during follicular growth (Fig. 1A), the periovulatory period (Fig. 1B), and the luteal period (Fig. 1C). Levels of mRNA for MMP-19 transiently increased at 12 h after eCG injection (fivefold higher compared to levels at 0 h) and then declined to the 0 h levels by 48 h post-eCG (Fig. 1A). During the hCG-induced periovulatory period, levels of MMP-19 mRNA began to increase at 8 h, reached the highest level at 12 h after hCG injection (11-fold higher than levels at 0 h post-hCG), and then declined by 48 h post-hCG (Fig. 1B). During the gonadotropin-induced luteal period of pseudopregnancy (Fig. 1C), MMP-19 mRNA levels were increased by 16 days after hCG administration, and the levels reached maximum at Day 21 (twofold higher than levels at Day 2).
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Localization of mRNA for MMP-19 During Follicular Growth, the Periovulatory Period, and the Luteal Period
The cellular localization pattern of mRNA for MMP-19 was examined in ovaries from both gonadotropin-treated immature rats and naturally cycling adult rats by in situ hybridization. When animals were treated with eCG to stimulate follicular growth, relatively low expression of MMP-19 mRNA was localized to the theca-interstitial layer of ovarian tissue sections obtained at 12 and 24 h post-eCG (data not shown), but then the expression of MMP-19 mRNA appeared to decrease by 48 h post-eCG (Fig. 2, A and B).
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During the hCG-induced periovulatory period, initial low expression of MMP-19 mRNA was observed in the theca-interstitial layer at 4 h after hCG (Fig. 2, C and D). By 8 and 12 h after hCG administration, high expression of MMP-19 mRNA was localized to the theca-interstitial layer of the ovary obtained at 8 and 12 h after hCG (Fig. 2, E–H). Furthermore, certain large preovulatory follicles expressed MMP-19 mRNA in the granulosa cell layer at 12 h after hCG administration (Fig. 2, I and J), although the intensity of hybridization signal for MMP-19 in granulosa cells was lower than that in the theca layer. After ovulation, MMP-19 mRNA was also localized to the newly forming CL (Fig. 2, K and L).
To confirm that the transient induction of mRNA for MMP-19 in periovulatory follicles seen in the hCG-induced immature rat ovulation model occurs in the cycling adult rat ovary in the natural setting, the ovaries collected from naturally cycling adult rats between the period of the endogenous gonadotropin surge and ovulation were also examined. A similar expression pattern of MMP-19 mRNA in preovulatory follicles was observed in naturally cycling rats. For instance, before the LH surge, MMP-19 mRNA was not detected in growing follicles (Fig. 3, A and B), but high expression of MMP-19 mRNA was localized to the majority of CL from previous estrous cycles. A few of the preovulatory follicles began to express MMP-19 mRNA in the theca layer as early as 4 h after the LH surge (Fig. 3, C and D). However, by 8 h after the LH surge, MMP-19 mRNA was highly expressed in the theca and, to a lesser extent, in the granulosa cell layers of periovulatory follicles. The thecal expression was as strong as that in CL from previous estrous cycles (Fig. 3, E and F). In ovaries obtained at 12 h after the LH surge, MMP-19 mRNA was localized to newly forming CL, although the expression was lower compared to that in the adjacent CL that was produced from previous estrous cycles (Fig. 3, G and H). Interestingly, the intensity of hybridization signal for MMP-19 between two adjacent CL from previous estrous cycles (Fig. 3, G and H) appeared to be different, implying a possibility of a differential expression pattern of MMP-19 mRNA at different stages of luteal development.
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Given the observations of luteal expression of MMP-19 mRNA in ovaries from cycling adult rats, we next examined changes in the expression pattern of MMP-19 mRNA in luteal tissues in rat ovaries collected at specific stages of luteal development throughout pseudopregnancy using gonadotropin-treated rats. The MMP-19 mRNA was detected uniformly throughout the forming CL (Fig. 4, A and B) or the mature CL (Fig. 4, C and D). The low levels of cellular expression of MMP-19 observed in CL at Day 2 corresponds to results from the Northern blot analysis. The strongest hybridization signal for MMP-19 mRNA was localized to regressing CL seen at the late luteal stages (Fig. 4, E and F), whereas expression of MMP-19 mRNA was not localized to follicles in ovarian tissue sections from pseudopregnant rats (Fig. 4, B, D, and F).
