DEVELOPMENTAL DYNAMICS 216:374–384 (1999) Expression of Cathepsin Proteinases by Mouse Trophoblast In Vivo and In Vitro SUZANNE AFONSO, LINDA ROMAGNANO, AND BRUCE BABIARZ* Department of Cell Biology and Neuroscience, Nelson Labs, Busch Campus, Rutgers University, Piscataway, New Jersey ABSTRACT Implantation and placentation in the mouse requires successful invasion of the uterine wall by primary and secondary trophoblast giant cells. Their invasive nature depends in part on the upregulation of proteinases for the phagocytosis and extracelluar digestion of maternal cells and matrix materials. The work reported here studies the expression of cathepsin proteinases during secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. Cathepsins B (CB), L (CL), and D (CD) were found to be upregulated during trophoblast differentiation in vivo at the message and protein level producing expression patterns equivalent to those of primary trophoblast. Invasive trophoblast cells expressed higher levels of the processed or active forms of the enzymes, coinciding with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials. Trophoblast differentiation in vitro showed a similar upregulation of cathepsin enzymes. The enzymes were localized to heterogeneous vesicles that resembled both lysosomes and heterophagic vesicles. The presence of a large lysosomal population within the giant cells was confirmed by vital staining with acridine orange. Analysis of trophoblast-conditioned media also demonstrated secreted forms of CB and CL. The results suggest that cathepsin enzymes may contribute to trophoblast invasion not only through the intracellular breakdown of molecules phagocytosed by trophoblast cells, but also by the extracellular digestion of matrix molecules and activation of other pro-enzymes. Dev Dyn 1999;216:374–384. r 1999 Wiley-Liss, Inc. Key words: mouse; implantation; placentation; trophoblast; cathepsin B; cathepsin L; cathepsin D These cells penetrate the basement membrane and invade the decidualizing stroma to establish the implantation site. Concurrently, embryonic cells facing the mesometrial pole of the uterus continue to divide forming the ectoplacental cone (EPC). Beginning on day 6.5, the EPC forms an outer layer of secondary trophoblast giant cells which invade into the mesometrial uterus to begin placenta formation. Invasion ceases on day 10.5, with chorioallantoic placental function beginning on day 11. This invasive process is mediated in part by a balanced synthesis of trophoblast-derived proteinases and decidua-derived proteinase inhibitors. Mouse trophoblast has been shown to produce a number of matrix metalloproteinases (MMPs), including stromelysin (Lefebvre et al., 1995), membrane-type MMP (Tanaka et al., 1998), and predominately MMP-9 or gelatinase B (Harvey et al., 1995; Alexander et al., 1996; Das et al., 1997). In vivo, MMP-9 is localized to invasive giant cells and enzyme activity is detected in trophoblast conditioned media in vitro (Behrendsten et al., 1992; Harvey et al., 1995; Das et al., 1997). Inhibition of MMP-9 activity results in incomplete uterine decidualization and failure of trophoblast invasion (Alexander et al., 1996). The serine proteinase, urokinasetype plasminogen activator (uPA), is also produced by mouse trophoblast giant cells in vivo and secreted by blastocyst outgrowths in vitro (Harvey et al., 1995; Teesalu et al., 1996; Zhang et al., 1996). Inhibitors for uPA and MMPs are not synthesized by the giant cells, but are detected as products of the uterine decidua. Both the tissue inhibitor of metalloproteinase-3 (TIMP-3) (Alexander et al., 1996; Leco et al., 1996; Das et al., 1997) and plasminogen activator inhibitor (Teesalu et al., 1996) are synthesized by decidual cells abutting the invasive trophoblast, suggesting they play a role in the control of enzyme activity during invasion. We have identified the cysteine proteinases and their inhibitors as another enzyme/inhibitor family that plays a critical role in implantation (Afonso et al., 1997). Uterine decidua was shown to upregulate the cysteine proteinase