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a Center for Research on Reproduction and Women's Health, Departments of Obstetrics and Gynecology and
b Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
c Division of Nephrology, Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215
d Department of Population Health and Reproduction, University of California, Davis, California 95616
e Department of Biochemistry and Molecular Biology, Albany Medical College, Albany, New York 12208
f Department of Biochemistry, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The apparent role for MMPs in remodeling of the fetal membranes at term raises the possibility that inhibitors of MMP action could be used to prevent premature membrane rupture in women at risk of this complication. To elucidate the biochemical events leading to physiological embryonic membrane remodeling before birth, and to facilitate the testing of drugs for their ability to block fetal membrane collagen catabolism and membrane rupture, we sought to develop a simple and inexpensive in vivo model. To this end, we examined the chicken egg as a novel system. Like mammalian fetuses, the chick embryo is surrounded by embryonic membranes. Here we report that an enzyme that can degrade fibrillar collagens, the chicken homologue of MMP-13, appears in chick embryonic membranes before hatching. ProMMP-13 is induced at a time when the fibrillar collagen content of the embryonic membranes is declining and there are striking structural alterations in the membranes. The gross structural changes in the membranes can be prevented by the introduction of an MMP inhibitor into the extraembryonic fluid, suggesting that MMP inhibitors could be used to prevent or retard fetal membrane rupture.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fertilized eggs from White Leghorn chickens were obtained on embryonic day (ED) 13 from a commercial supplier (B&E Eggs, Stevens, PA) and maintained at 37°C. Chicks normally hatch during the night of ED 20 or the morning of ED 21. To collect fetal membranes, the shells were gently removed between 0900 and 1000 h on ED 17–20 and at 1700–1800 h on ED 20. Extraembryonic fluid was collected into a syringe through a 21-gauge needle before dissection of the membranes. The chorioallantoic membrane adjacent to the shell was discarded, and the allantoic membrane covering the embryo was collected for analysis.
In some experiments, batimastat (BB-94; 4-[N-hydroxyamino]-2R-isobutyl-3S-[thienylthiomethyl]-succinyl-L-phenylalanine-N-methylamide) [9], supplied by British Biotech (Oxford, UK) was administered daily at a dose of 40 mg/kg into the extraembryonic fluid (amniotic cavity) using a 3.81-cm 22-gauge needle. The needle hole in the shell was closed with Elmer's glue. The drug was suspended in dimethylsulfoxide (DMSO). Equivalent volumes of the vehicle (100 µl) were injected into control eggs. Treatments were begun on ED 16, and embryonic membranes were collected on ED 20 between 0900 and 1100 h.
The embryonic membranes were generally collected from 3–4 eggs. Experiments were repeated on at least two separate occasions using tissues collected from different eggs to establish the generality of the results.
Histological Studies
Membranes were fixed in either neutral buffered formalin or Bouin's solution and were paraffin-embedded for routine histological evaluation with hematoxylin and eosin staining.
Changes in Embryonic Membrane Fibrillar Collagen Content
Thirty milligrams wet weight of membrane tissue was dissolved in 0.5 M acetic acid containing 1 mM PMSF and 5 mM EDTA and was extracted for 2 days at 4°C. The supernatants were collected, and then collagens were precipitated with 10 vol ethanol. The protein pellets were dried and then dissolved in SDS-PAGE loading buffer containing 10% ß-mercaptoethanol. Equal aliquots of the extracts were subjected to SDS-PAGE, and the gels were silver-stained.
Hydroxyproline Assay
For the determination of hydroxyproline-containing peptides [10], 50 µl of extraembryonic fluid was dried in plastic tubes in an oven at 100°C. Fifty microliters of 4 N NaOH was added, and the tubes were then placed into boiling water for 90 min. Fifty microliters of 1.4 N citric acid was then added to bring the pH to 6.0. One milliliter of chloramine-T solution was added, and the tubes were incubated at room temperature for 20 min. One milliliter of aldehyde/perchloric acid solution was then added, and the tubes were incubated at 65°C. After 15 min, absorbance at 550 nm was determined. Samples were assayed in triplicate.
