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Endocrinology 148(3):1226 –1234 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-0669
Functional Corpora Lutea Are Formed in Matrix Metalloproteinase Inhibitor-Treated PlasminogenDeficient Mice Patrik Wahlberg, Ida Bode´n, Josefin Paulsson, Leif R. Lund, Kui Liu, and Tor Ny Department of Medical Biochemistry and Biophysics (P.W., I.B., J.P., K.L., T.N.), Umeå University, SE-901 87 Umeå, Sweden; and Finsen Laboratory (L.R.L.), Rigshospitalet, Strandboulevarden 49, DK-2100 Copenhagen, Denmark Corpus luteum (CL) formation involves dramatic tissue remodeling and angiogenesis. To determine the functional roles of the plasminogen activator and matrix metalloproteinase (MMP) systems in these processes, we have studied CL formation and function in plasminogen (plg)-deficient mice, with or without treatment with the broad-spectrum synthetic MMP inhibitor galardin. Both the adult pseudopregnant CL model and the gonadotropin-primed immature mouse model were used. We found that CL formed normally not only in plasminogen-deficient mice and in galardin-treated wild-type mice, but also in galardin-treated plg-deficient mice, suggesting that neither of the plasminogen activator and MMP systems is essential for CL formation. Nevertheless, in plg-deficient mice, serum progesterone levels were reduced by approximately 50%, and the progesterone levels were not re-
T
HE OVARY IS a heterogenous organ containing follicles and corpora lutea (CL) at various stages of development. After ovulation, a CL is formed from a ruptured follicle in a process that involves extensive tissue remodeling and angiogenesis (1–3). In this process, the surrounding vascular network invades the forming CL, which transforms the CL into one of the most vascularized organs of the body (2, 4). The CL produces progesterone, which prepares the uterus for embryo implantation. However, if implantation is unsuccessful, the CL first loses its capacity to produce progesterone (functional luteolysis) and then undergoes structural regression, during which the CL degenerates structurally (2, 5, 6). Numerous studies in various species have shown that extracellular matrix-degrading proteases from the plasminogen activator (PA) and matrix metalloproteinase (MMP) systems are expressed during CL formation (1, 3, 7–14), suggesting that these two protease systems are important for the formation of CL. However, there have been very few functional studies and, therefore, the importance of these proFirst Published Online November 22, 2006 Abbreviations: CL, Corpus luteum; hCG, human chorionic gonadotropin; MMP, matrix metalloproteinase; PA, plasminogen activator; plg, plasminogen; PMSG, pregnant mare serum gonadotropin; psp, pseudopregnant; StAR, steroid acute regulatory protein; TIMP, tissue inhibitor of metalloproteinase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; uPA, urokinase type PA. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
duced further by galardin treatment. When CL from plg-deficient mice were stained for several molecular markers for CL development and regression, they appeared healthy and vascularized, and were indistinguishable from CL from wildtype mice. This implies that the reduced progesterone levels were not caused by impaired CL formation. Taken together, our data suggest that neither plasmin nor MMPs, alone or in combination, are required for CL formation. Therefore, the tissue remodeling and angiogenesis processes during CL formation may be mediated by redundant protease systems. However, the reduced serum progesterone levels in plg-deficient mice suggest that plasmin, but not MMPs, plays a role in maintenance of luteal function. This role may be performed through proteolytic activation of growth factors and other paracrine factors. (Endocrinology 148: 1226 –1234, 2007)
tease systems for CL formation and function has not been clearly established. Plasminogen (plg)-deficient mice exhibit reduced fertility (15) despite the fact that they ovulate at a relatively normal rate (16). Therefore, it is possible that CL formation and/or function are compromised in these mice. In addition, most MMP-deficient mice that have been studied have been fertile, except for the fact that the MT1-MMP-deficient mice die before reaching the reproductive age (17, 18). Thus, the combined roles of MMPs in CL formation and function are still unclear. In addition, using an effective broad-spectrum MMP inhibitor galardin (GM6001) and plg gene-deficient mice, recent studies have revealed a functional overlap between the PA and MMP systems in the tissue remodeling processes of skin wound healing and placentation (19, 20), but not in ovulation (21). Taken together, this indicates that the cooperation between the PA and the MMP systems may operate in a tissue-specific manner (21). The aim of this study has been to investigate the functional roles of plasmin and MMPs in CL formation and function, and to assess whether there is a functional synergy between these two protease systems in the CL. Our data suggest that neither plasmin nor MMPs are essential for CL formation, whereas plasmin may play a role in the control of progesterone production by the CL. Materials and Methods Materials Pregnant mare serum gonadotropin (PMSG), human chorionic gonadotropin (hCG), paraformaldehyde, and levamisole were purchased
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from Sigma (St. Louis, MO). SuperFrost Plus microscope slides were purchased from Menzel-Glaser (Braunschweig, Germany). Restriction enzymes, Taq polymerase, 4-nitro blue tetrazolium chloride, 5-bromo4-chloro-3-indolyl-phosphate, anti-digoxigenin AP Fab fragments, and digoxigenin-labeled UTP were obtained from Roche Molecular Biochemicals (Mannheim, Germany).
