Expression and function of matrix ... - Semantic Scholar

Development 122, 1723-1736 (1996) Printed in Great Britain © The Company of Biologists Limited 1996 DEV4680

1723

Expression and function of matrix metalloproteinases and their inhibitors at the maternal-embryonic boundary during mouse embryo implantation Caroline M. Alexander1,*, Elizabeth J. Hansell1, Ole Behrendtsen1, Margaret L. Flannery1, Nerendra S. Kishnani2,†, Susan P. Hawkes2 and Zena Werb1,‡ 1Laboratory

of Radiobiology and Environmental Health and Department of Anatomy and 2Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA *Present address: Department of Newborn Medicine, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA †Present address: Department of Pharmacy, State University of New York, Buffalo, NY 14214, USA ‡Author for correspondence (e-mail: [email protected])

SUMMARY Gelatinase B, a matrix metalloproteinase (MMP) of high specific activity, is highly expressed and activated by mouse blastocysts in culture, and inhibition of this enzyme activity inhibits lysis of extracellular matrix (Behrendtsen, O., Alexander, C. M. and Werb, Z. (1992) Development 114, 447-456). Because gelatinase B expression is linked to invasive potential, we studied the expression of gelatinase B mRNA and protein in vivo, in implanting trophoblast giant cells, and found that it was expressed and activated during colonization of the maternal decidua. mRNAs for several other MMPs (stromelysin-1, stromelysin-3 and gelatinase A) and MMP inhibitors (TIMP-1 and TIMP-2) were expressed in the undifferentiated stroma toward the outside of the decidua, and TIMP-3 mRNA was expressed in primary and some mature decidual cells during their differentiation. Both mRNA and TIMP-3 protein were present at high concentrations transiently, and declined from 6.5 days post coitum onward, as the cells underwent

apoptosis during the main period of gelatinase B expression and ectoplacental growth and expansion. To assess the function of MMPs during implantation and decidual development, we either injected a peptide hydroxamate MMP inhibitor into normal mice or studied transgenic mice overexpressing TIMP-1. In both cases, decidual length and overall size were reduced, and the embryo was displaced mesometrially. Embryo orientation was less strictly regulated in inhibitor-treated deciduae than in control deciduae. Morphogenesis and development of oil-induced deciduomas were also slowed in the presence of the inhibitor. We conclude that administration of MMP inhibitors retards decidual remodeling and growth, and we suggest that the MMPs expressed in precursor stromal cells promote their differentiation and expansion.

INTRODUCTION

tissue without an epithelium-lined lumen, but with an embryo at the core. The extraembryonic tissues of the mouse blastocyst attach and ingress into the stromal/ decidual mass, dividing, expanding and differentiating. At 6.5 days p.c., local blood vessels lose patency, and blood drains into the lumen that surrounds the ectoplacental cone (El-Shershaby and Hinchliffe, 1975; Welsh and Enders, 1991b). At 7.5 days p.c., the integrity of the circulation is restored. Large blood sinuses form, and active angiogenesis is apparent at the polar (mesometrial) end of the embryo. The embryo grows, remodeling the surrounding yolk sac with a thick basement membrane (Reichert’s membrane). Gradually, the supporting decidual cells atrophy to become residual at 12 days p.c. (Abrahamsohn and Zorn, 1993). During these migratory, invasive and remodeling reactions, there is clearly a requirement for ECM metabolism and modulation. We have studied the expression of several matrix metalloproteinases (MMPs) and their inhibitors during implantation to test the hypothesis that this class of enzyme is functionally involved. Previously, we showed that the

The process of mouse embryo implantation starts 5.0 days post coitum (p.c.), when regularly spaced blastocysts lodge in clefts in the convoluted epithelial surface of the antimesometrial side of the asymmetric uterine wall (reviewed by Cross et al., 1994). Expansion of the stroma closes down the uterine lumen (Weitlauf, 1994). The embryos adhere via the mural trophoblast cell surface, and then the uterine epithelium sloughs off, starting closest to the embryo and spreading to the uterine glands (El-Shershaby and Hinchliffe, 1975; Schlafke and Enders, 1975; Welsh and Enders, 1991a). Differentiation of the uterine mesenchymal cells closest to the implantation site during decidualization is accompanied by a transition of basic cellular characteristics from stromal to para-epithelial: Cells acquire extensive cell-cell contacts (including gap junctions), swell up to 10 times their stromal cell volume, become polyploid and lay down a basement membrane-like extracellular matrix (ECM) around each cell. Decidual cells are cohesive; each implantation site becomes a separable mass of decidual

