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Ovary |
a Department of Obstetrics & Gynecology, University of Kentucky, Lexington, Kentucky 40536
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, follicle, granulosa cells, ovary, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Associated with these repetitive cycles of luteal development and regression is extensive connective tissue remodeling [1, 2]. Remodeling of the connective tissue and extracellular matrix occurs during luteal formation as well as regression. There is negligible remodeling during the midluteal period, when steroid production is maximal. Recent evidence suggests that luteal tissue remodeling is regulated, in part, by a family of proteolytic enzymes referred to as matrix metalloproteinases, or MMPs [3–5]. Throughout the body, the action of the MMPs is closely regulated in the extracellular matrix by specific inhibitors [6, 7]. Of central importance are the tissue inhibitors of metalloproteinases, or TIMPs. Currently, four distinct TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) have been identified and characterized based on their molecular weight, biological activity, or cDNA cloning [6, 7]. The TIMPs differ in their regulation, affinity for the MMPs, and their site of action [6, 7]. For example, TIMP-1, -2, and -4 are secreted and act in the extracellular space whereas TIMP-3 is bound to the extracellular matrix [7]. Although bound to the extracellular scaffolding, TIMP-3 is capable of inhibiting MMP activity, thus providing an additional level of MMP regulation by acting at the site of MMP action [8, 9].
The TIMPs may play a role in luteal function by regulating MMP action during luteal connective tissue turnover or by providing homeostasis during the midluteal period. Additionally, the TIMPs may impact luteal physiology through actions other than as proteinase inhibitors. For example, the TIMPs have been reported to stimulate cell growth [10], impact angiogenesis [11, 12], induce apoptosis [13–16], and regulate ovarian steroidogenesis [17–19]. All of these physiological actions are important for overall luteal function. Support for the concept that the TIMPs have a role in luteal action is evident from changes in the patterns of TIMP expression throughout pseudopregnancy or pregnancy in the rodent [20–22]. The levels of TIMP-1 mRNA are elevated during early luteal formation in the rat [20, 21], decline during the midluteal period of pregnancy or pseudopregnancy to remain low in the rat [20, 21] and mouse [22]. The expression of TIMP-3 mRNA increases during the mid to latter stages of pseudopregnancy [21]. However, to begin to fully understand the function of the TIMPs in luteal formation, maintenance, and demise, it is important to know where these inhibitors are present and how their expression patterns change. Therefore, we investigated the cellular localization for the mRNA of TIMP-1, TIMP-2, and TIMP-3 throughout pseudopregnancy in order to begin to understand their potential role in regulating luteal tissue remodeling, cellular proliferation, and/or steroidogenesis associated with the development, maintenance, and regression of the corpus luteum in the rat. The mRNA for TIMP-4 was not examined in the current study due to the extremely low levels of expression and the lack of significant changes after hCG (K. Simpson, personal communication).
A subsequent and separate series of experiments was conducting based on previous observations that, following a hCG stimulus, TIMP-3 mRNA was induced in the granulosa cells of certain large preovulatory follicles whereas adjacent follicles lacked TIMP-3 mRNA [23]. Furthermore, expression of granulosa cell TIMP-3 mRNA appears to be associated with healthy follicles [24]. In an attempt to understand the relationship between TIMP-3 mRNA expression and the status of whether these follicles were destined to become CL, markers of follicular maturity were compared with the presence of TIMP-3 mRNA during the preovulatory and early luteal periods.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature female Sprague-Dawley rats (15 days old, purchased and shipped with the mother; Harlan Sprague-Dawley Inc., Indianapolis, IN) were housed in controlled environmental conditions under the care and supervision of a licensed veterinarian. All animal procedures for these experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee (IACUC). Rats were maintained on a 14L:10D cycle and provided water and rat chow ad libitum. For the pseudopregnant studies, rats were injected with eCG (10 IU s.c., generously provided by A.F. Parlow, National Institute of Diabetes & Digestive & Kidney Diseases' National Hormone and Peptide Program) between 0900 and 1000 h on the morning of Day 22–23 of age to stimulate folliculogenesis. Forty-eight to 50 h after eCG administration, animals were injected with hCG (10 IU s.c.; Sigma Chemical Co., St. Louis, MO) to induce ovulation and pseudopregnancy. Ovulation in this model occurs at 12–16 h after hCG ([25], personal observation). Animals were killed at 1, 2, 4, 8, 12, and 14 days after hCG administration. These time points represent luteal formation (Days 1 and 2), luteal maintenance when progesterone production is maximal (Days 4 and 8), and luteal regression (Days 12 and 14). Ovaries were removed, cleaned, embedded in OCT (VWR Scientific, South Plainfield, NJ), and frozen for the cellular localization of TIMP mRNA as described below.