Animals C57BL/6J mice were obtained from Bomholtgård Breeding and Research Center (Ry, Denmark). The plg-deficient mice have been described previously (15). Heterozygous plg gene-deficient breeding pairs were used to generate homozygous, heterozygous, and matched wildtype control mice. The mice were genotyped with a chromogenic enzyme activity assay that determines plg levels in blood plasma (16). The mice were housed under controlled environmental conditions with free access to water and food. Illumination was on between 0600 –1800 h. Experimental protocols were approved by the regional ethical committee of Umeå University.
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In situ gelatinase zymography In situ gelatinase zymography was performed on 10-m cryosections as described by Leco et al. (24). Briefly, the sections were covered with a layer of 0.5% low-melting-point agarose gel containing substrate buffer and 100 g/ml fluorescein-conjugated DQ gelatin from Molecular Probes Inc. (Eugene, OR), overlayed with coverslips, and incubated in a humidity chamber at 37 C for 24 h. As a negative control, 10 mm EDTA, which chelates Zn2⫹ and Ca2⫹, was included in the substrate buffer (data not shown). Fluorescence images were taken with a Leica DC300F digital camera attached to a Leica DM LB microscope (Leica, Wetzlar, Germany).
Staining of blood vessels
The adult psp mouse model has been described previously (22). One female mouse, 8 –12 wk old, was housed with one vasectomized male mouse until a vaginal copulation plug was detected, which indicated d 1 of pseudopregnancy. Mice were killed at d 3 and 6 of pseudopregnancy by decapitation. Blood samples were collected for measurement of plasma progesterone levels, and the ovaries were collected, frozen in Tissue-Tec OCT compound, and stored at ⫺80 C.
To detect blood vessels, immunohistochemistry was performed on 10-m cryostat sections of ovaries. A monoclonal antibody to CD31 protein, obtained from PharMingen (San Diego, CA), was used at a concentration of 10 g/ml. The bound primary antibody was detected with an avidin-biotin complex staining system from Santa Cruz Biotechnology (Santa Cruz, CA). The slides were mounted with Mount Quick solution and images were taken with a Leica DC300F digital camera attached to a Leica DM LB microscope (Leica), a setup that was also used to take images of the other stainings. For determination of blood vessel density, sections from ovaries at d 6 of pseudopregnancy were stained for CD31 protein and examined through a 10 ⫻ 10 eyepiece grid with a ⫻40 objective. At this magnification, each grid covers an area of 0.0625 mm2 (0.25 ⫻ 0.25 mm). Each square of the grid that contained a stained cell was counted as a “hit,” giving a maximum possible score of 100. Three mice in each group were used, and three randomly selected CL from each mouse were counted.
Gonadotropin-induced CL formation in immature mice
Synthesis of RNA probes
Twenty-five-day-old female mice were treated ip with 1 IU PMSG and 46 h later with 5 IU hCG, which was designated as d 0 of CL development. Starting from d 2, the mice were treated with 50 g ovine prolactin sc twice a day at 0800 and 1600 h. This protocol yielded CL that were viable for at least 6 d, as measured by serum progesterone levels and the expression of several markers for CL development and regression. Four other protocols involving only PMSG and hCG treatment without prolactin supplement did not lead to the formation of stable CL (data not shown). Using this protocol, mice were killed at d 3 by decapitation and blood samples and ovaries were collected.