Key words: metalloproteinase, TIMP, mouse, implantation, trophoblast, decidua, apoptosis, transgenic embryo, uterus

1724 C. M. Alexander and others expression of gelatinase B is upregulated in parallel with the differentiation of trophoblast in cultures of mouse blastocysts (Behrendtsen et al., 1992). We assayed the lytic properties of giant cells in culture and found that both the MMP inhibitor TIMP-1 and an anti-gelatinase B antibody inhibited the clearing of subjacent matrix by trophoblast cells (Behrendtsen et al., 1992). Gelatinase B has a wide substrate specificity (Birkedal-Hansen et al., 1993), and the murine enzyme has a high specific activity and has been correlated with the invasive behavior of a number of sarcoma, melanoma and carcinoma cell lines (Bernhard et al., 1990). Matrix metalloproteinases are clearly implicated in invasive, erosive and remodeling reactions by three factors. First, TIMP1 and TIMP-2 produce significant alteration in the invasive properties of various normal and pathologic cell types (Alexander and Werb, 1992; Davies et al., 1993; Khokha, 1994; Montgomery et al., 1994). Second, their expression correlates with the expression of erosive pathologies such as arthritic conditions (Singer et al., 1995). Third, their substrate specificities in vitro suggest that these enzymes can cleave many ECM target proteins (Birkedal-Hansen et al., 1993). MMPs and their inhibitors tend to be co-expressed, leading to the hypothesis that the net extracellular active proteinase concentration determines the invasive or lytic properties of a cell. However, the normal function of this class of enzymes in vivo remains undetermined. We have studied the expression and function of MMPs and TIMPs at the maternal-fetal interface during mouse embryo implantation. MATERIALS AND METHODS Materials Outbred CF-1 and CD-1 mice were obtained from Charles River (Wilmington, MA). UltraSpec RNA purification reagent (Biotecx, San Antonio, TX), Duralon membranes and QuikHyb (Stratagene, San Diego, CA) were used to prepare RNA blots. The MMP inhibitor, 3-(N-hydroxycarbamoyl)-2(R)-isobutylpropionyl-L-tryptophan methylamide (MPI; Grobelny et al., 1992) and an MMP inhibitor control (N-(tert-butyloxycarbonyl)-L-leucine-L-tryptophan methylamide; MIC) were gifts from Glycomed Corp., Alameda, CA (courtesy of R. Galardy). The derivation of the rabbit antibody to gelatinase B was described by Behrendtsen et al. (1992). IgG fractions of high-titer rabbit bleeds were purified by protein A-Sepharose chromatography. Rabbit antiserum to mouse placental lactogen-1 was kindly provided by Dr Frank Talamantes (Colosi et al., 1987). Decidual samples Female mice were mated and checked for vaginal plugs in the morning (midnight = 0 days p.c.). They were killed by cervical dislocation, and uteri were removed at the time points indicated. The uterus was cut open, and deciduae were teased out. Subfractions of decidua, for evaluation of mRNA or protein content, were obtained by pinning the mesometrium downward, cutting out the top third of the exposed antimesometrial decidua and removing it to liquid nitrogen. The opposite, mesometrial third of the decidua was also saved. After all the decidual tissue was removed, the residue was called the uterine sheath. Deciduae were processed for morphologic evaluation or in situ hybridization by fixing in 4% paraformaldehyde for 4-16 hours and paraffin embedding. Immunocytochemistry Paraformaldehyde-fixed deciduae were embedded in paraffin, and 6mm sections were rehydrated, incubated in 0.1 M glycine for 3

