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Regular Article |
a State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China
b Laboratory of Reproductive Endocrinology, Harbin Medical University, Harbin 150086, China
ABSTRACT
The extracellular matrix proteolytic machinery has long been recognized as one of the most important mechanisms for regulating trophoblast invasion. Matrix metalloproteinases (MMPs) are a group of proteases involved in this process, and their activities are regulated by tissue inhibitors of MMPs (TIMPs). In this study, we collected rhesus monkey uteri on Days 12, 18, and 26 of pregnancy and examined the mRNA expression of MMP-2, -9, -14, and TIMP-1, -2, -3, as well as the activities of MMP-2 and -9 by using in situ hybridization and gelatin zymography, respectively. The results showed that MMP-2 and -9 were expressed earlier than MMP-14 and TIMPs in the pregnant endometrium. MMP-14 and TIMP-2 mRNAs appeared in perivascular decidual cells earlier than MMP-2 mRNA. On Day 26 of pregnancy, placental villi expressed little MMP-2, -14, and TIMP transcripts but abundant MMP-9 mRNA. Furthermore, MMP-2, -9, -14, and TIMP-1, -2, -3 were highly expressed on the fetal-maternal border but were absent in the myometrium. TIMP-3 mRNA in the endometrium was specifically localized to some cells lining the outer membrane of several groups of arterioles. Combined with the results obtained by gelatin zymography, we found that active MMP-2 existed in the endometrium throughout these three phases, while MMP-9 showed considerable activities only on Days 18 and 26 of pregnancy. The data suggest key roles for MMP-2 and -9 in invasion of trophoblast cells into the endometrium and the development of the placenta and might indicate that these processes are regulated by MMP-14 and TIMP-1, -2, and -3.
decidua, implantation/early development, syncytiotrophoblast, trophoblast, uterus
INTRODUCTION
Blastocyst implantation that begins with attachment to the uterus by the trophoblast and terminates with formation of the placenta is a complex series of events in early pregnancy. Dramatic structural changes and extensive tissue remodeling occur in both the uterus and the placenta during this period. Many proteolytic pathways, especially the metalloproteinase pathway, have been reported to be related to this process [1, 2].
Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix (ECM), and their substrates range from collagens, proteoglycans, to many glycoproteins. This family of enzymes has 23 identified members thus far [3–6], and most of them share many characteristics, such as a common mode of activation and inhibition by their native inhibitors, tissue inhibitors of MMPs (TIMPs) [3]. Thus, the net MMP activity is the result of a balance between the levels of activated MMPs and those of TIMPs. Among these MMPs, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) have been more extensively investigated because of their accepted roles in early implantation. MMP-2 and -9 derive from different genes, but they have similar structure and substrate specificity. MMP-2 and -9 are synthesized as latent proenzymes and must be activated in order to show their proteolytic activities and degrade various components of ECM including type IV, V, VII, and X collagens, fibronectin, and gelatin etc. [7]. MMP-14—membrane type 1-MMP (MT1-MMP) [8]—is another key member of MMPs family. Main functions of MMP-14 range from degradation of a number of ECM components, to the activation of proMMP-2. MMP-14 has also been found to be an important enzyme involved in tumor metastasis and angiogenesis [9, 10].
TIMPs play pivotal roles at the site undergoing extensive structural remodeling too. This family has 4 members—TIMP-1, -2, -3, and -4 [11–14]. The most widely appreciated function of TIMPs is confined to their inhibitory effect on activated MMPs. Further studies show that TIMP-1 and TIMP-2 can bind to pro-MMP-2 and pro-MMP-9, respectively, thus stabilizing these proenzymes and to some degree promoting their activation [15]. Effects of TIMPs on stimulating cell growth were initially recognized when TIMP-1 and TIMP-2 were reported to have erythroid-potentiating activities [16].
Numerous studies in mouse [17], primate [18], and human [3] have shown that MMPs and TIMPs are key regulators of blastocyst implantation, with MMPs promoting and TIMPs hindering the invasion process of trophoblast cells.
The early rhesus monkey placenta shows dramatic invasive capacities. During this process, cytotrophoblast and syncytiotrophoblast lining placental villi invade the endometrium [19–21]. This kind of invasion shares many characteristics with that of malignant tumor cells. But unlike tumor invasion, trophoblast invasion is confined spatially to uterus and temporally to early pregnancy [22, 23]. This precisely controlled process might be achieved through the release of inhibitors against proteolytic enzymes such as TIMP-1, -2, and -3 [17, 24] by the endometrium and the placenta.