A plasmid containing steroid acute regulatory protein (StAR) cDNA was obtained from Dr. Stocco, Texas Technical University (Lubbock, TX). PCR primers selecting the regions 226 –721 and 702-1193 of the StAR gene were purchased from DNA Technology A/S (Aarhus, Denmark). PCR fragments were ligated into pGEMT vectors and sequenced to confirm their correct identity. Plasmids containing StAR, MMP-12 (25), and rat LH receptor (26) fragments were linearized before being transcribed in vitro to generate both the sense and antisense strands of the probe. During transcription, the probes were labeled with digoxigenin using the DIG RNA Labeling Kit from Roche Molecular Biochemicals.
Pseudopregnant (psp) mouse model
In situ hybridization Treatment with a MMP inhibitor The hydroxamate-based MMP inhibitor galardin, also known as GM6001 (23), was dissolved in 4% carboxymethyl cellulose in 0.9% NaCl to a final concentration of 20 mg/ml. In adult psp mice, 100 mg/kg of the drug was administered ip once daily, starting in the morning the day a vaginal plug was detected. In immature gonadotropin-primed mice, 200 mg/kg of the drug was administered ip once daily, starting at the time of hCG administration. The inhibitor reaches the ovary at a concentration that is sufficient to efficiently suppress gelatinase activity, but has only a minor effect on the ovulation rate (21). The same volume of the carrier compound (carboxymethyl cellulose) was used as a control.
In situ hybridization was performed on 10-m cryostat sections using digoxigenin-labeled antisense riboprobes as previously described (27, 28). Sense strands of the probes were used in parallel as a background control.
Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining TUNEL staining was performed on 10-m cryosections with the In Situ Cell Death Detection Kit/POD from Roche Diagnostics (Mannheim, Germany). The slides were mounted with Mount Quick solution and images were taken as described above.
Gelatin substrate assay
Progesterone measurement
Twenty-five-day-old female mice were induced to ovulate with 5 IU PMSG and 46 h later with 5 IU hCG. Galardin (200 mg/kg) or the carrier compound was injected at the same time as hCG. Mice were killed 24 h after the hCG treatment. Ovaries (n ⫽ 10) with visible ova in the oviduct were selected for preparation of ovarian extracts, which was performed as described previously (21). Fifteen-microgram aliquots of ovarian extracts were tested for gelatinolytic activity using an assay from Chemicon (Temacula, CA) that detects degradation of biotinylated gelatin. Immediately before use, MMPs in ovarian extracts were activated with 2.5 mm aminophenylmercuric acetate at pH 7.0 –7.5. Gelatinase activity in these activated extracts was then measured according to the manufacturer’s instructions.
Progesterone was extracted from blood plasma with diethyl ether. The ether phase was evaporated and resolved in Assay Buffer from the DELFIA Progesterone Kit (Wallac, Turku, Finland). The progesterone concentration was then determined by using the kit according to the instructions of the manufacturer.
