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Ovary |
Departments of Animal Science3
Physiology,4 Laboratory of Mammalian Reproductive Biology and
Genomics,5 Michigan State University, East Lansing, Michigan 48824-1225
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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follicle, granulosa cells, ovary, ovulation, theca cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The matrix metalloproteinases (MMPs) are a large family of at least 25 metal dependent enzymes that play a role in cell remodeling, extracellular matrix (ECM) degradation, and tissue repair [3, 4]. The collagenases (MMP-1, MMP-8, MMP-13, MMP-18) are best noted for their ability to degrade fibrillar collagens, such as type I and III collagen [5]. Ovulation is dependent on weakening of the type I and III collagen-rich ECM in the theca externa and tunica albuginea layers of the preovulatory follicle wall [1], and there is evidence that selective collagen degradation occurs at the apex of the follicle [6, 7]. Experiments in rats and sheep indicate that preovulatory follicular collagenolytic activity is increased after the gonadotropin surge [7, 8]. This activity is most likely attributable to members of the collagenase subfamily of MMPs. Therefore, hormonal regulation of specific members of the collagenase subfamily of MMPs may play a key role in mediating follicle wall degradation prior to ovulation. As an initial step toward understanding the regulation and potential role of specific collagenases in follicle rupture, we determined the intrafollicular localization and effect of the preovulatory gonadotropin surge on MMP-1 and MMP-13 mRNA and protein in bovine preovulatory follicles.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All procedures described where animals were used were approved by the All University Committee on Animal Use and Care at Michigan State University (Protocol #04/98-056-00 and #03/01-051-00).
Experimental Model and Tissue Collection
Follicular development and timing of the preovulatory gonadotropin surge were synchronized in Holstein cows using the Ovsynch (GnRH-7d-PGF2
-36h-GnRH) procedure [9, 10]. Briefly, GnRH is injected to start a new wave of follicular growth and thus a new dominant follicle. Seven days later, PGF2 is given to regress corpora lutea (CL). A second GnRH injection is given to induce a gonadotropin surge resulting in ovulation of the dominant follicle an average of 28 h later [9]. Daily ultrasound analyses were performed after the first GnRH injection until the time of follicle collection to verify follicle synchrony and to exclude animals that turned over a new follicular wave prior to the second GnRH injection. Ovaries containing ovulatory follicles or new CL were collected by colpotomy at 0, 6, 12, 18, 24, and 48 h (CL) after the second GnRH injection (n = 5– 6 per time point).
For mRNA quantification and Western blot analysis, ovaries containing the ovulatory follicle or new CL (mRNA only) were collected at 0, 6, 12, 18, 24, and 48 h (n = 5–6 per time point) following the second GnRH injection. Follicles were dissected and processed as described previously [11].
Collection of tissues for immunohistochemical analyses was as follows. Crossbred beef heifers (n = 6) received two injections of PGF2 (25 mg, 11 days apart) to synchronize estrus. Daily ultrasound analyses were performed from the time of the second PGF2 injection until the time of follicle collection to map initiation of the first follicular wave and identify the dominant follicle. On Day 6 postestrus, animals received two injections of PGF2 (15 mg each, 12 h apart) to regress the CL. Approximately 30 h after the first PGF2 injection on Day 6, animals were treated with 100 µg GnRH to induce a gonadotropin surge. Ovaries containing the ovulatory follicle were collected by colpotomy at 0 and 20 h after GnRH injection (n = 3 each). Ovulatory follicles were dissected from the ovary, cut into 4- to 5-mm2 pieces, fixed overnight in neutral buffered formalin, and embedded in paraffin.
