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Original Studies |
Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology, Edinburgh, United Kingdom EH3 9EW
Address all correspondence and requests for reprints to: Dr. W. Colin Duncan, Medical Research Council Reproductive Biology Unit, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9EW. E-mail: c.duncan{at}ed-rbu.mrc.ac.uk
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Introduction |
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The activity of MMPs is controlled at several levels, including synthesis as proenzymes, enzyme activation, and the production of specific tissue inhibitors (9, 10). Tissue inhibitors of metalloproteinases (TIMPs) are of particular interest, as TIMP-1 is one of the major products of the corpus luteum. It is produced in large amounts by the corpus luteum of many species, including the rat (11), sheep (12), cow (13), pig (14), monkey (15), and human (16). In addition, it has recently been reported that TIMP-2 is produced by corpora lutea of rats (11), sheep (17), and cows (18), and that TIMP-3 can also be detected in rat ovaries (11).
TIMPs bind to and inhibit metalloproteinase enzymes with a one to one stoichiometry (10). As TIMP-1, in particular, is produced in large amounts throughout the normal luteal phase (16), it is not clear how metalloproteinase enzymes function in an environment containing large amounts of specific inhibitor. This study aimed to investigate the expression and localization of the common MMPs and their specific tissue inhibitors in the human corpus luteum throughout the normal luteal phase and the effect of luteal rescue with exogenous hCG, to mimic the hormonal changes of early pregnancy.
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Materials and Methods |
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All reagents were obtained from Sigma Chemical Co. (Poole, UK),
unless otherwise stated. Prof. M. R. Waterman of Vanderbilt
University (Nashville, TN) provided the antibody to 17
-hydroxylase.
The probes to MMP-2 (gelatinase-A), MMP-9 (gelatinase-B), TIMP-1, and
TIMP-2 were provided by British Biotech Pharmaceuticals (Oxford, UK).
Probes for TIMP-3 and MMP-1 (interstitial collagenase) were purchased
from University Technologies International (Calgary, Canada). The
reverse zymography kit was also obtained commercially from University
Technologies International. All restriction enzymes and ribonucleic
acid (RNA) polymerases were obtained from Promega (Southampton, UK).
Human placental tissue was obtained from the local maternity
hospital.
Collection of corpora lutea
Corpora lutea were enucleated at the time of hysterectomy in women undergoing surgery for benign conditions as described previously (16). In all women, only one corpus luteum was identified on the surface of one of the ovaries. In each case this corpus luteum was removed and studied as described below. All women were healthy, aged 32–45 yr, with regular menstrual cycles and had not received any form of hormonal treatment for at least 3 months before taking part in the study. The date of the LH surge was determined by estimation of LH concentrations in serial early morning urine samples collected before operation (19). On this basis, three corpora lutea classified as early luteal (LH+1 to LH+5), three as midluteal (LH+6 to LH+10), and three as late luteal (LH+11 to LH+14) were investigated. In addition, three women were given im injections of hCG (Profasi, Serono Laboratories, Welwyn Garden City, UK) from LH+7 in daily doubling doses, starting at 125 IU, for 6–8 days until surgery. This regimen has been shown to reproduce the hormonal changes of early pregnancy (20). An additional corpus luteum was obtained from a woman who had received hCG for 8 days to achieve luteal rescue, but the operation was postponed. This corpus luteum was collected 3 days after the final hCG injection.
At operation, the whole corpus luteum was enucleated from the ovary by blunt dissection, and the ovary was oversewn. The tissue was immediately divided into radial blocks to ensure that the whole thickness of the gland was represented in any piece. Two pieces of tissue were rapidly snap-frozen in liquid nitrogen and stored at -70 C for subsequent protein and RNA extraction. One piece was frozen in embedding medium (Tissue-Tek OCT compound, Miles, Elkhart, IN) and stored at -70 C. Serial frozen sections (6 µm) were cut onto ribonuclease-free slides coated with poly-L-lysine (50 µg/l) and stored at -70 C until use. In each case, an endometrial biopsy was fixed in 4% paraformaldehyde and processed into paraffin wax for luteal phase dating by tissue morphometry (21). Plasma was taken before surgery, and the progesterone concentration was measured using a standard RIA (22). This study was approved by the Reproductive Medicine Branch of the South-East of Scotland ethics committee, and informed consent was obtained from all patients before tissue collection.