Results In vivo inhibition of ovarian MMPs by galardin treatment
We have recently shown that galardin can efficiently inhibit ovarian MMPs during ovulation (21). To confirm that
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galardin can also inhibit MMPs during CL formation, we measured in vitro gelatinase activity in ovarian extracts and also performed in situ gelatinase zymography on frozen ovary sections. Mice were induced to ovulate with PMSG and hCG, and galardin or the carrier compound was injected at the same time as hCG treatment. Twenty-four hours after hCG treatment, ovarian extracts were prepared and activated with aminophenylmercuric acetate to convert proMMPs to their active form before gelatinase activity was measured. As shown in Fig. 1, less than 5% of the gelatinolytic activity remained in extracts from galardin-treated mice relative to extracts from carrier-treated mice. This indicates that sufficient amounts of galardin are present in the ovary to inhibit MMPs during CL formation. To confirm that galardin can inhibit MMPs in the forming CL, we also performed in situ gelatinase zymography on ovarian sections from mice 24 h after hCG/galardin treatment. In ovaries from carrier-treated mice, gelatinase activity was detected inside the forming CL. This had a speckled pattern (Fig. 2A). The gelatinolytic activity was virtually abolished in ovaries from galardin-treated mice (Fig. 2B). In situ collagenase zymography was also performed in a separate experiment. However, only very faint collagenolytic activity could be detected (data not shown), which may be explained by our previous findings that there is no detectable expression of collagenase mRNAs in the forming CL in the mouse (12, 25). Reduced serum progesterone levels in plg-deficient mice
To further evaluate the functional importance of plasmin and MMPs for CL formation and function, we studied CL formation in plg-deficient mice using a physiological adult psp mouse model. The mice were treated either with galardin or the inactive carrier substance, starting at d 1 of pseudopregnancy until the mice were killed at d 3 and 6. Blood
FIG. 1. Ovarian gelatinolytic activity is inhibited by galardin in vivo. Immature female wild-type mice were induced to ovulate with PMSG and hCG. At the time of hCG injection, galardin (200 mg/kg) or the same volume of the carrier compound was injected into the mice. They were killed 24 h after hCG treatment, and ovarian extracts were prepared and analyzed for gelatinolytic activity. The bars represent mean ⫾ SEM of relative gelatinolytic activity, where the activity level in extracts from carrier-treated mice was set to 100%.
Wahlberg et al. • CL Formation in Protease-Deficient Mice
FIG. 2. Galardin treatment inhibits in situ gelatinolytic activity in the forming CL. Immature female wild-type mice were induced to ovulate with PMSG and hCG. At the time of hCG treatment, galardin (200 mg/kg) or the same volume of carrier was injected into the mice. They were killed 24 h after hCG treatment, and in situ zymography was performed on 10-m frozen sections. Gelatinolytic activity appears as green fluorescence. The pictures show representative results from carrier-treated mice (A) and galardin-treated mice (B).
samples and ovaries were collected and the luteal functions were assessed by weighing the ovaries, counting the number of CL, and determining the serum progesterone levels. At d 3 of pseudopregnancy (Table 1), which is an early stage of the functional CL, carrier-treated plg-deficient (plg⫺/⫺) mice exhibited 18% fewer CL than carrier-treated wild-type (plg⫹/⫹) mice (7.3 ⫾ 0.5 in carrier-treated plg⫺/⫺ mice vs. 8.9 ⫾ 0.5 in carrier-treated plg⫹/⫹ mice; P ⫽ 0.036). No significant difference in ovarian weight was seen between different plg genotypes. In carrier-treated plg-deficient mice, the serum progesterone levels were 54% lower than those in carrier-treated plg wild-type mice (P ⫽ 0.0065). The serum progesterone levels were reduced by 37% in carrier-treated plg heterozygous (plg⫹/⫺) mice compared with carriertreated plg⫹/⫹ mice (P ⫽ 0.022). In all three plg genotypes, galardin treatment had no further influence on the progesterone levels measured (Table 1). At d 6 of pseudopregnancy (Table 2), no significant difference in the number of CL or ovarian weight was seen in different plg genotypes. At this time point, the serum progesterone levels were not significantly lower in carriertreated plg heterozygous mice than in carrier-treated wildtype mice (22%; P ⫽ 0.26). However, in carrier-treated plgdeficient mice, the serum progesterone levels were reduced by 45% (P ⫽ 0.017). As at d 3, galardin treatment had no significant effect on the number of CL, on ovarian weight, or on serum progesterone levels, regardless of the plg genotype. In galardin-treated plg heterozygous mice, the serum progesterone was reduced by 41% relative to carrier-treated wild-type mice (P ⫽ 0.007). The serum progesterone levels in galardin-treated plg-deficient mice were reduced by 50% relative to carrier-treated wild-type mice (P ⫽ 0.013). Healthy and vascularized CL form in psp plg-deficient mice treated with galardin
To assess whether the observed reduction in serum progesterone levels in the plg-deficient mice was a result of defective CL formation, ovary sections were stained for several molecular markers of CL development and regression. As markers for the development of a functional CL, we used
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TABLE 1. Comparison of ovarian function at d 3 of pseudopregnancy Genotype
Plg⫹/⫹ Plg⫹/⫹ Plg⫹/⫺ Plg⫹/⫺ Plg⫺/⫺ Plg⫺/⫺
Inhibitor treatment
Galardin Galardin Galardin
No. of mice
No. of CL per mouse
Ovary weight per mouse (mg)
Serum progesterone (nM)
10 10 10 10 10 12
8.9 ⫾ 0.5 7.7 ⫾ 0.5 8.1 ⫾ 0.2 8.7 ⫾ 0.6 7.3 ⫾ 0.5 6.9 ⫾ 0.5
8.9 ⫾ 0.7 8.6 ⫾ 0.4 7.3 ⫾ 0.2 8.3 ⫾ 0.8 7.6 ⫾ 0.7 7.1 ⫾ 0.5
549.6 ⫾ 67.5 558.1 ⫾ 79.5 346.2 ⫾ 40.2a 380.5 ⫾ 53.5 251.8 ⫾ 69.2 262.3 ⫾ 39.8
Values represent mean ⫾ SEM. a n ⫽ 9 (insufficient amounts of serum from one mouse).