During the course of these and previous studies [23], we observed an LH/hCG induction of TIMP-3 mRNA in the granulosa cells of certain large preovulatory follicles (12 h after hCG) or early CL (24 h after hCG), whereas granulosa cells of adjacent follicles did not express TIMP-3 mRNA. In an attempt to understand the relation between TIMP-3 mRNA expression and the status of these follicles, a separate series of experiments was performed where expression for markers of follicular maturity were compared with the presence of TIMP-3 mRNA. Immature 22– to 23-day-old rats were injected with eCG (10 IU s.c.) followed by hCG (10 IU s.c.) 48–50 h later. Ovaries were collected at 0, 4, 8, 12, and 24 h after hCG. The 0–12 h after hCG time points are referred to as preovulatory while the 24-h time point is referred to as the early luteal period. Tissues were sectioned, and serial sections were processed for the localization of TIMP-3, aromatase, cholesterol side-chain cleavage, or LH receptor mRNA.
In Situ Hybridization
In situ hybridization was performed as previously described [24] using plasmids containing murine TIMP-1, TIMP-2, or TIMP-3 cDNA (supplied by Kevin Leco, University of Western Ontario). In situ hybridization for markers of follicular maturity were performed using plasmids for aromatase, P450 side-chain cleavage (supplied by Joanne Richards, Baylor University), and LH receptor (supplied by Ok-Kyong Park-Sarge, University of Kentucky). Plasmids were linearized using the appropriate restriction enzymes, and the antisense and sense cRNA probes for the TIMPs (TIMP-1, -2, and -3), LH receptor, aromatase, and side-chain cleavage were synthesized from the corresponding linearized plasmid and labeled with [
35S]uridine 5'-triphosphate using a Maxiscript in vitro RNA transcription kit from 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 at 10 µm and mounted on Probe-On Plus slides (Fisher Scientific, Pittsburgh, PA). Tissues were fixed in 4% paraformaldehyde, washed, and dehydrated in ethanol. Each cRNA probe was allowed to hybridize to the mRNA overnight in hybridization buffer containing 1 x 106 cpm of probe per slide at 55°C in a humidified chamber. Approximately 18–20 h later, slides were washed extensively to remove nonspecifically bound TIMP cRNA followed by RNase A treatment (100 µg/ml in Tris-EDTA buffer) for 30 min at 45°C. Slides were again washed extensively, dehydrated in ethanol, and air dried. Sections were processed for autoradiography using Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) and stored at 4°C for various times up to 4 wk. For visualization of the in situ reaction product, slides were developed in Kodak D19 (1:1), fixed in Kodak Rapid Fixer, and stained with Gill 2 hematoxylin solution (Fisher Scientific). Tissues were examined with a Nikon Eclipse E800 microscope (Nikon Corp., Melville, NY) under bright- and darkfield optics. A sense cRNA probe, used as a control for nonspecific binding, was included for each time point for the different TIMPs.
One ovary from a minimum of three animals was used for in situ hybridization (n = 3). For each TIMP, at least 16 tissue sections per ovary were analyzed for the antisense cRNA probe, making a total of 48 tissue sections analyzed for each time point for each mRNA examined. For the control sense probe, four sections per ovary were analyzed such that a total of 16 sections per time point were examined for each TIMP.
Morphometric Analysis
To determine the changes in TIMP mRNA expression patterns during pseudopregnancy, morphometric analysis was performed using Metamorph software (version 4.6 r9, Universal Imaging Corporation, West Chester, PA) as previously described [23]. Briefly, 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 on areas not containing tissue was subtracted from the entire image. The remaining mRNA in situ reaction product was then calculated in the different regions of the CL and expressed as a percentage of the area analyzed that contained reaction product. This calculation does not allow an absolute quantitation of the amount of mRNA within the analyzed ovarian cellular compartment. The total number of CL analyzed at each time period from at least three different ovaries is indicated in Table 1. Homogeneity of variance for the in situ reaction product was assessed by Levene test, and subsequently, differences in mRNA expression were determined by one-way analysis of variance (ANOVA) 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. Differences in expression patterns of the TIMPs between the encircling band surrounding the CL (i.e., outside of the CL) versus the body of the CL (i.e., inside of the CL) were analyzed with a Student t-test.