minutes and incubated in blocking solutions (5% nonfat milk powder, 1 mg/ml ovalbumin and 5% sheep serum in phosphate-buffered saline; PBS) for 30 minutes each. Blocking serum was removed, rabbit polyclonal antibody to gelatinase B or mouse placental lactogen-1 (diluted to 50 mg/ml in 0.1% bovine serum albumin) was added, and sections were incubated for 1 hour at ambient temperature. Slides were washed in PBS and incubated for 30 minutes at ambient temperature in 5% sheep serum. Sections were incubated with biotinylated sheep anti-rabbit secondary antibody (Sigma #B9140), diluted 1:100 in 0.1% bovine serum albumin for 1 hour at ambient temperature, washed in PBS and then incubated with streptavidin/alkaline phosphatase (Vector Labs SA-5100), diluted 1:100, for 1 hour at ambient temperature. The alkaline phosphatase was developed in substrate from Vector Labs kit SK-5100. Controls used in place of the specific primary antiserum were preimmune serum or normal rabbit serum (Sigma Chemical Co., St. Louis, MO). Slides were counterstained with methyl green. In situ hybridization Paraffin sections (8 mm) were hybridized with 35S cRNA probes as described by Frohman et al. (1990), with a few modifications. The Probe On capillary gap system with Probe On Plus slides was used for hybridization incubations. To decrease solution viscosity, we included 0.1% Brij in all aqueous solutions and 2% chondroitin sulfate and 1% dextran sulfate in the hybridization solution. Hybridization was performed at 55˚C for all probes except TIMP-1 (50˚C). Nearadjacent sections were probed with at least three different anti-sense cRNAs. Unique expression of each probe served as a positive control for nonspecific signal. In some cases, near-adjacent sections were probed with sense cRNAs confirming the positive signal. A nonspecific signal, with sense and anti-sense probes, was frequently seen to be associated with erythrocytes, which are ineffectively removed from implantation sites, even with perfusion fixation. Therefore, all silver grains were examined at high magnification to confirm their association with cells other than erythrocytes. Each slide had at least two adjacent sections, and duplicate slides were used for each probe. Slides were coated with nuclear track emulsion (NTB2 Kodak or K.5D Ilford), and duplicate slides were exposed for two time points (4 days to 3 weeks). After development, slides were counterstained with hematoxylin and eosin or the intercalating DNA dye, Hoechst 33258 (Grevin et al., 1993). The following mouse anti-sense probes were used: gelatinase B (pSP65 92b; Reponen et al., 1994), spanning nt 1917-2240, transcribed with SP6 polymerase; gelatinase A (pSP65 72; Reponen et al., 1992), spanning nt 604-1165, transcribed with SP6 polymerase; stromelysin-1 (pTRM11; Hammani et al., 1992), spanning nt 3115-4051, transcribed with T7 polymerase; stromelysin3 (H. Luk and Z. Werb, unpublished data), a 383-bp probe generated by polymerase chain reaction (PCR) of uterus RNA with redundant oligos spanning nt 1094-1476, transcribed with T7 polymerase; TIMP-1 (pTIMP-8; Gewert et al., 1987), encoding the entire cDNA, transcribed with T3 polymerase; TIMP-2 (Alexander and Werb, 1992), a 360-bp probe spanning amino acid sequence 73-194, generated by PCR of mouse 3T3 fibroblast RNA, transcribed with T3 polymerase; TIMP-3 (Leco et al., 1994), a 2.4-kb cDNA including 160 bp of the 5′ noncoding region, together with the complete coding region and 3′ noncoding region, transcribed with T7 and hydrolyzed to 300 nt. Localization of apoptotic cells DNA in nuclei of cells in 8-mm paraffin sections was labeled at free 3′OH termini by using digoxigenin-labeled dUTP and terminal deoxynucleotidyl transferase (TUNEL) according to the manufacturer’s instructions (Oncor, Gaithersburg, MD). Free 3′OH termini are present at strand breaks produced by endonuclease cleavage during apoptosis. Sections were incubated in fluorescein isothiocyanatelabeled anti-digoxigenin antibody and visualized using fluorescence microscopy. Nuclei were counterstained with propidium iodide. The