Gelatin zymography
Protein was extracted from corpora lutea in 0.1% (wt/vol) SDS at 4 C. The protein content of the sample after sonication was measured using the method of Bradford (23). Seventy-five micrograms of protein in sample buffer [10% (vol/vol) glycerol, 1% (wt/vol) SDS, and 0.04% (vol/vol) bromophenol blue] were applied, without heating or reduction, to an 11% (wt/vol) polyacrylamide gel containing 1 mg/mL gelatin and 0.1% (wt/vol) SDS. After electrophoretic separation of proteins, the gels were incubated in 2.5% Triton X-100 for 30 min to remove the SDS. The gels were then incubated for 16 h at 37 C in 50 mmol/L Tris-HCl (pH 7.6) containing 0.2 mol/L NaCl, 5 mmol/L CaCl2, and 0.02% (wt/vol) Brij 35. The gels were stained in staining solution [30% (vol/vol) methanol, 10% glacial acetic acid, and 0.5% (wt/vol) Coomassie brilliant blue G250] and then destained in the same solution in the absence of dye.
Reverse zymography
Reverse zymography using 75 µg of each protein sample was performed using a commercial kit. Briefly, 12% (wt/vol) polyacrylamide gels containing 0.1% (wt/vol) SDS, 1 mg/mL gelatin, and a solution of secreted metalloproteinases (as supplied) were prepared. After electrophoresis, the gels were washed overnight in a solution of 2.5% Triton X-100, 50 mmol/L Tris-HCl (pH 7.5), and 5 mmol/L CaCl2. The gels were rinsed in water and incubated in 50 mmol/L Tris-HCl (pH 7.5) and 5 mmol/L CaCl2 with gentle shaking for 24 h at 37 C. Staining and destaining were carried out as described above, and bands corresponding to TIMP-1, TIMP-2, and TIMP-3 were identified by reference to the standards supplied with the kit.
Northern blot analysis
Total cellular RNA was isolated by the method of Chomczynski and Sacchi (24) using a commercial kit, and its concentration was determined by absorption at 260 nm. Total RNA (20 µg) was denatured, electrophoresed in a 1.5% formaldehyde-agarose gel, and transferred to a nylon membrane (Amersham International, Aylesbury, UK) by capillary action in 20 x SSC (1 x SSC is 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7). Northern blot analysis was conducted as described previously (16) using [32P]deoxy-CTP-labeled complementary DNA probes. The complementary DNA probes were derived from the following plasmids: a 0.7-kb fragment of human MMP-1 in pBluescript, a 1.6-kb fragment (6–1576 bp) of human MMP-2 in pGEM 4Z, a 1.3-kb fragment (759–2105 bp) of human MMP-9 in pGEM4Z, full-length human TIMP-1 in pGEM4Z, full-length human TIMP-2 in pGEM4Z, and a 0.2-kb fragment (400–600 bp) of human TIMP-3 in pBluescript. After washing (16), the blots were laid onto a phosphor screen for 48–72 h and visualized using a PhosphorImager computer (Molecular Dynamics, Maidstone, UK). The blots were then stripped (14) and reprobed with a 32P end-labeled oligonucleotide that hybridizes to 18S RNA, as described previously (25). The molecular size of the bands was calculated with reference to a standard RNA mol wt marker (Promega) run in an adjacent lane.