Generation of cDNAs
Bovine complementary DNAs (cDNA) were generated by the reverse transcription polymerase chain reaction (RT-PCR) using bovine CL RNA and 
20 nucleotide primers that correspond to 100% conserved sequences present in the nucleotide sequence of analogous cDNAs. To generate MMP-1 and MMP-13 cDNAs, a 356-base-pair (bp) partial cDNA encoding for MMP-1 (corresponding to nucleotides 576–931 of bovine MMP-1 cDNA [Genbank accession #X58256]) and a 376-bp partial cDNA encoding for MMP-13 (corresponding to nucleotides 872–1247 of bovine MMP-13 cDNA [Genbank accession #AF072685]) were amplified, ligated into pBluescript plasmids (Stratagene, La Jolla, CA) and subjected to fluorescent dye terminator sequencing to verify identity. A second 334-bp MMP-1 partial cDNA encoding a portion of MMP-1 upstream of the previously mentioned MMP-1 cDNA (corresponding to nucleotides 119–452 of bovine MMP-1 cDNA [Genbank accession #X58256]) was also generated for use in subsequent experiments.
Characterization of MMP-1 and MMP-13 mRNA
Total RNA was isolated using the Trizol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's instructions. Poly A+ RNA was isolated from total RNA using a mRNA isolation kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions to increase sensitivity for MMP-13 Northern blots. To determine transcript size and number and to optimize specificity of hybridization conditions, approximately 15 µg pooled total RNA or 6 µg poly A+ RNA (MMP-13) from each sample analyzed were subjected to electrophoresis through 1% agarose-formaldehyde gels. RNA was then capillary transferred to nylon membranes (BioRad, Richmond, CA) and UV cross-linked. For quantification of MMP-1 mRNA abundance, 6 µg total RNA isolated from each sample were spotted in duplicate onto nylon membranes (BioRad) using a dot blot apparatus (BioRad). Membranes were allowed to dry briefly and then UV cross-linked.
Membranes were prehybridized at 42°C overnight in either 50% formamide, 5x SSC (saline-sodium citrate buffer; 1x SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0), 5x Denhardt's (1x is 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 50 mM NaPO4, 0.1% SDS, and 250 µg herring sperm DNA/ml prehybridization buffer (MMP-1) or Ambion Ultrahyb (Ambion, Austin, TX) containing 250 µg herring sperm DNA/ml (MMP-13). Subsequent hybridizations took place at 42°C for 18 h in 50% formamide, 5x SSC, 1x Denhardt's, 20 mM NaPO4, 0.1% SDS, 10% dextran sulfate (MMP-1), or Ambion Ultrahyb (MMP-13) with the addition of 100 µg herring sperm DNA/ml hybridization buffer and >1 x 106 cpm/ml 32P-labeled cDNA. Specific 32P-labeled MMP-1, MMP-13, and ribosomal protein L-19 (RPL-19) cDNA probes were prepared by PCR using procedures described previously [12]. RPL-19 was used for normalization purposes. Membranes were washed in 1x SSC, 0.1% SDS, 0.1% sodium pyrophosphate for 20 min at 42°C followed by one 20-min wash in 0.1x SSC, 0.1% SDS, 0.1% sodium pyrophosphate at 42°C, one 20-min wash at 47°C, and one 20-min wash at 50°C in the same buffer. Following washing, filters were exposed to a PhosphorImager cassette. After exposure (2–24 h) the cassette was scanned using a PhosphorImager (BioRad). After Northern analyses, size of RNA transcripts was determined based on relative migration of RNA molecular weight markers (Roche). After hybridizations for MMP-1 and MMP-13, the membranes were then stripped and reprobed with the 32P RPL-19 cDNA. Previous experiments demonstrated that RPL-19 mRNA abundance in bovine preovulatory follicles and new CL is not regulated by the gonadotropin surge [12]. Relative densitometric units for MMP-1 were quantitated and adjusted relative to RPL-19 mRNA expression using Molecular Analyst Version 1.5 software (BioRad). Preliminary Northern blot experiments demonstrated that hybridization and washing conditions used in subsequent dot blot analyses were specific and yielded hybridization to distinct transcripts of appropriate size for each mRNA of interest. Preliminary experiments also demonstrated that an increase in hybridization intensity was detected following hybridization of each cDNA to increasing amounts of sample RNA (1–10 µg).
