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. 2013 Sep 18;89(5):121. doi: 10.1095/biolreprod.112.106989

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle1

Katherine L Rosewell 3, Feixue Li 4, Muraly Puttabyatappa 3, James W Akin 5, Mats Brännström 6, Thomas E Curry Jr 3,2
PMCID: PMC4434989  PMID: 24048576

ABSTRACT

Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Keywords: follicle, granulosa cell, human, ovary, ovulation, protease inhibitor, theca cell

INTRODUCTION

Before follicular rupture and ovulation can take place, there are substantial alterations in the interaction between the follicle and its surrounding matrix. Such interactions are controlled in part by the delicate balance between the matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases, or TIMPs. There are four isoforms of TIMP (TIMP1–4) and, while they all inhibit MMPs, they have differing affinities for specific members of the MMP family as well as different sites of action [1]. TIMPs also inhibit members of the ADAMs (a disintegrin and metalloprotease) and ADAMTS (ADAM with thrombospondin motifs) families [2, 3], which is particularly relevant in light of the finding that ADAMTS1 plays a key role in the ovulatory process [4]. TIMP3 exhibits the broadest spectrum of inhibition of these peptidases [5, 6] and is, therefore, considered to be a major regulator of MMP activity in vivo [7].

TIMPs have other biological activities outside of their classical action as metalloproteinase inhibitors. TIMPs are complex molecules that can have opposing effects on cell function depending on their tissue localization and the hormonal environment. Many of these cell functions (proliferation, angiogenesis, and apoptosis) are crucial for ovulation and luteinization. For example, TIMPs can increase cell proliferation of many different cell types, including both normal [8, 9] and malignant [10] cells. In contrast, TIMP1 can decrease the cell growth of mammary epithelial cells [11, 12]. TIMPs 1–3 are anti-angiogenic [13–15]; in particular, Kang et al. [16] demonstrated that TIMP3 was able to inhibit tumor angiogenesis and endothelial cell proliferation. Qi et al. [17] found that TIMP3 is able to block the ability of VEGF to bind to its receptor (VEGFR2), inhibiting both downstream signaling mechanisms and angiogenesis. In contrast to its anti-angiogenic actions, TIMP3 also stabilizes the vascular network, thereby preventing the regression of newly formed blood vessels [18]. Thus, TIMP3 has diverse actions depending upon the cell type, tissue, or physiological setting.

TIMPs have both pro- and antiapoptotic activity. TIMP3 is able to promote apoptosis in a variety of cancer cell lines ranging from the noninvasive MCF-7 cells to the highly invasive HT1080 cells [19]. Overexpression of TIMP3 promotes apoptosis in melanoma cells [20] by its effect on the stabilization of several death receptors and the subsequent activation of the caspase cascade [21]. In contrast, TIMP1 is able to inhibit apoptosis in several breast cancer cell lines [22] by activating downstream signaling pathways and promoting cell survival [23, 24].

It has become well established that MMPs and TIMPs play a role in the restructuring of the ovary during follicular growth and in the changes leading to ovulation and the formation of the corpus luteum [25, 26]. Since TIMP3 is able to act on the broadest range of proteinases of any of the TIMPs and it is the only TIMP bound to the extracellular matrix [27–29], it is a prime candidate to provide inhibitory action in the ovarian follicle and surrounding stroma of the ovary. Timp3 mRNA in the rat ovary reaches its highest level on proestrus and drops significantly by 1100 h on the day of estrus [30]. In the rat, Timp3 mRNA is present in the theca and stroma at the beginning of the periovulatory period, and as the time of ovulation approaches, Timp3 expression increases in the granulosa cells of the preovulatory follicle [31]. However, nothing is known about TIMP3 throughout the periovulatory period in the female human.

The cellular location and timing of both mRNA and protein expression of TIMP3 in the rodent has led us to consider its role in the human follicle during the periovulatory period. TIMP3 is perfectly poised to impact the ovulatory process in humans. We hypothesized that TIMP3 would increase prior to ovulation in order to keep MMP activity in check and prevent wholesale destruction of the follicle wall. Therefore, in the present study, we focused on expression and localization of TIMP3 across the periovulatory period in the human.

MATERIALS AND METHODS

Materials

Unless otherwise noted, all chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Molecular biological enzymes, oligonucleotide primers, culture media, SYBR Green ER, and Trizol were purchased from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Reagents for immunohistochemistry were purchased from Biocare (Concord, CA) unless otherwise noted.

