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Regulation and Localization of Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in the Mouse Ovary during Gonadotropin-Induced Ovulation¹

Department of Medical Biochemistry and Biophysics, Ume University, S-90187 Ume, Sweden

Address all correspondence and requests for reprints to: Dr. Tor Ny, Department of Medical Biochemistry and Biophysics, Ume University, S-90187 Ume, Sweden. E-mail: tor.ny{at}medchem.umu.se

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
At the time of ovulation, proteolytic degradation of the follicular wall is required to release the mature oocyte. Extracellular proteases, such as serine proteases and matrix metalloproteinases (MMPs), are thought to play important roles in this process. In this study we have examined the regulation of 11 MMPs and 3 tissue inhibitors of metalloproteinases (TIMPs) during gonadotropin-induced ovulation in the mouse. Northern blot hybridization showed that messenger RNA for several MMPs and TIMPs, including gelatinase A, MT1-MMP, stromelysin-3, MMP-19, TIMP-1, TIMP-2, and TIMP-3, were present at detectable levels in the mouse ovary. In addition, ovarian extracts contained gelatinolytic activities corresponding to the inactive proforms of gelatinase A and gelatinase B. Most of the MMPs and TIMPs were expressed at a constitutive level throughout the periovulatory period. However, MMP-19 and TIMP-1 revealed a different expression pattern; they were both induced 5–10 times by hCG and reached their maximum levels at 12 h after hCG treatment, corresponding to the time of ovulation. At this time point, MMP-19 and TIMP-1 messenger RNA were localized to the granulosa and thecal-interstitial cells of large preovulatory and ovulating follicles. This temporal and spatial regulation pattern suggests that MMP-19 might be involved in the tissue degradation that occurs during follicular rupture and that TIMP-1 could have a role in terminating MMP activity after ovulation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
EXTRACELLULAR proteolysis generated by the matrix metalloproteinases (MMPs) has been associated with many physiological and pathological processes, such as embryo implantation, wound healing, inflammation, tumor invasion, and ovulation (for reviews, see Refs. 1, 2, 3). The MMPs are a family of extracellular proteinases, which to date consists of 19 members that together have enzymatic activity against virtually all components of the extracellular matrix. All MMPs share a similar domain structure, including a zinc-binding site in the catalytic domain. They are synthesized in a latent proform that requires activation through proteolytic cleavage and can be inhibited by chelating agents and tissue inhibitors of metalloproteinases (TIMPs) (for reviews, see Refs. 4, 5, 6).

Mammalian ovulation is a process triggered by the preovulatory surge of LH from the pituitary gland, which results in liberation of the mature ovum from the preovulatory ovarian follicle. This process requires proteolytic degradation of basement membranes and the connective tissue that constitutes the follicular wall (7, 8, 9). Over the years, several lines of indirect evidence have suggested that both MMPs and the plasminogen activator (PA) system are important for generating the proteolytic activity needed at the time of ovulation. Specifically, 1) an increase in both PA and MMP activity has been found in the rat ovary before ovulation (10, 11, 12, 13, 14); 2) intrabursal injection of plasmin inhibitors and antibodies against tissue-type PA partially block gonadotropin-induced ovulation in rats (15, 16); and 3) synthetic MMP inhibitors can suppress ovulation in perfused rat ovaries (17, 18). However, more recent studies, using gene-deficient mouse strains, have suggested that the PA system is less important for ovulation than previously anticipated. Thus, only a slight reduction in ovulation efficiency was found in mice deficient for either both PAs or plasminogen during gonadotropin-induced ovulation (19) (Ny, A., G. Leonardsson, A.-C. Hägglund, P. Hägglöf, V. A. Ploplis, P. Carmeliet, and T. Ny, unpublished data), which may merely be due to a delayed maturation of these mice manifested in their reduced body weight.