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Effects of Prostaglandin Synthesis Inhibition and Progesterone-Receptor Antagonists on MMP-19 mRNA Expression in the Rat Ovary
The LH surge-induced production of progesterone and prostaglandins by preovulatory follicles is essential for ovulation [26]. Furthermore, the transient expression of mRNA for both progesterone receptor [27] and cyclooxygenase-2 [28] peaks around 4 h after hCG injection, whereas MMP-19 mRNA expression is highest at 12 h post-hCG. Therefore, in the present study, we tested whether the induction of MMP-19 mRNA in periovulatory follicles is mediated by the activation of the progesterone receptor or the action of prostaglandins during the early preovulatory period. To ensure the effectiveness of an inhibitor of prostaglandin synthesis (indomethacin, 1 mg/rat [29]) or antagonists for the progesterone receptor (ZK98299 and RU486, 1 mg/rat [30]), these agents were administered 1 h before hCG injection. The dosages of indomethacin and RU486 used in the present study have been shown to completely block ovulation in rats [29, 30], which was confirmed in the present study by counting none or only a few oocytes in the oviduct after the expected time of ovulation. The time intervals of tissue collection at 6 and 12 h after hCG injection were selected both because the increase in MMP-19 mRNA levels could be detected as early as 6 h post-hCG and because the expression reached the maximal level at 12 h post-hCG injection in vivo.
In rats treated with an antiovulatory dose of indomethacin, the ovarian level of MMP-19 mRNA was not different from that in the controls at 6 and 12 h post-hCG (P > 0.05) (Fig. 5A). Likewise, neither ZK98299 nor RU486 had any effect on the hCG-stimulated increase in MMP-19 mRNA levels in the gonadotropin-primed immature rat ovary (P > 0.05) (Fig. 5B).
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Effects of LH on Expression of mRNA for MMP-19 in Granulosa and Theca-Interstitial Cell Cultures
To determine whether the transient increase in levels of MMP-19 mRNA after hCG injection in vivo can be mimicked in vitro, theca-interstitial and granulosa cells isolated from eCG-primed immature rat ovaries (48 h post-eCG) were cultured in the absence or presence of a luteinizing dose of LH (100 ng/ml). In cultured granulosa cells, LH treatment increased levels of MMP-19 mRNA in a time-dependent manner. Levels were higher in granulosa cells cultured with LH than in control cultures during the first 12 h. The highest expression was observed at 12 h, and the levels decreased between 12 and 48 h of culture (Fig. 6A).
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In cultured theca-interstitial cells, MMP-19 mRNA levels were increased in a time-dependent manner in both control and LH-treated cultures (Fig. 6B). Expression peaked at 12 h and decreased between 12 and 48 h of culture. In contrast to the response in cultured granulosa cells, LH treatment did not have any stimulatory effect on MMP-19 mRNA expression in theca-interstitial cell cultures above that seen in the control culture at any of the culture times. To assure that the LH treatment was effective in the theca-interstitial cell cultures, androstenedione secretion by the cultured cells was measured by radioimmunoassay (Direct Androstenedione; Diagnostic Products). As expected, LH stimulated androstenedione production by 10-fold compared to control theca-interstitial cells collected at 12 h after cultures (0.14 ± 0.03 vs. 1.48 ± 0.1 ng/ml).
Intracellular Signaling Mechanism of MMP-19 mRNA Induction In Vitro
The ovulatory LH stimulus activates both protein kinase (PK) A and PKC signaling pathways in preovulatory granulosa cells in vitro [31, 32]. In the present study, to investigate which intracellular signaling pathway is involved in the up-regulation of MMP-19 mRNA in response to an ovulatory dose of LH, granulosa cells from eCG-primed immature rat ovaries were cultured in the absence or presence of forskolin and/or phorbol 12-myristate 13-acetate (PMA) to mimic the PKA and PKC signaling of an ovulatory LH stimulus, respectively. The cells were cultured for 12 h, the culture period of maximal MMP-19 mRNA expression in preovulatory follicular cells both in vivo and in vitro. As expected, a luteinizing dose of LH stimulated MMP-19 mRNA expression in cultured granulosa cells (Fig. 7). Treatment with various doses of forskolin (1–50 µM) stimulated MMP-19 mRNA expression. All doses of forskolin used in the granulosa cell cultures increased the levels of MMP-19 mRNA to values either equivalent to or higher than that of the LH treatment (Fig. 7). Likewise, MMP-19 mRNA expression was also stimulated by treatment with PMA (10–100 nM), similar to the levels observed with the LH treatment (Fig. 7). When the cells were cultured with forskolin (10 µM) plus PMA (20 nM), the level of MMP-19 mRNA was increased to a level similar to that with LH treatment, but lower than that with the same dose of forskolin alone (P < 0.05) (Fig. 7).