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DNA Fragmentation Assay
As TIMP-3 has been reported to induce apoptosis [13–16], experiments were conducted to correlate the presence of apoptosis with TIMP-3 mRNA expression during the latter stages of pseudopregnancy, Days 12 and 14. Serial sections of the ovaries used for in situ hybridization for TIMP-3 mRNA were also examined for apoptosis by testing for DNA fragmentation using the terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick end-labeling (TUNEL) method. The frozen tissue sections (10 µm) were prepared by fixation in 4% paraformaldehyde and rinsing in PBS. DNA fragmentation was determined using the ApoAlert DNA Fragmentation Assay from Clontech (Palo Alto, CA). After performing the assay according to the manufacturer's instructions, the slides were mounted with VectaShield Mounting Medium with propidium iodide (Vector, Burlingame, CA). Tissues were analyzed on a Nikon Eclipse E800 microscope for fluorescent detection of apoptosis. Follicles or corpora lutea that demonstrated at least five cells displaying fluorescence were classified as undergoing apoptosis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Situ Localization of TIMP-1 mRNA> During Pseudopregnancy
At Day 1 of pseudopregnancy, intense reaction product for TIMP-1 mRNA was observed surrounding the developing CL and in the stroma (Fig. 1, A and B). TIMP-1 mRNA was also present within the developing CL, although the percentage of the area occupied by in situ reaction product within the CL was less (26.5% ± 5.9%, n = 21, P < 0.05) than that observed surrounding the CL (57.8% ± 6.1%, n = 21). The expression of TIMP-1 mRNA in granulosa cells of adjacent follicles (5.8% ± 1.5%, n = 8) was lower than that observed within the forming CL and approximated background levels (Fig. 1, A and B). In the 2-day-old CL, the intensity of the band of TIMP-1 mRNA surrounding the CL was reduced (31.6% ± 6.4% versus 57.8% ± 6.1% of the area occupied by reaction product, n = 21; Fig. 1, E and F), and by Day 4, the mRNA expression surrounding the CL was lost, although low levels of TIMP-1 mRNA expression were found within the CL (Fig. 1, I and J). The localization pattern of TIMP-1 mRNA was unchanged throughout the remainder of pseudopregnancy, with high levels of expression found associated with the stroma and surrounding certain follicles. However, the percentage of the area of the CL occupied by in situ reaction product (Table 1) declined within the CL at Days 8 (Fig. 1, M and N), 12 (Fig. 1, Q and R), and 14 (Fig. 1, U and V). Negative control sections (i.e., hybridized with sense cRNA probes) demonstrated only background levels of expression for all of the different mRNAs examined (Fig. 2).
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In Situ Localization of TIMP-2 mRNA> During Pseudopregnancy
The pattern of expression for TIMP-2 mRNA exhibited some similarities to that seen for TIMP-1 mRNA. At Day 1 of pseudopregnancy, intense reaction product for TIMP-2 mRNA was observed surrounding the developing CL and was present throughout the stroma (Fig. 1C). TIMP-2 mRNA was also present within the developing CL and the percent of the area occupied by in situ reaction product was less than that observed in the adjacent stroma (8.1% ± 1.3%, n = 21 versus 35.5% ± 3.3%, n = 11). Whereas the expression of TIMP-1 mRNA was homogenous throughout the CL, TIMP-2 mRNA was present in a punctate pattern within the CL, with high levels of expression present near the center of the developing CL (Fig. 1, C and G). The granulosa cells of adjacent follicles did not express observable levels of TIMP-2 mRNA. The localization pattern of TIMP-2 mRNA was unchanged throughout the remainder of pseudopregnancy, with intense reaction encircling the CL and high levels of expression found associated with the stroma and surrounding developing follicles (Fig. 1, K, O, S, and W). The percent of the area occupied by TIMP-2 mRNA was low and declined during pseudopregnancy (Table 1). Of interest was the appearance of TIMP-2 in the center of forming CL (Fig. 1G) as well as in regressing CL on Days 12 (Fig. 1S) and 14 (Fig. 1W).