To further investigate the role of matrix-degrading proteases in the ovary, in this study we examined the regulation of 11 MMPs and 3 TIMPs during gonadotropin-induced ovulation in the mouse.

	Materials and Methods

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Materials
PMSG, hCG, and paraformaldehyde were purchased from Sigma Chemical Co. (St. Louis, MO). McCoy’s 5A medium (modified without serum) was obtained from Life Technologies, Inc. (Gaithersburg, MD). Tissue-Tek OCT compound was purchased from Miles, Inc. (Elkhart, IN). The riboprobe in vitro transcription system was obtained from Promega Corp. (Madison, WI). Antidigoxigenin antibodies, digoxigenin-labeled UTP, and purified human gelatinase A were purchased from Roche Molecular Biochemicals (Mannheim, Germany). SuperFrost Plus microscope slides were purchased from Menzel-Glaser (Braunschweig, Germany). The Ultraspec TM RNA Isolation System was obtained from Biotex Laboratories, Inc. (Houston, TX). Purified murine gelatinase B was obtained from Chemicon International (Temecula, CA).

Animals
Immature female mice (C57BL/6J) were obtained from Bomholtgrd Breeding and Research Center Ltd. Boommice (Ry, Denmark). The mice had free access to water, and standard mouse pellets were available ad libitum (Lactamin, Stockholm, Sweden). A 12-h light, 12-h dark cycle was maintained with the light cycle initiated at 0600 h. Experimental protocols were approved by the regional ethical committee of Ume University (A10/96). Twenty-five-day-old mice (weight, 10 g) were injected with 1.5 IU PMSG to stimulate follicle growth and 48 h later with 5 IU hCG to induce ovulation. Mature 8- to 10-week-old mice and rats were examined for stage of the estrous cycle by daily examination of vaginal smears. Animals were killed by cervical dislocation, and ovaries were collected. The ovaries used for in situ hybridization were directly embedded in Tissue-Tek OCT compound, frozen in pre-cooled 2-methylbutane, and stored at -80 C.

Isolation of mouse probes
The full-length complement DNA (cDNA) for neutrophil collagenase (MMP-8) (20), the TIMP-1 cDNA fragment (nucleotides -22–663) (21), and the TIMP-2 cDNA fragment (nucleotides 500–788) (22) were gifts from Drs. C. Lopez-Otín, G. Opdenakker, and K. Tryggvason, respectively. The mouse gelatinase A (MMP-2) cDNA fragment (nucleotides 915-1415) (23), gelatinase B (MMP-9) cDNA fragment (nucleotides 411–932) (24), collagenase-3 (MMP-13) cDNA fragment (nucleotides 795-1280) (25), stromelysin-1 (MMP-3) cDNA fragment (nucleotides 730-1190) (26), stromelysin-2 (MMP-10) cDNA fragment (nucleotides 647-1175) (27), stromelysin-3 (MMP-11) cDNA fragment (nucleotides 250–685) (28), MT1-MMP (MMP-14) cDNA fragment (nucleotides 935-1464) (29), matrilysin (MMP-7) cDNA fragment (nucleotides 400–920) (30), metalloelastase (MMP-12) cDNA fragment (nucleotides 269–769) (31), and TIMP-3 cDNA fragment (nucleotides 227–802) (32) were obtained by RT-PCR and ligated into pGEM vectors. To isolate the mouse equivalent of human MMP-19, the mouse EST section of GenBank at NCBI was searched for similarities to human MMP-19. A partial cDNA clone (accession no. AA611442) of 387 bp was found that showed 83% identity to the human MMP-19 (33) and was identified as mouse MMP-19 (Pendas, A. M., personal communication). The cDNA for the EST sequence was isolated by RT-PCR. In the RT reaction, RNA prepared from mouse ovaries was used. The DNA sequences of all probes isolated by RT-PCR were verified by DNA sequencing. Before transcription, plasmids were linearized such that antisense or sense RNA probes could be obtained. For Northern blot analysis, transcription was performed using [-³²P]UTP and an in vitro transcription system from Promega Corp. The specific activities of the probes were 2–5 x 10⁸ cpm/µg RNA. The riboprobes used for in situ hybridization were labeled with digoxigenin-labeled UTP and the appropriate RNA polymerase.