inhibitor cystatin C in response to invading primary and secondary trophoblast. A concomitant INTRODUCTION Implantation of the mouse embryo involves a controlled invasion of the uterine wall by the extraembryonic trophoblast. The process is initiated by the attachment of the blastocyst to the uterine epithelium on day 4.5 of development and the differentiation of an outer layer of invasive primary trophoblast giant cells. r 1999 WILEY-LISS, INC. Grant Sponsor: National Institutes of Health; Grant Number: HD20581. *Correspondence to: Bruce Babiarz, Department of Cell Biology and Neuroscience, Nelson Labs, Busch Campus, Rutgers University, Piscataway, NJ 08855. E-mail: firstname.lastname@example.org Received 25 March 1999; Accepted 2 September 1999 CATHEPSIN EXPRESSION IN MOUSE TROPHOBLAST 375 Fig. 1. Northern analysis of cathepsins B, L, and D expression in developing placenta. Total RNA was analyzed from EPCs from days 7.5 and 8.5, placentae from days 9.5 to11.5, and primary trophoblast from day 8.5. (a) Hybridization with CB probe detected a single transcript at 2.2 kb (double headed arrow). Analysis by scanning densitometry showed an approximate 3⫻ increase in message by day 11.5. (b) Hybridization with CL probe detected a single band at 1.7 kb (double headed arrow). Scanning densitometry showed that the levels were highest in EPCs from days 7.5 and 8.5 and decreased 4⫻ by day 10.5. (c) Hybridization with CD probe detected a single transcript at 2.2 kb (double headed arrow). Analysis by scanning densitometry showed an approximate 3⫻ increase in message by day 9.5. For each, the blots were stripped and reprobed with the 18s rRNA control probe (*, lower panels). increase in the cysteine proteinases cathepsin B (CB) mRNA and cathepsin L (CL) protein was observed in the differentiated trophoblast giant cells (Afonso et al., 1997). In addition, treatment of pregnant females with E-64, a synthetic cysteine proteinase inhibitor, resulted in a failure of implantation and normal uterine decidualization. The work reported here extends our observations on cathepsin synthesis during trophoblast differentiation in vivo and in vitro. The cathepsin proteinases are the major lysosomal enzymes, and include the cysteine proteinases CB and CL, and the aspartyl proteinase cathepsin D (CD) (Barrett et al., 1986; Everts et al., 1996). All three enzymes are synthesized in a proform which can be activated to a number of lower molecular weight species (Barrett et al., 1986; Berquin and Sloane, 1995). Increased levels of cathepsins, within the lysosomal compartment and as secreted enzymes, have been associated with tumor progression and aggressiveness in many types of cancers (Berquin and Sloane, 1995; Keppler and Sloane, 1996; Ren and Sloane, 1996; Garcia et al., 1990; Roger et al., 1994). They are also upregulated in a number of normal tissues involved in phagocytosis, matrix remodeling, and apoptosis (Briozzo et al., 1991; Page et al., 1992; Gu et al., 1994; Kakegawa et al., 1995; Sinha et al., 1995). We have used Northern and Western blotting, in situ hybridization, and immunohistochemistry to study the expression of CB, CL, and CD during the periimplantation and early placentation stages (gestational days 7.5 to 11.5) in vivo. The work analyzes secondary trophoblast differentiation, and compares the expression patterns to fully differentiated day 8.5 primary trophoblast giant cells. The enzymes were further studied during secondary trophoblast differentiation in vitro using EPC outgrowths. 