Relative levels of MMP-13 mRNA in periovulatory follicular and luteal RNA samples were determined by semiquantitative multiplex RT-PCR analysis as described by Kurebayashi et al. [13] with modifications described here. A 1-µg aliquot of each RNA sample was treated with RNase-free DNase I (Life Technologies), and a 91-ng fraction was utilized in first-strand cDNA synthesis with Superscript II reverse transcriptase according to the manufacturer's instructions. Synthesized cDNA was then used as a template for coamplification of a 245-bp partial cDNA for bovine MMP-13 and a 360-bp cDNA encoding for bovine RPL-19. Negative control cDNA synthesis reactions were conducted in the absence of reverse transcriptase and used as template in PCR to verify absence of genomic DNA contamination. Ratios of primer sets between MMP-13 and RPL-19 and number of cycles were determined to amplify both products logarithmically at relatively similar efficiencies without competition. PCR reactions for each sample were carried out in duplicate. A portion of each reaction was run on an agarose gel, and following ethidium bromide staining, intensity of amplified products was determined with image analysis software (Quantity One, BioRad). A linear relationship between amount of product loaded on the gel and signal detected with the image analysis program (below saturation) was established in preliminary experiments. Relative expression levels are reported as the density of the amplification product for MMP-13 divided by that obtained for RPL-19. Intra- and interassay coefficients of variation were <6%.
Western Blot Analysis of MMP-1 and MMP-13
Homogenates from the apex and base of preovulatory follicles (collected at 0, 6, 12, 18, and 24 h relative to GnRH injection to induce the preovulatory gonadotropin surge) were prepared separately as described previously [11] and used in Western blot experiments to quantify relative amounts of MMP-1 and MMP-13 protein in the apex and base of preovulatory follicles. The optimal protein amount for quantification was determined by Western analysis of increasing protein concentrations of pooled follicle homogenates from each time point. For each antibody used, an increase in signal intensity was obtained with increasing protein concentrations. Thirty micrograms of protein from each individual sample were separated on 10% SDS-polyacrylamide gels and then electroblotted to polyvinylidene fluoride (PVDF) membranes (Bio-Rad) at 100 V for 1 h. Membranes were blocked in 5% BLOT-QuickBlocker (GenoTech, St. Louis, MO) with 0.1% Tween-20 in TBST (150 mmol/L NaCl, 50 mmol/L Tris, 0.1% Tween-20, pH 7.5) for 1 h at room temperature, followed by incubation with primary antibody in 1 x femto TBST (GenoTech) with 0.5% BLOT-QuickBlocker overnight at 4°C. The concentration of primary antibody used for MMP-1 (rabbit anti-human polyclonal MMP-1 antibody; Chemicon, Temecula, CA), MMP-13 (rabbit anti-human MMP-13 polyclonal antibody; Chemicon), and actin (mouse anti-human actin monoclonal antibody; Chemicon) were 0.02 µg/ml, 0.04 µg/ml, and 1:500 000 (v/ v), respectively. After primary antibody incubation, membranes were washed five times for 5 min each in TBST, followed by incubation with goat anti-rabbit (1:2500 [v/v]; Amersham Biosciences UK, Buckinghamshire, England) or goat anti-mouse (1:10 000; GenoTech) peroxidase-conjugated antibodies for 1 h at room temperature. After four TBST washes, immunoreactive proteins were visualized using chemiluminescence HRP immunodetection system (GenoTech). After incubation with MMP-1 or MMP-13 antibodies, membranes were stripped using GenoTech Re-Probe buffer and then reprobed with the actin antibody. Relative molecular mass of immunoreactive proteins detected was determined based on relative migration of protein standards (Broad Range SDS PAGE Standards, Bio Rad). No signal was detected when duplicate membranes were incubated with equal amounts of rabbit IgG or mouse IgG. After development, films were scanned, and density of each band was analyzed using computer-aided densitometry. Relative densitometric units representing MMP-1 and MMP-13 immunoreactivity were adjusted relative to densitometric units for actin immunoreactivity. Densitometric units for the 40 000- and 38 000-Mr bands for MMP-1 were combined prior to statistical analysis because resolution was not always sufficient to reliably quantitate each band separately in each sample and/or because both bands were not always detectable in each sample.