RNA preparation and analysis
Total RNA from mouse ovaries was isolated with the Ultraspec TM RNA Isolation System. For Northern blot analysis, total RNA was fractionated by agarose gel electrophoresis in the presence of formaldehyde and transferred to Hybond-N filters (Amersham Pharmacia Biotech, Aylesbury, UK) according to the supplier’s instructions. The prehybridization and hybridization were performed as previously described (12). To verify the accuracy of the probes and the hybridization conditions, positive controls consisting of RNA prepared from control tissues, known to express the different MMPs and TIMPs, were included in the Northern blot hybridizations. The relative abundance of specific messenger RNAs (mRNAs) was analyzed with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and normalized to the relative abundance of GAPDH mRNA in the corresponding samples.

In situ hybridization
The in situ hybridization was performed as previously described (34, 35) with digoxigenin-labeled riboprobes.

Preparation of ovarian extract
Ovaries were dissected free of adhering tissue, washed several times in McCoy’s 5A medium, and kept at -80 C. Eight to 16 ovaries taken at the same time after the hormone treatment were directly transferred to a precooled homogenizer, and ovarian extracts were prepared on ice as previously described (12), followed by centrifugation at 14,000 rpm for 10 min at 4 C. The supernatant was removed, and the protein concentration was determined using the bicinchoninic acid protein assay. To convert the inactive precursor form of MMPs to their active form, ovarian extracts were treated with the organomercurial compound aminophenylmercuric acetate (APMA) at a concentration of 2.5 mM (pH 7.0–7.5) for 30 min at 37 C (36).

Gelatin zymography
Samples of ovarian extracts (15 µg total protein) prepared from mice from each time point were analyzed by SDS-PAGE zymography with 7.5% PAGE gels containing gelatin (1.8 mg/ml) as previously described (37). The electrophoresis was performed at 20 mA until the dye front reached the bottom of the gel (1.5 h). After electrophoresis, gels were incubated in 2.5% (vol/vol) Triton X-100 twice for 20 min each time to remove SDS and then incubated in a buffer containing 50 mM Tris (pH 7.5), 5 mM CaCl₂, 1% Triton X-100, and 0.02% NaN₃ for 50 h.

Data analysis
All experiments for Northern blot analysis and gelatin zymography were repeated at least three times. Each time ovaries from five or six mice were used per time point. All experiments for in situ hybridization were repeated at least three times, and each time ovaries from two different mice were used per time point. The relative amount of mRNA is expressed as the mean ± SEM of three individual experiments.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of MMP and TIMP mRNAs in the mouse ovary
To determine the expression patterns of different MMPs and TIMPs in the mouse ovary, immature 25-day-old female mice were injected with PMSG and 48 h later with hCG. PMSG stimulates follicle development, and the subsequent hCG administration induces ovulation that takes place 10–14 h after hCG injection. At different time points after the hormone treatment, ovaries were removed, and total RNA was isolated and analyzed by Northern blot hybridization. The RNA hybridization probes were obtained by in vitro transcription of cDNA fragments, corresponding to 11 MMPs and 3 TIMPs. To verify the accuracy of the probes and the hybridization conditions, positive controls, consisting of total RNA prepared from tissues known to express the different MMPs and TIMPs, were included in the Northern blot hybridizations (data not shown).