376 AFONSO ET AL. RESULTS Northern Blot Analysis cDNA probes to mouse CB, CL, and CD were used to analyze mRNA synthesis in EPC and placental trophoblast tissues. A single 2.2 kb CB transcript was expressed in EPCs from days 7.5 to 8.5 and placentae from days 9.5 to 11.5 (Fig. 1a). Scanning densitometry of autoradiograms indicated that CB levels steadily increased throughout this time period, with an approximate 3-fold increase by day 11.5. The expected 1.7 kb CL transcript consistently showed a biphasic expression pattern with the highest message levels in day 7.5 and 8.5 EPCs and at the end of the invasive period in day 11.5 placentae (Fig. 1b). For CD, a single 2.2 kb transcript was detected and, like CB, showed an approximate 3-fold increase from days 7.5 to 11.5 (Fig. 1c). For all three, Northern analysis of differentiated day 8.5 primary trophoblast giant cells showed high levels of synthesis of similar sized transcripts for all three enzymes (Fig. 1). Western Blot Analysis Specific antibodies to CB, CL, and CD were used to identify the protein forms synthesized by trophoblast tissues. Western analysis identified CB bands at 34kDa and 30kDa in day 7.5 EPCs (Fig. 2a), representing the proform and active form, respectively (Berquin and Sloane, 1995). In comparison, day 8.5 primary giant cells showed greater amounts of these forms, plus higher molecular weight species of 43 kDa and 37 kDa (Fig. 2a). Continued differentiation of secondary trophoblast resulted in the increased production of the 30 kDa protein on day 9.5. With the completion of the invasive phase of secondary trophoblast and onset of placental function on day 11.5, the active 30 kDa protein was decreased, and the 34 kDa proform became the predominate CB species (Fig. 2a). CL western blots showed a consistent upregulation of protein during EPC development with three different proteins detected at 38 kDa, 34 kDa and 29 kDa (Fig. 2b). Day 8.5 primary trophoblast synthesized high levels of all three proteins (Fig. 2b). Based on work with mouse (Hamilton et al., 1991) and rat (Conliffe et al., 1995) placentae, the 38 kDa protein has been identified as the proform and the 34 kDa and 29 kDa proteins as the processed and lysosomal forms respectively. CD western blots also showed a consistent upregulation of protein during trophoblast differentiation (Fig. 2c). All samples expressed a 60 kDa protein (Fig. 2c), most likely representing the proform of CD (Barrett et al., 1986). On day 7.5 low levels of 46 kDa, 42 kDa, and 38 kDa proteins were observed which were all upregulated with further secondary giant cell differentiation (Fig. 2c). These proteins represent mature (46 kDa) and further processed forms (42 kDa and 38 kDa) of the enzyme (Moulton and Khan, 1992; Compaine et al., 1995). With the onset of placental function on day 11.5, a downregulation of the 38 kDa form was observed (Fig. 2c). Day 8.5 primary trophoblast giant cells displayed a pattern and intensity Fig. 2. Western blot analyses of cathepsins B, L, and D in primary trophoblast, EPCs from days 7.5, placentae from days 9.5 and 11.5, and day 8.5 primary trophoblast. (a) CB antibody detected bands at 34 and 30 kDa on day 7.5. The 30 kDa band predominated in day 9.5 samples and on day 11.5, the 34 kDa band was most prominent. Additional bands at 43 and 37 kDa were observed in day 8.5 primary trophoblast samples, as well as intensely staining of 34 and 30 kDa bands. (b) CL antibody detected three protein forms in all samples at 38kDa, 34 kDa and 29 kDa which were upregulated from days 7.5 to 11.5. (c) CD antibody detected a 60 kDa protein of 60 kDa in all samples. An upregulation of 46 kDa and 42 kDa protein occurred in day 9.5 and 11.5 placenta. Day 8.5 primary trophoblast and day 9.5 secondary trophoblast showed an additional form at 38 kDa. CATHEPSIN EXPRESSION IN MOUSE TROPHOBLAST 377 Fig. 3. In situ hybridization of cathepsins B, L, and D. Day 7.5 (a–c) CB mRNA was localized to the primary giant cells (pGC) (b) and secondary giant cells (sGC) surrounding the epc core (c). Similar localization of CL and CD was observed (not shown). Day 9.5 (d–e) CL mRNA was localized to primary giant cells (d) and to the secondary giant cells of the developing placental rudiment (e). Similar localization of CB and CD was observed (not shown). Day 11.5 (f–g). CL (not shown) and CB (f) expression were limited to the placental secondary giant cells, whereas CD (g) was also expressed in the spongiotrophoblast layer (stb). lab, labyrinthine trophoblast. (a), (e), (f), (g), 100⫻; (d) 200⫻; (b), (c), 400⫻. similar to the later stages of placental development except for downregulation of the 38 kDa form (Figure 2c). CD, in