MMP function during murine implantation 1725 number of positive nuclei per unit area was determined on a Macintosh 7100/80 AV computer using the public domain NIH Image program (U.S. National Institutes of Health; available from the Internet by anonymous FTP from zippy.nimb.nih.gov). Unit area was designated as number of square pixels, and images were scanned at 300 pixels/inch. RNA blot analysis Tissue was homogenized in UltraSpec reagent according to the manufacturer’s instructions with an Omnigene Polytron; RNA was separated on 1.2% gels and transferred to Duralon membranes. RNA blots were hybridized by standard techniques. Substrate gel electrophoresis (zymography) Lysates of decidual fractions were prepared by homogenizing tissue pieces in RIPA buffer (1:4 (wt:vol) in 150 mM NaCl, 1.0% NP40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM TrisHCl buffer, pH 8.0) at 4˚C. Lysates were centrifuged at 14,000 g for 15 minutes, and the insoluble, ECM-enriched fractions were washed in RIPA buffer, aliquoted, and stored at −80˚C or immediately processed. No difference between fresh samples and once-thawed samples was seen. Pellets were resuspended in substrate gel sample buffer for 10 minutes at 37˚C. Zymography was described by Behrendtsen et al. (1992), and reverse zymography was described by Staskus et al. (1991). Briefly, gelatin (a general proteinase substrate) was included in the SDS-polyacrylamide gel mixture at 1 mg/ml. Samples were not denatured with heat or reducing agents, but were mixed with sample buffer containing SDS and loaded on 10% or 15% separating gels. After separation, proteins were renatured by incubating the gel in 2.5% Triton X-100 and then incubating in Tris-HCl buffer, pH 8.0, containing Ca2+ at 37˚C to allow gelatinolysis for 1648 hours. Gels were stained with Coomassie Blue. Clearing of background gelatin by proteinases was revealed as clear bands. For reverse zymography, a crude mixture of gelatinases from medium conditioned by Rous sarcoma virus-transformed chick embryo fibroblasts at an empirically derived concentration was copolymerized with the gel mixture. Inhibitor activities were revealed as dark bands against the partially cleared background. SDS-polyacrylamide gel electrophoresis of the same extracts revealed which bands were protein bands with no inhibitor activity. Generation of deciduomas Pseudopregnant females were generated by mating with vasectomized males and were induced to decidualize by the transfer of 30 ml of peanut oil with a 25-gauge needle on a 1-ml syringe into the uterus of anesthetized females at 4.25 days p.c. Treatment with MMP inhibitors The control compound (MIC) or the proteinase inhibitor (MPI) was Fig. 1. Expression of gelatinase B by trophoblast giant cells. Gelatinase B was identified in sections of mouse implantation sites at (A) 5.5 days p.c., (B) 6.5 days p.c., (C, D) 7.5 days p.c., and (E) 8.5 days p.c., by means of an immunopurified antibody (A,C,E) or in situ hybridization analysis (B). (D) A control section, adjacent to the section shown in C, was stained with preimmune IgG. Arrows in A and B indicate cells positively stained for gelatinase B that were morphologically identified as primary trophoblast giant cells; small arrow indicates nonspecific association of silver grains on red blood cells by the gelatinase B probe (see Materials and Methods). Insets in A, C and E show the positively stained cells at 4× magnification, to allow evaluation of their size and morphology. Inset in B shows an overview of the embryo at the same lower magnification as A, C and D. Images are bright-field photomicrographs. Red signal in panels A, C, D, and E is alkaline phosphatase substrate reaction; nuclei are counterstained with methyl green. In B, silver grains show as black against hematoxylin and eosin counterstain. Bars indicate 56 µm.

injected intraperitoneally at 100 mg/kg body weight as a 20 mg/ml slurry in 4% carboxymethyl cellulose in 0.9% saline. Injections began on the evening of day 3 p.c. (late day 3.5) and were administered every 12 hours thereafter until the mice were killed at 6.5 days p.c. Experiments with β-actin-TIMP-1 transgenic mice The derivation of transgenic mice expressing human TIMP-1, driven by the β-actin promoter, has been described by C.M. Alexander, E.W.

1726 C. M. Alexander and others Howard, M.J. Bissell and Z. Werb (unpublished data). Homozygous mice on the CD-1 background were used in the present study. Timed matings, decidual harvest and tissue lysate methods for control CD-1 mice were as described earlier. The β-actin promoter directed the ubiquitous expression of TIMP-1 in all organs, and serum levels of about 50 ng/ml. No gross changes were induced by expression of this transgene, and fertility of these mice was comparable to that of controls.

RESULTS Gelatinase B is expressed in trophoblast giant cells during implantation Gelatinase B synthesis is induced in cultures of differentiating trophoblast cells (Behrendtsen et al., 1992). To show that this enzyme is also expressed in vivo during implantation, we localized mRNA and protein in sections of decidua by using in situ hybridization and immunocytochemistry with an immunopurified antibody, respectively (Fig. 1). This antigelatinase B antibody was found to be function-perturbing in our previous study (Behrendtsen et al., 1992), specifically inhibiting the lysis of a subjacent ECM by cultured trophoblast cells. Primary giant cells around the implantation site at 5.5 days p.c. (Fig. 1A) stained positively with anti-gelatinase B