Zymography
Membranes were homogenized in two volumes of RIPA buffer (10 mM Tris, pH 7.4, 1% Nonidet NP-40, 0.1% SDS, 0.1% deoxycholic acid, 150 mM NaCl, 1 mM EDTA, and 10 µg/ml aprotinin). Equal amounts of extract protein and equal volumes of extraembryonic fluid were analyzed by substrate gel zymography in 8% polyacrylamide gels impregnated either with gelatin (1 mg/ml) or with casein (1 mg/ml) [7]. After electrophoresis, gels were washed in 2.5% Triton X-100 for 30 min and then incubated overnight at 37°C in 50 mM Tris buffer containing 0.15 M NaCl and 30 mM CaCl2. The gels were then stained with Coomassie Blue R-250 dye. To establish that the gelatinolytic activities represented MMPs, the gels were incubated as described above but in the absence or presence of EDTA (10 mM).
Cloning of a Chicken MMP-13 cDNA
Total RNA was prepared from chicken embryonic membranes collected on the afternoon of Day 20 (1700 h). Two and one half micrograms of RNA was reverse-transcribed into first-strand cDNA using Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD). Four microliters of the total reaction mixture was subjected to 35 cycles of polymerase chain reaction (PCR) amplification (1 min denaturation at 94°C, 1 min annealing at 54°C, and 1 min extension at 72°C followed by 7 min extension at 72°C) using degenerate primers designed from the human collagenase-3 (MMP-13) sequence. The primer set used consisted of forward primer: 5'-CNNACGNTGCTGGN-GTNCC-3'; reverse primer: 5'-TGNCCAGAACTTCA-GTGNGC-3', where N is either A, C, G, or T. After amplification, 40 µl of the total 100-µl reaction mixture was separated in a 1.2% agarose gel, and products were visualized by ethidium bromide staining. The anticipated product of 0.4 kilobases (kb) was isolated and cloned into the PCR 2.1 cloning vector. The identity of the amplified product was determined by DNA sequence analysis using an automated DNA sequencer. The DNA sequences were compared with sequences deposited in GenBank. One clone of the 12 clones analyzed had a sequence that was 78% identical to human MMP-13 and was considered to be the chicken homologue of MMP-13. With use of the same method of reverse transcription (RT)-PCR and subcloning, another 680-base pair (bp) cDNA was obtained with a 5'-primer (5'-GGTCAGATGATTCTAGAGGGT-3') designed from the original 400-bp cDNA clone and a 3'-primer (5'-GACAGCATCTACTTTGTCGCC-3') designed from sequences of human, rat, and mouse MMP-13. The sequence of this 680-bp cDNA was 80% identical to human MMP-13. We also cloned a third fragment of 534 bp using the method described above with 33 cycles of PCR amplification (30 sec at 94°C for denaturation, 30 sec at 50°C for annealing, and 30 sec at 72°C for extension, followed by 7 min extension at 72°C). The 5'-primer (5'-ATGGACACAGGCTACCCCAAGTTC-3') was designed from the 680-bp segment cloned above, and the 3'-primer (5'-TGTGGTTCCAGCCACGCATAGTCA-3') was designed from the human, rat, and mouse proMMP-13 sequences. The DNA sequences of the three cDNAs were aligned and translated with Macvector software (IBI, New Haven, CT). The amino acid sequences were compared with the previously published human [11], rat [12], and mouse [13] MMP-13 deduced amino acid sequences using SeqVu 1.0.1.
Expression of MMP-13 and MMP-2 mRNAs
Total RNA was isolated from embryonic membranes collected on different days using the TRIzol Reagent (GIBCO BRL) according to procedures recommended by the manufacturer. Equal amounts of RNA were size-fractionated in 0.8% agarose gels and transferred to nylon membranes according to previously described methods [6]. The membranes were probed with cDNAs for chicken MMP-13 and MMP-2, and human 18S rRNA.