situ hybridization was performed on sections through the implantation site. The patterns of expression for all three enzymes were similar during trophoblast development (Fig. 3). Both primary and secondary trophoblast giant cells expressed mRNA for all three enzymes from days 7.5 to 9.5 (Fig. 3a–e). In the day 11.5 placenta CL and CB were restricted to the outer layer of giant cells, while CD transcripts were also expressed in the spongiotrophoblast layer (Fig. 3f, g). In Vivo Localization EPC differentiation produces a number of different trophoblast tissues, including giant cells, spongiotrophoblast, and labyrinthine trophoblast. To elucidate the cell-type specific expression patterns of CB, CL, and 378 AFONSO ET AL. Fig. 4. Immunolocalization of cathepsins B, L, and D. Day 7.5 (a–c) CB was localized to the giant cells (a) and, at higher magnification, labeled cytoplasmic vesicles in primary (b) and secondary (c) giant cells. Similar localization of CL and CD was observed (not shown). Day 9.5 (d–e) CL was localized to cytoplasmic vesicles in primary giant cells (d) and secondary giant cells of the developing placental rudiment (e). Similar localization of CB and CD was observed (not shown). Day 11.5 (f–g). CL (not shown) and CB (f) expression were limited to the placental secondary giant cells, whereas CD (g) was also expressed in the spongiotrophoblast layer (stb). lab, labyrinthine trophoblast. (a) 100⫻; (b), (c), 200⫻; (d), (e), (f), (g), 400⫻. Immunohistochemical analyses using specific antisera to CB, CL, and CD localized the proteins in a pattern similar to message localization. Both primary and secondary giant cells stained for all three enzymes at day 7.5 of development (Fig. 4a). Higher magnification showed intense staining of cytoplasmic vesicles in invasive trophoblast giant cells (Fig. 4b, c). Giant cells remained positive for all three enzymes on days 9.5 (Fig. 4d, e) and 11.5 (Fig. 4f), with the spongiotrophoblast expressing CD (Fig. 4g). EPC outgrowths. After 48 hr in culture (equivalent to day 9.5 of development), during which large outgrowths of differentiated secondary trophoblast develop, levels of CB, CL, and CD were all increased relative to day 7.5 EPCs (Fig. 5). Some differences were observed compared to day 9.5 secondary giant cells in vivo (see Fig. 2). For CB the 34 kDa proform predominated with only a slight expression of the active 30 kDa form (Fig. 5a). CL expression showed a pattern most similar to day 9.5 secondary giant cells in vivo (Fig. 5b). For CD the 38 kDa and 34 kDa processed forms were not observed (Fig. 5c). Additionally, analysis of conditioned media from 48 hr outgrowths showed that trophoblast secreted the proforms of both CB (46 kDa) and CL (38 kDa), but secreted CD could not be detected (Fig. 5a–c). Analysis of EPC Outgrowths To determine if the upregulation of cathepsins associated with trophoblast differentiation in vivo could be duplicated in vitro, we analyzed protein expression in CATHEPSIN EXPRESSION IN MOUSE TROPHOBLAST Fig. 5. Western blot analysis of cathepsins B, L, and D in EPC outgrowths. Lane 1, day 7.5 EPC lysates; Lane 2, 48 hr EPC outgrowth lysates; Lane 3, EPC conditioned media. (a) CB showed an upregulation of the 34 kDa protein in outgrowths. A 46 kDa proform was detected in conditioned media. (b) CL showed an upregulation of the 38 and 29 kDa proteins in outgrowths. A 38 kDa proform was detected in conditioned media. (c) CD showed a decrease of the 60 kDa proform and a large increase in the 46 and 42 kDa proteins in outgrowths. The processed 38 kDa form was reduced. No CD was detected in conditioned media. The enzymes were localized in outgrowths using immunofluorescence. For each enzyme heterogeneous cytoplasmic granules, arranged in a perinuclear fashion, were labeled (Fig. 6a–f). Comparison with phase images suggested that these vesicles included both homogenous lysosomes and larger heterophagic vesicles. The presence of a large population of lysosomes in secondary trophoblast cells was confirmed by vital staining with acridine orange (Fig. 6g, h). DISCUSSION This work has