In situ hybridization
Isotopic in situ hybridization was performed on frozen sections using 35S-labeled riboprobes. Antisense and sense riboprobes incorporating 35S-labeled UTP (Amersham International) were synthesized using a commercial kit (Promega). The riboprobes were generated from the above plasmids using the following restriction enzymes and RNA polymerases: MMP-1, HindIII with T7 polymerase (antisense), and NotI with T3 polymerase (sense); MMP-2, EcoRI with T7 polymerase (antisense), and HindIII with SP6 polymerase (sense); MMP-9, EcoRI with T7 polymerase (antisense), and PstI with SP6 polymerase (sense); TIMP-1, KpnI with T7 polymerase (antisense), and HindIII with SP6 polymerase (sense); and TIMP-2, HindIII with SP6 polymerase (antisense), and EcoRI with T7 polymerase (sense).
In situ hybridization was conducted according to the method described previously (15) at 55 C using 1 x 106 cpm 35S-labeled antisense riboprobe. The 35S-labeled sense riboprobe (1 x 106 cpm) was added to serial sections as a negative control. After washing under increasingly stringent conditions (15), the slides were dipped in photographic emulsion (Kodak NTB-2, IBI, Cambridge, UK) and incubated at 4 C for 21 days in the dark. After developing (Kodak D19) and fixing (Kodak Unifix) at 15 C in the dark, the sections were washed in water, counterstained with hematoxylin, dehydrated through graded alcohols, and mounted (Pertex, Cellpath, Hemel Hempstead, UK).
Immunohistochemistry
Frozen sections were fixed at 4 C in 15% (vol/vol) aqueous
picric acid containing 2% (wt/vol) paraformaldehyde, pH 7.4, for 10
min and washed in phosphate-buffered saline for 20 min at 4 C.
Nonspecific binding was blocked using a goat serum solution [normal
goat serum (SAPU, Carluke, UK) diluted 1:5 in Tris-buffered saline with
5% (wt/vol) BSA]. The primary antibody to 17-hydroxylase was
diluted to a concentration of 1:1500 in Tris-buffered saline and
applied to the section for 20 h at 4 C. Antibody binding was
visualized with an avidin-biotin-alkaline phosphatase complex (AB-AP
kit, Dako, High Wycombe, UK) using biotinylated goat antirabbit Igs
(Dako) as the secondary antibody. Coloration was achieved using a
substrate that produced a red end product (Alkaline Phosphatase
Substrate Kit I, Vector Laboratories, Peterborough, UK). Sections were
counterstained with hematoxylin, dehydrated, and mounted as described
above.
Analysis of results
The intensities of the 92- and 66-kDa bands detected by zymography were measured by computer-aided densitometric image analysis (NIH Image 1.55, NIH, Bethesda, MD) after image capture and inversion. Northern blot band intensity was measured using the PhosphorImager computer. To correct for minor differences in loading, the ratio of the relative band intensity to the 18S band intensity was used for data analysis. One-way ANOVA was used to investigate differences in expression throughout the luteal phase. The rescued corpora lutea were compared to the late luteal corpora lutea using an unpaired t test. A commercial software package was used for statistical analysis (StatView 4.0, Abacus Concepts, Berkeley, CA).
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Results |
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The classification of the corpora lutea by serial urinary LH measurement agreed with the luteal phase dating of endometrial biopsies using the method of Li et al. (21). The plasma progesterone concentrations were 35.3 ± 9.8 nmol/L in the early luteal samples, 41.0 ± 9.9 nmol/L in the midluteal samples, and 19.2 ± 12.9 nmol/L in the late luteal samples. After luteal rescue by exogenous hCG, plasma progesterone concentrations had increased to 52.6 ± 1.5 nmol/L. The plasma progesterone concentration in the postrescue sample was 9.16 nmol/L.