Immunohistochemistry
Immunohistochemistry was performed using a VECTASTAIN Elite ABC Kit (Vector Labs, Burlingame, CA) according to the manufacturer's instructions. Briefly, approximately 5-µm serial sections were prepared and mounted onto 3-aminopropyltriethoxysilane-coated microscope slides. Paraffin sections were deparaffinized in xylene and then rehydrated in graded alcohol. Antigen retrieval was performed by boiling the sections in antigen retrieval citra solution (BioGenex Laboratories, San Ramon, CA) for 15 min and allowing the slides to cool for 30 min at room temperature. The slides were then treated for 10 min with 3% hydrogen peroxide in deionized water to eliminate endogenous peroxidase activity. The sections were then serially incubated with normal goat serum for 30 min at RT, mouse anti-human MMP-1 monoclonal antibody (100 µg/ml; Oncogene, Boston, MA) at 1:50 dilution or mouse anti-human MMP-13 monoclonal antibody (200 µg/ml; Chemicon) at 1:300 dilution for 1 h at RT, and biotinylated anti-mouse IgG for 30 min at RT, followed by incubation with ABC reagent for 30 min at RT. Intervening washes were performed after each antibody incubation. The sections were developed using a DAB Substrate Kit (Vector Labs) for 2–10 min and were then counterstained with VECTOR Hematoxylin QS (Vector labs) and mounted with Permount (Fisher Scientific, Fair Lawn, NJ). Parallel controls were set up including sections incubated without primary antibody or with equivalent amount of normal mouse IgG (Vector Labs).
Statistical Analyses
Differences in mRNA and protein abundance were determined by one-way ANOVA using the General Linear Models procedure of the Statistical Analysis System (SAS; Version 8.0). When heterogeneity of variance was observed, data were log transformed prior to statistical analysis. Individual comparisons of mean RNA and protein concentrations were performed using the Fisher protected least significant differences test, and results are reported as mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Relative abundance of MMP-1 mRNA in bovine periovulatory follicular and luteal tissue was regulated by the preovulatory gonadotropin surge (Fig. 1). The MMP-1 cDNA hybridized specifically to two MMP-1 mRNA species of 2.4 and 1.8 kilobases (kb) (Fig. 1A). Differential up-regulation of the 2.4- and 1.8-kb transcripts was observed. Expression of the 2.4-kb transcript was predominant at the 6- and 12-h time points (data not shown). In contrast, the 1.8-kb transcript was induced and predominant at the 48-h time point (in new CL), where abundance of the 2.4-kb transcript was low (Fig. 1A). Both MMP-1 transcripts were also detected when a second MMP-1 cDNA, located 5' to the original cDNA, was utilized as the probe in Northern analysis (data not shown). Relative abundance of MMP-1 mRNA (both transcripts combined, as determined by dot blot analysis) was greater at 6, 12, and 48 h compared to the 0-h (pregonadotropin surge) time point (P < 0.05).
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Expression of MMP-13 mRNA During the Periovulatory Period
Expression of MMP-13 mRNA was also regulated by the preovulatory gonadotropin surge (Fig. 2). The MMP-13 cDNA hybridized specifically to a 3.0-kb transcript (Fig. 2A). Relative levels of MMP-13 mRNA were low and thus determined using semiquantitative multiplex RT-PCR analysis (Fig. 2B). MMP-13 mRNA abundance was higher (P < 0.05) at 6, 24, and 48 h post-GnRH injection compared to the 0-h time point (Fig. 2C).