shown that the cathepsin proteinases CB, CL, and CD are specifically upregulated in second- 379 ary trophoblast giant cells during mouse implantation and placentation. CB and CD message levels increased approximately 3-fold from days 7.5 to 11.5. Previous work (Afonso et al, 1997) suggested that CB mRNA was maintained at a constant level, however the larger samples collected for this work showed a consistent upregulation. CL message showed a biphasic pattern with the highest transcription levels observed early in the invasive period (days 7.5 to 8.5), followed by a decrease on days 9.5 and 10.5 and a slight increase on day 11.5. Previous analysis of CL message in mouse placentae from days 7 to18 did not show this biphasic pattern in early development, but did so between days 12 and 15 (Hamilton et al., 1991). In either case, the patterns do not parallel the observed protein expression, suggesting that CL regulation is complex, encompassing transcriptional and post-transcriptional controls. For both primary and secondary trophoblast, single transcripts were observed for each enzyme in the size range observed in a number of other tissue types (Barrett et al., 1986; Berquin and Sloane, 1995). Altered transcripts of CB and CL have been associated with metastatic cancer cells (Qian et al., 1989; Chauhan et al., 1993). Despite the invasive nature of mouse trophoblast these altered forms were not expressed. Protein analysis also showed an upregulation of all three enzymes during EPC development. Developmental stages containing the most invasive secondary giant cells (e.g., day 9.5) produced the highest amounts of processed or active forms, similar to the invasive day 8.5 primary giant cells. These processed proteins represent the lysosomal enzymes (Barrett et al., 1986). Like other trophoblast proteinases, such as gelatinase B (Alexander et al., 1996) and uPA (Teesalu et al., 1996), the proteins, as well as the messages, were localized to the outer, more mature trophoblast cells. Intracellularly, the enzymes were localized to numerous, heterogeneous cytoplasmic granules. Vital staining of EPC outgrowths confirmed the presence of a large population of lysosomes within these cells. Electron microscopy analyses have also detailed the accumulation of lysosome-like bodies and heterophagosomes in mouse trophoblast during invasion in vivo (Olovsson and Nilsson, 1993). The predominance of the processed and/or active forms of the enzymes within day 8.5 primary and day 9.5 secondary trophoblast most likely represents the increase in the lysosomal component of these cells. This coincides with the period of trophoblast phagocytosis of maternal blood, decidual cells, and matrix materials (Bevilacqua and Abrahamsohn, 1989). At the beginning of placental function (day 11), decreases are observed in the active or processed forms of CB and CD, coinciding with the switch to the noninvasive phenotype of the placental giant cells. Analyses of EPC cultures showed that secondary giant cells displayed some differences in processing CB and CD enzymes compared to in vivo samples. Decreased amounts of the active and/or processed forms were observed. This may be due to the absence in the 380 AFONSO ET AL. Fig. 6. Immunofluorescent localization of cathepsins B, L, and D in EPC outgrowths. Cathepsins B (a–b), L (c–d), and D (e–f) were all localized to heterogeneous cytoplasmic vesicles located in a perinuclear array in differentiated secondary giant cells. Vital staining with acridine orange (g–h) identified a similar heterogenous population of lysosomes. n, nuclei. All images at 400⫻. culture system of the maternal cells and matrix normally phagocytosed, changing lysosomal enzymatic contents or demands. These cultures did secrete proforms of CB and CL suggesting an extracellular role for these enzymes. The proforms of CB and CL have been shown to be activated by acidic extracellular microenvironments (Kakegawa et al., 1995) and by interaction with cathepsin proteinases (Wiederanders and Kirschke, 1989; McDonald and Emerick, 1995; van der Stappen et al., 1996), plasminogen activator (Dalet-Fumeron et al., 1996) and neutrophil elastase (Burnett et al., 1995). Both CB and CL are capable of digesting matrix molecules including laminin, collagen IV, and fibronectin (Lah et al., 1989; Guinec et al., 1993), all of which are found within the decidual environment (Wewer et al., 1986). CB and CL also activate other proteinases involved in matrix degradation. CB activates the metalloproteinase, stromelysin (Murphy et al., 1992) and CL activates pro-urokinase-type plasminogen activator (Goretzki et al., 1992). All of these enzymes have been shown to be produced by mouse trophoblast (Harvey et al., 1995; Alexander et al., 1996; Teesalu et al., 1996). CATHEPSIN EXPRESSION IN MOUSE TROPHOBLAST Soluble cysteine proteinases can also liberate proteolytically-active fibronectin fragments suggesting that these enzymes can activate latent proteinases from the basement membrane to initiate a novel proteolytic cascade (Guinec et al., 1993). Therefore, cysteine proteinases may contribute to invasion by the digestion of matrix molecules and the extracellular activation of other pro-enzymes, as well as through the intracellular breakdown of molecules phagocytosed by trophoblast cells. The process of embryo implantation most likely requires a complex proteinase cascade involving interactions between cysteine, serine, and metalloproteinases. The importance of individual enzymes is reflected in the implantation blocking effects in vivo of the cysteine proteinase inhibitor E-64 (Afonso et al., 1997) and the peptide hydroxamate MMP inhibitor (Alexander et al., 1996). The complexity is supported by the viability of mouse models which overexpress TIMP-1 (Alexander et al., 1996) and possess targeted mutations in urokinasetype and tissue-type plasminogen activator (Carmeliet et al., 1994). Clearly determining the regulation of the proteolytic cascade, including the interactions between multiple families of enzymes and inhibitors, will be necessary to understand the process of embryo implantation. EXPERIMENTAL PROCEDURES Materials Proteinase inhibitors, SDS-PAGE supplies, secondary antibody conjugates and in situ hybridization reagents were from Sigma (St. Louis, MO). All culture media, serum, media supplements, and agarose were from Gibco BRL (Grand Island, NY). Tri-Reagent was from Molecular Research Center, Inc. (Cincinnati, OH) and the Megaprime random priming kit was from Amersham (Arlington Heights, IL). GeneScreen Plus nylon membrane, Renaissance chemiluminescence kit, and [µ-32P]dATP were from DuPont NEN (Boston, MA). The Lowry-based DC Protein Assay kit was from BioRad (Hercules, CA) and the Western blotting medium was Immobilon-P from Millipore (Bedford, MA). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). The digoxigenin labeling and immunodetection kits were from Boehringer Mannheim (Indianapolis, IN). The DAB (3–38 diaminobenzidine) substrate kit, Vectastain ABC Elite kit and DAB enhancing solution were from Vector (Burlingame, CA). Preprocathepsin B cDNA (inserted into pGEM2 vector) was the generous gift of S.J. Chan, University of Chicago, IL. Mouse cathepsin L cDNA (inserted into the pBS KSvector) was purchased from University Technologies International, Inc. (Calgary, Alberta, Canada). The cathepsin D cDNA (inserted in Bluescript KS ⫹ vector) was from American Type Culture Collection (Rockville, MD). The IgG fraction of normal rabbit serum and the 18s rRNA probe were kindly provided by D.T. Denhardt, Rutgers University, NJ. The cathepsin B antiserum was kindly provided by B. Sloane, Wayne State University, Michigan. Cathepsin L antiserum was the generous 381 gift of R. Hamilton, Iowa State University, IA. The cathepsin D antiserum was a generous gift from D. Sensibar, Northwestern University Medical School, Illinois. Mice and Tissue Collection Naturally-mated mice (CF1, Charles River) were sacrificed on days 7.5 to11.5 of development (the morning the vaginal plug was observed was designated as day 0 of development). In all cases, the uteri were removed and transferred to sterile Dulbeccos Modified Eagles Medium (DMEM) with HEPES