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Regular Article |
a Department of Obstetrics and Gynecology, University of Kentucky, Lexington, Kentucky 40536
ABSTRACT
The matrix metalloproteinase (MMP) system consists of a proteolytic component, the metalloproteinases, and an associated class of tissue inhibitors of metalloproteinases (TIMPs). We investigated the cellular localization of the TIMPs and the gelatinase family of MMPs throughout the latter stages of follicular growth and during the periovulatory period. Immature female rats were injected with eCG, and ovaries were collected at the time of eCG administration (0 h) and at 6, 12, 24, or 36 h after eCG injection (i.e., follicular development group). A second group of animals (periovulatory) was injected with eCG followed by hCG 48 h later, and ovaries were collected at 0, 12, and 24 h after hCG. Ovaries were processed for the cellular localization of gelatinase or TIMP mRNA or gelatinolytic activity. Gelatinase mRNA (MMP-2 and MMP-9) was localized to the theca of developing follicles and to the stroma. Following a hCG stimulus, MMP-2 mRNA increased as the granulosa cells of preovulatory follicles underwent luteinization during formation of the corpus luteum (CL). MMP-9 mRNA remained predominately in the theca during this period. In situ zymography for gelatinolytic activity demonstrated a pattern of activity that corresponded with the localization of MMP-2 and MMP-9 mRNA around developing follicles. Gelatinolytic activity was observed at the apex of preovulatory follicles and throughout the forming CL. The mRNA for TIMP-1, -2, and -3 was localized to the stroma and theca of developing follicles. TIMP-3 mRNA was present in the granulosa cells of certain follicles but was absent in granulosa cells of adjacent follicles. At 12 h after hCG, luteinizing granulosa cells expressed TIMP-1 and TIMP-3 mRNA, but TIMP-2 mRNA was at levels equivalent to the background. In the newly forming CL at 24 h after hCG administration, the luteal cells expressed TIMP-1, -2, and -3 mRNA, although the pattern of cellular expression was unique for each of the TIMPs. These findings demonstrate that the MMPs and TIMPs are in the cellular compartments appropriate for impacting the remodeling of the extracellular matrix as the follicle grows, ovulates, and forms the CL.
corpus luteum, follicle, interstitial cells, ovary, ovulation
INTRODUCTION
Initiation of follicular growth at the primordial follicle stage begins with activation and proliferation of the granulosa cells, differentiation of a thecal cell layer from the ovarian stroma, and deposition of a basement membrane between the granulosa and thecal compartments. With continued growth and development, the resulting mature graafian follicle is approximately 400-fold larger than its original primordial follicle progenitor. The follicle rests in an extracellular environment composed, in part, of collagen, laminin, and fibronectin [1–4]. To accommodate the dynamic and extensive growth of the follicle, there must be concomitant remodeling of the granulosa cell basement membrane and the extracellular matrix surrounding the follicle. During the ovulatory process, extensive remodeling is again apparent in the dissolution of the granulosa cell basement membrane [5, 6] and fragmentation of the extracellular matrix of the follicular wall [7–10], allowing oocyte release. Following ovulation, the ruptured follicle is transformed into a corpus luteum (CL) by extensive cellular reorganization and neovascularization as the fluid-filled antral cavity is infiltrated by blood vessels, fibroblasts, and thecal and granulosa cells [11–13]. We hypothesized that this follicular growth, follicular rupture, and early luteal formation occurs, in part, through the action of the matrix metalloproteinases (MMPs) and their inhibitors.
The MMP system is involved in connective tissue remodeling processes throughout the body. This system comprises both proteolytic enzymes and their associated inhibitors. The proteolytic component of this system, the MMPs, consists of over 20 structurally related enzymes and includes four major classes: the collagenases, the gelatinases, the stromelysins, and the membrane-associated metalloproteinases, as well as enzymes with characteristics that preclude their inclusion within these four classes [14, 15]. The gelatinase class consists of two distinct MMPs, MMP-2 or 72-kDa gelatinase and MMP-9 or 92-kDa gelatinase. These enzymes have a potent ability to bind to and cleave gelatin and therefore act to degrade major constituents of basement membranes, including type IV collagen, laminin, and fibronectin.