antiserum. At this stage, trophoblasts emigrate out of the primitive trophectoderm surrounding the embryo, displace uterine epithelial cells, cross the underlying basement membrane, and differentiate to form a shell of primary giant cells embedded in maternal decidua. Accumulation of gelatinase B protein was paralleled by synthesis of gelatinase B mRNA in these cells; in situ hybridization of sections from 6.5day-p.c. implantation sites (Fig. 1B) showed that every trophoblast giant cell, identified by morphologic criteria, was positive for gelatinase B mRNA expression. Immunostaining of 7.5- and 8.5-day-p.c. deciduae (Fig. 1C,E) showed that, as the ectoplacental cone grew, the population of trophoblast giant cells synthesizing gelatinase B increased, all around the margins of the expanding ectoplacental cone. Placental lactogen-1, a marker of trophoblast differentiation (Cross et al., 1994, 1995), was also present in the giant cells (data not shown). The ingression, growth and differentiation of the ectoplacental cone into the decidua mostly occurs after the uterine epithelium has undergone apoptosis and after the underlying basement membrane has been remodeled and penetrated by reactive endothelial cells (Welsh and Enders, 1991a). Therefore, in contrast to primary trophoblast giant cells, secondary giant cells may not be functionally dependent on the expression of invasive, lytic properties. Immature cells in the ectoplacental cone did not stain positively for gelatinase B

A

Fig. 2. Expression of mRNAs for MMPs and TIMPs in trophoblast and decidua. (A) mRNA was isolated from whole decidua without removal of the embryos (decidua + embryo) and from the uterine sheath residue (uterus), and blots were hybridized with cDNAs for TIMP-1, TIMP-2, TIMP-3, gelatinase A and gelatinase B, together with an endothelial cell marker, PECAM-1. Internal controls were actin and ethidium bromide (EtBr) staining of rRNA bands. (B,C) Bands on RNA blots were quantified and normalized by using scanning densitometry. Data for TIMP-1, TIMP-2 and gelatinase A are shown in B, and data for TIMP-3 and gelatinase B are shown in C. RNA blots were probed in duplicate for each of two preparations at 5.5 and 7.5 days p.c. and each of four preparations at 6.5 days p.c. Error bars represent standard error of the mean.

MMP function during murine implantation 1727 (Fig. 1C), confirming that expression of this enzyme is a marker of terminal differentiation of trophoblast giant cells. TIMPs and MMPs have distinct distributions and temporal regulation in the implantation site To determine which MMPs and TIMPs were poised to contribute functionally to the remodeling processes characteristic of the implantation site, we probed RNA extracted from a timed series of deciduae (and embryos) and residual uterine tissue for their expression (Fig. 2). TIMP-1 increased steadily throughout the time course under study (5.5 to 9.5 days p.c.), whereas TIMP-2 expression was approximately constant (Fig. 2A,B).

TIMP-3 mRNA showed a striking temporal regulation, highly induced by 6.5 days p.c., decreasing at 7.5 days p.c., and ceasing at 8.5 days p.c. (Fig. 2A,C). PECAM-1, a marker of endothelial cells (Baldwin et al., 1994) and therefore of vascularization, was high and constant in decidual tissue and little expressed in the fibrous uterine sheath. Gelatinase A expression declined by 6.5 days p.c. (Fig. 2A,B), but gelatinase B (like TIMP-1) increased; it was low but detectable at 5.5 and 6.5 days p.c., increased at 7.5 days and was highest at 9.5 days p.c. (Fig. 2A,C). Gelatinase B expression paralleled the increasing number of differentiated trophoblast giant cells. Interestingly, the peak expression of TIMP-3 preceded the accumulation of gelatinase B and the

Fig. 3. Expression of MMP and TIMP activities during implantation. (A) Lysates of decidual and uterine tissues, isolated at 5.5-9.5 days p.c., were analyzed by gelatin zymography. Clearing of the gelatin substrate (white bands) indicates enzyme activity. Gelatinase A (gel A), activated gelatinase A (gel A*), gelatinase B (gel B), activated gelatinase B (gel B*) and a 25×103 Mr MMP are evident. (B) A zymographic separation of enzyme activities in 6.5-day lysates was incubated with (+) or without (−) the metalloproteinase inhibitor 1,10-phenanthroline. (C) The same lysates were analyzed by reverse zymography to identify MMP inhibitor activities at the various stages. Inhibitor activities appear as dark bands. TIMP-1 (T1) and TIMP-2 (T2) standards were from medium conditioned by mouse 3T3 cells (Std). TIMP-3 (T3) was identified by comigration with chicken TIMP-3. (D) Reverse zymographic analysis of subfractions of decidua shows the regionalization of expression of the various inhibitors and enzymes. Decidua (DEC) was dissected from the uterine sheath (UT) at 6.5 days p.c. Two subfractions were dissected out of decidua: a portion of antimesometrial tissue (AMD), enriched in the peri-implantation zone, and another distal fraction from the mesometrial pole (MESO). (E) Reverse zymographic analysis of subfractions of deciduomas shows that deciduomas expressed enzymes and inhibitors in similar quantities and regionalized patterns. UT, uterine sheath; MESO, mesometrium; PX, proximal tissue excised from the perilumenal region of the deciduoma; DIS, distal tissue excised from the outer region of the deciduomal tissue adjacent to the uterine sheath. (F) Zymographic analysis shows that activation of gelatinase B was lower in deciduomal tissue (lanes 1 and 3) than in decidual samples (lanes 2 and 4). Lysates were prepared at 0.25 mg wet weight of tissue/ml, and the insoluble fractions were resuspended in 1 volume of sample buffer. The equivalent of 0.25 mg of protein was loaded onto each lane.