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Relative Amounts of MMP-1 and MMP-13 Proteins in the Apex and Base of Bovine Preovulatory Follicles
Western blot analysis was used to determine effects of the gonadotropin surge on relative abundance of MMP-1 and MMP-13 proteins in the apex and base of bovine preovulatory follicles (Fig. 3). The MMP-1 antibody specifically recognized two prominent immunoreactive proteins of 40 000 and 38 000 Mr present in bovine preovulatory follicle extracts (Fig. 3, A and B). Additional minor bands of 52 000 and 49 000 Mr were also detected (data not shown). Relative amounts of MMP-1 protein in the apex of preovulatory follicles were transiently increased at 6 h post-GnRH injection (Fig. 3A; P < 0.05). Relative amounts of MMP-1 protein in the base of preovulatory follicles were also increased (Fig. 3B; P < 0.05) in response to the gonadotropin surge. The increase in MMP-1 protein was noted at 6, 12, and 18 h post-GnRH injection and thus of longer duration than that observed in the follicular apex.
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In contrast to MMP-13 mRNA abundance, which was low, MMP-13 protein was readily detectable in homogenates of the apex and base of preovulatory follicles. The MMP-13 antibody specifically recognized a single immunoreactive protein of 42 000 Mr. Relative amounts of MMP-13 protein were also increased in the apex and base of preovulatory follicles exposed to the gonadotropin surge (Fig. 3, C and D; P < 0.05). Specifically, MMP-13 protein was increased in the apex of follicles collected 24 h after GnRH injection (Fig. 3C), whereas MMP-13 protein was elevated in the base of preovulatory follicles collected at all four time points after the gonadotropin surge (Fig. 3D).
Intrafollicular Localization of MMP-1 and MMP-13 Proteins
MMP-1 and MMP-13 immunoreactivities were localized in bovine preovulatory follicle sections collected at 0 and 20 h relative to injection of GnRH to elicit a gonadotropin surge (Fig. 4). The intrafollicular cell types for which each MMP was localized were consistent between the two time points. We did not attempt to quantify changes in intensity of staining in the various cell types in response to the gonadotropin surge. MMP-1 immunoreactivity was localized to both the granulosal and the thecal layers of bovine preovulatory follicles collected at 0 h (Fig. 4, A and C) and 20 h (Fig. 4B) relative to GnRH injection, with minimal specific staining detected in the adjacent ovarian stroma. MMP-13 immunoreactivity was also detected in the granulosal and thecal layers, with more prominent staining detected in the thecal layer (Fig. 4, E–G). A low but detectable level of staining for MMP-13 was also detected in the adjacent ovarian stroma. No significant staining was detected when sections were incubated in the absence of primary antibody (data not shown) or when sections were incubated with appropriate concentrations of normal mouse IgG (negative control; Fig. 4, D and H).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, MMP-1 mRNA abundance was increased in response to the preovulatory gonadotropin surge, subsequently declined prior to ovulation, but was increased in new CL collected approximately 48 h after GnRH administration (20 h after expected time of ovulation [9]). Up-regulation of interstitial collagenase mRNA in PMSG-stimulated rat ovaries following administration of an ovulatory stimulus has also been reported [16], with a similar relative temporal expression pattern prior to ovulation as denoted in the present studies. In contrast, MMP-1 mRNA abundance in monkey preovulatory granulosal cells is also elevated after administration of an ovulatory dose of hCG [17], but levels remain high in samples collected near the time of ovulation (36 h post-hCG administration). While up-regulation of MMP-1 mRNA is also seen during the ovulatory process in the previously mentioned species, observation of two distinct MMP-1 mRNA transcripts has not been reported previously. A 1.7-kb transcript is present in rat ovaries collected during the preovulatory period [16]. In the present studies, the 2.4-kb bovine MMP-1 transcript was increased during the preovulatory period, whereas the 1.8-kb transcript was undetectable at all preovulatory time points but was up-regulated in new CL collected 48 h following the gonadotropin surge. While there is evidence of posttranscriptional regulation of MMP-1 mRNA (stability) in other systems [18], additional studies will be required to precisely delineate the nature of the two MMP-1 transcripts identified.