Healthy and vascularized CL form in gonadotropin-primed plg-deficient mice treated with galardin
in situ hybridization for LH receptor (26) and StAR (29) mRNAs. StAR mediates the transport of cholesterol across the mitochondrial membrane, which is the rate-limiting step in progesterone biosynthesis, and therefore, it may be a useful marker for the development of a functional CL (30). To determine the degree of vascularization of the CL, we stained sections with an antibody to CD31 protein. As markers of CL regression, we used TUNEL staining to detect apoptotic cells (31) and in situ hybridization for MMP-12 mRNA. Specific signal for MMP-12 appears as black dots as it is expressed in invading macrophages, which may participate in the destruction of the CL (12). Blue staining for MMP-12 in follicles in Figs. 3 and 4 was found to be unspecific background. As shown in Fig. 3, A–E, healthy CL from carrier-treated wild-type mice at d 6 of pseudopregnancy were fully vascularized (Fig. 3A) and they expressed high levels of LH receptor and StAR mRNAs (Fig. 3, B and C). They contained few or no apoptotic cells (Fig. 3D) and did not express MMP-12 mRNA (Fig. 3E). Likewise, CL from carrier-treated plg-deficient mice were fully vascularized (Fig. 3F). Quantification of blood vessel density revealed no significant difference between CL from carrier-treated wild-type mice and carrier-treated plg-deficient mice (92.1 vs. 90.7; P ⬎ 0.05). CL from carrier-treated plg-deficient mice also expressed high levels of LH receptor and StAR mRNAs (Fig. 3, G and H). They contained few or no apoptotic cells (Fig. 3I) and did not express MMP-12 mRNA (Fig. 3J). Finally, as shown in Fig. 3, K–O, the CL from galardin-treated plg-deficient mice also appeared indistinguishable from those from carrier-treated wild-type mice. The density of blood vessels in CL from these mice was found to be the same as that in CL from carriertreated wild-type mice (91.3 vs. 92.1; P ⬎ 0.05). The same expression pattern was also seen in psp mice at d 3 (data not shown).