Sample Collection

Human granulosa and theca cells from periovulatory follicles were collected as previously described [32]. The study was approved by the regional human ethics committee of Gothenburg University, and informed, written consent was obtained from all patients enrolled in the study. Briefly, the dominant follicle was collected from patients undergoing laparoscopic tubal sterilization, which was timed to take place at one of four different periovulatory periods (preovulatory, early ovulatory, late ovulatory, and postovulatory groups), as described below. To obtain high-quality patient material, only women with proven fertility, regular menstrual cycles, and without hormonal medications for at least 3 mo were included. The women were monitored with repeated transvaginal ultrasound (TVU) for two to three cycles to enable planning of surgery at a stage when the dominant follicle was preovulatory. During the menstrual cycle of the laparoscopic sterilization with tissue collection, TVU was performed every 1–2 days to enable the surgery to be planned at the accurate stage of the menstrual cycle. The ovary containing the dominant follicle was identified by TVU. At the time of surgery, 5 ml of blood was collected by venipuncture for hormonal analysis.

For samples collected at the preovulatory phase, surgery was performed prior to the luteinizing hormone (LH) surge, when the dominant follicle was ≥14 mm and ≤17.5 mm. The remaining patients, who had dominant follicles between 14 and 20 mm, received an injection of human chorionic gonadotropin (s.c.; 250 μg recombinant hCG [rhCG]; Ovitrelle; Merck Serono, Geneva, Switzerland) to mimic the endogenous LH surge and underwent surgery after varying lengths of time following rhCG injection: early ovulatory phase, 12 to ≤ 18 h; late ovulatory phase, >18 to ≤ 34 h; and postovulatory phase, >44 to ≤70 h. Frequent TVU examinations after rhCG administration have determined that rupture occurs approximately 36–38 h after rhCG [32, 33]. Samples for the measurement of serum levels of progesterone and estradiol were taken during surgery for later confirmation of each patient's ovulatory phase category, and these samples were assayed (Architect; Abbott Diagnostic Division, Abbott Park, IL) at the in vitro fertilization (IVF) lab at Sahlgrenska Hospital. The entire intact dominant follicle was excised with scissors from the ovary and placed inside a laparoscopic sac to be retrieved through the suprapubic trocar incision, which had been extended to around 30 mm, and processed intact for immunohistochemistry or bisected for the collection of granulosa and theca cells.

The intact follicles for immunohistochemistry were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned at 7μm. For cell isolation, granulosa cells were collected by bisecting the follicle to release the loosely attached cells. The mural granulosa cells were then gently scraped from the follicle wall and pooled with the loosely attached granulosa cells. Theca cells were harvested mechanically from the remnant of the follicle by separating the theca interna cell layer from the theca externa layer. This theca interna cell layer also contains vascular cells, leukocytes, and fibroblasts [34]. For clarity we refer to these as “theca cells.” The cells were frozen in liquid nitrogen for subsequent processing and analysis of TIMP3 mRNA expression by real-time RT-PCR. Theca cells were obtained from all four periovulatory phases, but granulosa cells could not be collected from the postovulatory group since the bulk of the granulosa cells had been extruded at follicular rupture; thus, there are only three time points for the granulosa cells.

Real-Time RT-PCR

Total RNA was isolated from granulosa or theca cells using Trizol reagent and reverse transcribed using SuperScript II and Oligo (dT)18 primer according to the manufacturer's protocol (Invitrogen Life Technologies, Inc.). Oligonucleotide primers for human TIMP3 (Forward 5′-ACGATGGCAAGATGTACACAGG-3′, Reverse 5′-GGAAGTAACAAAGCAAGGCAGG-3′), and human L32 (Forward 5′-GAAGCCCAAGATCGTCAAAA-3′, Reverse 5′-AGGATCTGGCCCTGGCCCTTGAATCT-3′), an internal endogenous control gene, were designed based on the previously reported sequences of human TIMP3 (GenBank, NM_000362) and L32 (GenBank, NM_013226) using PRIMER3 (SDSC Biology Workbench version 3.2, San Diego, CA) software. The real-time PCR reactions contained 10% of the RT reaction product, 0.4 μM of forward and reverse primers, 0.3 μl of 1:10-diluted ROX reference dye (provided with SYBR Green ER qPCR SuperMix Universal kit), and SYBR Green SuperMix. PCR reactions were performed on an Mx3000P QPCR System (Agilent Technologies, Inc., Santa Clara, CA). The thermal cycling steps included 2 min at 50°C to permit optimal AmpErase uracil-N-glycosylase activity; 10 min at 95°C for initial denaturation; and then each cycle of 30 sec at 95°C, 30 sec at 55°C, and 40 sec at 72°C for 40 cycles; followed by 1 min at 95°C, 30 sec at 58°C, and then 30 sec at 95°C for ramp dissociation. The performance of the assay mix and endogenous control was tested through analysis of the amplification plots and plate sample values generated in the MxPro RT-PCR analysis program (Agilent Technologies) following each RT-PCR reaction. The relative amount of TIMP3 mRNA in each sample was calculated following the ΔΔCT method and normalized to the endogenous reference gene, L32.