The activity of MMPs is rigorously controlled at multiple levels, including inhibition of enzyme activity in the extracellular space by MMP inhibitors. Two major classes of MMP inhibitors are generally distinguished, serum borne and tissue derived [14, 16, 17]. The serum-borne inhibitors include the macroglobulins, such as 
2-macroglobulin, which have broad proteolytic inhibitor capacity. Although the macroglobulins are typically classified as serum-derived inhibitors, they are produced and found in the ovary [18, 19]. The second class of inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are locally produced and share similarities in their structural conformation. These similarities include internal disulfide bonds that are important for the overall protein structure and a highly conserved N-terminal domain critical for functional MMP inhibition [16, 17, 20]. Four distinct TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) have been identified based on their molecular weight and biological action. These inhibitors differ in their regulation, enzyme specificity, and mode of action [16, 17]. TIMP-1 is a secreted glycoprotein that binds to the active form of MMPs and to the latent form of MMP-9. TIMP-2 is also a secreted inhibitor, has been shown to be differentially regulated from TIMP-1, and is proposed to act selectively on different MMPs [16, 17, 21]. For example, TIMP-2 has a high affinity for the latent and active forms of MMP-2 and plays a critical role in the activation of this MMP. Unlike TIMP-1 or TIMP-2, TIMP-3 is secreted and then bound to the extracellular matrix. TIMP-3 has been suggested to act as an additional regulatory stop point for MMP action [22, 23]. TIMP-4 has recently been cloned, and preliminary studies suggest that it has many traits similar to those of TIMP-2 [17, 24]
In the ovary, MMPs and TIMPs have been postulated to play a critical role in extracellular matrix remodeling associated with ovulation and luteal formation and regression [25]. Little is known, however, about the cellular localization of the MMP system during this periovulatory period [26, 27]. We hypothesized that MMPs and their inhibitors would be localized to areas where extracellular matrix remodeling occurs during the extensive cellular proliferation, angiogenesis, and growth of the follicle [1, 2]. In support of this postulate is the observation that mRNA expression levels for the MMPs [28] and TIMPs [29] change in association with gonadotropin-induced follicular development. To test this hypothesis, we induced follicular growth in immature rats and examined the changes in gelatinase and TIMP cellular localization during follicular growth, follicular differentiation associated with an ovulatory stimulus, and early luteal formation.
MATERIALS AND METHODS
Materials
Equine CG was graciously supplied by Dr. A.F. Parlow and the National Hormone and Pituitary program. The fluorescein-labeled gelatin substrate was a generous gift from Dr. Stephen Palmer (RW Johnson Pharmaceutical Research Division, Johnson & Johnson Laboratories, Raritan, NJ). Murine cDNAs for MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 were kindly provided by Dr. Dylan Edwards (University of East Anglia, Norwich, England). All chemicals were from Sigma Chemical Co (St. Louis, MO) except where otherwise noted.
Animals
Immature female Sprague-Dawley rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and kept in environmentally controlled conditions under the supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee. Rats were maintained on a 14L:10D cycle and provided water and rat chow ad libitum. Between 0900 and 1000 h on Day 23 of age, rats were injected s.c. with 10 IU of eCG to induce follicular development. In one group (follicular development), animals were killed at the time of eCG administration (0 h) and at 6, 12, 24, or 36 h after injection of eCG. A second group of animals (periovulatory) was injected with eCG and then with hCG (10 IU) 48 h later. Rats were killed at the time of hCG administration (0 h) and at 12 and 24 h after hCG. At necropsy, the ovaries were removed, cleaned, weighed, and snap frozen in optimal cutting temperature medium (VWR Scientific, South Plainfield, NJ) for either 1) the cellular localization of gelatinase or TIMP mRNA or 2) the localization of gelatinolytic activity.
In Situ Hybridization
In situ hybridization was performed using plasmids containing murine MMP-2, MMP-9, TIMP-1, TIMP-2, or TIMP-3 cDNA. Plasmids were linearized using the appropriate restriction enzymes. The antisense and sense riboprobes for the MMPs and TIMPs were synthesized from the corresponding linearized plasmid and labeled with [-35S]rUTP (ICN, Costa Mesa, CA) using the Maxiscript in vitro RNA transcription kit (Ambion, Austin, TX). After cRNA synthesis, the probes were purified over G-50 Sephadex Quick Spin Columns (Roche Molecular Biochemicals, Indianapolis, IN).
