Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print October 14, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.005678
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BIOLOGY OF REPRODUCTION 68, 214–221 (2003)
DOI: 10.1095/biolreprod.102.005678
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

Localization and Expression of Tissue Inhibitor of Metalloproteinase-4 in the Immature Gonadotropin-Stimulated and Adult Rat Ovary1

Kristen S. Simpsona, Carolyn M. Komar3,a, and Thomas E. Curry, Jr2,a

a Department of Obstetrics and Gynecology, University of Kentucky, Lexington, Kentucky 40536-0298


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tissue inhibitors of metalloproteinases (TIMPs) are important regulators of the matrix metalloproteinases (MMPs), proteolytic enzymes essential for controlling the coordinated tissue remodeling that takes place in the ovary. In the present study, we characterized the ovarian expression pattern of TIMP-4. The localization of TIMP-4 mRNA was determined by in situ hybridization in adult cycling rats. TIMP-4 mRNA on the day of estrus was expressed in a punctate pattern in stroma and in corpora lutea (CL) from previous cycles but not in newly formed CL or follicles. At metestrus, TIMP-4 mRNA was present in certain CL from the current and previous cycles and continued to exhibit a punctate pattern of expression in the stroma. By diestrus, TIMP-4 mRNA was detected in the thecal layer surrounding follicles, and a relatively high level of expression was observed in a punctate pattern within new and previous CL and in the stroma. TIMP-4 mRNA was also observed in the thecal layer at proestrus, but the punctate pattern within CL and stroma was absent. To correlate the changes in cellular localization with changes in overall TIMP-4 levels, ovarian mRNA and protein levels were examined in adult cycling rats and in gonadotropin-stimulated immature rats. In cycling rats, there was no change in mRNA or protein levels across the cycle, although there was a trend towards higher levels during estrus (P = 0.08). In gonadotropin-treated rats, there was an increase in TIMP-4 mRNA 48 h after eCG administration with a corresponding doubling of TIMP-4 protein. Although TIMP-4 mRNA and protein tended to decline after hCG treatment, this trend was not significant (P = 0.08). These findings indicate that TIMP-4 could play an important role in regulating MMPs in a localized manner in follicles and CL throughout the cycle.

corpus luteum, follicular development, granulosa cells, ovary, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mammalian ovary undergoes dynamic structural changes during the reproductive cycle. The tissue restructuring necessary to accommodate follicular growth and development, rupture of the follicle and release of the oocyte, and the subsequent formation of the corpus luteum (CL) requires extensive remodeling of the extracellular matrix (ECM). This remodeling is postulated to occur through the action of the matrix metalloproteinases (MMPs), a family of structurally related enzymes capable of breaking down components of the ECM. Various members of the MMP family have been identified in ovarian tissue and play a role in tissue remodeling (reviewed in [1, 2]).

Because the integrity of the ECM is important in the physiology and function of tissues, MMP activity is regulated at several levels, including transcription, translation, activation, and inhibition [3]. The inhibition of MMP activity is implemented by two classes of inhibitors, serum borne (such as {alpha}-macroglobulins) and tissue derived (tissue inhibitors of metalloproteinases [TIMPs]) [3, 4]. There are four members of the TIMP family (TIMP-1 through TIMP-4), each of which can inhibit MMP activity by forming noncovalent bonds with the MMPs in a 1:1 molar ratio [4]. Although capable of binding to most MMPs, each TIMP exhibits a greater affinity for and thus inhibition of specific MMP family members [5]. In addition, some TIMPs are capable of binding to both the active and latent forms of the MMPs. For example, TIMP-1 binds with high affinity to proMMP-9, whereas TIMP-2 shows a greater affinity for proMMP-2 [6, 7].