MP-13 mRNA expression was also increased in bovine preovulatory follicles following the gonadotropin surge. More specifically, MMP-13 mRNA abundance was maximal at 6 and 24 h post-GnRH injection but reduced at 48 h (new CL) relative to the 24-h time point, indicating that temporal expression of MMP-13 mRNA in bovine preovulatory follicles is distinct from that observed for MMP-1. Preovulatory regulation of MMP-13 mRNA has been examined previously in mouse and rat ovaries. In contrast to results observed in the bovine model, MMP-13 mRNA is undetectable by Northern analysis of preovulatory mouse ovarian RNA [19]. Messenger RNA for MMP-13 is highly expressed in the rat ovary, particularly on proestrus and estrus [20]. Expression of MMP-13 mRNA is also increased (48 h post-eCG administration) during rat folliculogenesis [21]. To our knowledge, up-regulation of MMP-13 mRNA in rat ovaries in vivo in response to an ovulatory stimulus remains controversial. Balbin et al. [20] did not find evidence of up-regulation of MMP-13 mRNA in ovaries of eCG-treated rats following hCG administration. However, MMP-13 mRNA was increased in perfused rat ovaries treated with LH and IBMX [22]. Our results clearly demonstrate that MMP-13 mRNA expression in bovine preovulatory follicles is increased in response to the gonadotropin surge.
Ovulation is dependent on localized degradation of the type I and III collagen-rich ECM present in the theca externa/tunica albuginea regions of the preovulatory follicle wall [1]. The observed intrafollicular localization of MMP-1 and MMP-13 in the thecal layer of bovine preovulatory follicles is consistent with a potential role for each enzyme in collagenolysis and the ovulatory process. In the present study, MMP-1 immunoreactivity was localized to both the granulosal and the thecal cell layers of bovine preovulatory follicles. In rabbits, MMP-1 was localized primarily to the thecal cells, interstitial tissue, and germinal epithelium of rabbit follicles throughout the preovulatory period, but staining increased at the apex of preovulatory follicles near the onset of ovulation and in the granulosal layer of newly ovulated follicles [23]. Immunoreactivity for MMP-13 was localized to both the thecal and the granulosal cells of bovine follicles in the present study but was more prominent in the thecal layer. MMP-13 was also localized primarily to the thecal layer and interstitial cells of rat ovaries [20]. The potential direct role of granulosal cell-derived MMP-1 and MMP-13 in the ovulatory process is not clear. However, a contribution to breakdown of the follicular basement membrane is possible. Both MMP-1 and MMP-13 can potentially cleave proteoglycan components of the basement membrane, and type IV collagen is a substrate for MMP-13 [24].
Relative amounts of MMP-1 protein were increased in both the apex and the base of bovine preovulatory follicles following the gonadotropin surge. The significance of the distinct 40 000- and 38 000-Mr immunoreactive forms of MMP-1 detected are unclear. Autolytic fragments of human collagenase with differing enzymatic activities have been described, but such fragments are of considerably smaller relative molecular mass than observed in the present studies [25]. Additional faint immunoreactive proteins of 52 000 and 49 000 Mr were also detected in the present studies. Such higher Mr proteins presumably correspond to the proforms of MMP-1 [25] but were present at levels too low to allow quantification. The significance of the transient increase in MMP-1 protein in the apex of bovine preovulatory follicles at 6 h after GnRH injection also is currently unclear. Morphological evidence of ECM degradation is generally not detectable until closer to the expected time of ovulation, although evidence of cell disassociation was noted in rat preovulatory follicles as early as 5 h after hCG stimulation [26]. Contributions of increased MMP-1 protein independent of collagenolysis cannot be excluded from consideration. MMP-1 can cleave noncollagenous components of the ECM, proteinase inhibitors, cytokines, and growth factor-binding proteins and can activate other MMPs [4, 27]. Determination of the exact contribution of MMP-1 to the ovulatory process in cattle will require further investigation.