Ovaries were sectioned as serial sections at 8–10 µm and mounted on Probe-On Plus slides (Fisher Scientific, Pittsburgh, PA). Tissues were fixed in 4% paraformaldehyde in PBS and then sequentially washed in PBS, 0.75% glycine, PBS, and 1.5% triethanolamine with 0.25% acetic anhydride before being dehydrated. Each MMP and TIMP probe was allowed to hybridize overnight in hybridization buffer (50% formamide, 10% dextran sulfate, 20 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 8.0, 300 mM NaCl, 1x Denhardt solution, 0.1 mg/ml salmon sperm DNA, 0.25 mg/ml of yeast total RNA, 0.25 mg/ml of yeast tRNA, 0.1% SDS, 0.1% sodium thiosulfate, and 100 mM dithiothreitol) containing probe at 1 x 106 cpm/slide in a humidified chamber at 60°C. Approximately 18–20 h later, slides were washed extensively to remove nonspecifically bound riboprobe. Tissues were washed with 2x standard saline citrate (SSC) buffer (1x SSC = 0.15 M NaCl and 15 mM sodium citrate; all washes in SSC also contained 10 mM sodium thiosulfate), followed by RNase A treatment (100 µg/ml in Tris-EDTA buffer) for 30 min at 45°C. Slides were then washed in 0.2x SSC, followed by a wash in 0.1x SSC for 1 h at 60°C before rinsing in deionized H2O, dehydrating in ethanol, and air drying. Sections were processed for autoradiography using Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) and stored at 4°C for 3–6 wk. For visualization of the in situ reaction product, slides were developed in Kodak D19 (1:1) and stained with Gill formulation no. 2 hematoxylin solution (Fisher Scientific). Tissues were examined with an Eclipse E800 Nikon microscope (Nikon Corp., Melville, NY) under bright- and darkfield optics. A sense riboprobe, used as a control for nonspecific binding, was included for each ovary and each time point for the different MMP or TIMP riboprobes.
One ovary from each of three of the animals was used for in situ hybridization. For each MMP or TIMP, 16 tissue sections per ovary were analyzed, making a total of 48 tissue sections analyzed for each time point for each mRNA examined. Four sections per ovary were analyzed using each MMP or TIMP sense probe so that a total of 16 sections/time point were examined.
Morphometric Analysis
To determine the changes in mRNA expression patterns, morphometric analyses were performed using Metamorph software (version 4.1.5; Universal Imaging Corp., West Chester, PA). Images of the in situ hybridization reaction were captured, and the image was thresholded using the Metamorph imaging system such that the overall background of silver grains in areas not containing tissue was subtracted from the entire image. The remaining mRNA in situ reaction product was then calculated in the granulosa cell layer, theca cell layer, or luteal cells with the Metamorph region tools and regions statistics software, which allow the granulosa, thecal, or luteal cell compartments to be outlined (trace region tool) and the amount of silver grains (representing the in situ reaction product) within the outlined compartment to be calculated. The mRNA in situ reaction product within the identified granulosa, theca, or luteal compartment was expressed as a percentage of the outlined area occupied by reaction product (percentage thresholded area in the regions statistics software). Thus, the outlined compartments with high levels of mRNA expression will be reflected by a higher percentage of the area occupied by the in situ reaction product. A preliminary experiment was performed to determine whether the changes in mRNA expression observed visually were correlated with the results of the morphometric analysis. Expression of mRNA for MMP-2 demonstrated no visible change in expression pattern in the granulosa and theca across follicular development (i.e., 0, 12, 24, 36, and 48 h post-eCG); this finding was confirmed by morphometric analysis (data not shown). Similarly, luteal expression of MMP-2 increased, as determined both observationally and morphometrically (Table 1). Thus, we analyzed only those mRNA expression patterns that demonstrated marked visual changes, such as those seen during the periovulatory period. The total number of follicles or CL analyzed at each time period from the three different ovaries is indicated in Table 1. Homogeneity of variance for the in situ reaction product was assessed by the Levene test, and differences in mRNA expression subsequently were determined by one-way analysis of variance using SPSS software (version 10.0.5; SPSS Inc., Chicago, IL). Post hoc group comparisons were performed using the Student-Newman-Keuls procedure, with P < 0.05 considered significant.