In addition to their classical role as MMP inhibitors, the TIMPs have other nonclassical actions. These nonclassical actions may be a direct action of the TIMPs or an indirect consequence of alteration of MMP action. For example, TIMP-1 and TIMP-2 act as growth factors [8–10] and modulate angiogenesis [11–13]. TIMP-1 also regulates steroidogenesis in cultured rat Leydig and granulosa cells [14, 15], and recent studies have shown that TIMP-2 is involved in the activation of proMMP-2 by forming a complex with membrane type 1 MMP (MT1-MMP; reviewed in [16]). Various studies have demonstrated that TIMP-3 plays a role in cell death by inducing apoptosis [5, 17–19], and it may play a role in cell cycle progression and differentiation [20]. Thus, the TIMPs may act not only as MMP inhibitors but as multifunctional proteins.

The newest family member, TIMP-4, was identified in 1996 by Greene and colleagues [21]. Data from human, rat, and mouse tissues show that TIMP-4 is more closely related to TIMP-2 and TIMP-3 than to TIMP-1 [21–23]. Liu and coworkers [24] have reported that TIMP-4 effectively inhibits the activity of MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, with preference for the gelatinases (MMP-2 and MMP-9). TIMP-4 binds with high affinity to proMMP-2 in a manner similar to that of TIMP-2 [25]. Because the distribution of TIMP-4 in various tissues differs from that for the other three TIMP family members, investigators have hypothesized that this inhibitor is an important modulator of ECM remodeling in a tissue-specific manner [21, 23].

TIMP-1, -2, and -3 are expressed in the rat and mouse ovary, and expression is influenced by the reproductive cycle and hormonal milieu [26–30]. TIMP-4 has been identified in the ovaries of mice [23], rats [31], horses [32], and humans [33], but very little is known about its cellular localization or pattern of expression. The following study was conducted to test the hypothesis that TIMP-4 expression in the ovary is regulated by the hormonal signals associated with the ovarian changes occurring in adult cycling and immature gonadotropin-stimulated rats.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Unless otherwise noted, all chemicals and reagents were purchased from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). All animal procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee.

Animals

Cycling rat model Adult female Sprague-Dawley rats (3 mo of age; Harlan Sprague-Dawley, Indianapolis, IN) were monitored for cyclicity by vaginal lavage. Once the rats exhibited at least three consecutive 4-day estrous cycles, the ovaries were collected (n = 3 or 4/time point) at 1000 to 1100 h on each day of the cycle; estrus, metestrus, diestrus, and proestrus. One ovary was frozen for RNA or protein analysis, and the other was frozen in optimal cutting temperature medium (VWR, Atlanta, GA) for RNA localization by in situ hybridization.

Gonadotropin-stimulated rat model Immature female Sprague-Dawley rats were injected with 10 IU of eCG at 21 days of age. To study the expression of TIMP-4 during follicular development, half of these animals were killed at 0 (no eCG), 6, 12, 24, or 48 h after treatment with eCG (n = 3–7/time point). To examine the expression of TIMP-4 during the periovulatory period, the remaining animals received 10 IU of hCG 48 h post-eCG to simulate the LH surge and induce ovulation. Animals were killed at 0 (48 h post-eCG, no hCG), 4, 8, 12, or 24 h post-hCG (n = 3–5/time point). At each time point, both ovaries were collected, frozen, and stored at -80°C.

In Situ Hybridization

TIMP-4 mRNA was localized in cycling rat ovaries on each day of the cycle by in situ hybridization as described previously [26]. Ovaries from cycling rats were sectioned at 10 µm and mounted onto Probe-On Plus slides (Fisher). Antisense and sense 35S-labeled riboprobes were synthesized from linearized plasmid containing mouse TIMP-4 (generously supplied by Dr. Kevin Leco, University of Western Ontario, London, ON, Canada) using the Maxiscript in vitro RNA transcription kit (Ambion, Austin, TX). Both the antisense and sense riboprobes were approximately 400 bases in length. Tissue sections were prepared as described previously and hybridized overnight at 60°C with 3 x 106 cpm antisense or sense probe per slide. The slides were then washed and processed for autoradiography using Kodak NTB2 emulsion (Eastman Kodak Co., Rochester, NY). After the tissue sections were exposed to emulsion at 4°C for 5–6 mo, the reaction product was visualized by development in Kodak D19 (1:1) and stained with hematoxylin. Tissues were examined with a Nikon Eclipse E800 microscope (Nikon, Melville, NY) under bright- and darkfield optics.