Western Blot Analysis of MMP-13 Expression
Extracts of embryonic membranes were prepared in RIPA buffer as described above. Equal amounts of protein (200 µg/lane) were subjected to Western blotting using a rabbit polyclonal antibody raised against rat MMP-13 as previously described [6]. The Amersham (Piscataway, NJ) enhanced chemiluminescence (ECL) system was used to detect antibody bound to antigen.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sequential sections of allantoic membranes collected on ED 17 to 21 were examined. On ED 17 and 18, the membranes showed little evidence of cellular degeneration (Fig. 1A). Blood vessels were evident, and mitotic figures were occasionally observed in the allantoic epithelium. On ED 19, pyknotic nuclei, some engulfed by mononuclear phagocytes, were observed. By ED 20, membrane fragmentation and loss of nuclear detail evidenced by nuclear swelling were common observations. In other areas, nuclear pyknosis was prominent (Fig. 1B). In some membrane regions, almost complete obliteration of cellular architecture was seen.
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Loss of Fibrillar Collagen from the Embryonic Membranes Before Hatching
The fibrillar collagen content of the embryonic membranes, determined by SDS-PAGE analysis of extracts from equal wet weights of membrane tissue, was reduced approximately 70% by ED 20 (Fig. 2). The collagen 
1(I) monomer, migrating at 180 kDa, and the less abundant 2(I) monomer migrating below the 1(I) monomer were both diminished on ED 20. Coincident with the decline in membrane fibrillar collagen, hydroxyproline-containing peptides increased in the extraembryonic fluid, presumably reflecting embryonic membrane collagenolysis. Absorbance values per 50 µl extraembryonic fluid were 0.148 ± 0.038; 0.175 ± 0.041; 0.206 ± 0.077; and 0.296 ± 0.080 on ED 17, 18, 19, and 20, respectively (values are means ± SD, n = 3).
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Appearance of MMP Activity in the Embryonic Membranes Before Hatching
A 55-kDa gelatinase activity was detected in extracts of membranes prepared from tissue collected on ED 20 (Fig. 3). This activity was not detected on ED 17–19. The 55-kDa lysis band was also detected in extraembryonic fluid collected on ED 19 and 20, but not on ED 17 or 18. Zymography also revealed the presence of 62- and 66-kDa gelatinase activities, which reflect MMP-2. The levels of MMP-2 activity in embryonic membrane extracts were relatively constant between ED 17–20. These activities were not detected in extraembryonic fluid on ED 17 and 18, but were present at low levels on ED 19 and at high levels on ED 20. On ED 19, an 80-kDa activity that may have represented activated MMP-9 was occasionally observed in embryonic membrane extracts. The gelatinolytic activities in the membrane extracts and extraembryonic fluid were inhibited when gels were incubated in the presence of 10 mM EDTA (Fig. 3), confirming that the enzymes were metalloproteinases.
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Stromelysin-1 (MMP-3) and MMP-13, in contrast to gelatinases like MMP-2 and MMP-9, efficiently hydrolyze casein [14–17]. Zymography carried out in casein-impregnated gels confirmed the appearance of the 55-kDa enzyme in membrane extracts on ED 20 and extraembryonic fluid on ED 19 and 20 (Fig. 4). As expected, MMP-2 activity was not detected in the casein zymograms. The ability of the 55-kDa enzyme to degrade both gelatin and casein suggested that it could be related to mammalian proMMP-3 or proMMP-13, enzymes with similar molecular weights [11–17].
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Cloning of a cDNA Encoding the Chicken Homologue of MMP-13
RT-PCR was used to clone cDNA fragments encoding the chicken homologue of MMP-13. The deduced amino acid sequence of the chicken open reading frame derived from the PCR-generated cDNAs was 70% identical to the sequences of rat [12], human [11], and mouse [13] MMP-13 (Fig. 5). The DNA sequence of the partial cDNA has been deposited in GenBank (Accession Number AF070478).