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In Situ Zymography
Fluorescently labeled gelatin substrate (1 mg/ml) was mixed (1:1) with 1% agarose melted in Tris buffer (50 mM Tris-HCl, pH 7.4, 10 mM CaCl2, and 0.05% Brij 35). The liquid gelatin mixture was spread on glass slides and allowed to gel at 4°C. Frozen sections (10 µm) of unfixed tissue were mounted on the gelatin substrate and coverslipped. Slides were incubated in a light-protected, humidified chamber at 37°C for approximately 18–20 h. Lysis of the substrate was assessed by examination with a fluorescent microscope. Enzymatic activity activates the quenched fluorescent substrate producing areas of fluorescence that can be seen against a black background [30]. The specificity of the reaction was determined by incorporating the MMP inhibitor ilomastat (Chemicon International, Temecula, CA) or EDTA (50 or 100 mM) with the gelatin substrate, which blocked MMP action.
RESULTS
In Situ Hybridization for MMP-2
Ovaries collected at the time of eCG administration demonstrated MMP-2 mRNA in situ reaction product in the theca interna and theca externa of developing follicles and in the connective tissue stroma outside of the theca externa between follicles (Fig. 1, A and B). In the hilar region, small luteinized follicles exhibited intense reaction product. These follicles have lost their granulosa cells and have undergone thecal hypertrophy [31]. During follicular growth (i.e., 0–48 h after eCG administration), MMP-2 mRNA continued to be present in the theca and stroma, with the appearance of mRNA in granulosa cells of large luteinizing follicles (Table 1). A representative photomicrograph of MMP-2 mRNA after 48 h of follicular growth is depicted in Figure 1, C and D. At all time points examined, expression of MMP-2 mRNA was greater in the theca than in the granulosa cell compartment (Fig. 1 and Table 1). Following an hCG stimulus, expression of MMP-2 mRNA was observed in the granulosa cells of preovulatory follicles, with a significant increase of MMP-2 in forming CLs at 24 h after hCG (Fig. 1, E and F, and Table 1). The difference in the appearance of MMP-2 mRNA expression between the two different CLs in Figure 1F may be related to the plane of section through the CL or the stage of luteal formation.
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In Situ Hybridization for MMP-9
The expression of MMP-9 mRNA at the time of eCG administration was localized primarily to the theca externa with expression also observed in the stroma. Intense expression of MMP-9 mRNA was observed associated with the vasculature in the hilar region (Fig. 2, A and B). With continued follicular growth following eCG, MMP-9 mRNA in situ reaction product remained in the theca and stroma with an intense band of hybridization encircling each follicle (Fig. 2, C and D). After hCG administration, the pattern of MMP-9 mRNA remained in the stroma encircling the developing CL (Fig. 2, E and F).
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In Situ Hybridization for TIMP-1
TIMP-1 mRNA in ovaries collected at the time of eCG administration was localized to the germinal epithelium, theca externa, and stroma and was especially abundant within the hilar region of the ovary (Fig. 3, A and B). During follicular growth (i.e., 0–48 h after eCG administration), the pattern of TIMP-1 expression remained in the germinal epithelium, the theca, and the stroma (only the follicular expression is depicted in Fig. 3, C and D). In situ reaction product in the oocyte and the granulosa cells approximated that observed outside of the tissue (i.e., background levels). In ovaries collected at 12 h after hCG administration, TIMP-1 mRNA was observed in the theca interna of putative preovulatory follicles (designated preovulatory based upon follicular size and morphology). Granulosa cells of these follicles exhibited moderate yet significant levels of TIMP-1 mRNA expression compared with granulosa cells of adjacent smaller antral follicles (Fig. 3, E and F, and Table 1). There was a dramatic increase in mRNA expression in luteinizing granulosa cells of the forming CL (Fig. 3, G and H, and Table 1). The forming CL also exhibited an intense reaction product encircling the CL.