buffer for dissection. After removal of uterine muscle, decidual capsules were split open and the embryos and extra-embryonic material teased free. Trophoblast tissue was collected by microdissection of the EPC from day 7.5 embryos (Romagnano and Babiarz, 1990), sheets of primary trophoblast were isolated from day 8.5 implantation sites, and whole placentae collected on days 9.5 to 11.5. To produce secondary trophoblast outgrowths, day 7.5 EPC rudiments were cultured in DMEM supplemented with 10% BCS. EPCs were allowed to differentiate for 48 hrs and collected with a cell scraper. EPC conditioned media was collected from 12 to 14 EPCs cultured in a single well of a 96-well plate. After 48 hr in DMEM with 10% BCS, cultures were rinsed in DMEM and incubated an additional 8 hr in 40 µl of DMEM supplemented with 0.2% lactalbumin hydrolysate (Behrendsten et el, 1992). Media was then collected for analysis. Northern Analysis Total RNA was isolated from EPCs and placentae using Tri-Reagent following manufacturer’s protocol. Microdissected tissues were pooled from over 800 day7.5 and -8.5 embryos to produce EPC and primary trophoblast RNA samples. For placentae, 200 day-9.5, 50 day-10.5, and 20 day-11.5 samples were collected from multiple litters. Northern analysis was followed exactly from Current Protocols (Ausubel et al., 1987). Briefly, total RNA (5–10 µg) was denatured with glyoxal and dimethyl sulfoxide and separated on a 1.2% agarose gel for 3 hr. RNA was transferred to a nylon membrane via overnight capillary blotting. Membranes were prehybridized for 3–4 hr at 42°C and hybridized overnight at 42°C in prehybridization solution containing 2–3 ⫻ 106 cpm/ml of randomly primed, ␣-32P labeled cDNA probes for CB, CL, or CD. After hybridization, membranes were washed and exposed to X-ray film for 24–48 hours at ⫺70°C. Subsequently, membranes were stripped and rehybridized with the 18s rRNA loading control probe. Using the Molecular Dynamics Personal Densitometer SI, scanning densitometry was employed to determine the relative intensity of hybridization signal and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The intensities were quantified by normalizing band intensity to the 18s rRNA. All experiments were repeated in triplicate and mean intensities ⫾ the standard deviation of the mean calculated. 382 AFONSO ET AL. Western Analysis Tissue samples were pooled from multiple litters/ cultures: day 7.5 (400 EPCs); day 8.5 EPCs and primary trophoblast from over 400 embryos, day 9.5 (120); day 10.5 (50); day 11.5 (20); and EPC outgrowths (120). Tissues were incubated for 30 min at 37°C in PBS/ 0.02% NP-40 with a proteinase inhibitor cocktail (0.01% PMSF, 10 µg/ml aprotinin and 10 µg/ml soybean trypsin inhibitor), sonicated on ice, and spun at high speed to remove unsolubilized material. Total protein from each sample was determined. Conditioned medium (30 µl) was collected and added to 10 µl of 4X treatment buffer. Samples (30–40 µg total protein or 20 µl of conditioned medium) were analyzed by SDS-PAGE on 5–15% gradient gels under reducing conditions and transferred to membranes using a semi-dry blotter. Membranes were blocked with 5% Carnation milk in PBS, then incubated with either rabbit anti-mouse cathepsin B (1:2,000), rabbit anti-mouse cathepsin L (1:1,000), or rabbit antimouse CD (1:1,000) diluted in 1% Carnation milk in PBS. After washing, the membrane was incubated with goat anti-rabbit IgG-peroxidase conjugate (1:5,000). All incubations were performed for 1 hr at room temperature. Bands were visualized by chemiluminescence. Experiments were repeated 3 times. In control blots, the Ig fraction of normal rabbit serum was used in place of the primary antisera and produced non-significant background (data not shown). In Situ Hybridization The CB cDNA plasmid was linearized with NheI or SalI, the CL cDNA plasmid with BamHI or HindIII and the CD cDNA plasmid with SpeI or SalI to provide template for antisense or sense riboprobes, respectively. Linearized template (1µg) was used to synthesize SP6, T7, or T3 polymerase-directed digoxigeninlabeled