One ovary from each of three animals was used for in situ hybridization. Sixteen tissue sections per ovary were hybridized with the antisense TIMP-4 probe, for a total of 48 tissue sections analyzed for each time point. Four sections per ovary were hybridized with the TIMP-4 sense riboprobe, so that a total of 12 sections per time point were examined.

Immunohistochemistry

Immunohistochemistry was performed as described by Bowen and Keyes [34] to determine the location of macrophage cells within the ovary on all days of the cycle. Serial sections (10 µm) of the ovaries used for in situ hybridization were processed for immunohistochemistry. Tissue sections were air dried and fixed in 95% ethanol, and the endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in methanol. After washing in PBS containing 1% BSA, the tissues were blocked by incubating in 10% normal horse serum in PBS with 1% BSA. The tissues were again washed in PBS with 1% BSA and then incubated with a mouse anti-rat monocyte/macrophage monoclonal antibody (ED1 antibody; Chemicon, Temecula, CA) for 30 min at 37°C in a humidified chamber. The antibody was diluted 1:200 in PBS with 1% BSA, 10% normal rat serum, and 10% normal horse serum. After incubation with the primary antibody, the tissues were washed with PBS with 0.1% BSA and subsequently incubated with biotinylated horse anti-mouse IgG (1:100 dilution in PBS with 0.1% BSA, 10% normal rat serum, and 10% normal horse serum: Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA) for 30 min at 37°C. Tissues were washed again in PBS with 0.1% BSA, and bound antibody was detected using the Vectastain Elite ABC kit peroxidase system and 3-amino-9-ethyl carbazole (AEC Substrate Kit for Peroxidase; Vector Laboratories) according to the manufacturer's instructions. Tissue sections were stained with hematoxylin and examined using a Nikon Eclipse E800 microscope. Appropriate binding of the antibodies was tested by replacing the primary or secondary antibodies with normal serum. No nonspecific binding was detected in any tissues.

RNase Protection Assay

Total RNA was isolated from ovaries using Trizol reagent (Life Technologies, Rockville, MD) according to the manufacturer's instructions and was quantified by spectrophotometry. The rat cDNA for TIMP-4 (a generous gift from Dr. Marsha Moses, Harvard Medical School, Boston, MA) [22] and mouse ribosomal protein L32 (kindly provided by Dr. O.-K. Park-Sarge, University of Kentucky, Lexington, KY) were linearized with the appropriate restriction enzymes. Antisense riboprobes were transcribed using the Maxiscript in vitro RNA transcription kit, [32P]UTP (10 mCi/ml; DuPont New England Nuclear, Boston, MA), and T7 RNA polymerase.

RNase protection assays were carried out as described previously [35]. Total RNA (3–6 µg) was hybridized with excess radiolabeled antisense riboprobe for 15–18 h at 50°C. Loading variation between samples was standardized by including L32 riboprobe in all hybridization reactions. Protected RNA fragments were analyzed by electrophoresis through a 5% acrylamide, 8 M urea gel. Quantification of band intensity in the gels was determined from an imaging plate using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The band intensity for TIMP-4 mRNA in each sample was normalized to the corresponding band for L32.

Western Analysis

Protein was extracted from ovarian tissue by standard protocols, quantified using the Bradford assay, and analyzed by Western blot analysis. For the Western blot analysis, protein (20–25 µg) from each sample was loaded onto 12.5% polyacrylamide gels, separated by electrophoresis, and subsequently transferred to nitrocellulose membranes (0.2 µm Optitran; Schleicher and Schuell, Keene, NH). Immunodetection of TIMP-4 was accomplished utilizing the Immun-Star Anti-Rabbit Chemiluminescent Detection Kit (BioRad, Hercules, CA). After equilibration in Tris-buffered saline with Tween (TBST; 10 mM Tris, 150 mM NaCl, 0.05% Tween-20, pH 8.0), the membranes were blocked with 0.2% nonfat dry milk in TBST and incubated with a rabbit anti-TIMP-4 antibody (Triple Point Biologics, Forest Grove, OR) at 1:10 000 for 1 h at room temperature. Following three washes in TBST, the membranes were incubated with the secondary antibody (1:3000) for 1 h at room temperature. After washing three times in TBST and incubating 5 min in the chemiluminescent substrate solution, membranes were exposed to X-OMAT autoradiography film (Kodak) for 15 sec to 3 min to ensure linearity of the signal. Specificity of the TIMP-4 antibody was verified by adding TIMP-4 peptide to compete with antibody binding.