In Situ Localization of TIMP-3 mRNA> During Pseudopregnancy
The pattern of expression for TIMP-3 mRNA was strikingly different than that observed for either TIMP-1 or TIMP-2 mRNA. At Day 1 of pseudopregnancy, intense reaction product for TIMP-3 mRNA was observed in the luteinizing granulosa cells of the developing CL, with negligible expression found in the stroma (Fig. 1D). In follicles, expression of TIMP-3 mRNA in the granulosa cells was low and approximated background levels. With continued luteal development, there was a homogenous, intense expression of TIMP-3 mRNA throughout the CL. Although the cellular pattern was unchanged throughout pseudopregnancy (Fig. 1, H, L, P, T, and X), there was a decrease in the percent of the area occupied by TIMP-3 mRNA at the end of pseudopregnancy (Table 1). TIMP-3 mRNA was lost in the center of regressing CL (Days 12 and 14) in areas where TIMP-2 mRNA expression was observed (Fig. 1, T and X).
Because TIMP-3 has been reported to induce apoptosis [13–16], experiments were conducted to determine whether the presence of TIMP-3 mRNA correlated with apoptotic changes in regressing CL. The expression of TIMP-3 mRNA is abundant in CL on Days 12 and 14 of pseudopregnancy (Fig. 1, T and X). Analysis of apoptosis in these regressing CL revealed the presence of TIMP-3 mRNA; however, there was a lack of fluorescent DNA fragmentation in these CL (Fig. 3). Apoptosis was present in adjacent follicles, demonstrating the ability of the assay to detect DNA fragmentation.
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In Situ Localization of TIMP-3 mRNA During Early> Luteal Formation
During the course of these and previous studies [23], we observed an LH/hCG induction of TIMP-3 mRNA in the granulosa cells of certain large preovulatory follicles, whereas granulosa cells of adjacent follicles contained minimal expression of TIMP-3 mRNA (Fig. 4G). In an attempt to understand the relation between TIMP-3 mRNA expression and the status of these follicles, a series of experiments was performed where expression for markers of follicular maturity were compared with the presence of TIMP-3 mRNA. At the 0-h time point (i.e., 48 h after eCG and prior to hCG), TIMP-3 mRNA was abundant in the stroma but was not as highly expressed in the granulosa cell layer (Fig. 4, A and B). In situ localization of the mRNA for aromatase, P450 side-chain cleavage, and the LH receptor confirmed that the follicles were estrogenic and not luteinized (Fig. 4, C, D, and E, respectively). By 12 h after hCG, TIMP-3 mRNA was observed in the luteinizing granulosa cells of certain follicles whereas granulosa cells of adjacent follicles were unlabeled (Fig. 4, F and G). The fact that the majority of these follicles were luteinizing is apparent from the appearance of P450 side-chain cleavage mRNA (Fig. 4I) and the loss of expression for aromatase and the LH receptor mRNA (Fig. 4, H and J). By 24 h after hCG, TIMP-3 mRNA was abundant in the granulosa-lutein cells of developing CL (Fig. 4, K and L). Luteinization was confirmed by the presence of P450 side-chain cleavage mRNA (Fig. 4N). However, a very small number of follicles or CL expressed P450 side-chain cleavage but did not exhibit TIMP-3 mRNA (Fig. 4, I and N, respectively). TIMP-3 mRNA was not observed in follicles that continued to express aromatase and LH receptor mRNA (Fig. 4, M and O).
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Of particular interest was the observation that, at 12 h after hCG, certain follicles had partial expression of TIMP-3 mRNA within the luteinizing granulosa cell compartment (denoted in Fig. 4G). These same follicles continued to express aromatase or LH receptor mRNA but in regions of the follicles that did not highly express TIMP-3 mRNA (Fig. 4, H and J).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The overall cellular pattern of luteal TIMP-2 mRNA did not change throughout pseudopregnancy, although there was a decline in the percentage of the luteal area expressing TIMP-2 mRNA. Previous findings have reported that levels of TIMP-2 mRNA, as determined by Northern analysis, are unchanged in ovaries collected throughout pseudopregnancy [21]. Thus, the abundant expression of mRNA for TIMP-2 surrounding the CL or in other cellular compartments, such as the theca or the stroma, may mask the decline in the percent of the area occupied by TIMP-2 mRNA observed in the CL in the present study. The current findings, however, reveal differences in the pattern of expression within the CL. The intense in situ reaction product around the forming CL and within the center of the CL would indicate that these are areas where TIMP-2 may regulate extensive tissue remodeling. Alternatively, the abundance of TIMP-2 mRNA may translate into a physiologic role in luteal function such as stimulating cell growth independent of its ability to act on MMPs, as has been reported for fibroblasts [31].