riboprobes with DIG RNA labeling mix. Riboprobe integrity was checked by Northern analysis. The procedure followed was described in SchaerenWiemers and Gerfin-Moser (1993). Briefly, samples were frozen in liquid nitrogen and stored at ⫺70°C. Cryosections (15µm) were cut and mounted on SuperFrost Plus slides, fixed with cold phosphate buffered 4% paraformaldehyde for 10 min, then acetylated with freshly prepared 0.1 M triethanolamine (pH 8), 0.25% acetic anhydride for 10 min. The slides were prehybridized at room temperature for 4–6 hours in hybridization buffer (50% formamide, 5X SSC, 2% blocking solution provided by Boehringer Mannheim, 0.1% N-laurylsarcosine, 0.02% SDS, 250 µg/ml tRNA and 500 µg/ml sheared salmon sperm DNA). Hybridization mixture was prepared by adding 200 ng of DIG riboprobe per ml of hybridization solution, heating to 85°C, then chilling on ice. Sections were hybridized overnight in a moist chamber at 72°C with 200 µl of hybridization mixture per slide. Subsequent to hybridization, slides were washed with 2X SSC buffer and incubated with antidigoxigenin antibody. Labeled probe was detected using a colorimetric immunodetection kit containing NBT (4-nitroblue tetrazolium chloride) and X-phosphate (BCIP, 5-bromo-4-chloro-3-indolyl-phosphate). For control experiments, tissue sections were incubated with sense strand cRNA or without probe (data not shown). Representative samples (3 capsules) were analyzed from 3 different litters for each cathepsin. Immunolocalization To localize CB, CL, and CD, decidual capsules were fixed with 4% paraformaldehyde, embedded in paraffin according to standard protocol and cut into 7 µm sections. The immunohistochemical ABC technique was performed using an antigen retrieval step (Rempel et al., 1994). After routine deparaffinization and rehydration, tissue sections were incubated in 3% hydrogen peroxide to inactivate endogenous peroxidases. The slides were placed in 10 mM sodium citrate buffer (pH 6.0) and boiled for 5 min in a microwave oven. The buffer was changed and the slides were boiled for an additional 5 min. Slides were allowed to cool to room temperature and then rinsed in PBS. All subsequent incubations were performed at room temperature. Sections were blocked with 10% goat serum in PBS for 30 min. After brief rinsing, sections were incubated overnight in a humidified chamber with anti-CB (1:500), anti-CL (1:600), or anti-CD (1:500) diluted in PBS (Rempel et al., 1994; Hamilton et al., 1991). After washing with PBS, sections were incubated for 30 min with goat-anti-rabbit IgG-biotin conjugate (1:200 in PBS), washed and then incubated for 45 min with ABC Vectastain solution. The signal was visualized using the DAB Substrate kit for peroxidase (Hamilton et al., 1991). To enhance the signal, sections were equilibrated in 0.05 M sodium bicarbonate (pH 9.6) for 10 min and then covered with DAB enhancing solution for 10 sec followed by rinsing in water to stop the reaction. The label was visualized by bright field using a Nikon Diaphot 300 inverted microscope. For in vivo experiments, 3 representative capsules were labelled from 3 different litters for each enzyme. To localize the enzymes in EPC outgrowths, 48 hr cultures were fixed with methanol and blocked in 5% Carnation milk in PBS. Samples were incubated for 1 hr in primary antisera diluted in 1% Carnation, washed, and incubated for an additional hour in goat anti-rabbit IgGFITC conjugate. The label was visualized by epifluorescence. Negative control experiments included incubation with the IgG fraction of normal rabbit serum or secondary antibody alone. To localize lysosomes, EPC outgrowths were washed with DMEM, incubated for 5 min at 37°C in 1 µg/ml acridine orange, and returned to fresh DMEM for an additional 15 min (Chen et al., 1985). Samples were observed immediately by FITC epifluorescence. For in vitro experiments, outgrowths from 3 litters were analyzed for each enzyme. ACKNOWLEDGMENTS The authors would like to thank Drs. S. J. Chan, D. T. Denhardt, B. Sloane, R. Hamilton, and D. 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