Immunohistochemistry

Sections containing human follicles were deparaffinized for 10 min in xylene, followed by a decreasing ethanol series, three washes in Tris-buffered saline (TBS), and then quenching for 5 min with Peroxidazed 1 to block endogenous peroxide activity. Antigen retrieval was done using DakoCytomation Target Retrieval solution (Dako North America, Inc., Carpinteria, CA) for 20 min at 100°C. The slides were allowed to come to room temperature, washed once in TBS, and then blocked for 10 min with Background Sniper. Sections were incubated with TIMP3 antibody (1:200 dilution [0.1 mg/ml]; Abcam, Cambridge, MA) at room temperature for 2 h, washed in TBS, and incubated with biotinylated secondary antibody for 20 min (Trekkie Biotinylated Rabbit Link). To amplify the reaction signal, the sections were treated with a conjugated streptavidin alkaline phosphatase (TrekAvidin-AP label) for 10 min before staining was visualized using a Vulcan Fast Red chromogen; sections were then counterstained with hematoxylin and coverslipped using VectaMount mounting media (Vector Laboratories, Burlingame, CA). In control sections, primary antibody was omitted.

IVF Cell Collection and Cell Viability Assay

Due to the scarcity of material from human follicles of the menstrual cycle, functional studies to examine TIMP3 action were performed with human granulosa-lutein cells from women undergoing IVF in our assisted reproductive technology program. This part of the study was approved by the human Institutional Review Board of the University of Kentucky. Women (n = 6) were treated with a gonadotropin-releasing hormone agonist (Lupron; TAP Pharmaceutical Products, Inc., Lake Forest, IL), given recombinant follicle-stimulating hormone (Gonal-f; Serono, Inc., Rockland, MA) to induce follicular growth, and monitored by TVU and serum estradiol levels. When the two largest follicles reached an average diameter of ≥18 mm, rhCG (250 μg; Ovitrelle) was administrated s.c., and granulosa-lutein cells were collected by TVU-guided aspiration 34–36 h after hCG administration. After removal of the oocytes, granulosa-lutein cells with associated red blood cells (RBCs) were pooled from multiple follicles of the same patient and placed in OptiMEM I media. The cells were then pelleted by centrifugation (2000 × g for 5 min) and resuspended in 1 ml of OptiMEM I media. The cell suspension was carefully layered on a 40% Percoll gradient prepared in Hanks Balanced Salt Solution and centrifuged at 1000 × g for 30 min to separate the RBCs from the granulosa-lutein cells, after which the RBC-free granulosa-lutein cells were recovered from the top of the gradient and washed twice in OptiMEM I media. Cells (2 × 104) were plated in a 96-well tissue culture plate in OptiMEM I containing ITS (insulin-transferrin-selenium; 1×) and 1.25% gentamycin (total volume per well was 200 μl). Twenty-four hours later, the media was changed, and cells were treated with increasing concentrations of recombinant TIMP3 (0, 0.01, 0.1, and 1 μg/ml) for 24 h. At the end of the treatment period, the fraction of viable cells was determined using an MTS assay kit (CellTiter Aqueous; Promega, Madison, WI) as per the manufacturer's instructions. Briefly, assays were performed by adding the CellTiter 96 Aqueous One reagent into the culture well and incubating at 37°C for 4 h, and then absorbance was read at 490 nm using an Infinite F200 plate reader (Tecan Group Ltd., Männedorf, Switzerland). The quantity of the formazan product is directly proportional to the number of live viable cells.