The present observations in the pseudopregnant ovary differ from the pattern of cellular localization of TIMP-2 mRNA observed in regressing CL collected from ovaries throughout the rat estrous cycle. In naturally cycling rats, TIMP-2 mRNA was higher in regressing CL than newly forming CL, with high levels of expression in certain cells and regions [24]. The difference in TIMP-2 mRNA expression may be related to the stage of luteal demise between the two models. In the pseudopregnant model, the CL becomes functional in terms of progesterone production, whereas in the cycling rat, the CL does not reach its full steroidogenic potential and undergoes regression. Furthermore, the CL from previous cycles may represent CL that are further advanced in their structural demise than the CL examined in the present study. The cellular pattern of TIMP-2 mRNA expression observed throughout pseudopregnancy differs from the luteal pattern in other species across the estrous or menstrual cycle. For example, in the bovine, TIMP-2 mRNA was low during the early luteal phase (Day 4), significantly increased between Days 10 and 15 of the estrous cycle, and then declined at Day 19 [32]. In sheep, TIMP-2 mRNA was highly abundant throughout the CL [33], and separation of ovine luteal cell populations by centrifugal elutriation demonstrated that the purified large luteal cells contained approximately 20-fold more TIMP-2 mRNA than the small luteal cells [26, 33]. In the human, TIMP-2 mRNA levels did not change in CL collected across the menstrual cycle [29]; however, the pattern of localization was different than that found for other species. TIMP-2 mRNA was present in the theca-lutein cells and the connective tissue surrounding the steroidogenic cells of the CL [29]. These differences in the pattern of expression for TIMP-2 mRNA observed in the current study versus previous reports in the rat, ovine, and human suggest diverse actions for TIMP-2 between the pseudopregnant state and in CL collected across the cycle.
The expression of TIMP-3 mRNA was examined in detail from the time of hCG administration through early luteal formation (24 h after hCG) as well as throughout pseudopregnancy. The rationale for investigating TIMP-3 during the transition between the final stages of follicular maturation and early luteal development was the intriguing observation that certain preovulatory follicles expressed TIMP-3 mRNA whereas adjacent follicles lacked this inhibitor [23]. We postulated that those preovulatory follicles that expressed TIMP-3 mRNA were follicles that would ovulate and form CL. This postulate was confirmed by comparing the transition and differentiation of preovulatory follicles with the expression of TIMP-3 mRNA. For example, in response to hCG, it would be expected that follicles selected to ovulate would lose expression of aromatase as the follicle switched from production of estradiol to progesterone synthesis, gain expression of side chain cleavage with the change in steroid production, and down-regulate LH receptor expression. This pattern of differentiation was precisely what was observed for preovulatory follicles expressing TIMP-3 mRNA. Further evidence that TIMP-3 mRNA is switched on in preovulatory follicles is the correlative report of TIMP-3 mRNA expression in granulosa cells of healthy follicles from rat ovaries collected throughout the estrous cycle [24]. Finally, the appearance of this inhibitor in the CL throughout pseudopregnancy and the finding that the CL is the predominant cellular source of this inhibitor supports the concept that TIMP-3 mRNA is turned on in healthy preovulatory follicles that then become CL. The role of TIMP-3 in this process of transition, however, is unknown.
The current data provide an interesting snapshot of the expression of TIMP-3 mRNA as the ovulatory follicle is transformed into a CL. At 12 h after hCG, these follicles would be in the early process of follicular rupture ([25], personal observations) and yet certain of these follicles exhibit a regional pattern of granulosa cell TIMP-3 mRNA expression. The reason for this partial expression of TIMP-3 mRNA in certain areas of the follicle is unknown and deserves further investigation to characterize the extent and location of this pattern of expression. One may speculate, however, that the region of the follicle adjacent to the cumulus oocyte complex may regulate the expression of TIMP-3 mRNA in a site-specific manner to control proteolysis or that TIMP-3 has additional actions other than as a proteinase inhibitor, such as regulating growth [10] or steroidogenesis [17] as attributed to the other TIMPs, in the ovulatory process.