In Table 1, the MMP and TIMP probes that were used for the Northern blot hybridization and their expression patterns during the periovulatory period are listed. As shown, the expression of several MMPs was below the detection level. These include the mRNAs encoding gelatinase B (MMP-9), collagenase-3 (MMP-13), neutrophil collagenase (MMP-8), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), matrilysin (MMP-7), and metalloelastase (MMP-12). Studies in the rat have previously shown that collagenase-3 mRNA is expressed in the ovary of adult cycling rats, but not during gonadotropin-induced ovulation (38). To determine whether the regulation of collagenase-3 was similar in the mouse ovary, we prepared total RNA from mouse and rat ovaries collected at different stages of the estrous cycle, and 20 µg were hybridized with the antisense collagenase-3 probe. As shown previously (38), a clear hybridizing signal was detected in the RNA prepared from rat ovaries in proestrus and estrus. However, no expression of collagenase-3 mRNA was detected in the mouse ovary at any stage of the cycle (data not shown).

Regulation of MMP and TIMP mRNAs during gonadotropin-induced ovulation
The relative mRNA expression and the regulation of MMPs and TIMPs detected in the mouse ovary are shown in Fig. 1. Of the MMPs studied, the mRNAs encoding gelatinase A and stromelysin-3 were the most abundantly expressed (Fig. 1A). MT1-MMP was also expressed at all time points at a clearly detectable level. However, the expression level of MT1-MMP was about half that of stromelysin-3. The mRNA expression of these three proteases remained at a relatively constant level throughout the periovulatory period, and no up-regulation was detected before ovulation. The only MMP that was regulated during the periovulatory period was MMP-19 (Fig. 1B). In mice treated only with PMSG, MMP-19 mRNA expression was low. However, after treatment with hCG, MMP-19 mRNA was up-regulated about 5–10 times, and the maximum level was reached at 12 h after hCG treatment, which correlates with the time of ovulation (Fig. 1B). Compared with gelatinase A, stromelysin-3, and MT1-MMP, the expression of MMP-19 mRNA was much lower even at its peak level, 12 h after hCG treatment.

View larger version (15K): [in this window] [in a new window]   Figure 1. Relative expression of MMP and TIMP mRNAs during gonadotropin-induced ovulation in the mouse. Total RNA, prepared from ovaries, was analyzed for MMP and TIMP mRNA expression by Northern blot analysis as described in Materials and Methods. The intensity of the hybridization signal was calculated from the ImageQuant program (Molecular Dynamics, Inc.). Relative amounts of mRNA are expressed as the mean ± SEM of three individual experiments. PMSG denotes ovaries treated with PMSG for 48 h. 4 h, 8 h, 12 h, 14 h, and 24 h denote the time after hCG treatment. A, Relative amounts of stromelysin-3, gelatinase A, and MT1-MMP mRNAs at the indicated time points. B, Relative amounts of MMP-19 mRNA at the indicated time points. C, Relative amounts of TIMP-1, TIMP-2, and TIMP-3 mRNAs at the indicated time points.

Localization of MMP and TIMP mRNA expression in the mouse ovary during gonadotropin-induced ovulation
The ovary is a heterogeneous organ consisting of follicles at different developmental and maturation stages. To localize the cellular sites of MMP and TIMP mRNA synthesis we have used in situ hybridization. A summary of the results described below is presented in Fig. 2.

View larger version (154K): [in this window] [in a new window]   Figure 2. In situ localization of MMP and TIMP mRNAs during the periovulatory period. Ovaries from immature mice treated with PMSG and hCG, as described in Materials and Methods, were removed at different time points after gonadotropin treatment. A series of 10-µm cryostat sections of ovaries were hybridized to digoxigenin-labeled gelatinase A, MT1-MMP, stromelysin-3, MMP-19, and TIMP-1 antisense probes. All sections in the first column were from ovaries treated with PMSG for 48 h. All sections in the second column were obtained from ovaries 4 h after hCG treatment. Sections in the third column were from ovaries treated with hCG for 12 h. Sections A–C were hybridized to an antisense gelatinase A probe. Sections D–F were hybridized to an antisense MT1-MMP probe. Sections G–I were hybridized to an antisense stromelysin-3 probe. Sections J–L were hybridized to an antisense MMP-19 probe. Sections M–O were hybridized to an antisense TIMP-1 probe. Scale bar, 300 µm.