Statistical Analysis

Results are expressed as mean ± SEM. Differences in mRNA or cell viability between groups were analyzed by one-way ANOVA, and post-hoc comparisons were performed when appropriate using least significant differences and Tukey honestly significant differences. P < 0.05 was considered significant.

RESULTS

Characteristics of the Patient Population

The characteristics of the patient population used for follicle collection are outlined in Table 1. The patients ranged from 30–39 yr of age; however, there was no difference in the age of the patients across the four periovulatory phase groups. Likewise, there was no difference in the length of the previous menstrual cycles or the day upon which surgery was performed. After administration of hCG, there was an expected increase in serum progesterone levels along with a peak in serum estradiol levels at the late ovulatory phase (Table 1). There was no difference in the size of the follicle at the time of collection or hCG administration.

TABLE 1.

Characteristics of the study population, with periovulatory samples of the menstrual cycle.*

graphic file with name i0006-3363-89-5-121-t01.jpg

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* 

Values are shown as mean ± SEM, and the ranges are given below.

† 

N/A = these women did not receive rhCG.

a,b 

Different superscripts denote a significant difference between groups (P < 0.05).

Expression of Human TIMP3 mRNA by Real-Time PCR

TIMP3 mRNA was present at all time points examined (pre-, early, late, and postovulatory follicles) in granulosa and theca cells. In granulosa cells, TIMP3 mRNA expression was increased 2-fold from the early to late ovulatory stage (Fig. 1; n = 5). In the theca, TIMP3 was constitutively expressed across the entire periovulatory period (Fig. 1).

FIG. 1.

FIG. 1

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Real-time PCR analysis for TIMP3 mRNA in granulosa and theca cells from human follicles collected across the periovulatory period. There was a significant increase (*) in TIMP3 mRNA expression in granulosa cells from the pre- to late ovulatory stage (P < 0.05, n = 5). There was no significant change in thecal TIMP3 expression across the periovulatory period (P = 0.23).

Localization of Human TIMP3 During the Periovulatory Period in Whole Follicles

TIMP3 protein was detected by immunohistochemistry using an antibody to the C-terminal region of the protein. In preovulatory follicles prior to exogenous rhCG, immunoreactive TIMP3 was detected in the theca cell layer as well as in a few of the granulosa cells (Fig. 2A). At 12–18 h after rhCG, the early ovulatory follicles exhibited TIMP3 staining in the thecal cell layer as well as in the granulosa cells adjacent to the basement membrane (Fig. 2B). Light staining was present in most of the antral granulosa cells at this time. By the time the follicle had reached the late ovulatory stage (18–34 h after rhCG), the thecal layer was heavily stained and there was positive immunoreaction product throughout the granulosa cell layer (Fig. 2C). There was no staining when the primary antibody to TIMP3 was omitted (Fig. 2D).

FIG. 2.

FIG. 2

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Immunohistochemistry of TIMP3 expression in follicles collected during the preovulatory (A), early (B), and late (C) ovulatory periods (n = 3–6 per group). TIMP3 immunostaining was visualized by using Vulcan Fast Red chromogen (bright pink); sections were counterstained with hematoxylin (blue nuclear staining). TIMP3 was expressed in the theca (black arrow) and in a few of the granulosa cells (red arrow) in the preovulatory follicle (A). Protein expression in the early ovulatory follicle (B) exhibited an intense pattern of staining in the theca (black arrow). The granulosa cells along the basement membrane were immunoreactive for TIMP3 (green arrow), while the antral granulosa cells were lightly stained (red arrow). In the late ovulatory period (C), TIMP3 was highly expressed in the ovulatory follicle in both the granulosa and theca cell layers. A serial section of a late ovulatory follicle adjacent to the section in C without primary antibody to TIMP3 did not demonstrate any specific staining (D). Original magnification ×10, bars = 100 μm.

TIMP3 is known to participate in vascular endothelial cell tube stabilization [18] to prevent the regression of newly formed vascular networks. The vascular supply plays a critical role in the selection of the dominant follicle that is destined to mature and ovulate [35]. We observed that TIMP3 was present in the vascular smooth muscle cells of certain vessels associated with the dominant follicle (Fig. 3). Sections from early ovulatory follicles show staining in the vascular beds of the theca (Fig. 3).

FIG. 3.

FIG. 3

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Immunohistochemistry of TIMP3 expression in a vascular bed of the theca in early ovulatory follicles, large arteries (A), and a smaller artery (B). TIMP3 was highly expressed in both the large and small arteries. A serial section adjacent to the section in A without primary antibody to TIMP3 did not demonstrate any specific staining (C). Original magnification ×10, bars = 100 μm.