Statistical Analysis

Differences in mRNA or protein levels determined by RNase protection assay or Western blot analysis, respectively, were analyzed by one-way ANOVA (n = 3–7), and post hoc comparisons were performed when appropriate using least significant differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two animal models were used in these studies to determine the temporal expression pattern of TIMP-4 mRNA and protein: adult cycling rats and immature gonadotropin-primed rats. By studying the adult cycling animal, we were able to examine TIMP-4 mRNA expression in the ovary during follicular development, ovulation, and CL formation and regression under natural conditions. Localization of TIMP-4 mRNA was examined only in ovarian sections from cycling animals because all aspects of ovarian remodeling are represented in these animals, including the regressing CL. Use of the immature gonadotropin-primed animals allowed examination of the changes in TIMP-4 expression during follicular development following induction of follicular growth with eCG. It also permitted examination of the changes occurring during follicle maturation, ovulation, and early CL formation following hCG administration (periovulatory period). Use of the immature rat model allowed synchronization of follicular growth and ovulation, while increasing the abundance of growing and preovulatory follicles.

To explore the cellular changes in TIMP-4 mRNA expression throughout the ovarian cycle, TIMP-4 mRNA was localized in the adult cycling rat ovary by in situ hybridization (Fig. 1). Because of the low levels of TIMP-4 mRNA, the tissues had to be exposed to photographic emulsion for an extended period of time (5–6 mo). Different generations of CL were identified, including CL from the current cycle (new CL) and regressing CL from previous cycles (previous CL) [26]. On estrus, CL from previous cycles exhibited a punctate TIMP-4 mRNA expression pattern within the CL, whereas newly formed CL and follicles contained only background levels of reaction product (Fig. 1, A–C). In addition, limited areas of the stroma surrounding these ovarian structures exhibited a punctate pattern of expression, suggesting that a specific cell type(s) is producing TIMP-4 mRNA in both the stroma and previous CL. By metestrus, a similar cellular pattern of expression was observed in some previous and new CL, whereas others had only background levels of hybridization (Fig. 1, D–F). One animal exhibited high levels of TIMP-4 mRNA in the cells of the vascular wall of large blood vessels (data not shown). High levels were also seen in the ovarian sections from one of the proestrus animals examined (Fig. 2, A and B). A distinct punctate pattern of TIMP-4 mRNA expression continued to be observed in the stroma on metestrus and diestrus (Fig. 1, G–I). Luteal expression of TIMP-4 on diestrus was particularly interesting; a high level of TIMP-4 mRNA was observed in a punctate pattern within new and previous CL. Also, thecal expression around follicles was observed on diestrus and proestrus (Fig. 1, J–L, and Fig. 2, A and B). However, the punctate pattern of hybridization within CL and stroma was no longer evident on proestrus (Fig. 2, A and B).



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  		FIG. 1. In situ hybridization analysis of TIMP-4 mRNA in the cycling rat ovary. Representative brightfield (A, D, G, and J) and darkfield microscopic images are of ovarian tissue sections hybridized with a TIMP-4 antisense (B, E, H, and K) or sense (C, F, I, and L) riboprobe. Ovarian tissues were collected on all 4 days of the estrous cycle; estrus, metestrus, diestrus, and proestrus. F, Follicle; nCL, newly formed corpus luteum; pCL, corpus luteum from a previous cycle. Bar = 500 µm (J)