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In Situ Hybridization for TIMP-2
TIMP-2 mRNA displayed a pattern of cellular localization similar to that of TIMP-1, with TIMP-2 mRNA observed in the theca externa and stroma during follicular growth (Fig. 4, A and B). One notable difference in TIMP-2 mRNA localization was that the expression in the theca included the theca interna, whereas TIMP-1 was confined to the theca externa. TIMP-2 mRNA was at background levels in the oocyte and granulosa cells. In large graafian follicles at 48 h after eCG, TIMP-2 mRNA remained in the thecal layer with low to background levels detected in the granulosa cells (Fig. 4, C and D). The pattern of TIMP-2 mRNA in preovulatory follicles at 12 h after hCG remained in the theca and stroma, with the luteinizing granulosa cells expressing low to undetectable levels (Fig. 4, E and F). However, in ovaries collected at 24 h after hCG, the forming CL expressed TIMP-2 mRNA in the luteinizing granulosa cells, whereas granulosa cells in adjacent follicles were unlabeled. Of interest was the pattern of expression with TIMP-2 mRNA present in the innermost cells encircling the centrum of the forming CL (Fig. 4, G and H).
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In Situ Hybridization for TIMP-3
At the time of eCG administration, TIMP-3 mRNA exhibited a pattern of cellular localization similar to that of the other TIMPs. TIMP-3 mRNA was detected in the theca interna, theca externa, and stroma, with low to undetectable expression in oocytes and granulosa cells (Fig. 5, A and B). With continued follicular growth, the pattern of TIMP-3 mRNA expression was unchanged, except for a shift in TIMP-3 mRNA expression to the granulosa cells as the follicle matured. This shift resulted in some follicles exhibiting TIMP-3 mRNA expression in the granulosa cells even though mRNA for this inhibitor was absent in this compartment in adjacent follicles (Fig. 5, C and D). This disparity in granulosa cell expression of TIMP-3 mRNA was more apparent at 12 h after hCG administration, with an increase in expression of granulosa cells of these follicles (Fig. 5, E and F, and Table 1). At 24 h after hCG, TIMP-3 mRNA was observed throughout the luteinizing granulosa cells in the forming CL, whereas the granulosa cells of adjacent follicles contained low to undetectable levels of TIMP-3 transcript (Fig. 5, G and H).
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In Situ Zymography
During the period of follicular growth, intense fluorescence indicative of gelatinolytic activity was detected in the thecal cells immediately adjacent to the granulosa cell basement membrane encircling follicles of various sizes (Fig. 6A). Intense fluorescence was not detected in the granulosa layer. In preovulatory follicles, gelatinase activity was detected surrounding the follicle primarily in the thecal layer (Fig. 6B). During the periovulatory period, intense fluorescence was observed in the apical region of preovulatory follicles at 12 h after hCG (Fig. 6C). At 24 h after hCG, moderate fluorescence was present in the cells throughout the forming CL (Fig. 6, D and E). Gelatinolytic activity was absent in adjacent tissue sections incubated in the presence of EDTA (Fig. 6F) or the MMP inhibitor ilomastat (data not shown).
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DISCUSSION
There is substantial evidence that the metalloproteinases and their inhibitors play a role in the events associated with the periovulatory and luteal periods; however, little is known about the cellular localization of this exquisite proteolytic system. Even less is known about the MMP system during follicular development. Because follicular growth requires extensive changes in the extracellular matrix, we hypothesized that follicular growth occurs through the actions of metalloproteinases and their inhibitors. Findings from the current study support a role for the MMP system in follicular development, ovulation, and early luteal development. The cellular localization pattern of the gelatinases and TIMPs is congruent with the changes in the extracellular matrix that occur in these specific ovarian compartments during follicular growth, follicular rupture, and luteal formation.
During the dynamic and extensive growth of the follicle, there is remodeling of the granulosa cell basement membrane and of the extracellular matrix surrounding the follicle [4]. The fact that such remodeling may occur through the action of the MMP system is supported by the current findings and by previous reports that the MMPs and their inhibitors are regulated by signals that induce follicular growth. For example, administration of eCG to immature rats stimulated an increase in mRNA expression for MMP-2, MMP-9, and TIMP-1 within 12–24 h after eCG administration and an increase in mRNA levels for MMP-13 at 36–48 h after gonadotropin treatment [28, 29]. In the goat, collagenase activity has been noted to increase with increasing follicular size [32]. The location of the MMP system and its responsiveness to hormonal stimulation, therefore, support a role for the MMPs and TIMPs in follicular development.