The expression pattern for stromelysin-3 mRNA is shown in Fig. 2, G–I. At all time points investigated, stromelysin-3 mRNA was only localized to the granulosa cells of small and middle-sized follicles. No expression could be detected in the large preovulatory follicles (Fig. 2, G–I).

As shown by Northern blot analysis, MMP-19 mRNA was the only MMP message that was up-regulated before ovulation (Fig. 1B). The expression pattern of MMP-19 is shown in Fig. 2, J–L. At 48 h after PMSG treatment, the expression of MMP-19 mRNA was below the detection level (Fig. 2J). However, after hCG injection, the expression of MMP-19 mRNA was induced, and at 4 h after hCG, the expression of MMP-19 was detected in the thecal-interstitial cells of follicles at different maturation stages (Fig. 2K). At 12 h after hCG treatment, corresponding to the time of ovulation, several large preovulatory follicles were found in the ovary. At this time point the expression of MMP-19 mRNA had increased even further and was mainly localized to the granulosa and thecal-interstitial cells of preovulatory and ovulating follicles (Fig. 2L). Expression of MMP-19 mRNA was also detected in small and middle-sized follicles at this time point, but only in the surrounding thecal-interstitial cells.

TIMP-1 mRNA was also dynamically regulated in the ovary during the periovulatory period. In ovaries from mice treated with PMSG for 48 h, no TIMP-1 mRNA expression could be detected (Fig. 2M). However, 4 h after hCG treatment TIMP-1 mRNA was induced. As shown in Fig. 2N, the expression of TIMP-1 was localized to the thecal-interstitial cells of most follicles and to the granulosa cells of the large preovulatory follicles. Within the preovulatory follicles, TIMP-1 mRNA expression was mainly localized to the mural granulosa cells located closest to the basement membrane, and the expression decreased toward the center of the follicle. At 12 h after hCG treatment, the granulosa and thecal-interstitial cells of large preovulatory follicles continued to express high levels of TIMP-1 mRNA (Fig. 2O). However, at this time point the expression was more uniformly distributed among the different populations of granulosa cells.

Regulation of gelatinolytic activity during the periovulatory period
To study the regulation of ovarian MMP activity during the periovulatory period, immature 25-day-old female mice were injected with PMSG and hCG. At different time points after hormone treatment, ovarian extracts were prepared and analyzed by gelatin zymography. Ovarian extracts from all time points contained a prominent gelatinolytic activity with a molecular mass of approximately 70 kDa (Fig. 3, lanes 1–5). The 70-kDa lytic zone comigrated with purified human gelatinase A (Fig. 3, lane 7) and was identified as the murine homolog of gelatinase A in a Western blot using antibodies directed against human gelatinase A (data not shown). As shown in Fig. 3, lanes 1–5, relatively high levels of gelatinase A activity were present at a constant level throughout the periovulatory period. In addition to gelatinase A activity, the ovarian extracts also contained a much less prominent lytic activity with a molecular mass of approximately 100 kDa. The 100-kDa lytic zone comigrated with purified murine gelatinase B (Fig. 3, lane 8), suggesting that this is gelatinase B. The level of gelatinase B activity was relatively constant during the periovulatory period, with a slight increase at 4 h after hCG treatment. As shown in Table 1, the mRNA encoding gelatinase B was undetectable in 20 µg total RNA prepared from mouse ovaries at different time points during the periovulatory period. However, a weak signal was obtained when 1.5 µg poly (A)⁺ RNA, prepared from ovaries collected 4 h after hCG, were analyzed by Northern blot hybridization using the gelatinase B probe (data not shown). As shown in Fig. 3, both gelatinase A and gelatinase B appeared to be present mainly in their inactive proform as the molecular masses of these activities were reduced by approximately 10 kDa after treatment with the MMP-activating agent APMA (Fig. 3, lane 6).