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Effects of Cycloheximide on LH-Induced MMP-19 mRNA in Cultured Granulosa Cells
To determine whether the LH or forskolin-induced increases in MMP-19 mRNA expression in cultured granulosa cells require de novo protein synthesis, granulosa cells were incubated for 12 h in the absence or presence of LH, LH plus cycloheximide (1 or 10 µg/ml; concentrations that blocked protein synthesis in cultured rat granulosa cells [33]), or forskolin (10 µM) plus cycloheximide. Cycloheximide treatment blocked both LH- and forskolin-induced increases in levels of MMP-19 mRNA in granulosa cell cultures (P < 0.05) (Fig. 8).
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We also tested whether LH-stimulated up-regulation of MMP-19 mRNA in cultured granulosa cells is mediated by the action of progesterone or prostaglandins. The granulosa cells were cultured in the absence or presence of LH, LH plus ZK98299 (1 or 10 µM; concentrations that inhibited hCG-induced pituitary adenylate cyclase-activating polypeptide [PACAP] mRNA expression in cultured rat granulosa cells [34]), or LH plus NS-398 (1 or 10 µM; a specific cyclooxygenase-2 inhibitor [35]). Neither ZK98299 nor NS-398 had any effect on the LH-stimulated increases in MMP-19 mRNA expression in granulosa cells cultured for 12 h (P > 0.05; data not shown).
We have confirmed the effectiveness of ZK98299 by assessing the levels of mRNA for PACAP, a progesterone receptor-regulated gene [34]. Similar to previous published data [34], ZK98299 treatment inhibited the LH-induced PACAP mRNA expression in granulosa cells cultured for 6 and 12 h (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the ovary, the growth of primordial follicles to the graafian follicle stage is accomplished by rapid cellular proliferation and extensive angiogenesis around the follicle, both of which require regulated remodeling of the ovarian ECM. The present findings demonstrate that gonadotropin-stimulated follicular growth corresponds to the transient increase in MMP-19 mRNA expression. For instance, levels of MMP-19 mRNA in the whole ovary, as well as the thecal expression of MMP-19 mRNA in growing follicles, were increased during the first 12–24 h after eCG administration. This is a time period of extensive follicular growth in gonadotropin-treated rat ovaries [36], suggesting the possible involvement of MMP-19 during follicular development. Previously, we reported similar increases in the levels of mRNA for collagenase-3 (MMP-13) and gelatinases (MMP-2 and -9) in immature rat ovaries during gonadotropin-induced folliculogenesis [36]. A time difference, however, exists between the expression of MMP-19 and the other MMPs, in that MMP-19 mRNA expression was increased during the first 12–24 h of follicular growth and the rise in mRNA levels for the gelatinases and collagenase-3 occurred 24–48 h after the initial eCG stimulus. These findings suggest that MMP-19, together with the gelatinases and collagenase-3, may participate in the remodeling process of follicular connective tissue in a stage-specific manner during follicular development in the rat ovary.
The rupture of ovulatory follicles requires degradation of ECM components of the follicular wall. The support for a role of MMP-19 in the ovulatory process comes from the present finding of a rapid and transient increase in MMP-19 mRNA expression in periovulatory follicles close to the time of ovulation in both gonadotropin-stimulated immature and naturally cycling adult rat ovaries. Consistent with these findings is a previous report showing dramatic up-regulation of MMP-19 mRNA expression in periovulatory follicles after injection of hCG to eCG-primed immature mice [19]. Furthermore, in the present study, cultured granulosa cells from eCG-primed immature rat ovaries were induced by an ovulatory dose of LH to increase the levels of mRNA for MMP-19 in a manner similar to that seen in vivo. These results provide support for the hypothesis that the LH surge induces the transient increase in MMP-19 mRNA expression in granulosa cells of rat periovulatory follicles.