Ability of Exogenous TIMP3 to Regulate Human Granulosa-Lutein Cell Viability

There are conflicting reports on the action of TIMP3 in other tissues, ranging from supporting cell growth [36] to inducing apoptosis [20]. As TIMP3 was stimulated by hCG in the present study, we assessed the ability of TIMP3 to induce changes in cell viability. Due to the difficulty in acquiring timed human follicles, these experiments were performed in granulosa-lutein cells collected immediately prior to ovulation (late ovulatory period) from patients in our IVF program. Treatment with 0.1 μg/ml TIMP3 significantly decreased the number of live cells by 35%, and 1 μg/ml TIMP3 further decreased viability by 60% (Fig. 4).

FIG. 4.

FIG. 4

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Addition of exogenous recombinant TIMP3 to human granulosa-lutein cells from IVF patients significantly decreased cell viability (n = 6). Granulosa-lutein cells were prepared from patients undergoing stimulation for IVF and treated with recombinant TIMP3, and cell viability was determined. Different letters denote significant differences between groups (P < 0.05).

DISCUSSION

In the current study, the use of a defined and unique patient population has for the first time allowed investigation and description of an increase in TIMP3 mRNA and protein expression in a tightly controlled manner across the human ovulatory cycle. There were no differences in the ages of the 35 women who participated in this study, their menstrual cycle length, the cycle day when the follicle was retrieved, or the size of the follicle at the time of rhCG administration. The measurement of the serum concentration of steroids at these defined stages gives us a clear indication that the rhCG administration results in similar steroidal profiles when compared to those after the natural gonadotropin surge. Additionally, the serum steroid profiles document that the follicles have responded appropriately to hCG treatment. For example, the increase in progesterone observed in the present study is similar to the rise previously reported in plasma and peritoneal fluid [37]. The rise of estradiol reached a significant peak during the late ovulatory phase several hours before ovulation; this is in agreement with our earlier studies [34] but in slight contrast to estradiol levels in plasma and peritoneal fluid, which did not differ before ovulation [37].

In the present study, the mRNA expression pattern in granulosa cells revealed a significant doubling of TIMP3 during the late ovulatory phase when compared to the preovulatory phase. This finding of an induction of TIMP3 after hCG is in agreement with the periovulatory increase observed in other species. In the rat, there is a marked induction of the expression of Timp3 mRNA in certain follicles at 12 h after hCG by in situ hybridization [31], and cultured rat granulosa cells exhibit a biphasic expression pattern for TIMP3 after hCG administration, with a peak at 2 h and another at 16 h [38]. In the mouse, there are conflicting reports of Timp3 mRNA expression, with Timp3 increasing during the early ovulatory period [39] or exhibiting low constitutive levels in the intact ovary [40, 41]. In the bovine follicle, TIMP3 mRNA increases approximately 2-fold at 12 h after GnRH injection [42]. Furthermore, it has been shown that TIMP3 mRNA is more abundant in healthy follicles [31, 43]; thus, this rise in TIMP3 in the human, bovine, and rat may be required for the final stages of follicular maturation in preparation for the matrix remodeling that takes place before ovulation and expulsion of the oocyte.

In addition to modulating the changes in the follicular matrix, this local production of TIMP3 may be important in mediating the inflammatory response associated with ovulation. For example, mice lacking TIMP3 are unable to mount an adequate immune response to an exogenous inflammatory stimulus (lipopolysaccharide administration) [44], and TIMP3 has been shown to control tumor necrosis factor (TNF)-mediated inflammation in the liver [44, 45]. TIMP3 controls the inflammatory process by its ability to inhibit TNF-α converting enzyme (TACE, also known as ADAM17 [45]). These observations have led to the suggestion that TIMP3 is a critical regulator of the release of membrane-bound mediators, which dictate the magnitude of the inflammatory response [44]. Although TIMP3 regulation of immune responses was not examined in the present study, it is possible that TIMP3 regulates proteolytic cascades associated with inflammation during the process of follicular rupture and oocyte release.