As a control model where galardin is administered to the mice before ovulation takes place, we used a gonadotropininduced CL formation model in immature mice. The formation of CL in these mice was investigated by sectioning ovaries and staining for different molecular markers as described in Materials and Methods. In carrier-treated wild-type mice, the CL were vascularized (Fig. 4A) and expressed high levels of LH receptor (Fig. 4B) and StAR (Fig. 4C) mRNAs. Few or no apoptotic cells were detected (Fig. 4D) and no MMP-12 mRNA was expressed (Fig. 4E). Likewise, in carrier-treated plg-deficient mice, CL were vascularized (Fig. 4F), expressed high levels of LH receptor (Fig. 4G) and StAR (Fig. 4H) mRNAs, contained few or no apoptotic cells (Fig. 4I), and expressed no MMP-12 mRNA (Fig. 4J). Finally, as was observed in psp mice, galardin-treated plg-deficient mice also showed an expression pattern that was indistinguishable from that in CL from carrier-treated wild-type mice (Fig. 4, K–O). In these mice (Table 3), there was no difference in the number of CL or in ovarian weight between the different plg genotypes, with or without galardin treatment. However, the serum progesterone levels were reduced in both plg heterozygous mice (19%; P ⫽ 0.40) and plg-deficient mice (33%; P ⫽ 0.10), but these differences were not statistically significant (P ⬎ 0.05). Galardin treatment did not further influence the serum progesterone levels significantly. Discussion
To study the importance of the PA and MMP systems in CL formation and function, we suppressed both plasmin and MMP activities by treating plg-deficient mice with the synthetic broad-spectrum MMP inhibitor galardin (GM6001)
TABLE 2. Comparison of ovarian function at d 6 of pseudopregnancy Genotype
Plg⫹/⫹ Plg⫹/⫹ Plg⫹/⫺ Plg⫹/⫺ Plg⫺/⫺ Plg⫺/⫺
Inhibitor treatment
Galardin Galardin Galardin
No. of mice
No. of CL per mouse
Ovary weight per mouse (mg)
Serum progesterone (nM)
10 11 9 12 9 9
8.3 ⫾ 0.4 9.3 ⫾ 0.4 7.6 ⫾ 0.2 7.9 ⫾ 0.3 8.0 ⫾ 0.3 7.6 ⫾ 0.7
7.9 ⫾ 0.6 8.4 ⫾ 0.6 7.8 ⫾ 0.4 8.0 ⫾ 0.4 7.9 ⫾ 0.6 7.9 ⫾ 0.7
660.5 ⫾ 82.0 530.5 ⫾ 49.1 513.2 ⫾ 97.6 386.5 ⫾ 43.5a 361.9 ⫾ 72.2b 327.5 ⫾ 87.6
Values represent mean ⫾ SEM. a n ⫽ 11 (insufficient amounts of serum from one mouse). b n ⫽ 8 (insufficient amounts of serum from one mouse).
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FIG. 3. Expression of markers for CL development and regression in plg-deficient psp mice. Tissue sections from psp mice at d 6 were stained as detailed in Materials and Methods. The in situ hybridization signal appears as blue-black. Immunostaining and TUNEL staining appear as red. A–E, CL from carrier-treated wild-type mice; F–J, CL from carrier-treated plg-deficient mice; and K–O, CL from galardin-treated plg-deficient mice. Sections were immunostained with an anti-CD31 antibody (A, F, and K), TUNEL stained (D, I, and N), and hybridized with antisense RNA probes for LH receptor (B, G, and L), StAR (C, H, and M), and MMP-12 (E, J, and O). Specific signal for MMP-12 appears as black dots. The horizontal black bar in O represents 200 m.
during CL development. Surprisingly, we found that healthy CL not only formed in plg-deficient mice and in wild-type mice that were treated with galardin, but they also formed in plg-deficient mice that were treated with galardin. This suggests that neither plasmin nor MMPs, alone or in com-
bination, are required for the development of a viable CL. However, we observed an approximately 50% reduction in serum progesterone levels in plg-deficient mice, independently of galardin treatment, which suggests a novel role for plasmin in the maintenance of CL function in the mouse.
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FIG. 4. Expression of markers for CL development and regression in plg-deficient gonadotropin-primed mice. Ovary sections from immature, gonadotropin-primed mice at d 3 were stained as detailed in Materials and Methods. The in situ hybridization signal appears as blue-black. Immunostaining and TUNEL staining appear as red. A–E, CL from carrier-treated wild-type mice; F–J, CL from carrier-treated plg-deficient mice; and K–O, CL from galardin-treated plg-deficient mice. Sections were immunostained with an anti-CD31 antibody (A, F, and K), TUNEL stained (D, I, and N), and hybridized with antisense RNA probes for LH receptor (B, G, and L), StAR (C, H, and M), and MMP-12 (E, J, and O). Specific signal for MMP-12 appears as black dots. The horizontal black bar in O represents 200 m.