Virtually nothing is known about the intracellular signaling pathways by which the LH surge up-regulates the expression of MMP-19 mRNA in follicular cells. The present results demonstrate that the stimulatory effect of LH on MMP-19 mRNA expression can be mimicked by treating granulosa cells with either forskolin, a direct activator of adenylate cyclase, or PMA, an activator of PKC. Interestingly, the response of granulosa cells to these agonists was somewhat unique, in that forskolin at 10 µM stimulated MMP-19 mRNA expression approximately threefold higher than LH treatment alone, but when the combination of the agonists (10 µM forskolin plus 20 nM PMA) was added to granulosa cell cultures, the level of MMP-19 mRNA was similar to that with LH treatment. The forskolin-plus-PMA combination has been frequently used to exert the acute effect of the LH surge [37]. Therefore, the present results could be interpreted as indicating that the collaborative action of the PKA and PKC pathways activated by the LH surge may be crucial for controlled, yet sufficient, expression of MMP-19 mRNA in granulosa cells of periovulatory follicles. Furthermore, that cycloheximide abolished the stimulatory effect of LH or forskolin on MMP-19 mRNA expression in cultured granulosa cells suggests that the up-regulation requires new protein synthesis. However, the new protein synthesized is not a cyclooxygenase (COX-2) or progesterone receptor, because neither blocking activation of progesterone receptors nor inhibiting prostaglandin synthesis had any effect on MMP-19 mRNA expression both in vivo and in vitro.
Intriguingly, the expression pattern of MMP-19 mRNA in cultured theca-interstitial cells is somewhat unique. Unlike the findings in the granulosa cell cultures, transient increases in MMP-19 mRNA levels were observed in untreated cells, and an ovulatory dose of LH (100 ng/ml) had no effect on MMP-19 mRNA expression above that seen in the control culture at any of the various culture periods. Similar autonomous, transient up-regulation of transcript has also been reported in theca-interstitial cell cultures for another gene of the MMP family [38]. For instance, the levels of mRNA for MMP-23 were dramatically increased in cultured theca-interstitial cells from immature rat ovaries. Interestingly, LH had little effect on MMP-23 mRNA expression in cultured theca-interstitial cells, whereas forskolin or 8-Br(bromo)-cAMP treatments reduced the levels of MMP-23 mRNA. Ohnishi et al. [38] suggested that the dosages of LH (5–50 ng/ml) used in their study might not have produced sufficient amounts of cAMP in cultured theca externa/fibroblasts to repress MMP-23 mRNA expression to a level similar to that of forskolin- or 8-Br-cAMP-treated cells. Therefore, it remains to be determined whether accumulation of higher cAMP content by stimulating the theca-interstitial cells in vitro with higher dosages of LH, or directly with forskolin or 8-Br-cAMP, would affect MMP-19 mRNA expression. In the present study, the cultured theca-interstitial cells responded to LH treatment by producing significantly higher levels of androstenedione compared to control cultures, indicating that the lack of an LH-induced response on MMP-19 mRNA expression is not caused by the destruction of LH receptors during the preparation of theca-interstitial cells. We also observed a similar pattern of spontaneous, transient up-regulation of MMP-19 mRNA expression in cultures of small pieces of residual ovarian tissue (data not shown), discounting the possibility that the dissociation of thecal tissue structure may cause the spontaneous up-regulation of MMP-19 mRNA expression in the control cultures. As possible endogenous repressors for MMP-19 mRNA expression by thecal cells in vivo, we examined the effect of estradiol and growth differentiation factor (GDF)-9 on MMP-19 mRNA expression by theca-interstitial cell cultures. Both estrogen (10 nM) and GDF-9 (100 ng/ml culture media) had no effect on thecal expression of MMP-19 mRNA at 12 h of culture (results from duplicated culture experiments; data not shown). Although the present data of theca-interstitial cultures did not show any correlation with these hormone treatments or tissue-preparation procedures, more studies are needed to investigate the regulatory mechanisms by which the autonomous accumulation of MMP-19 mRNA occurs in cultured theca-interstitial cells to uncover the in vivo regulation of MMP-19 mRNA in theca cells of preovulatory follicles.