Identification of metalloproteinases and their tissue inhibitors
Three distinct bands of gelatinase activity at 92, 72, and 66 kDa
were detected in the human corpus luteum by gelatin zymography (Fig. 1
). These are consistent with MMP-9 and
the latent and active forms of MMP-2, respectively (4, 26). Reverse
zymography demonstrated a band of inhibition of gelatinase activity at
approximately 28 kDa and a lighter band at 21 kDa (Fig. 2). These correspond to TIMP-1 and
TIMP-2, respectively (4, 27). An additional band at 24 kDa was seen in
human placental tissue, but was absent from corpora lutea. This is
consistent with TIMP-3 (27), which is produced by decidual tissue (28).
TIMP-1 and TIMP-2 could be detected in samples taken from different
stages of the luteal phase and after luteal rescue with exogenous hCG
(Fig. 2). The activities of MMP-2 and MMP-9 changed over the luteal
phase (Fig. 3). MMP-9 activity peaked in
the early and late luteal phase and was lowest in the midluteal phase
(P < 0.05). In contrast, MMP-2 activity increased
throughout the luteal phase to a maximum in the late luteal phase
(P < 0.05). Luteal rescue with hCG resulted in lower
MMP-2 activity than during the late luteal phase in the absence of hCG
(P < 0.05). When the corpus luteum was rescued with
hCG, and trophic support was withdrawn (in the postrescue sample),
large amounts of MMP-2 activity were clearly identified by zymography
(Fig. 1).
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A single band of approximately 0.9 kb corresponding to TIMP-1 (16, 29) was detected in human corpora lutea by Northern blotting (Fig. 4). This confirms our previously reported
results (16). Northern blotting for TIMP-2 resulted in a single band of
3.6 kb (Fig. 4). This is consistent with the transcript size for TIMP-2
messenger RNA (mRNA) in the human (30). Several mRNA species
corresponding to TIMP-3 (28) were detected in the placenta, but were
not seen in the human corpus luteum (data not shown). As we have
previously reported (16), there were no significant differences in the
level of TIMP-1 expression throughout the luteal phase or after luteal
rescue with hCG (Fig. 5). Likewise,
TIMP-2 expression did not change throughout the luteal phase or after
luteal rescue (Fig. 5).
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). There were
no differences in MMP-1 expression throughout the luteal phase or after
luteal rescue with exogenous hCG (Fig. 6).
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mRNA for TIMP-1, TIMP-2, MMP-1, MMP-2, and MMP-9 were localized in
human corpora lutea by isotopic in situ hybridization. Each
of these mRNA species had a specific pattern of localization that
persisted throughout the normal luteal phase and after luteal rescue
with exogenous hCG. In agreement with our previous findings, TIMP-1 was
highly expressed in the granulosa-lutein cells of the corpus luteum
(Fig. 7, a and b) (16). In contrast,
TIMP-2 was localized to different regions of the corpus luteum. TIMP-2
was expressed at the periphery of the granulosa-lutein cells (Fig. 7c).
Comparison with serial sections immunostained for 17-hydroxylase to
identify the thecal-lutein cells showed that TIMP-2 was expressed by
the thecal-lutein cells (Fig. 7d). In addition, TIMP-2 was expressed in
the fibrous connective tissue surrounding the steroidogenic cells (Fig. 7c).
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Only very low levels of expression were seen in the area of the gland
containing steroidogenic cells (Fig. 8, a and b). MMP-2 was localized
to the thecal-lutein cells and surrounding connective tissue (Fig. 8c).
MMP-2 could also be detected around the vasculature and occasionally in
cells within blood vessels. Little MMP-2 expression could be detected
in the granulosa-lutein cell population. The distribution of MMP-9 was
different, as expression was localized to individual cells within the
gland (Fig. 8d). Although these cells were concentrated along the
thecal-lutein cell layer and vascular connective tissue, some cells
within the granulosa-lutein compartment were also found to express
MMP-9 (Fig. 8d). This pattern of expression of MMPs and TIMPs was
consistent and was seen in all serial sections examined (Fig. 9).