The formation of a CL from a ruptured follicle involves extensive tissue remodeling and angiogenesis. In this process, the vascular network in the theca layer invades the forming CL and transforms it into one of the most vascu-
larized organs of the body (2, 4). Meanwhile, granulosa cells and theca cells differentiate into luteal cells, and fibroblasts migrate into the forming CL to provide a network of supportive tissues (32). During the past two decades, proteolysis
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Wahlberg et al. • CL Formation in Protease-Deficient Mice
mediated by the PA and MMP systems has been implicated as an important mediator of CL formation (1, 5, 14). For example, studies in rodents have revealed that both tissue type PA and urokinase type PA (uPA), as well as some MMPs, are expressed in the forming CL, suggesting that these protease systems have an important role in the rapid tissue remodeling and angiogenesis processes during CL formation (7, 9, 12). Many studies have shown that several MMPs are expressed during CL formation; however, the functional role of MMPs in luteal physiology is not well understood. Mice deficient in a single MMP gene are generally fertile and have mild or no aberrant phenotypes for ovarian functions (1, 14). However, one exception is the MT1MMP-deficient mice whose general health is too severely compromised to permit normal reproduction (17, 18). Our data from the current study have revealed that healthy and fully vascularized CL can form when both the PA and MMP systems are compromised, which is surprising in light of the results of previous studies that suggest that these protease systems have important roles in the CL (1, 3, 5, 14). However, over 600 genes for proteases have been found in the mouse genome (33), and therefore, the PA and MMP systems are only a subset of all proteases. Thus, it is possible that the proteases that are most important for CL formation and function are from different classes than those included in this study. Moreover, several different protease systems with overlapping functions may act in concert to ensure that this crucial biological system is functional. To suppress MMP activity in this study, we used the broad-spectrum, hydroxamate-based MMP inhibitor galardin (GM6001) (23). In vitro and in vivo control experiments performed in our laboratory have shown previously that galardin is distributed to the ovary in sufficient amounts to efficiently suppress gelatinase activity during ovulation (21). To confirm that this is the case also during CL formation, by the use of a gelatinase activity assay and also in situ gelatinase zymography, we have shown in this study that galardin treatment can also efficiently suppress gelatinase activity during CL formation. Because galardin is a broad-spectrum MMP inhibitor with a general inhibitory mechanism against all MMPs, therefore, the suppression of gelatinase activity observed should reflect a general suppression of MMP activities. Even so, we cannot exclude the possibility that there may still have been some residual activity from other MMPs that could not be measured in this study. In the present study, we observed a slightly reduced number of CL in psp plg-deficient mice. We believe that this effect does not reflect defective CL formation per se, but is secondary to the tendency of a slightly reduced ovulation rate in
plg-deficient mice (16). Nevertheless, in such mice, we observed an approximately 50% reduction in serum progesterone levels in the psp mice, suggesting that plasmin may be of importance for progesterone synthesis or metabolism. This reduction in serum progesterone levels remained significant when taking the slightly reduced number of CL into account (data not shown). Interestingly, heterozygous plgdeficient mice with half of the normal serum plg level also showed reduced serum progesterone levels. This suggests that the effect of plg deficiency may be dose-dependent. By staining ovary sections for various molecular markers of CL development and regression, we found that CL from plg-deficient mice were indistinguishable from those from wild-type mice. The observed effect of plg deficiency on serum progesterone levels does not appear to be due to impaired CL formation. Our findings suggest instead that plasmin may play a novel role in the maintenance of luteal function. This effect may be exerted through proteolytic activation, or inactivation of growth factors and other ovarian paracrine factors. For example, IGF binding proteins, which can inhibit IGF signaling by sequestering IGFs, are expressed in the mouse CL (34). Plasmin and some MMPs can cleave some of the IGF binding proteins, thereby releasing IGFs that may subsequently stimulate progesterone production (35– 37). However, our preliminary findings indicate that there is no change in the activation of the IGF receptor during CL formation in plg-deficient mice compared with wild-type mice (data not shown). In a previous study, we observed no significant difference in serum progesterone levels in psp uPA-deficient mice, although uPA was the only PA expressed in the mature mouse CL (12). This suggests that tissue type PA, the mRNA of which is only expressed in the forming CL, may functionally compensate for the lack of uPA to activate plg in the mouse CL. Two recent studies have shown that treatment of plgdeficient mice with the broad-spectrum MMP inhibitor galardin has a dramatic effect in addition to that of plg deficiency (19, 20). When plg-deficient mice are treated with galardin, skin wound healing is completely inhibited, whereas the wound can heal at a reduced rate in carriertreated plg-deficient mice and in galardin-treated wild-type mice (19). In this study, treatment of wild-type mice with galardin had no significant effect on CL formation or function. This suggests that although the MMPs may have important roles in CL formation and function, they are not essential. Surprisingly, the treatment of plg-deficient mice with galardin had no obvious effect on the formation of a healthy CL. In galardin-treated plg-deficient mice the CL
TABLE 3. Comparison of ovarian function at d 3 of gonadotropin-induced CL life Genotype
Plg⫹/⫹ Plg⫹/⫹ Plg⫹/⫺ Plg⫹/⫺ Plg⫺/⫺ Plg⫺/⫺
Inhibitor treatment
Galardin Galardin Galardin
Values represent mean ⫾
SEM.