View larger version (43K): [in this window] [in a new window]   Figure 3. MMP activity in ovarian extracts during gonadotropin-induced ovulation. Samples of ovarian extracts (15 µg total protein), from different time points after gonadotropin treatment, were analyzed by gelatin zymography. Lane 1 represents ovarian extract from mice treated with PMSG for 48 h. Lanes 2–5 represent ovarian extract from mice collected at 4, 8, 12, and 24 h, respectively, after hCG injection. Lane 6 is ovarian extract from the 4 h time point activated by APMA. Lane 7 represents 5 mU purified human gelatinase A, and lane 8 represents 8.05 ng purified murine gelatinase B. Migration patterns of pro and activated forms of gelatinase A and gelatinase B are indicated with arrows.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Both the development of ovarian follicles and the breakdown of the follicular wall at the time of ovulation require remodeling and degradation of extracellular matrix components (7, 8, 9, 11, 42). Consistent with this, studies in the rat have shown that gelatinolytic and collagenolytic activities as well as mRNA coding for MMPs are present in the ovary (13, 43, 44, 45, 46, 47). The expression kinetics and cellular distribution of the MMPs suggest that they generate proteolytic activity that plays a role in follicular development and the degradation of the follicular wall at the time of ovulation. In the present study we show for the first time the expression and regulation of MMPs in the mouse ovary during ovulation. Different MMPs and TIMPs were expressed in a coordinated and cell-specific manner in response to physiological signals, and the expression pattern and kinetics imply that individual MMPs may have different roles during the follicular life cycle. For seven of the MMPs included in our study we were unable to detect the corresponding mRNA by Northern blot hybridization, using 20 µg total RNA from mouse ovary. However, this does not exclude the possibility that these MMPs may be expressed in the ovary at a low level and play a role in proteolytic processes.

Of the different MMPs and TIMPs that were included in this study, MMP-19 and TIMP-1 had expression kinetics and tissue distribution that most clearly support the hypothesis that they have a role in the follicular wall degradation at the time of ovulation. In mice treated with PMSG for 48 h, only relatively low levels of MMP-19 and TIMP-1 mRNA was found in the ovary. However, after an ovulatory dose of hCG, both MMP-19 and TIMP-1 mRNA were dramatically induced, and just before ovulation, their expression was mainly localized to granulosa and thecal-interstitial cells of large preovulatory and ovulating follicles. MMP-19 is a novel member of the MMP family, and it appears to be the first identified member of a new MMP subfamily, as it lacks a number of structural features characteristic of the different subgroups of MMPs (33). To date, very little is known about the substrate specificity of MMP-19, but preliminary data indicate that MMP-19 has stromelysin-like activity (33). Although the relative expression of MMP-19 mRNA in the ovary was lower than that of the other MMPs studied, the temporal and spatial regulation of MMP-19 suggests that it might be involved in the tissue degradation that occurs during follicular rupture, and TIMP-1 could have a role in terminating MMP activity after ovulation.

MT1-MMP is a membrane-associated MMP, which in vitro can activate progelatinase A to its active form (39). Although MT1-MMP and gelatinase A had distinct expression patterns and were expressed at relatively constant levels throughout the periovulatory period, their expression patterns in different ovarian compartments suggest that they may play a role in degradation of the follicular wall at the time of ovulation. Gelatinase A mRNA was expressed in the thecal-interstitial cells of both developing and large preovulatory follicles just before ovulation. MT1-MMP had a more complex expression pattern; however, just before ovulation MT1-MMP mRNA was expressed in both granulosa and thecal-interstitial cells of preovulatory follicles. Previous studies in the rat reveal a regulation and cellular expression pattern of gelatinase A and MT1-MMP similar to that found here for the mouse (47). The expression kinetics and tissue distribution of cells expressing MTI-MMP and gelatinase A in preovulatory follicles, therefore, suggest the possibility that progelatinase A is activated by MTI-MMP in the thecal-interstitial tissue just before ovulation, and the resulting proteolytic activity may play a role in follicular wall degradation. However, both MT1-MMP and gelatinase A were also expressed in the ovary during follicular development, suggesting that these MMPs may also be involved in tissue-remodeling processes during follicular maturation.