The present study was undertaken to compare the localization of MMP-2, -9, -14, and TIMP-1, -2, -3 transcripts, and to investigate activities of MMP-2 and -9, by using in situ hybridization and gelatin zymography on serial sections of rhesus monkey uteri samples during early pregnancy.
MATERIALS AND METHODS
Animals, Tissue Collection, and Processing
Animal care and handling were in accordance with the Policy on the Care and Use of Animals of the Ethical Committee, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences. Proven fertile male and female rhesus monkeys were housed separately in the Center for Medical Primate, Institute of Medical Biology, Chinese Academy of Medical Sciences, and were fed with regular monkey pellets and water ad libitum. Female monkeys for this study were selected using the criteria of regular menstrual cycles and a history of previous pregnancy. They were allowed to cohabit with the male for 2 days from the anticipated time of ovulation based on the records of the previous menstrual cycles. The anticipated day of ovulation (Day 12 of the menstrual cycle) was designated as Day 0 of pregnancy. Prediction of pregnancy was made on the basis of the presence of energetic spermatozoa in vaginal smears, changes in the uterus structure through ultrasonic diagnosis, as well as the existence of monkey chorionic gonadotropin (mCG) in the urine. On estimated Days 12 (n = 2), 18 (n = 2), and 26 (n = 2) of pregnancy, a rhesus monkey was laparotomized under ketamine hydrochloride anesthesia, and the uterus was removed, trimmed laterally, and opened. The implantation sites were located and cut into two parts. One part was embedded in embedding medium (Triangle Biomedical Sciences, Durham, NC) for frozen tissue specimens and the other was immediately frozen in liquid nitrogen and stored at -80°C until analyzed.
Immunohistochemistry
A biotin-avidin-peroxidase complex (ABC) method (Vectastain ABC kit, cat. no. PK-4002; Vector Labs., Burlingame, CA) was adopted to localize vimentin immunohistochemically in the cryosections of rhesus monkey uteri samples. Briefly, cryosections (10 µm) were fixed in 4% paraformaldehyde for 10 min and washed with PBS (150 mM NaCl, 10 mM Na2HPO4, 1.5 mM NaH2PO4, pH 7.5). They were then sequentially incubated at room temperature with normal horse serum (Vector Labs.) for 20 min, mouse anti-vimentin antibody (diluted 1:50, cat. no. sc-6260; Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 30 min, biotinylated anti-mouse IgG (Vector Labs.) for 30 min, methanol containing 0.3% H2O2 for 30 min, and ABC reagent (Vector Labs.) for 40 min. Intervening PBS washes were necessary after each incubation. Final visualization of antigen was achieved with diaminobenzidine (DAB)/H2O2 solution. The sections were counterstained with Mayer hematoxylin and mounted for photography.
In Situ Hybridization
The plasmids used in this study were kindly provided by Dr. Kevin Leco (Department of Pharmacology and Therapeutics, University of Calgary, Calgary, Canada), Dr. Akiko Okada (Institute de Genetique et de Biologie Moleculaire et Cellulaire, Université Louis Pasteur, Paris, France), and Dr. Yanling Wang (Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China). Probe labeling was performed according to the instructions that came with the digoxigenin RNA labeling mix (cat. no. 1 277 073; Boehringer-Mannheim, Indianapolis, IN). In situ hybridization was conducted by the method of Braissant and Wahli [25] with slight modifications. In brief, adjacent cryosections (10 µm) were fixed for 15 min in 4% paraformaldehyde and incubated in PBS containing 0.1% active diethylpyrocarbonate (DEPC) for 30 min at room temperature. The slides were then prehybridized with a hybridization solution without probes (50% deionized formamide, 5x SSC [1x SSC is 0.15 M NaCl, 0.015 M sodium citrate], and 120 µg/ml salmon sperm DNA) for 2 h at 50°C. The labeled probes, at a dilution of 400 ng/ml, were denatured at 70°C for 10 min. Hybridization reaction was carried out at 50°C for 18 h. The slides were then washed for 30 min in 2x SSC at room temperature, 1 h in 2x SSC at 65°C, 1 h in 0.1x SSC at 65°C. These were followed by incubation for 2 h at room temperature with anti-digoxigenin-alkaline phosphatase (diluted 1:3000, cat. no. 1093 274; Roche Diagnostics Ltd., Hong Kong, China). The slides were rinsed in buffer 1 (100 mM Tris, 150 mM NaCl, pH 7.5) for 30 min to remove excess antibody. Color development was carried out using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP; Boehringer-Mannheim). Sense probe hybridization was used as a control for background levels.