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Discussion |
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In the small numbers we analyzed, the expression of TIMP-2 did not change during the functional luteal phase or after luteal rescue with exogenous hCG. TIMP-2 expression was found to change during the luteal phase in ovine corpora lutea (17). Smith et al. reported that TIMP-2 expression was maximal in the early luteal phase and significantly lower in the late luteal phase (17). In the cow, TIMP-2 expression was reported to increase significantly from the early to the midluteal phase (18), and expression was increased after PG-induced luteolysis (13). Further evidence of a species difference is that the primary TIMP-2 transcript size in the sheep corpus luteum is 1.0 kb (17), whereas in the human corpus luteum and other tissues (30) the size is 3.5 kb. In the human corpus luteum, control of tissue remodeling during the functional luteal phase does not appear to be related to alterations in the levels of expression of TIMPs.
Collagenase (MMP-1) and gelatinases A and B (MMP-2 and MMP-9) are expressed in the human corpus luteum. MMP-2 and MMP-9 have previously been detected by zymography in homogenates of rat ovaries (26), bovine corpus luteum (34), and luteinized human granulosa cells (35, 36). MMP-1, MMP-2, and MMP-9 mRNAs have been described in the pseudopregnant rat ovary (37). Collagen and other components of the ECM are an integral part of the structure of the corpus luteum (3, 38). The human corpus luteum expresses enzymes with the capacity to proteolytically break down these components of the ECM.
The expression and activity of MMPs in the corpus luteum changed during the luteal phase. MMP-2 expression and activity were maximal in the late luteal corpus luteum. This is consistent with a role in tissue remodeling associated with luteolysis. In the rat, PRL-induced structural luteolysis was associated with the activity of metalloproteinase enzymes, particularly MMP-2 (26). Interestingly, Aston et al. recently reported that MMP-2 activity increased with length of time of culture of luteinized granulosa cells (36). The major MMP secreted from ovine luteal explants was MMP-2 (39). Expression of MMP-2 in the corpus luteum may be associated with the tissue remodeling at the time of luteolysis.
In contrast, high levels of MMP-9 activity were also detected in the early luteal phase. It is possible that MMP-9 is involved in the extensive tissue remodeling that occurs during the formation of the corpus luteum from the ruptured follicle (3). A role of MMP-9 in the formation of the corpus luteum is supported by the finding that it is the primary metalloproteinase detected in follicle explants (39). In addition, MMP-9 is the major MMP secreted into the culture medium of luteinized bovine (38) and human granulosa cells (35, 36). Dispersed luteal cells from 4-day-old bovine corpora lutea had both MMP-2 and MMP-9 activities, but MMP-9 activity decreased with duration of culture (34), and MMP-9 was seen in the medium of cultured human granulosa cells only during the first 2 days of culture (35). This provides preliminary evidence that MMP-9 may have a role in ovulation and the tissue remodeling associated with the formation of the corpus luteum.
Compared to that during the late luteal phase, exposure of the corpus luteum to hCG during luteal rescue was associated with reduced expression and activity of MMP-2. This is clearly different from the process of ovulation, when LH/hCG stimulates an increase in MMP-1 and MMP-2 expression (5, 40, 41). Follicular levels of MMP-2 increase between the LH surge and ovulation (39). In cultures of luteinized granulosa cells, hCG also was shown to reduce the expression of MMP-2 and MMP-9 (36, 42). Human granulosa cells cultured on a thin layer of ECM are lost from culture in the absence of gonadotropin (36). These cells are released from culture via an active process suppressed by hCG (43). One of the effects of hCG during maternal recognition of pregnancy appears to be the inhibition of metalloproteinase expression.