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  		FIG. 2. In situ hybridization analysis of TIMP-4 mRNA in a proestrus rat ovary. Representative brightfield (A) and darkfield (B) microscopic images are of a proestrus ovary hybridized to a TIMP-4 antisense riboprobe. TIMP-4 mRNA expression is visible in the vascular cells of the wall of large blood vessels (arrows) and in the thecal layer around follicles (arrowhead). Bar = 500 µm (A)

To ascertain whether macrophages were expressing TIMP-4 mRNA, macrophages were identified in adjacent ovarian sections on all days of the cycle. The expression of TIMP-4 mRNA did not correspond to the presence of immunoreactive macrophages. For example, macrophages were highly abundant in several regressing CL on metestrus, but the TIMP-4 mRNA expression pattern was not consistent with the location of macrophages (Fig. 3).



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  		FIG. 3. Comparison of TIMP-4 mRNA expression and the immunolocalization of macrophage cells in a metestrus rat ovary. Representative brightfield (A) and darkfield (B) microscopic images are of a metestrus ovary analyzed by in situ hybridization using a TIMP-4 antisense riboprobe. A serial section of the ovary (C) was analyzed by immunohistochemistry to localize macrophage cells. The dark red staining (arrows), which is especially evident in some of the corpora lutea, shows the location of antibody binding and, thus, the location of macrophages. F represents the same follicle in each panel; CL indicates the same corpus luteum in each ovary. Bar = 500 µm (C)

The temporal pattern of TIMP-4 mRNA expression in whole ovarian tissue homogenates was determined using RNase protection assays because TIMP-4 was not detectable by Northern analysis. Figure 4A (top) shows a representative RNase protection assay for TIMP-4 mRNA in whole ovarian tissue collected at estrus, metestrus, diestrus, and proestrus. Although the levels of TIMP-4 mRNA did not change significantly in naturally cycling animals throughout the estrous cycle (Fig. 4A, bottom; P = 0.081), the mRNA levels on estrus were 40% higher than those on metestrus.



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  		FIG. 4. TIMP-4 mRNA and protein levels in the rat ovary during the estrous cycle. A) Top panel shows an autoradiograph of a representative RNase protection assay demonstrating the protected fragment of mRNA for TIMP-4 and ribosomal protein L32 from individual ovaries collected on estrus (E), metestrus (M), diestrus (D), and proestrus (P). Bottom panel shows a graph of the relative levels (mean ± SEM) of TIMP-4 mRNA in total RNA isolated from ovaries collected on each day of the cycle (n = 3 or 4/time point). Relative levels were calculated by correcting for the amount of L32 within each sample. B) Top panel shows an autoradiograph of a representative Western blot depicting immunolabeled TIMP-4 from individual ovaries collected on estrus, metestrus, diestrus, and proestrus. Bottom panel shows a graph of TIMP-4 protein levels (mean ± SEM, n = 3/time point)

Western analysis utilizing an antibody generated against amino acids 207–225 [24] of human TIMP-4 detected a single protein of approximately 28 kDa in rat ovarian tissue samples. The rat ovarian protein approximates the predicted size of human TIMP-4 at 22–24 kDa [24] and may be a glycosylated form of the human protein. No change in TIMP-4 protein levels was observed during the estrous cycle (Fig. 4B; P = 0.069), although protein levels on estrus were 57% higher than those on metestrus, and the pattern of protein abundance mimicked the pattern of mRNA expression.

In the gonadotropin-stimulated model, levels of mRNA for TIMP-4 increased during the later stages of follicular development (Fig. 5A). At 48 h post-eCG, levels of mRNA for TIMP-4 were greater than those at 0, 6, and 12 h post-eCG. The expression of TIMP-4 protein during follicular development mirrors the pattern seen for its mRNA (Fig. 5B). Although not significantly different, the expression of TIMP-4 protein tended to increase toward a peak at 48 h post-eCG, with a twofold increase in TIMP-4 protein between 0 and 48 h.