Gelatin Zymography
Protein extraction was performed according to the method provided with the Trizol reagent (cat. no. 15596; Gibco-BRL Life Technologies Inc., Rockville, MD). Protein extract (50 µg) was mixed with 4x sample buffer (8% SDS [w:v], 0.04% bromophenol blue [w:v], 40% glycerol [v:v], 0.25 M Tris), then subjected to electrophoresis in a 10% polyacrylamide gel containing 0.5 mg/ml gelatin (Difco Laboratories, Detroit, MI). The gel was washed in 2.5% Triton X-100, 50 mM Tris-HCl, at pH 7.5 for 1 h to remove SDS and incubated for 18 h in calcium assay buffer (50 mM Tris, 200 mM NaCl, 10 mM CaCl2, 1 µM ZnCl2, 1% Triton X-100, pH 7.5) at 37°C. After staining with 0.2% Coomassie brilliant blue R-250 in 50% methanol and 10% acetic acid, the gel was destained with 10% acetic acid. Estimation of molecular weight was possible due to the concurrent electrophoresis of molecular weight markers.
Statistics
Activities of MMP-2 and -9 detected by gelatin zymography were quantified by computer-aided densitometry (Personal Densitometer SI; Molecular Dynamics Inc., Sunnyvale, CA). Values are means ± SEM of three experiments, and data were analyzed for statistical differences with Student t-test to verify differences between individual groups. (Compared with P12: *P < 0.05; **P < 0.01.)
RESULTS
Localization of Vimentin
Figure 1 shows the typical structure and orientation of the uterine compartments on Days 12, 18, and 26 of pregnancy, according to the immunohistochemical localization of vimentin. Villous mesenchyme, general decidual cells in the endometrium, and myometrium were vimentin-positive, while cytotrophoblast and syncytiotrophoblast lining the villi, extravillous trophoblast cells, glandular epithelium, and luminal epithelium of the endometrium were vimentin-negative (Fig. 1, a–c).
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In Situ Hybridization Results of MMP and TIMP mRNAs in Pregnant Uteri
The in situ hybridization results are summarized in Tables 1–3. Figures 2–4 show mRNA expressions of MMP-2, -9, and -14, respectively, in the placental villi, pregnant endometrium, myometrium, as well as on the fetal-maternal border. None of these transcripts were detected in the myometrium on Day 12 (Figs. 2a, 3a, and 4a), Day 18 (Figs. 2b, 3b, and 4b), and Day 26 (Figs. 2c, 3c, and 4c) of pregnancy.
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Faint but clear MMP-2 mRNA was detected in the decidual cells adjacent to the luminal epithelium of the endometrium on Day 12 of pregnancy, but MMP-2 mRNA expression in the other compartments of the endometrium was barely detectable (Fig. 2a). On Day 18 of pregnancy, MMP-2 mRNA expression in the walls of some arterioles situated adjacent to the implantation site was the most abundant, and MMP-2 transcripts were highly expressed in placental villi as well (Fig. 2b). On Day 26 of pregnancy, placental villi had no signal for MMP-2 mRNA, but the trophoblast shell expressed abundant MMP-2 mRNA. Perivascular decidual cells also synthesized numerous MMP-2 mRNA (Fig. 2c).
Distribution of MMP-9 mRNA is much different from that of MMP-2 mRNA (Fig. 3). MMP-9 mRNA was visualized in the upper glandular epithelium and luminal epithelium as early as Day 12 of pregnancy (Fig. 3a). Its expression was transferred from the epithelium to the general decidual cells on Day 18 of pregnancy, and the placental villi expressed MMP-9 mRNA as well (Fig. 3b). On Day 26 of pregnancy, expression of MMP-9 mRNA in the trophoblast shell and the endometrial compartments was relatively faint, but intense expression of MMP-9 mRNA was detected in the placental villi (Fig. 3c).
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MMP-14 expression is similar with MMP-2 to some degree (Fig. 4). Almost no MMP-14 mRNA was detected on Day 12 of pregnancy (Fig. 4a). As was MMP-2 mRNA, MMP-14 mRNA was intense in the walls of some arterioles situated adjacent to the implantation site on Day 18 of pregnancy. MMP-14 transcripts also concentrated in the placental villi and perivascular decidual cells (Fig. 4b). Placental villi on Day 26 of pregnancy exhibited little expression of MMP-14 mRNA, but the trophoblast shell exhibited numerous MMP-14 transcripts. Perivascular decidual cells also synthesized MMP-14 mRNA (Fig. 4c).