1728 C. M. Alexander and others maximal invasive activity of the secondary trophoblast giant cells after 8.5 days p.c. Stromelysin-1 and stromelysin-3 showed low and constant expression (data not shown). Enzyme and inhibitor activities are detectable in decidual lysates We then determined gelatinolytic enzyme and inhibitor activities by zymography. Gelatinase A was found in the decidual extracts in both precursor (72×103 Mr) and activated (62×103 Mr) forms (Fig. 3A). Its activity was highest in 5.5-day-p.c. samples and declined thereafter; this pattern corresponds with mRNA expression for this enzyme (Fig. 2A). The proportion of activated enzyme was constant throughout. A 25×103 Mr gelatinase was expressed in uterus and in undifferentiated decidual tissue (5.5 days p.c.) but disappeared upon differentiation of the decidua between 5.5 and 6.5 days p.c., making it an effective marker of this transition of stromal cells. This 25×103 Mr enzyme was not an MMP, because it was not inhibited by 1,10-phenanthroline (Fig. 3B). Surprisingly, when normalized to wet weight of tissue, gelatinase B activity was similar in decidua and uterus throughout the time course. In fact, extracts of oil-induced deciduoma, which does not contain embryonic trophoblast, also yielded similar quantities of gelatinase B activity (Fig. 3F). The gelatinase B activity seen by zymography therefore did not correlate with data from RNA blots or with the embryonic trophoblastspecific signal observed by immunostaining or by in situ

hybridization (Figs 1, 2). The discrepancy between expression patterns of mRNA and activity may be due to the release of preformed gelatinase B protein from the intracellular granules of infiltrating neutrophils or monocytes (Shapiro et al., 1995) or to its immobilization on cell surfaces or on the ECM surrounding cells in the decidua and uterus (Menashi et al., 1995). However, the implantation reaction was accompanied by the specific activation of gelatinase B (Fig. 3A). Distribution of the enzyme between inactive and active forms in extracts of whole decidua embryo varied; the proportion of activated gelatinase peaked at 7.5 days p.c. and declined until the last time point examined (9.5 days p.c.). Activated gelatinase B was not present in the non-decidual uterine tissue (Fig. 3D). Dissection of the decidua into mesometrial and antimesometrial regions (see below for diagram) showed that gelatinase B activation was specific to the antimesometrial peri-implantation domain and not to the mesometrial domain (Fig. 3D). Deciduomal tissue lysates typically showed less activation of gelatinase B than did corresponding decidual lysates (Fig. 3F), except when deciduomas had bloody cores. In that case, the amount and activation of gelatinase B were greatly increased. Reverse zymography of inhibitor activities confirmed that TIMP-3 was the major MMP inhibitor present in decidual extracts at 5.5-8.5 days p.c. (Fig. 3C). This is not surprising, given the fact that TIMP-3 binds to ECM much more avidly than do TIMP-1 and TIMP-2, increasing its effective half-life. Low levels of TIMP-1 and TIMP-2 were also detected, as well as

Fig. 4. Localization of expression of gelatinase B and TIMP-3 to the periimplantation zone. 7.5-day-p.c. deciduae were analyzed by in situ hybridization for expression of TIMP-3 and gelatinase B on near-adjacent sections. Bright-field images were used for ease of alignment of both signals, and an example was chosen that contained twin embryos. Overlaid signals are drawn in cartoon form (top panel) based on the observation of both signals in near-adjacent sections taken across the entire peri-implantation zone. Trophoblast giant cells expressing gelatinase B (lower left panel) are clearly surrounded by decidual cells expressing high levels of TIMP-3 (lower right panel). Insets (location arrowed in the main panel) show the morphology of the cells expressing gelatinase B and TIMP-3. In the cartoon, the arrows drawn outward from trophoblast indicate the direction of growth of the placenta, and the long arrow on the left indicates the direction of the wave of differentiation of decidual cells, from the antimesometrial pole to the mesometrial pole.