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  		FIG. 5. TIMP-4 mRNA and protein levels in the rat ovary during follicular development. A) Top panel shows an autoradiograph of a representative RNase protection assay demonstrating the protected fragment of mRNA for TIMP-4 and ribosomal protein L32 from individual ovaries collected 0, 6, 12, 24, or 48 h after treatment with eCG. Bottom panel shows a graph of the relative levels (mean ± SEM) of mRNA for TIMP-4 in total RNA (n = 5–7/time point). Relative levels were calculated by correcting for the amount of L32 within each sample. B) Top panel shows an autoradiograph of a representative Western blot depicting immunolabeled TIMP-4 from individual ovaries collected from rats 0, 6, 12, 24, or 48 h after treatment with eCG. Bottom panel shows a graph of TIMP-4 protein levels (mean ± SEM, n = 3/time point). Treatments with different superscripts are significantly different (P < 0.05)

During the periovulatory period, levels of mRNA for TIMP-4 did not change significantly after an injection of hCG to stimulate ovulation, although there was a trend towards a decline in TIMP-4 levels (Fig. 6A; P = 0.076). No significant change was observed in the expression of TIMP-4 protein after treatment with hCG, but protein levels tended to decline following an hCG stimulus (Fig. 6B), mirroring the downward trend in TIMP-4 mRNA expression.



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  		FIG. 6. TIMP-4 mRNA and protein levels in the rat ovary during the periovulatory period. A) Top panel shows an autoradiograph of a representative RNase protection assay demonstrating the protected fragment of mRNA for TIMP-4 and ribosomal protein L32 from individual ovaries collected 0, 4, 8, 12, or 24 h after treatment with hCG. Bottom panel shows a graph of the relative levels (mean ± SEM) of mRNA for TIMP-4 in total RNA (n = 4 or 5/time point). Relative levels were calculated by correcting for the amount of L32 within each sample. B) Top panel shows an autoradiograph of a representative Western blot depicting immunolabeled TIMP-4 from individual ovaries collected from rats 0, 4, 8, 12, or 24 h after treatment with hCG. Bottom panel shows a graph of TIMP-4 protein levels (mean ± SEM, n = 3/time point)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current studies are the first to characterize the expression of TIMP-4 in the ovary during the dramatic tissue remodeling associated with follicular development, ovulation, and CL formation and regression. TIMP-4 has been detected previously in the ovaries of mice [23], rats [31], horses [32], and humans [33]. The present study builds upon these early reports and provides evidence that the location of TIMP-4 mRNA expression changes during the rat estrous cycle. Furthermore, the levels of TIMP-4 mRNA are regulated during follicular growth and may reflect an involvement in the cyclic control of MMP activity within the ovary.

A role for TIMP-4 in ovarian function is supported by the unique localization of TIMP-4 mRNA within the naturally cycling ovary. Of particular interest was the distinct pattern of TIMP-4 mRNA in CL and stroma and the fact that this pattern changes across the estrous cycle. For example, TIMP-4 mRNA was present in a punctate pattern within both previous and new CL on metestrus and diestrus, only in previous CL on estrus, and in neither type of CL on proestrus. The expression of TIMP-4 mRNA observed in CL and stromal cells on diestrus was completely gone by proestrus. The type of cell(s) expressing this level of TIMP-4 mRNA, its function at this time of the cycle, and the reason for its disappearance by proestrus are unknown.

Further supporting a role for TIMP-4 in ovarian function are the findings that TIMP-4 mRNA increased following an eCG stimulus, and protein levels tended to mirror the changes in mRNA. Following an ovulatory dose of hCG, both TIMP-4 mRNA and protein decreased about 50%, although this change was not significant. Udoff et al. [31] also reported a similar decline (approximately 65% decrease) in TIMP-4 mRNA by 4 h post-hCG in eCG/hCG-stimulated immature rats. In the cycling animal, both the mRNA and protein levels tended to increase between proestrus and estrus, which is a dynamic period of ovarian remodeling marked by the final stages of follicular maturation and ovulation and the beginning of CL formation. Although many of the changes in TIMP-4 mRNA and protein expression were not significant during follicular growth or during the periovulatory period, dynamic changes in cellular expression can be masked by examining changes in intact whole ovarian tissue. This may be true especially for TIMP-4 because the level of mRNA expression is extremely low. Because of this low level of expression, TIMP-4 mRNA was undetectable by Northern analysis of 25 µg of total RNA, required a higher specific activity probe, and necessitated an extended period of exposure for both RNase protection assay and in situ hybridization. For example, typical exposure times for the other TIMPs in our laboratory range from 2 to 4 wk, whereas exposure for TIMP-4 mRNA in the current study was 6 mo. Thus, the data from the in situ hybridization and quantitative mRNA studies suggest that ovarian TIMP-4 mRNA is less abundant than are the other TIMPs.