No. of mice
No. of CL per mouse
Ovary weight per mouse (mg)
Serum progesterone (nM)
7 8 15 16 12 10
10.3 ⫾ 1.2 10.3 ⫾ 0.9 10.1 ⫾ 1.3 9.3 ⫾ 0.8 9.4 ⫾ 0.9 9.9 ⫾ 1.0
8.5 ⫾ 0.4 8.9 ⫾ 0.4 7.5 ⫾ 0.3 7.9 ⫾ 0.3 7.6 ⫾ 0.4 8.4 ⫾ 0.3
389.8 ⫾ 81.6 316.5 ⫾ 39.0 314.7 ⫾ 46.5 352.4 ⫾ 65.0 259.3 ⫾ 33.3 217.2 ⫾ 25.0
Wahlberg et al. • CL Formation in Protease-Deficient Mice
appeared healthy and vascularized, which suggests that in contrast to what has been observed in other tissues (19, 20), there is no functional overlap between the PA and MMP systems during CL formation. Similarly, no such synergy was observed when we treated plg-deficient mice with galardin during gonadotropin-induced ovulation (21). Taken together, these findings suggest that the tissue remodeling processes in the ovary may be different from those in other organs, possibly by involving additional or different protease classes. It has been reported that physiological inhibitors of MMPs, such as tissue inhibitors of metalloproteinase (TIMPs), may be important for CL formation and function. In a report by Nothnick (38), TIMP-1-deficient mice were found to have reduced serum progesterone levels during CL formation. Because we did not observe any significant change in serum progesterone levels in galardin-treated mice in our study, we speculate that the observed differences in TIMP-1-deficient mice may reflect an MMP-independent role for TIMP-1, perhaps by directly stimulating progesterone production (39). It is noteworthy that when plg-deficient mice were treated with galardin, there was no significant additional reduction of the serum progesterone levels, indicating that there is no synergy between the PA and MMP systems in the maintenance of the CL function. In summary, we have shown that, in contrast to previous findings in other tissue remodeling processes, viable CL can form when both the PA and MMP systems are compromised. However, plasmin may play a role in normal progesterone production. We suggest that there may be functional redundancies between various protease systems that act in concert, to ensure proper development and function of the CL. Acknowledgments Prolactin was provided kindly by Dr. A. F. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases National Hormone and Peptide Program, Harbor-UCLA Medical Center (Torrance, CA). StAR cDNA cloned in the eukaryotic vector pCMV5 was provided kindly by Prof. D. Stocco of the Department of Cell Biology and Biochemistry, Texas Technical University. Received May 18, 2006. Accepted November 10, 2006. Address all correspondence and requests for reprints to: Prof. Tor Ny, Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden. E-mail:
[email protected]. This work was supported by the Swedish National Cancer Foundation (4438-B04-05XBB), the Swedish Research Council (Research Grants 521-2005-6701 and 621-2005-5233), the Cancer Research Foundation of Umeå, the Swedish Society for Medical Research, and the JC Kempe Memorial Fund in Umeå. Disclosure Statement: The authors have nothing to disclose.
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