MMP function during murine implantation 1729 some higher molecular mass complexes, which are likely to be TIMP-3 dimers or complexes of MMPs with TIMPs. Between 5.5 and 6.5 days p.c., TIMP-3 activity was highly upregulated. By 8.5 days p.c., TIMP-3 disappeared from the extracts, closely following the mRNA expression time course. Analysis of the dissected antimesometrial peri-implantation zone showed that it was relatively enriched in TIMP-3, whereas the uterus and mesometrium contained higher levels of TIMP-2 (Fig. 3D). Deciduomal tissue showed the same pattern of localization of TIMP-3 protein expression by zymography. The innermost tissue around the lumen showed loss of expression of the 25×103 Mr enzyme activity and high TIMP-3 activity (Fig. 3E). TIMP-3 and gelatinase B are in distinct domains We used in situ hybridization to precisely locate cells expressing TIMP-3 and gelatinase B, which were shown by microdissection and zymography of decidual extracts to be enriched in the peri-implantation zone. In 6.5-day-p.c. (data not shown) and 7.5-day-p.c. decidua (Fig. 4), gelatinase B-positive giant cells at the margins of the trophoblast were bounded by primary decidual cells expressing high levels of TIMP-3 mRNA. Using enlargement of stromal cells as a morphologic marker of decidualization, we found that expression of TIMP3 at this stage of development was limited to differentiating

decidual cells, which peak at 6.5 days p.c. and are gone by 8 days. Similarly, TIMP-3 mRNA responded in parallel with a wave of decidual differentiation that was initiated at the antimesometrial pole of the implantation chamber and spread toward the mesometrial pole and outward through the spiny layer and the zone of vascularization (data not shown). Apoptosis in the peri-implantation decidua occurs in regions of low TIMP-3 expression Mature decidual cells are present transiently during implantation, undergoing differentiation followed by apoptosis to allow expansion of the growing embryo and placenta. To offer an explanation for the transient induction of TIMP-3 during terminal decidual differentiation, we examined the relationship of TIMP-3 expression to the initiation of an apoptotic program, determined by staining for DNA fragmentation in situ. Several cross-sections of deciduae, 6.5 to 8.5 days p.c., stained by the TUNEL method, were captured and overlaid to show the accumulated distribution (Fig. 5). The distribution of apoptotic cells in single sections, compared with the TIMP-3 mRNA distribution seen by in situ hybridization on near-adjacent sections, is shown in Fig. 5A,B. The decidual stroma at 6.5 days p.c. showed a low rate of apoptosis (1.03±0.24 cells/unit area) and the TIMP-3-positive zone had an even lower rate (0.63±0.07).

Fig. 5. Apoptosis in periimplantation decidua. Apoptotic nuclei (labeled with fluorescein by the TUNEL procedure, shown as dark green signal) were overlaid with the TIMP-3 mRNA expression pattern (obtained by in situ hybridization, shown as the white, dark-field signal of the silver grains) by using Adobe Photoshop (version 3.0) for decidual sections at 6.5 days p.c. (A) and 7.5 days p.c. (B) Data for A were derived from nearadjacent sections, and data for B were obtained from matched sections. The number of apoptotic nuclei per unit area (square pixels) was calculated (see Materials and methods) and used to generate color-coded zones with similar * apoptotic indices (except in the embryo proper). These zones are indicated on the cartoons (A,B), redrawn from sections (C-E). The cumulative signal from two or four overlaid sections containing apoptotic nuclei is shown: (C) 6.5 day p.c. (n=2); dark yellow, 12.8±7.2 nuclei per unit area; mid-yellow, 1.03±0.24; pale yellow (*), 0.63±0.07. (D) 7.5 day p.c. (n=2); dark yellow, 45±5.6; mid-yellow, 0.57±0.10. (E) 8.5 day p.c. (n=4); dark yellow, 2.6±1.8; mid-yellow, 0.3±0.14. Bars indicate: A, 174 µm; B, 220 µm; C, 290 µm; D, 350 µm; E, 435 µm.