The presence of low levels of TIMP-4 mRNA as observed in the present study in an environment where the other TIMPs are highly expressed raises the interesting question as to the potential role of TIMP-4 in ovarian function. Although we did not address this question in the current study, we can speculate on potential mechanisms for TIMP-4 based upon investigations in other tissues. One potential role for TIMP-4 is the classical role as an MMP inhibitor. The TIMPs bind to the MMPs and inhibit their activity. However, there are differences in the affinities of the TIMPs for the various MMPs, implying selectivity toward specific MMPs. For example, TIMP-4 can inhibit MT1-MMP, whereas TIMP-1 has little to no effect on this MMP [36]. TIMP-4 also has can increase the degradative capacity of MT1-MMP by preventing its autocatalytic processing into the cell [37]. Because there are no reports of the comprehensive comparison of the biological efficacy of the inhibitors toward the different MMPs [5], the possibility exists that TIMP-4, although at low levels in the ovary, has a high affinity for a specific proteinase in ovarian remodeling.

An alternative possibility for TIMP-4 in ovarian function is that TIMP-4 may be regulated in a manner different from that of other TIMPs, allowing it to be turned on by specific signals to control MMP activity at certain focal areas in the ovary. Support for this concept comes from the recent functional characterization of the TIMP-4 promoter and the distinct cellular expression of TIMP-4 mRNA observed in the present study. Young and colleagues [38] have demonstrated that the TIMP-4 promoter is distinct from other members of the TIMP family, supporting the possibility of a unique spatiotemporal expression pattern of this inhibitor relative to the other TIMPs. Of interest in the current study was the expression of TIMP-4 mRNA in the thecal layer surrounding follicles on diestrus and proestrus, which suggests that TIMP-4 may play a role in follicles, perhaps regulating ECM remodeling during the final stages of follicular maturation. This concept is supported by the increase in TIMP-4 mRNA observed 48 h after eCG injection in these studies. During all stages of equine follicular development, TIMP-4 protein was present in the theca and granulosa layers and in the ECM and fibroblast cells of the stromal region, further supporting a possible role for TIMP-4 in follicular growth [32]. Because the current findings of TIMP-4 mRNA in the rodent ovary were in contrast to the localization of TIMP-4 protein in the horse, we attempted to determine the cellular localization of TIMP-4 protein in rat ovarian tissue sections by immunohistochemistry. However, we were unable to block the antibody with TIMP-4 peptide and, thus, could not ascertain whether the localization of TIMP-4 protein was similar to that of its mRNA.

Another hypothesis for ovarian TIMP-4 action is that TIMP-4, like TIMP-2, is involved with regulating the amount of active MMP-2 available. TIMP-2 contributes to the activation of proMMP-2 by forming a complex with both the pro-form of MMP-2 and MT1-MMP, which allows the subsequent activation of MMP-2 by MT1-MMP (reviewed in [16]). Like TIMP-2, TIMP-4 has the ability to bind to proMMP-2 [25] and MT1-MMP [37]. However, although the complex formation of TIMP-2, MT1-MMP, and proMMP-2 results in the cleavage and activation of the pro-form of MMP-2, TIMP-4 does not enhance proMMP-2 activation. Rather, when it binds to MT1-MMP, it competitively inhibits TIMP-2 binding and activation of MMP-2 [37]. Hernandez-Barrantes et al. [37] suggested that the activation of MMP-2 may be dependent upon the balance of TIMP-2 and TIMP-4. TIMP-2 mRNA is expressed in the theca of follicles on all days of the cycle [26]. Thus, the balance of TIMP-2 and TIMP-4 in the theca may play an important role in the regulation of MMP-2 activation.