The mRNAs for the gelatinases in the current study were localized predominately in the theca surrounding the follicle, a region that undergoes remodeling during follicular growth. Although very little mRNA for the gelatinases was present in the granulosa cells of growing healthy follicles, there was an increase in the expression of MMP-2 following hCG administration. This increase in MMP-2 mRNA in luteinizing granulosa cells corresponds with the increase in mRNA following hCG administration as revealed by Northern analysis [33, 34] and suggests that the change in cellular function and/or the change in the extracellular environment results in a switch in the regulation and expression of this member of the gelatinase family. The increase in MMP-2 could be involved in degradation of the granulosa cell basement membrane or in the development of the extensive vascularization of the forming CL. An additional role for the gelatinases may be associated with follicular luteinization and atresia. The presence of MMP-2 mRNA in small and large luteinizing follicles in the present study and the observation in the ewe of an increase in MMP-2 and MMP-9 activity associated with follicular atresia following hypophysectomy [35] suggest that MMPs may play a role in atresia.
In the current study, we utilized in situ zymography to determine the cellular localization of gelatinolytic activity. The rationale for such an approach is that MMPs are regulated at multiple levels, including the level of transcription, synthesis of proteinases, activation of the proteinase from a latent form, and inactivation in the extracellular space by MMP inhibitors. Although the mRNA levels are an index of the changes in MMP expression, we explored whether the cellular localization of mRNA corresponded with gelatinolytic activity. We found that this activity did parallel the cellular localization of gelatinase mRNA. Because the in situ zymography technique utilizes a fluorescently labeled type IV collagen substrate, this novel approach to examination of gelatinolytic activity is unable to distinguish between the activities of the different gelatinolytic proteinases. Messenger RNA for MMP-2 and MMP-9 was present in the thecal layer surrounding the follicle, and gelatinolytic activity was observed in these regions where the basement membrane and extracellular matrix must be remodeled for follicular growth to occur. During the periovulatory period, the localization of gelatinolytic activity at the follicular apex indicates that MMP action is involved in the extracellular matrix remodeling requisite for follicular rupture, as suggested by numerous investigators [8, 25–27]. The subsequent expression of MMP-2 mRNA and gelatinolysis in the forming CL further supports a role for this class of MMPs in luteal formation through changes in the extracellular matrix associated with cellular proliferation and neovascularization.
The localization of TIMP mRNA in the present study corresponds, in part, with previous observations of TIMPs from isolated cells, immunohistochemistry, and in situ experiments. The TIMPs are synthesized in the theca, stroma, interstitial tissue, and germinal epithelium during follicular development [36–39]. Immunoreactive TIMP-1 protein has been observed in the thecal cells, the interstitial blood vessels, and the surface epithelium of the rat [40] and in the granulosa and theca layers of healthy follicles and the oocyte of the ewe [41]. To explore the correlation between the cellular changes in mRNA expression and changes in inhibitor protein, we attempted to perform immunohistochemistry and Western blot analysis for the three different TIMPs. Although various commercial sources of the TIMP antibodies were utilized, we were unable to block the immunoreaction product for TIMP-1 and TIMP-3 with purified protein. Further questions arose regarding the specificity of the antibodies when we observed multiple bands by Western analysis and TIMP-1 reaction product in our TIMP-1-deficient mice. As such, the specificity of the antibodies is questionable, and the correlative data for the immunohistochemical localization and changes in TIMP protein are not presented.
Unique to the current study was the localization of TIMP-2 and TIMP-3 mRNA during the periovulatory period. The switch in expression of TIMP-2 mRNA from very low to undetectable levels in the granulosa cells of preovulatory follicles to a high punctate pattern in the developing CL suggests that this inhibitor has a role in luteal formation. Another intriguing observation was the moderate expression level of TIMP-3 mRNA in the granulosa cells of certain antral follicles, whereas granulosa cells in adjacent follicles were devoid of TIMP-3 mRNA expression. Because TIMP-3 has been reported to be associated with apoptosis [42, 43], we recently investigated the expression of TIMP-3 in relation to follicular apoptosis in ovaries from cycling rats. Follicles with low to undetectable levels of granulosa cell TIMP-3 mRNA were apoptotic, whereas adjacent follicles exhibiting moderate expression of TIMP-3 in granulosa cells did not contain cells undergoing apoptosis [44]. Therefore, the differential expression of TIMP-3 mRNA may be related to the health status of the follicle. However, these findings are in contrast to previous reports that TIMP-3 is positively correlated with apoptosis. For example, rat aortic smooth muscle cells transfected to overexpress TIMP-3 mRNA undergo apoptosis [42], and TIMP-3 mRNA was observed in atretic murine follicles [43]. These differences in the association of TIMP-3 with apoptosis may be tissue specific or species specific or may be related to the high levels or TIMP-3 present in transfected cells.