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Rat Anti-MMP-13 Antiserum Detected a 55-kDa Protein in Chick Embryonic Membrane Extracts on ED 20
With knowledge of the high degree of amino acid sequence identity of chicken and rat MMP-13, we used a well-characterized polyclonal antiserum generated against rat MMP-13 to perform Western blotting on extracts of chicken embryonic membranes. This antiserum detected a 55-kDa protein on ED 20, corresponding to the 55-kDa gelatinase/caseinase (Fig. 6). The anti-rat MMP-13 also cross-reacted with a recombinant peptide derived from expression of chicken MMP-13 sequences in Escherichia coli (data not shown). These observations support the idea that the 55-kDa gelatinase/caseinase represents proMMP-13.
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Regulated Expression of the MMP-13 and MMP-2 in Embryonic Membranes
To determine whether MMP-13 mRNA is regulated in a pattern consistent with the zymographic findings, we performed Northern analysis on total RNA isolated from embryonic membranes between ED 18 and ED 20. Northern blots using the chicken MMP-13 cDNA as probe revealed the presence of a 23-kb message (Fig. 7). This transcript was barely detectable on ED 18, was prominent on ED 19, and reached its greatest abundance on ED 20. MMP-2 mRNA was also detected in chicken embryonic membranes, but there was no major change in the abundance of this message between ED 18 and ED 20. Thus, the changes in MMP-13 mRNA abundance roughly paralleled the changes in 55-kDa gelatinase/caseinase activity, with increases in MMP-13 transcripts preceding the marked rise in enzyme activity on ED 20. Moreover, the pattern of MMP-2 mRNA expression correlated well with the zymographic measurements of this activity in membrane extracts, showing no major changes between ED 18 and ED 20.
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Effects of Batimastat on the Histology of the Embryonic Membranes
We carried out a preliminary experiment to test the utility of the chicken egg system for assessment of the effects of drugs that inhibit MMPs on membrane structure. To determine whether MMP inhibitors can prevent the structural changes in the chick embryonic membranes that take place before hatching, we injected batimastat, which reversibly inhibits members of the MMP family, into the extraembryonic fluid of chick eggs daily between ED 16 and ED 19, and examined membrane histology on ED 20 as an endpoint for drug action. Histological examination indicated that batimastat treatment prevented the structural changes in the embryonic membranes, including membrane fragmentation and nuclear pyknosis, that are characteristically present by ED 20 (Fig. 8). In contrast, the expected disruption of the membranes was apparent in the DMSO-treated control eggs. The batimastat and DMSO treatments did not have gross effects on the embryos since the chicks were viable at the time of membrane collection. To document that batimastat blocked MMP activity in vivo, the level of hydroxyproline-containing peptides in extraembryonic fluid was determined as an index of collagen breakdown. Mean hydroxyproline levels were lower in the extraembryonic fluid of batimastat-treated eggs, in the range found on ED 18, compared to those in eggs treated with DMS0, which were similar to levels found in untreated eggs on ED 20 (batimastat: 0.187 absorbance units/50 µl extraembryonic fluid; DMS0: 0.292; average of two separate experiments with pooled extraembryonic fluid from 3 to 4 eggs in each treatment group).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The MMP appearing in the chicken membranes and extraembryonic fluid by ED 19–20 was tentatively identified as the chicken homologue of proMMP-13, on the basis of its apparent molecular weight, its ability to hydrolyze gelatin and casein, cross-reactivity with an antiserum generated against rat MMP-13, and the increase in transcripts encoding a protein with high amino acid sequence identity to mammalian proMMP-13 in association with the appearance of enzymatic activity. The possibility remains that the 55-kDa gelatinase/caseinase represents proMMP-3. This enzyme does not degrade native type I collagen. It could, however, participate in the catabolism of collagen fragments generated by the action of MMP-13 and by its ability to activate procollagenases. Since the amino acid sequences of mammalian MMP-3 and MMP-13 are sufficiently different, it is unlikely that the partial chicken cDNA sequence we have obtained represents a MMP-3 homologue. However, no cDNA clones currently exist encoding the chicken homologue of MMP-3, so we cannot conclusively rule out this possibility, nor can we determine whether chicken MMP-3 mRNA increases in the embryonic membranes before hatching. If the zymographically detected activity turns out to represent proMMP-3, then our findings demonstrate that two different MMPs are induced in embryonic membranes before hatching: MMP-3 and MMP-13.