Similar to the other TIMPs, TIMP-4 may also have nonclassical actions other than proteinase inhibition. The family of TIMPs may sometimes act as growth factors, regulators of steroidogenesis [14, 15], modulators of angiogenesis [11–13], or inducers of apoptosis [5, 17–19]. TIMP-4 inhibits cell invasiveness [39] and induces apoptosis in transformed cells. Tummalapalli and coworkers [40] reported that TIMP-4 controlled normal cardiac fibroblast transformation and induced apoptosis in transformed cells, which led to the suggestion that reduced levels of TIMP-4 elicit cellular transformation and may lead to adverse ECM degradation. Further, TIMP-4 is believed to play an important role in regulating angiogenesis; expression of TIMP-4 increases following vascular injury, and TIMP-4 has the ability to reduce the migration of vascular smooth muscle cells [41].

All of these reported actions of TIMP-4 in other tissues may have a physiological foundation for controlling ovarian ECM remodeling during the normal ovarian cycle. The ovary undergoes dramatic remodeling processes during each estrous cycle, including rapid cellular proliferation, differentiation, and migration and ECM breakdown and angiogenesis. Perhaps TIMP-4 is necessary to control these processes in the ovary. In particular, it may play an important role in the CL and stroma, where the pattern of expression suggests that TIMP-4 mRNA is being expressed by a specific cell type(s). TIMP-4 may be involved in the controlled removal of luteal tissue, preventing the growth and proliferation of abnormal cells, or inducing apoptosis. Because of the cellular pattern of TIMP-4 mRNA expression in the ovary, we examined the localization of macrophages in serial ovarian sections using a macrophage-specific antibody to determine whether macrophages were the source of TIMP-4 expression. Macrophage location did not correspond to the areas of TIMP-4 mRNA production. However, the expression of TIMP-4 in the cells of the vascular wall in some animals suggests that TIMP-4 may be produced by endothelial cells. Further investigation is needed to determine the exact type of cell(s) responsible for TIMP-4 mRNA synthesis.

In the present study, TIMP-4 expression was characterized within the ovary for the first time. TIMP-4 mRNA increased during the later stages of follicular development in the rat, but no significant changes in whole ovarian mRNA or protein expression were detected during the periovulatory period or during the cycle. However, there was an apparent tendency for TIMP-4 mRNA and protein to decrease post-hCG in gonadotropin-treated animals and to reach a peak on estrus in cycling animals. Most striking were the changes in mRNA localization that occurred across the cycle, with relatively high levels of TIMP-4 mRNA present in a specific cell type(s), primarily in CL and stroma. These results show that TIMP-4 mRNA and protein are produced within the ovary and that TIMP-4 may play an important role in localized ovarian compartments. Further studies are needed to determine the cell type(s) that are producing TIMP-4 and to determine the role of TIMP-4 in ovarian function.


   ACKNOWLEDGMENTS
 
The authors are indebted to Dr. Preston Alexander (Triple Point Biologics, Forest Grove, OR) for the generous gift of the TIMP-4 reagents utilized in this study, his intellectual insight into various issues, and his ongoing contributions to our understanding of the TIMP field. The authors also acknowledge Dr. Kevin Leco for the mouse cDNA for TIMP-4 and Dr. Marsha Moses for the rat cDNA for TIMP-4.


   FOOTNOTES
 
1 This work was supported by the Lalor Foundation (C.M.K.) and grants to T.E.C. (NIH HD23195 and NCRR P20 RR15592). Back

2 Correspondence: Thomas Curry, Jr., Department of Obstetrics and Gynecology, Chandler Medical Center, Room MS 331, University of Kentucky, 800 Rose St., Lexington, KY 40536-0298. FAX: 859 323 1931; tecurry{at}uky.edu Back

3 Current address: Department of Animal Science, Iowa State University, 2356F Kildee Hall, Ames, IA 50011-3150 Back

Received: 18 March 2002.

First decision: 13 April 2002.

Accepted: 7 August 2002.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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