Relative amounts of MMP-13 protein were also increased in both the apex and the base of bovine preovulatory follicles following the gonadotropin surge. To our knowledge, regulation of MMP-13 protein in response to the gonadotropin surge has not been reported previously. The increased MMP-13 protein (42 000 Mr) presumably corresponds to the active form of the enzyme [28]. In the follicular apex, MMP-13 protein was elevated at the 24-h time point, approximately 4 h before the expected time of ovulation. This result is consistent with a potential contribution of MMP-13 to the follicular collagenolysis characteristic of the ovulatory process. However, MMP-13 protein was also elevated in the base of preovulatory follicles at 6 h post-GnRH injection and remained elevated through the 24-h time point. Unfortunately, 48-h (new CL) samples for protein analysis were not collected in the present studies, so it is unclear whether increased amounts of MMP-13 protein are maintained in new CL. The difference in temporal regulation of MMP-13 protein in the apex versus the base of preovulatory follicles can most likely be attributed to the higher basal MMP-13 expression observed in the apex at the 0-h (pregonadotropin surge) time point. A potential role for MMP-13 in mediating the ECM remodeling characteristic of follicular growth and expansion has been proposed previously [21].
Results of the present studies demonstrate that both MMP-1 and MMP-13 are regulated by the preovulatory gonadotropin surge and thus support a potential contribution of both MMPs to the ovulatory process. However, results do not support a requirement for differential spatial up-regulation of MMP-1 or MMP-13 (in the follicular apex vs. the base) in the preovulatory collagenolysis characteristic of the ovulatory process. Previous studies demonstrated that preovulatory collagenase activity is regulated by the gonadotropin surge. Collagenolytic activity is increased in rat ovaries [8, 29, 30] and sheep preovulatory follicles [7] in response to an ovulatory stimulus, but the specific MMPs that mediate the preovulatory increase in collagenolytic activity generally are not known. Furthermore, there is evidence that selective collagen degradation occurs at the apex of the follicle, which could be attributable to collagenase activity. Espey showed that the follicle wall becomes thinner and the concentration of collagen fibrils is decreased at the apex as ovulation approaches [6]. Results of electron microscopic analysis of sheep follicles showed a decrease in collagen fibrils at the apex 24 h after a GnRH-induced gonadotropin surge [7]. In the present studies, both MMP-1 and MMP-13 proteins were increased (in bovine preovulatory follicle homogenates) in response to the preovulatory gonadotropin surge. However, we found no evidence of up-regulation of MMP-1 and MMP-13 specifically in the apex of bovine preovulatory follicles. Increased amounts of MMP-1 and MMP-13 proteins were also detected in homogenates of the base of preovulatory follicles. Thus, differential regulation of MMP-1 and MMP-13 expression within the apex versus the base of preovulatory follicles likely does not explain the spatial regulation of ECM proteolysis at the follicular apex characteristic of the ovulatory process. Furthermore, we cannot infer whether the increases in MMP-1 and MMP-13 protein detected in response to the gonadotropin surge were derived from the thecal layer, granulosal layer, or both.
In summary, results of the present studies indicate that the preovulatory gonadotropin surge results in increased MMP-1 and MMP-13 mRNA and protein expression in bovine preovulatory follicles and that the temporal regulation of each MMP during the preovulatory period is distinct. Further studies will be required to determine the specific intrafollicular signaling pathways that mediate the gonadotropin surge-induced increases in MMP-1 and MMP-13 mRNA and protein and whether gonadotropin surge-induced up-regulation of each MMP is obligatory for follicle rupture.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: George W. Smith, Department of Animal Science, Michigan State University, 1230D Anthony Hall, East Lansing, MI 48824-1225. FAX: 517 353 1699; smithge7{at}msu.edu
Received: 1 January 2004.
First decision: 22 January 2004.
Accepted: 9 April 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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