1730 C. M. Alexander and others Inside the decidual cells was a ring of dying uterine epithelial cells, just behind the shell of primary giant trophoblast cells. The apoptotic index for this peri-embryonic zone was 12.8±7.2. At 7.5 days p.c., apoptosis was low in the remaining region of TIMP-3 and in the decidual stroma outside this zone. However, apoptosis in the uterine layer of cells next to the embryo, consisting of dead and dying primary decidual cells, had increased (45±5.6). By 8.5 days p.c., when most of the TIMP-3-expressing decidual cells had disappeared, the peri-embryonic apoptotic embryonic rate had decreased, except around the ectoplacental cone, and the decidual rate was negligible. Thus, cells that synthesized TIMP-3 showed low rates of cell death, whereas cells no longer expressing TIMP-3 in proximity to gelatinase B synthesis showed high rates of cell death. MMPs and TIMPs have distinct spatial distributions in the implantation site The biochemical data shown here indicate that TIMP-3 and gelatinase B are only two of a number of contributors to the proteolytic environment of the implantation site. Accordingly, we used in situ hybridization to determine the spatial localization of other MMPs and TIMPs at 7.5 days p.c. Gelatinase A was expressed in the zones of the decidua that were not differentiated (Fig. 6A), in striking contrast to the embryonic trophoblast-specific expression of gelatinase B (Fig. 6B). The low level of gelatinase B mRNA in decidua did not localize to any specific site. Because the number of undifferentiated decidual cells declines as implantation progresses, the localization of gelatinase A explains the decrease in total activity observed by zymographic analysis of decidual extracts during the implantation period. Stromelysin-1 (Fig. 6F) and stromelysin-3 (Fig. 6E) were also expressed in the progressively smaller zone of undifferentiated cells toward the outer margin of the decidua. TIMP-1 (Fig. 6C) was expressed in undifferentiated zones, and TIMP-2 (Fig. 6H) was expressed in a similar, but relatively larger, domain. A summary diagram (Fig. 7) illustrates the bat-wing shape of the zone of differentiating maternal cells in sections of the 7.5-day decidua. We conclude that the expression of the enzymes gelatinase A, stromelysin-1, stromelysin-3 and the inhibitors TIMP-1 and TIMP-2 described here was typical of undifferentiated stroma and was excluded from the bat-wing domain. Metalloproteinase inhibitors alter decidualization and implantation in vivo To probe the function of MMPs during the implantation reaction, we inhibited MMP activity by one of two methods. Either we injected the MMP inhibitor

MPI intraperitoneally twice daily for 3 days, using MIC for control experiments, or we overexpressed TIMP-1 under the control of a β-actin promoter by transgenic means. Deciduae from mice injected with the control compound MIC were indistinguishable from those from uninjected mice. Normal deciduae had an elongated egg shape, and the embryos were displaced toward the antimesometrial end (Fig. 8D). The position of the mural tip of the egg cylinder was offset to 65% of the total decidual length (Table 1). In contrast, deciduae of mice injected with the inhibitor MPI were difficult to dissect out. They adhered to the uterine wall and tended to split into two halves, spilling out the embryos. After the deciduae were teased out of the uterine sheath, parts of uterine tissue were left adhering to them (indicated by dots outlining the decidua proper in Fig. 8A-C). We conclude that the boundary that usually separates the decidua from the uterine myometrium did not form effectively in MPI-treated mice. The deciduae were round instead of egg-shaped, giving a higher width-to-length ratio than controls (Table 1). The width of MPI-treated deciduae was unaffected, but they were shorter (83% of control), and their cross-sectional area, measured through the midplane, was

Fig. 6. Localization of expression of mRNAs for other MMPs and inhibitors. 7.5-day-p.c. deciduae were analyzed by in situ hybridization for the expression of (A) gelatinase A, (B) gelatinase B, (C) TIMP-1, (D) TIMP-3, (E) stromelysin-3, (F) stromelysin-1 and (H) TIMP-2. (G) A hematoxylin and eosin stain of a near-adjacent section. Bar indicates 56 µm.

MMP function during murine implantation 1731 Table 1. Morphometric analysis of implantation sites at 6.5 days p.c. in mice treated with metalloproteinase inhibitors or mice transgenic for TIMP-1 expression Mice* MIC (control) (n=29 deciduae/5 mice) MPI (n=38 deciduae/7 mice)

Cross-sectional area of decidua†

Length of decidua†

Embryo position‡

Decidual shape (width/length)

Resorption sites§ (% total sites)

1±0.027

1±0.038

0.65±0.01

0.71±0.01

0

0.83±0.045 (P