Stromelysin-3 mRNA was one of the most abundantly expressed MMPs at all time points studied, but compared with the other MMPs it has a distinct expression pattern. During both follicular development and ovulation, stromelysin-3 expression was only localized to granulosa cells of small and middle-sized follicles. No expression could be detected in large preovulatory follicles, which expressed gelatinase A, MT1-MMP, MMP-19, and TIMP-1 at the time of ovulation. This expression pattern, therefore, suggests that stromelysin-3 is not involved in follicular rupture. During other tissue-remodeling processes, stromelysin-3 mRNA expression has been found in areas where extensive cell death, apoptosis, occurs, for example during metamorphosis of the intestine and tail in the frog (48), during mammary gland involution in the mouse (28), and during limb, tail, and snout morphogenesis in the developing mouse embryo (49). Consistent with this, our preliminary in situ 3'-end labeling analysis indicates that stromelysin-3 expression is localized to follicles undergoing apoptosis. It is, therefore, possible that stromelysin-3 is involved in extracellular matrix-remodeling processes during follicular atresia (Hägglund, A.-C., and T. Ny, unpublished data).

Of the MMP inhibitors studied, TIMP-1 mRNA was expressed at the highest level, and it had had an expression pattern consistent with the hypothesis that it controls proteolytic activity during ovulation. This result is consistent with a previous study in the rat (50) indicating that the regulation of TIMP-1 during gonadotropin-induced ovulation is very similar in mouse and rat. TIMP-2 and TIMP-3 were also expressed in the mouse ovary, but these inhibitors were not induced by gonadotropins, and they were expressed at a lower level than TIMP-1.

The regulation of MMP activity in cells and tissues occurs at many levels: 1) the transcription of specific MMP and TIMP genes is regulated by growth factors, cytokines, and hormones; 2) MMPs are synthesized in inactive proforms that require activation at sites of action; and 3) MMPs can be inhibited by their specific inhibitors, TIMPs. Therefore, the presence of mRNAs coding for specific MMPs does not necessarily indicate the presence of any MMP activity. However, the study of MMP activity in vivo is complicated, because there is a lack of sensitive assays. In this study we have used gelatin zymography, which mainly detects the gelatinases, to assay MMP activity. During the whole periovulatory period, both gelatinase A and gelatinase B activities were detected in the ovarian extracts. However, both of these MMPs appeared to be present mainly in their inactive proform. In addition, no increase in activity was detected before ovulation.

In conclusion, our results show that several MMPs and TIMPs are expressed in a distinct and cell-specific manner during gonadotropin-induced ovulation. In particular, the up-regulation of MMP-19 and TIMP-1 suggests that these proteins may be involved in the tissue degradation that occurs during follicular rupture. Future studies using gene-deficient mice may reveal whether MMP-19 has a unique role in the degradation of the follicular wall at the time of ovulation.

	Acknowledgments

 
We thank Drs. K. Tryggvason, C. Lopez-Otín, and G. Opdenakker for the mouse TIMP-2 cDNA, neutrophil collagenase cDNA, and TIMP-1 cDNA, respectively. We also thank James Snell for critically reading this manuscript.

	Footnotes

 
¹ This work was supported by the Swedish Medical Research Counsel (K97–13X-09709–07A), the Swedish Cancer Society (3912-B97–01XAB), Cancerforskningsfonden in Ume (LP1177/95), the J. C. Kempes Foundation in Ume, and Svenska Sällskapet för Medicinsk Forskning.

Received January 21, 1999.

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