MMP-1 and MMP-2 had similar cellular localizations in the human corpus luteum. They were expressed in the connective tissue stroma, the vascular pedicles, and the thecal-lutein cell layer. Fibroblasts and endothelial cells are sources of MMPs (44), and they are likely to express MMP-1 and MMP-2 in the corpus luteum. In the endometrium (7) and in ovarian cancers (8), cells of the stroma also have been shown to express these enzymes. Although the expression of MMP-2 was maximal in the late luteal phase, its localization in the corpus luteum was not affected. This suggests that the source of MMP during luteolysis is the periphery of the gland. In contrast, MMP-9 mRNA was localized to single cells in steroidogenic and nonsteroidogenic cell layers. The identity of these cells is uncertain, but they are probably white blood cells. Polymorphonuclear leukocytes express MMP-9 (45), and we found that expression was often associated with blood vessels. Cells of the immune system, including macrophages, are also constituents of the human corpus luteum (46) and may be a source, or stimulator, of MMP expression.
It is unclear whether MMPs are expressed by the granulosa-lutein cells of the corpus luteum. Few grains were localized to this cell layer, and when present, they were in isolated individual cells. This finding is contrary to reports using cultures of luteinized granulosa cells (35, 36). In vitro MMP-9 expression falls with continuing culture. This has led some researchers to suggest that MMP-9 activity is related to leukocytes that accompany the granulosa cells in the first few days of culture (35). However, it is thought that bovine and human granulosa cells and bovine luteal cell dispersates in culture secrete MMP-2 (34, 35, 38). Although it is possible that MMP-2 activity in these cultures results from white cell or thecal contamination, it is likely that granulosa-lutein cells have the potential to express MMPs and are induced to do so in culture. However, it is clear that the main site of MMP-2 expression in the corpus luteum is not the granulosa-lutein cells.
TIMP-1 and TIMP-2 have different cellular localizations in the corpus luteum. TIMP-2 was localized to the thecal-lutein cells and the surrounding connective tissue stroma. Smith et al. found TIMP-2 in the theca of the ovine follicle (17). This is consistent with the primary localization of TIMP-2 in the follicle being maintained in the mature corpus luteum. The localization of TIMP-2 was similar to those of MMP-1 and MMP-2. This suggests that TIMP-2 may have a role in the local regulation of these enzymes in the corpus luteum. Indeed, it has been suggested that TIMP-2 displays a preference for MMP-2 (47). However, as we have previously reported (16), the localization of TIMP-1 is different. It is possible that TIMP-1 has other roles in addition to inhibition of metalloproteinases in the corpus luteum (17, 48, 49). However, the lack of significant ovarian disturbance in mice without a functional TIMP-1 gene (50) means that the role of high TIMP-1 expression in granulosa-lutein cells is not clear.
It was not clear how MMPs could function in the corpus luteum, which expresses large amounts of the specific inhibitor TIMP-1 (13, 16). We have shown that MMPs are expressed in different areas of the corpus luteum than TIMP-1. In addition, where MMPs were expressed in the granulosa-lutein cellular layer, expression was in foci of individual cells. The localization of MMPs seems to be a key factor in their activity in the corpus luteum.
In conclusion, the expression of MMP-2 in the late luteal phase may indicate a role for this enzyme in the tissue remodeling associated with luteolysis. One function of hCG during luteal rescue is to prevent this increase in MMP expression. As TIMP-1 and TIMP-2 change little, it is likely that control of MMP activity in the corpus luteum involves changing MMP, rather than TIMP, expression. MMPs are localized in different areas than TIMP-1, and where they are expressed in the same area, they are expressed in foci. This may explain how MMPs can function in the background of large amounts of TIMP-1.
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Acknowledgments |
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-hydroxylase, and Dr.
G. F. Erickson for providing his protocol for in situ
hybridization. |
Footnotes |
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2 Present address: Department of Obstetrics and Gynecology,
University of Sydney, Westmead Hospital, Sydney, Australia.
Received December 31, 1997.
Revised March 30, 1998.
Accepted April 1, 1998.
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43-subunit (N) disrupts tissue remodeling and the increase in
matrix metalloproteinase-2 during ovulation. Endocrinology. 136:2657–3664.
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