In addition to its action on inflammatory pathways, TIMP3 may act on ADAM17 (TACE) to regulate the balance between normal cellular function and apoptosis. TIMP3 induces apoptosis in a number of cancer cell lines (reviewed in Brew and Nagase [29]), which results from TIMP3 inhibition of ADAM17 with subsequent stabilization of TNF receptors on the cell surface. In previous experiments that reported on TIMP3 function, the levels of the inhibitor were increased either by overexpression constructs or adenoviral vectors, which provide model systems to examine TIMP3 action in the context of elevated levels of the inhibitor. However, a delicate equilibrium must exist between the proteinase and its inhibitor to allow appropriate remodeling of the extracellular matrix while maintaining the balance between the extent and location of proteolytic degradation. The results of the present study suggest that it is unlikely that the role of TIMP3 in the periovulatory follicle is to induce apoptosis, since TIMP3 is increased in luteinizing granulosa cells, which represent healthy cells transitioning from a proliferative to a differentiated state. This is supported by observations in the rat that Timp3 mRNA was associated with healthy follicles [43]. When the balance, however, was shifted toward increasing levels of TIMP3, apoptosis increased in human granulosa-lutein cells as well as cultured rat granulosa cells [38], supporting previous reports that with high levels, TIMP3 is proapoptotic [29].

The model system for these experiments allowed for the separation of thecal cells and granulosa cells. However, the mechanical separation of the theca also may have included the carryover of some granulosa cells or immune cells and fibroblasts that are also part of the thecal layer [34]. To address this possibility, the localization of TIMP was explored. During the early stages of the ovulatory process, immunoreactive TIMP3 was localized to the theca as well as to some of the granulosa cells adjacent to the basement membrane. During the late ovulatory phase, there was abundant TIMP3 staining throughout the thecal layer and granulosa cell layers. These findings are in agreement with the finding of Ripley et al. [46], who reported an increase in TIMP3 immunostaining correlating with follicular development, tumor stage, and tumor progression in the human ovary. In this previous study, staining intensity was highest in the theca interna and luteal cells. An elevated thecal expression of TIMP3 is also seen in other species, such as porcine [47], equine [48], and bovine [42]. This temporal progression of TIMP3 expression in the follicle may allow for the protection of the different cellular layers of the follicle from degradation by matrix metalloproteinases during specific phases of growth and maturation while ensuring the viability of those cells for luteinization as the corpus luteum forms.

In some patients, the immunostaining of TIMP3 in the theca did not correlate with the pattern of thecal cell mRNA expression wherein TIMP3 mRNA was abundant but TIMP3 was not highly expressed. It is well documented that there are discrepancies between mRNA levels and protein expression [49]. These discrepancies between TIMP3 mRNA and protein may reflect endocytosis of TIMP3. For example, TIMP3 secreted from chondrocytes can be removed from the extracellular space or from conditioned media by endocytosis through a process mediated by a low-density lipoprotein receptor-related protein (LRP) [50] and, therefore, the levels of TIMP3 protein may not reflect mRNA expression.

TIMP3 protein was also localized to the vascular smooth muscle cells of some of the vessels, particularly during the early ovulatory time period. Development of the thecal vascular bed is an important step in the health and growth of the dominant follicle [51]. What is surprising about these observations is that TIMP3 has been shown to be a potent inhibitor of angiogenesis by suppressing VEGF-mediated angiogenesis. It is possible that in the early preovulatory stage, TIMP3 could inhibit angiogenesis to prevent premature invasion of vessels into the granulosa cell layer. Alternatively, TIMP3 could be acting to maintain the existing vasculature in preparation for the dynamic changes in angiogenesis that occur during transformation of the ovulatory follicle to the corpus luteum, as TIMP3 is known to participate in vascular endothelial cell tube stabilization to prevent the regression of newly formed vascular networks [18].

In summary, the present study demonstrates for the first time gonadotropin-induced increases in TIMP3 mRNA and protein in the human periovulatory follicle. The increase in this inhibitor may act to regulate changes in the remodeling of the follicular extracellular matrix or other TIMP3-mediated cellular processes associated with ovulation. Tipping the balance toward high levels of TIMPs leads to a decrease in granulosa-luteal cell viability, highlighting the delicate equilibrium required between proteinase and inhibitor in normal ovarian cell function.

ACKNOWLEDGMENT

The authors would like to thank Dr. Linah Al-Alem and Ms. Lauren McCord for critically reading the manuscript.

Footnotes

1

Supported by grants to T.E.C. (NIH 5R01HD057446-02 and NCRR P20 RR15592) and a grant from the Swedish Research Council (11607-18-3) to M.B.

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