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Figures 5–7 show the mRNA expression patterns of TIMP-1 (Fig. 5), TIMP-2 (Fig. 6), and TIMP-3 (Fig. 7). TIMP-1, -2, and -3 transcripts were all absent in the myometrium on Days 12, 18, and 26 of pregnancy (Figs. 5–7) and the endometrium on Day 12 of pregnancy (Figs. 5a, 6a, and 7a). On Day 18 of pregnancy, placental villi expressed little TIMP-1 and -2 mRNAs (Figs. 5b and 6b), but considerable TIMP-3 mRNA (Fig. 7b). General decidual cells expressed TIMP-1 mRNA (Fig. 5b), but only a small number of cells lining the outer membrane of several groups of arterioles in the endometrium expressed TIMP-3 mRNA (Fig. 7b). TIMP-2 mRNA was distributed in extensive endometrial compartments, including vessel walls, perivascular decidual cells, and the glandular epithelium (Fig. 6b). Furthermore, TIMP-1, -2, and -3 mRNAs were hardly detectable in the placental villi on Day 26 of pregnancy (Figs. 5c, 6c, and 7c). Some cells in the trophoblast shell expressed TIMP-3 mRNA, but no TIMP-1 and -2 mRNAs. TIMP-1 and -2 transcripts in the endometrium were highly expressed in the perivascular decidual cells, and they were also abundant in the decidual cells adjacent to the fetal-maternal border (Figs. 5c and 6c). Distribution of TIMP-3 transcripts on Day 26 of pregnancy was similar to that on Day 18 of pregnancy (Fig. 7c).
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Gelatin Zymography
We extracted proteins from the endometrium samples on Days 12, 18, and 26 of pregnancy and subjected them to gelatin zymographic assay in order to evaluate the activities of MMP-2 and -9 during early pregnancy (Fig. 8). Two distinct bands of gelatin activity at 72 kDa and 64 kDa were detected in the extracts of rhesus monkey endometrium on Day 12 of pregnancy. These are in line with latent and active MMP-2. On Days 18 and 26 of pregnancy, three bands of gelatin activity at 92 kDa, 72 kDa, and 64 kDa were detected, and these are consistent with MMP-9 and the latent and active forms of MMP-2. Furthermore, the band of 92 kDa on Day 18 of pregnancy was more evident than those on Days 12 and 26 of pregnancy. Compared to the 72-kDa MMP-2, 64-kDa MMP-2 was always predominant in the endometrium on Days 12, 18, and 26 of pregnancy, and the band of 64-kDa MMP-2 on Day 26 of pregnancy was much stronger than that on Day 18 of pregnancy.
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DISCUSSION
We have investigated mRNA expression of MMPs (MMP-2, -9, and -14) and TIMPs (TIMP-1, -2, and -3) and examined the activities of MMP-2 and -9 in rhesus monkey endometrium and placenta on Days 12, 18, and 26 of pregnancy, with special attention to their expression patterns in the placenta, fetal-maternal border, endometrial compartments, and the myometrium.
Placental villi intrusion, together with other processes leading to the development of decidua and placenta must involve extensive ECM degradation. Our results showed that MMP-2 and -14 mRNAs were highly expressed in the placental villi on Day 18 of pregnancy, which makes them the most likely candidates to be involved in the placental villi intrusion that occurred at this time point. Furthermore, placental villi on Day 26 of pregnancy was a foci for MMP-9 mRNA, rather than MMP-2, -14 and TIMP-1, -2, and -3 mRNAs. The high level of MMP-9 transcripts in the placental villi suggests a critical role for MMP-9 in villous processes at this time point. This result agrees with Librach et al. who found that invasiveness displayed by human cytotrophoblast cells depended upon the production of MMP-9 [26].
On Day 26 of pregnancy, mRNAs for MMP-2, -9, and its possible regulators—MMP-14, TIMP-1, -2, and -3—were highly expressed on the fetal-maternal border, with MMP-2, -9, -14, and TIMP-3 in the trophoblast shell, and TIMP-1 and -2 in the maternal decidual cells. Their simultaneous syntheses on the fetal-maternal border suggest that their restricted and balanced expressions are important for the rapid matrix remodeling and controlled invasion in placental tissue [3]. The existence of TIMP-3 in the trophoblast shell also indicates that the trophoblast cells have an inherent capacity to limit invasion into the endometrium.