The CL undergoes dynamic morphological and functional changes during luteal development and regression, both of which require extensive and controlled tissue remodeling. Previous studies have shown distinct expression patterns of mRNA for several MMPs and tissue inhibitors of MMPs (TIMPs), such as gelatinase A, collagenase-3, MT1-MMP, and TIMP-1, during the period of luteal formation and/or regression in the rat ovary [39, 40]. The current in situ hybridization data are, to our knowledge, the first to document changes in the cellular localization pattern of MMP-19 mRNA expression at different stages of luteal development in both gonadotropin-induced pseudopregnant and cycling rat ovaries. The highest expression of MMP-19 mRNA was localized to the regressing CL. This finding corresponds to results from our Northern blot analysis, which showed increased levels of mRNA for MMP-19 in rat ovaries obtained at the late stage of gonadotropin-induced pseudopregnancy, the time when functional luteal regression is evident [41]. Although the mechanisms responsible for the up-regulation of MMP-19 in regressing CL have yet to be determined, the present finding of high MMP-19 mRNA expression in the regressing CL suggests that MMP-19 may play a role in luteal demise. The present results also provide evidence for species difference between rats and mice, in that MMP-19 mRNA expression was up-regulated in periovulatory follicles and regressing CL in rat ovaries but was detected only in periovulatory follicles [19], not in CL of adult pseudopregnant ovaries [42], in mice.
In conclusion, MMP-19 mRNA expression is transiently increased in growing follicles and periovulatory follicles as well as in the regressing CL in rat ovaries. The present experimental data show that the transient up-regulation of MMP-19 mRNA in periovulatory follicles can be mimicked in vitro by exposing granulosa cells to a luteinizing dose of LH, suggesting a direct involvement of LH on MMP-19 mRNA accumulation in granulosa cells of periovulatory follicles. Further studies will be needed to identify specific substrates for MMP-19 in ovarian tissues and, hence, to reveal the precise roles of this MMP during follicular development, the ovulatory process, and luteolysis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Misung Jo, Department of Obstetrics and Gynecology, Chandler Medical Center, 800 Rose Street, Room MS 331, University of Kentucky, Lexington, Kentucky 40536-0298. FAX: 859 323 3761; mjo2{at}uky.edu
Received: 9 May 2004.
First decision: 28 May 2004.
Accepted: 15 July 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the rat. J Reprod Fertil Suppl 1989 37:233-240[Medline] knockout mouse uterus. Proc Natl Acad Sci U S A 1999 96:3646-3651This article has been cited by other articles:
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  L. L Espey, R. A Garcia, H. Kondo, B. Ishizuka, S. Yoshioka, S. Fujii, S. Hampton, and J. S Richards Expression of paralogs of cytochrome P45021a1 pseudogene (Cyp21a1-ps) and endogenous retrovirus SC1 (SC1) in the rat ovary during the ovulatory process J. Endocrinol., July 1, 2008; 198(1): 231 - 241. [Abstract] [Full Text] [PDF]
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 H Kliem, H Welter, W D Kraetzl, M Steffl, H H D Meyer, D Schams, and B Berisha Expression and localisation of extracellular matrix degrading proteases and their inhibitors during the oestrous cycle and after induced luteolysis in the bovine corpus luteum Reproduction, September 1, 2007; 134(3): 535 - 547. [Abstract] [Full Text] [PDF]
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 D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]
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D. C Woods and A L Johnson Protein kinase C activity mediates LH-induced ErbB/Erk signaling in differentiated hen granulosa cells Reproduction, April 1, 2007; 133(4): 733 - 741. [Abstract] [Full Text] [PDF]
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 A. P. Hall, F. R. Westwood, and P. F. Wadsworth Review of the Effects of Anti-Angiogenic Compounds on the Epiphyseal Growth Plate Toxicol Pathol, February 1, 2006; 34(2): 131 - 147. [Abstract] [Full Text] [PDF]
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 F. X. Donadeu and M. Ascoli The Differential Effects of the Gonadotropin Receptors on Aromatase Expression in Primary Cultures of Immature Rat Granulosa Cells Are Highly Dependent on the Density of Receptors Expressed and the Activation of the Inositol Phosphate Cascade Endocrinology, September 1, 2005; 146(9): 3907 - 3916. [Abstract] [Full Text] [PDF]
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 K. Ogiwara, N. Takano, M. Shinohara, M. Murakami, and T. Takahashi Gelatinase A and membrane-type matrix metalloproteinases 1 and 2 are responsible for follicle rupture during ovulation in the medaka PNAS, June 14, 2005; 102(24): 8442 - 8447. [Abstract] [Full Text] [PDF]
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