The mammalian MMP-13 proenzymes have molecular weights ranging from 55 to 60 kDa. The activated enzymes have a molecular weight of 45–48 kDa. The increased activity we detected in membrane extracts and extraembryonic fluid by zymography has an apparent molecular weight of 55 kDa, which suggests that it represents the proMMP-13. The proenzyme is activated by the SDS-PAGE procedure, allowing its detection by zymography. However, because we have not obtained a cDNA encoding the full coding sequence of chicken MMP-13, we cannot compare the zymographic results with the predicted molecular weight of the proenzyme. Since fibrillar collagen is depleted from the chick membranes by ED 20, some active MMP-13 is presumably generated. MMP-2 as well as the recently discovered membrane-associated MMP, MMP-14, can activate proMMP-13 [21]. These enzymes may participate in a cascade of MMP activation leading to embryonic membrane extracellular matrix catabolism.
MMP-13 can catalyze the first step in the breakdown of fibrillar collagens, cleaving through the fibrillar collagen triple helix to produce fragments that are subsequently further degraded by gelatinases (e.g., MMP-2 and MMP-9) [15, 22–24]. MMP-13 has also been shown to activate proMMP-9 [25]. The increase in MMP-13 mRNA on ED 19 and 20 may reflect induction of gene transcription or the influx of MMP-13-expressing cells into the chick embryonic membranes before hatching. In the rat amnion, MMP-13 is also induced in epithelial cells and amnion fibroblasts before the start of active labor. In human fetal membranes, MMP-1, an interstitial collagenase, is increased with labor and membrane rupture [26]. Thus, in addition to the structural changes in mammalian and chick membranes before birth, there are related biochemical and molecular changes. It should be recognized that a decline in embryonic membrane fibrillar collagens could also result from changes in collagen synthesis [27, 28] as well as changes in the levels of endogenous inhibitors of MMPs (tissue inhibitors of matrix metalloproteinases), which might favor activation of MMPs [29].
A major goal of the present research was to develop a simple and inexpensive in vivo model for exploring the biochemistry of embryonic membrane remodeling before birth and to test the ability of compounds to prevent structural changes in the membranes. The chicken egg represents a self-contained system in which the process of birth (hatching) is determined by the chicken embryo and its membranes. This simple system circumvents the complexities of maternal-fetal interactions in mammalian parturition. Because of the prominent catabolism of fibrillar collagens and the central role of MMPs in collagen metabolism, we conducted preliminary experiments to examine the effects of a potent MMP inhibitor on membrane structure. The chicken genome contains genes that are highly homologous to those encoding mammalian MMPs, including MMP-2, MMP-13, and MMP-19 [30], so MMP inhibitors active on mammalian enzymes are likely to block the function of their chicken counterparts. The introduction of batimastat into the extraembryonic fluid prevented the characteristic morphological alterations in the chick membranes seen on ED 20. These preliminary studies suggest that the chicken egg can be a novel model system for exploring the activities of MMP inhibitors.
In summary, we have described morphological, biochemical, and molecular changes in chick embryonic membranes before hatching that parallel alterations occurring in mammalian fetal membranes before active labor begins. These changes in the fetal membranes appear to be part of a preparatory process for the onset of labor [31]. The chick egg may help us elucidate some of the molecular events surrounding membrane rupture and serve as a valuable in vivo system for examining the action of inhibitors of extracellular matrix catabolism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jerome F. Strauss, III, 778 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104. FAX: 215 573-5408; jfs3{at}mail.med.upenn.edu
Accepted: August 26, 1998.
Received: June 16, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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