The cellular localization of the TIMPs corresponded with the cellular localization of the gelatinases in many areas. Although the MMPs and the TIMPs are in similar cellular compartments, the results of the in situ zymography experiments indicate that the balance between enzyme and inhibitor favors the MMPs because gelatinolytic activity is observed. Other investigators have proposed that an imbalance in expression between the MMPs and TIMPs occurs at the time of ovulation in favor of the MMPs to allow proteolytic degradation of the follicular apex [45, 46]. During follicular development, the abundance of MMPs would allow local degradation of the extracellular matrix as the follicle grows, and the TIMPs may act to provide proteolytic homeostasis and maintain control of MMP action.
The TIMPs in the different ovarian compartments may have actions outside of their classically described roles as MMP inhibitors. TIMPs have been reported to be multifunctional through their ability to regulate growth and steroidogenesis. For example, TIMPs have been noted to promote embryo growth and development [47], have erythroid potentiating action [48–50], are anti-angiogenic agents [51, 52], stimulate cell growth in a variety of tissues [53], influence apoptosis [54], and recruit quiescent cells into the cell cycle [55]. Therefore, the TIMPs may act as autocrine/paracrine factors in ovarian processes involving cellular proliferation, differentiation, neovascularization, or steroidogenesis. Support for a multifunctional role in the ovary is provided by findings that a TIMP-1:procathepsin L complex stimulates steroid production in the ovary [56], TIMP-1 stimulates granulosa cell estradiol production [57], and TIMP-1-deficient mice have altered serum levels of estradiol and progesterone during the estrous cycle [58]. The TIMPs may therefore act to regulate cellular growth or differentiation associated with follicular development, ovulation, and luteal formation.
Although the definitive role of the MMP system in follicular growth is still unclear, it is readily apparent that the MMPs and TIMPs are in the appropriate cellular compartments and are associated with an increase in follicular growth in various species. Such observations have led Garcia and colleagues to propose that the MMP system may regulate normal follicular maturation and atresia to achieve the appropriate number of ovulatory follicles [32]. Alternatively, the concept that MMP inhibitors exist only to regulate ovarian proteolysis may be overly simplistic. Ovarian TIMPs may be multifunctional, acting as autocrine/paracrine factors in cellular proliferation, differentiation, neovascularization, and/or steroidogenesis during follicular growth, ovulation, and luteal formation.
ACKNOWLEDGMENTS
The authors acknowledge Dr. A.F. Parlow and the National Hormone and Pituitary program for the generous provision of eCG and Dr. Steven Palmer for the provision of the fluorescein labeled type IV collagen for the in situ zymography studies.
FOOTNOTES
First decision: 5 January 2001.
1 This work was supported by NIH grant HD23195 to T.E.C. 
2 Correspondence: Thomas E. Curry, Jr., Department of Obstetrics and
Gynecology, University of Kentucky Medical Center, 800 Rose Street,
Room C-355, Lexington, KY 40536-0293. FAX: 859 323 1931; tecurry{at}pop.uky.edu
Accepted: April 20, 2001.
Received: November 28, 2000.