In rhesus monkeys, blastocyst implantation that occurs on Day 9.5 of pregnancy [27] is a very complex process involving many proteolytic procedures. The existence of MMP-2 and -9 mRNAs on Day 12 of pregnancy strongly suggests their possible roles in this process. Further investigation by gelatin zymography showed that of the gelatinases secreted by the pregnant endometrium on Day 12 of pregnancy, only MMP-2 was active, which suggests that MMP-2 is the exclusively functional gelatinase and it appears in the endometrium within 3 days of the onset of blastocyst implantation, so MMP-2 is likely to be the primary mediator of invasion of the blastocyst into the endometrium on Day 12 of pregnancy. This result is similar to that of Xu et al. [28], who found that MMP-2, but not MMP-9, played an important role in human embryo implantation in the first trimester.
MMP-14 and TIMP-2 transcripts in perivascular decidual cells were expressed earlier than MMP-2 mRNA. Preferential expressions of MMP-14 and TIMP-2 imply MMP-14's regulatory effects on MMP-2 expression and hint that this process is mediated by TIMP-2. This finding is in line with previous findings by Bjorn et al. [29] and is also in agreement with those of Kinoh et al. [30] in mouse and Hernandez-Barrantes et al. [8] in monkey BS-C-1 cells. The zymography result that the active form of MMP-2 was significantly increased on Day 26 of pregnancy, compared with that on Day 18 of pregnancy, further supports the previous hypothesis.
Both MMP-2 and -14 mRNAs were highly expressed in the walls of some arterioles situated adjacent to the implantation site on Day 18 of pregnancy. The existence of active MMP-2 in these compartments suggests that it may function in degrading components of basement membranes (BMs), which provides the trophoblast with a mechanism for breaching the endothelial BM and the boundary of smooth muscle [18, 31]. Besides, on Day 26 of pregnancy, MMP-2, TIMP-1, and TIMP-2 transcripts were all highly expressed in perivascular decidual cells. This result suggests a possible regulatory effect of TIMP-1 and -2 on MMP-2 expression, because full enzyme activity of MMPs in vivo depends on the availability of TIMP-1 or -2 [32]. TIMP-1 and -2, as the inhibitors of MMPs, are also speculated to be responsible for protecting vessels and maintaining blood vessel integrity, which is very important during early pregnancy.
Another important finding of this study is the expression of TIMP-3. TIMP-3 mRNA was detected only in the outer membrane of some groups of arterioles in the endometrium on Days 18 and 26 of pregnancy. This result contrasts with the finding of Hurskainen et al. [3] in human endometrium, which showed that decidual cells and endothelial cells of spiral arteries were strongly labeled for TIMP-3 mRNA. We are still unclear why TIMP-3 mRNA has this specific distribution, but it does indicate that TIMP-3 may play a unique role in maintaining blood vessel integrity, and it also provides insights into other functions of TIMP-3.
MMP-2, -9, -14 and TIMP-1, -2, -3 were all absent in the myometrium on Days 12, 18, and 26 of pregnancy. These results are inconsistent with those obtained in rats, which showed that TIMP-1 and -2 mRNAs were expressed in large amounts in the circular smooth muscle of the myometrium on Day 7 of pregnancy (unpublished results). These differences may be the result of the early phases of gestation examined in this study, compared with the total 165 days of rhesus monkey pregnancy and the fact that the myometrium has not established a mechanism for slowing trophoblast progress.
In conclusion, our investigations implicate the participation of MMP-2, -9, -14 and TIMP-1, -2, -3 in rhesus monkey trophoblast cell invasion and the development of a placenta. These observations will permit further investigation of the precise mechanisms of MMPs and TIMPs on rhesus monkey blastocyst implantation.
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ACKNOWLEDGMENTS
The authors are grateful to Prof. Rujin Zou, Ms. Donghong Tang, and Ms. Xiaomei Sun for assistance with tissue collection. We thank Dr. Akiko Okada and Dr. Yanling Wang for the gifts of plasmids containing TIMP-2 cDNA and MMP-14 cDNA, respectively. We also appreciate Dr. Qingyuan Sun's helpful discussions and careful reading of this manuscript.
FOOTNOTES
First decision: 5 January 2001.
1 This study was supported by the Special Funds for Major State Basic Research Project (G1999055903), the Climbing Project of China (970211019-3), and the Knowledge Innovation Project of the Chinese Academy of Sciences. 
2 Correspondence: Cheng Zhu, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 19 ZhongGuanCun Road, Beijing 100080 China. FAX: 86 10 62529248; zhuc{at}panda.ioz.ac.cn
Accepted: February 9, 2001.
Received: November 30, 2000.
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