The pattern of cellular localization of TIMP-3 mRNA throughout pseudopregnancy demonstrates a high level of expression throughout the CL that is relatively unchanged. Quantitative examination of TIMP-3 mRNA levels in intact ovaries by Northern analysis has revealed an increase in expression throughout pseudopregnancy [21]. For example, during Days 1 and 2 of luteal formation, TIMP-3 mRNA levels were low, reached peak values at Day 8 of pseudopregnancy, and remained elevated thereafter [21]. This pattern of quantitative changes by Northern analysis would, at first inspection, appear to be incongruent with the current findings that the percentage of the luteal area occupied by TIMP-3 mRNA is unchanged throughout the first 12 days of pseudopregnancy. The measurement of the area occupied by TIMP-3 mRNA in situ reaction product does not take into account the size of the CL, which in turn would impact the overall levels of mRNA expression. For example, as the CL is forming, there are fewer luteal cells present; thus, the quantitative levels of TIMP-3 mRNA expression are lower. By Day 8, the CL is fully formed, has increased in size, and TIMP-3 mRNA is expressed throughout the CL, resulting in an overall increase in the levels of TIMP-3 mRNA. An attractive hypothesis is that the expression of this matrix metalloproteinase inhibitor throughout the CL may act to protect the CL from proteolytic degradation.
Unlike TIMP-1 and TIMP-2, which are found circulating in the extracellular fluid, TIMP-3 is bound to the extracellular matrix and acts locally to regulate MMP action. The presence of TIMP-3 mRNA, therefore, may maintain the structural integrity of the CL. Another possible role for TIMP-3 is the induction of apoptosis [13–16]. The presence of this inhibitor at the end of pseudopregnancy could play a role in the apoptotic changes associated with luteal demise [34, 35]. The current findings would suggest that the presence of TIMP-3 mRNA and the anticipated presence of TIMP-3 protein does not induce apoptosis in the CL at Days 12 or 14 of pseudopregnancy, as illustrated by the lack of TUNEL staining in these CL.
Although unique patterns of cellular localization for the mRNA for the TIMPs were observed throughout pseudopregnancy, the question remains as to what these patterns represent and the function of each of the different TIMPs in these ovarian compartments. These patterns of expression may indicate the site of action of the TIMPs in their classical role as MMP inhibitors in maintaining proteolytic homeostasis in the CL. The differences in the patterns of expression may be related to differences in the specificity of the TIMPs for the MMPs or the site of action of the various TIMPs either in the extracellular space of in the connective tissue matrix. Alternatively, the patterns of expression may reflect actions other than their role as proteinase inhibitors to regulate luteal physiology. Such nonclassical actions include stimulating cell growth in a variety of tissues [10], including the embryo [36], acting as antiangiogenic agents [11, 12], and inducing apoptosis [13–16]. The involvement of TIMPs in these processes, either directly or indirectly by mediating MMP action, has led to the postulate that TIMPs act as autocrine/paracrine factors in cellular proliferation, differentiation, and neovascularization. It is possible that the TIMPs may impact ovarian function through these nonclassical actions as evidenced by the findings that a TIMP-1-like protein stimulates steroid production in the testis and ovary [17], that granulosa cell estradiol production is stimulated by TIMP-1 in vitro [18], and that TIMP-1-deficient mice have altered serum levels of progesterone and estradiol during the estrous cycle [19]. However, the decline in TIMP-1 mRNA during the midluteal period in the present and former studies [20, 21] could be interpreted to mean that TIMP-1 does not play a major regulatory role in luteal progesterone production in the pseudopregnant model. Irrespective, the cellular localization of the various TIMPs in the present study may be related to their roles as matrix metalloproteinase inhibitors or their localization may reflect their roles in regulating cellular proliferation, differentiation, or steroidogenesis associated with luteal formation and function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Thomas E. Curry, Department of Obstetrics & Gynecology, 800 Rose Street, Room C-355, University of Kentucky, Lexington, KY 40536. FAX: 859 323 1931; tecurry{at}pop.uky.edu
Received: 15 January 2002.
First decision: 30 January 2002.
Accepted: 3 July 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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