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Q. Li, L. J Bakke, J R. Pursley, and G. W Smith Localization and temporal regulation of tissue inhibitors of metalloproteinases 3 and 4 in bovine preovulatory follicles Reproduction, November 1, 2004; 128(5): 555 - 564. [Abstract] [Full Text] [PDF]
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M. Jo, M. C. Gieske, C. E. Payne, S. E. Wheeler-Price, J. B. Gieske, I. V. Ignatius, T. E. Curry Jr., and C. Ko Development and Application of a Rat Ovarian Gene Expression Database Endocrinology, November 1, 2004; 145(11): 5384 - 5396. [Abstract] [Full Text] [PDF]
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 W. M. Frederiks and O. R.F. Mook Metabolic Mapping of Proteinase Activity with Emphasis on In Situ Zymography of Gelatinases: Review and Protocols J. Histochem. Cytochem., June 1, 2004; 52(6): 711 - 722. [Abstract] [Full Text] [PDF]
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M. Jo, L. E. Thomas, S. E. Wheeler, and T. E. Curry Jr Membrane Type 1-Matrix Metalloproteinase (MMP)-Associated MMP-2 Activation Increases in the Rat Ovary in Response to an Ovulatory Dose of Human Chorionic Gonadotropin Biol Reprod, April 1, 2004; 70(4): 1024 - 1032. [Abstract] [Full Text] [PDF]
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R. Wu, K. H. Van der Hoek, N. K. Ryan, R. J. Norman, and R. L. Robker Macrophage contributions to ovarian function Hum. Reprod. Update, March 1, 2004; 10(2): 119 - 133. [Abstract] [Full Text] [PDF]
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K. A. Young and R. L. Stouffer Gonadotropin and Steroid Regulation of Matrix Metalloproteinases and Their Endogenous Tissue Inhibitors in the Developed Corpus Luteum of the Rhesus Monkey During the Menstrual Cycle Biol Reprod, January 1, 2004; 70(1): 244 - 252. [Abstract] [Full Text] [PDF]
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 D. L. Russell, K. M. H. Doyle, S. A. Ochsner, J. D. Sandy, and J. S. Richards Processing and Localization of ADAMTS-1 and Proteolytic Cleavage of Versican during Cumulus Matrix Expansion and Ovulation J. Biol. Chem., October 24, 2003; 278(43): 42330 - 42339. [Abstract] [Full Text] [PDF]
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T. E. Curry Jr. and K. G. Osteen The Matrix Metalloproteinase System: Changes, Regulation, and Impact throughout the Ovarian and Uterine Reproductive Cycle Endocr. Rev., August 1, 2003; 24(4): 428 - 465. [Abstract] [Full Text] [PDF]
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 Y. Hirota, Y. Osuga, O. Yoshino, K. Koga, T. Yano, T. Hirata, E. Nose, T. Ayabe, A. Namba, O. Tsutsumi, et al. Possible Roles of Thrombin-Induced Activation of Protease-Activated Receptor 1 in Human Luteinized Granulosa Cells J. Clin. Endocrinol. Metab., August 1, 2003; 88(8): 3952 - 3957. [Abstract] [Full Text] [PDF]
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F. Gaytan, C. Bellido, M. Gaytan, C. Morales, and J. E. Sanchez-Criado Differential Effects of RU486 and Indomethacin on Follicle Rupture During the Ovulatory Process in the Rat Biol Reprod, July 1, 2003; 69(1): 99 - 105. [Abstract] [Full Text] [PDF]
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 S. J. Yan and E. A. G. Blomme In Situ Zymography: A Molecular Pathology Technique to Localize Endogenous Protease Activity in Tissue Sections Vet. Pathol., May 1, 2003; 40(3): 227 - 236. [Abstract] [Full Text] [PDF]
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K. S. Simpson, C. M. Komar, and T. E. Curry Jr Localization and Expression of Tissue Inhibitor of Metalloproteinase-4 in the Immature Gonadotropin-Stimulated and Adult Rat Ovary Biol Reprod, January 1, 2003; 68(1): 214 - 221. [Abstract] [Full Text] [PDF]
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T. E. Curry Jr and S. E. Wheeler Cellular Localization of Tissue Inhibitors of Metalloproteinases in the Rat Ovary Throughout Pseudopregnancy Biol Reprod, December 1, 2002; 67(6): 1943 - 1951. [Abstract] [Full Text] [PDF]
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F. Gaytan, E. Tarradas, C. Bellido, C. Morales, and J. E. Sanchez-Criado Prostaglandin E1 Inhibits Abnormal Follicle Rupture and Restores Ovulation in Indomethacin-Treated Rats Biol Reprod, October 1, 2002; 67(4): 1140 - 1147. [Abstract] [Full Text] [PDF]
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J. Zhang and L. A. Salamonsen In Vivo Evidence for Active Matrix Metalloproteinases in Human Endometrium Supports their Role in Tissue Breakdown at Menstruation J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2346 - 2351. [Abstract] [Full Text] [PDF]
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